WO2023147678A1 - Method for synthesising the compound desmethylxestospongin b (dmxeb) - Google Patents

Method for synthesising the compound desmethylxestospongin b (dmxeb) Download PDF

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WO2023147678A1
WO2023147678A1 PCT/CL2022/050013 CL2022050013W WO2023147678A1 WO 2023147678 A1 WO2023147678 A1 WO 2023147678A1 CL 2022050013 W CL2022050013 W CL 2022050013W WO 2023147678 A1 WO2023147678 A1 WO 2023147678A1
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Prior art keywords
dmxeb
compound
reacted
desmethylxestospongin
thf
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PCT/CL2022/050013
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Spanish (es)
French (fr)
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Julio César CARDENAS MATUS
Zakarian ARMEN
Masa PODUNAVAC
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Universidad Mayor
University Of California Santa Barbara
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Priority to PCT/CL2022/050013 priority Critical patent/WO2023147678A1/en
Publication of WO2023147678A1 publication Critical patent/WO2023147678A1/en

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    • 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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/537—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines spiro-condensed or forming part of bridged ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings

Definitions

  • the present invention relates to the chemical industry and a product for use in the pharmaceutical area.
  • the present invention relates to a method for obtaining a scalable synthesis of the compound desmethylxestospongin B (dmXeB), by means of a strategy that allows the preparation of xestospongins with variable stereochemistry and oxidation to C9/9'.
  • dmXeB was established to be an effective inhibitor of constitutive calcium transfer from the endoplasmic reticulum (ER) to mitochondria, and its ability to induce selective cancer cell death in a variety of cell lines, including cancer, was subsequently confirmed. metastatic, while normal cells are hardly affected.
  • Xestospongins are a group of natural compounds isolated from the marine sponge Xestospongia sp. Xestospongin B (XeB) that specifically inhibits the IP3R receptor, altering mitochondrial function, causing the selective death of cells of tumor origin.
  • XeB Xestospongin B
  • the availability of natural XeB is scarce, so the total synthesis of this compound currently appears to be a necessity.
  • said problem of the technique is solved since the natural product desmethylxestospongin B (dmXeB) is synthesized, assuming that the methyl group does not affect the inhibition of IP3R receptors and therefore, it will continue to have an antitumor effect.
  • the synthesis is based on the application of the Ireland-Claisen rearrangement, macrolactamization and, in the final part, the installation of an oxaquinolizine unit through a lactam reduction.
  • Xestospongins together with araguspongins, are a group of natural products isolated from marine sponges of Xestospongia sp. Structurally, these compounds are made up of two oxaquinolizidine units linked by saturated alkyldiene chains to form a macrocyclic nucleus, and they have variable oxidation and stereochemistry at C9/C9' as shown below.
  • IP3Rs inositol-1,4,5-triphosphate receptors
  • One of the objectives of the present patent application was to evaluate the effect of desmethylxestospongin B on mitochondrial respiration in breast cancer cell lines, and the resulting selectivity in inducing cancer cell death while leaving normal cells nearly unaffected. without affecting.
  • xestospongin B XeB
  • dmXeB desmethylxestospongin B
  • Xe C and Ar B are more accessible, they show lower specificity with notable side effects on calcium homeostasis, and no data are available for Ar C.
  • the Baldwin synthesis is based on the macrocyclic dimehzation of long-chain aliphatic 1,3-alkanols derived from pyridine, followed by reduction of the pyridine nucleus.
  • substitution at C9/9' is controlled thermodynamically by providing mixtures of stereoisomers at C9/9' (mixtures of Xe C, Xe A and Ar B).
  • No means of introducing the C9/9' hydroxyl substitution, or any other substitution at these positions, has been reported. Therefore, this approach has not provided access to XeB, dmXeB and other members of this group of natural products in higher oxidation states.
  • the highlights of the Hoye synthesis are the dimehzation of aldehydes attached to 1,3-aminoalkanols via a thiophene linker.
  • the C9/9' stereochemistry is controlled thermodynamically, providing a mixture of Xe A and Xe C. No means of introducing any substitution, including OH substitution, have been reported. Again, this approach has no demonstrable ability to provide access to XeB, dmXeB, and other members of this group of natural products in higher oxidation states.
  • Figures 1 a and 1 b show the increase in cellular calcium mediated by IP3Rs in response to 100 pM ATP in MDA-MB-231 cells treated for 1 h with ethanol (control) versus treated with 5 pM XeB ( Figure 1a). , and versus dmXeB ( Figure 1b). Mean ⁇ SEM of 3 independent experiments. In each experiment, 100 cells were analyzed. It is observed that control cells respond to ATP by increasing cell calcium from 0 to 50 (figure 1 a), and from 0 to 80 (figure 1 b), while cells subjected to XeB (figure 1 a) and dmXeB ( figure 1 b), do not show increases in calcium in response to ATP, remaining at 0. This indicates that XeB and dmXeB have effectively blocked the IP3Rs.
  • FIGS. 2a and 2b These figures show the Basal Oxygen Consumption Rate (OCR) of MDA-MB-231 ( Figure 2 a) and MCF10A ( Figure 2 b) cells incubated for 24 hours with increasing concentration of XeB (gray light) or dmXeB (dark gray). The black bar represents OCR basal cells without treatment (control). Mean ⁇ SEM (Mean Standard Error) of 3 independent experiments with 10 replicates each. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 compared to the respective control. It is observed that by increasing the concentration of XeB and dmXeB, the Oxygen Consumption Rate (OCR) decreases, indicating a reduction in cell metabolism, versus the control where no decrease is observed.
  • OCR Basal Oxygen Consumption Rate
  • Figures 3a and 3b show both cell lines, MDA-MB-231 and MCF10A (control). Both were treated with increasing concentrations of XeB (Figure 3a) and dmXeB ( Figure 3b) for 24 h. Cell death was determined by propidium iodide incorporation by flow cytometry. Mean ⁇ SEM of 3 independent experiments, each in triplicate. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, compared to the respective control. It is observed that as the concentration of both XeB ( Figure 3a) and dmXeB ( Figure 3b) increases, cell death increases significantly, especially in MDA-MB-231 cancer cells.
  • Figures 4a and 4b show 4 cell lines, MCF10A (control), BT-549, MCF-7, ZR-75-1. All were treated with increasing concentrations of XeB ( Figure 4a) and dmXeB ( Figure 4b) for 24 h. Cell death was determined by propidium iodide incorporation by flow cytometry. Mean ⁇ SEM of 3 independent experiments, each in triplicate. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, compared to the respective control. In both cases, a selective death of the cancer lines BT -549, MCF-7 and ZR-75-1 versus MCF10A (control without cancer) is observed.
  • Figure 5 These figures show a MDA-MB-Cell Migration Assay 231.
  • This figure shows photos of Plates from six experiments with MDA-MB-231 cells treated with: 0 pM (Control), 5pM and 7.5 pM XeB (left side) and 5pM and 7.5 pM dmXeB ((right side). It is observed that the migration of these cells decreases in a similar way with dmXeB and XeB.
  • Figure 6 These figures show a BT-549 Cell Migration Assay.
  • This figure shows photos of Plates from six experiments with BT-549 cells treated with: 0 pM (Control), 5pM and 7.5 pM XeB (left side), and 5pM and 7.5 pM dmXeB ((right side). It is observed that the migration of these cells decreases in a similar way with dmXeB and XeB.
  • Figure 7 show an invasion assay in a collagen matrix.
  • Groups of MDA-MB 231 tumor cells spheroids
  • spheroids were embedded in a collagen matrix and then treated for 0, 24, 48, 72 and 96h with 5 pM and 7.5 pM dmXeB or XeB.
  • the tumor cells invade the neighboring collagen matrix, which is observed as an increase in the perimeter of the spheroid. This increase in the perimeter and therefore of the invasion is inhibited by the presence of dmXeB and XeB.
  • the present invention discloses a method for the synthesis of the compound Desmethylxestospongin B, and its use as an inhibitor of tumor growth and metastasis.
  • This patent application provides a method to synthesize dmXeB (final product), for which an esterification of azidoalcohol 4 with 2-(benzyloxy ⁇ ) -5-chloropentanoic acid 5 or 5-chloropentanoic acid is performed to obtain intermediates 2 and 3 via Ireland-Claisen rearrangement to establish stereogenic centers at C9' and C9, respectively.
  • This synthesis design has the advantage of flexibility in accessing 1-oxaquinolizidine alkaloids with variable C9/9' substitution (with or without OH groups), as well as variable stereochemistry at the same positions.
  • figure 2a it may not all (S)-6 be transformed into compound 7. In that case a copper compound is used that uses this remaining (S)-6 and combines it with 8 to give 9 .
  • intermediate 15 is reacted with pyridine, CH2CI2, and 5-chloropentoyl chloride to obtain 16, then 16 is reacted with LDA, TBSCI, HMPA, THF, to obtain 17, then 17 is reacted, to obtain intermediary 3, under conditions according to the following scheme,
  • intermediates 2 and 3 are reacted with EDC, HCI, HOBT, CH2CI2, to obtain intermediate 18, then intermediate 18 is reacted with SnCh, PhSH, EtsN, CH3CN, to obtain intermediate 19, then 19 is reacted with LiOH, H2O, MeOH, THF, and then with HATU, Pr2Net, DMF, to obtain intermediate 1, then intermediate 1 is reacted with L ⁇ N(S ⁇ Me3 )2, THF, to obtain intermediate 20, then intermediate 20 is reacted, with (n-Bu)4NF, THF, and then with Li, NH3, THF, to obtain the final product dmXeB, under conditions according to the following scheme,
  • ester 16 gave carboxylic acid 17 (87% yield, 6:1 dr), which, after forming the methyl ester with MeaSiCHIX, was preempted to amino ester 3 via azide reduction with SnCh/PhSH (82% performance).
  • Fragment ligation was achieved by amide formation from acid 2 and amine 3, followed by another azide reduction under the same conditions, ester hydrolysis, and macrolactamization. At this stage, the minor diastereomers formed during ICR were separable and macrocyclic bis(lactam)1 was isolated in 35% yield as a single diastereomer.
  • the normal cell line MCF10A and a cancer cell line MDA-MB-231 were treated with increasing concentrations of dmXeB and XeB for 24 h, and oxygen consumption rates (OCR) were determined using the Seahorse system. As shown in Figures 2a and 2b, both compounds decrease OCR in a similar dose-dependent manner.
  • dmXeB induces selective cell death in breast cancer cell lines.
  • Several breast cancer cell lines representing the heterogeneity of breast cancer and the normal cell line MCF10A were treated with different concentrations of XeB or dmXeB for 24 h and cell death was measured by propidium iodide incorporation via cell cytometry. flow.
  • 7.5 pM XeB induced a significant increase in cell death in MDA-MB-231 cells, without affecting the normal MCF10A cell line.
  • XeB is still more effective in MDA-MB-231 cells, but some effects in MCF10A cells become measurable, indicating reduced selectivity.
  • dmXeB indeed shows some increased selectivity, inducing cell death in MDA-MB-231 at 5 pM, which is maintained at 10 pM without any effect on normal MCF10 cells ( Figures 3a and 3b).
  • MDA-MB-231 cells are even more sensitive, with almost 100% of the population killed, while MCF10A shows an initial increase in cell death.
  • representative cell lines of the luminal A (MCF-7) and luminal B (ZR-75-1) breast cancer cell lines XeB and dmXeB show selective cell death at concentrations between 5 and 10 pM. Similar to observations with the MDA-MB-231 cell line, dmXeB at a concentration of 20 pM caused almost complete cell death. in cell lines ZR-75-1 and MCF-7, while it also affected the normal cell line MCF10A to a much lesser extent. BT-549 cells are more resistant to both compounds, showing only selectivity at 7.5 pM.

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Abstract

The present invention relates to a method for synthesising the compound desmethylxestospongin B (dmXeB), wherein an esterification of azidoalcohol 4 with 2-(benzyloxy)-5-chloropentanoic acid 5 or 5-chloropentanoic acid is performed to obtain intermediates 2 and 3 via Ireland-Claisen rearrangement to establish stereogenic centres at C9' and C9, respectively; next, ω-amino acid precursors 2 and 3 are combined by sequential amide formation to obtain bis-lactam-1; and subsequently, by means of final hydrogenation of the double bonds of bis-lactam-1 in ethyl acetate, the compound dmXeB is obtained.

Description

Método para sintetizar el compuesto desmetilxestospongina B (dmXeB). Method for synthesizing the compound desmethylxestospongin B (dmXeB).
CAMPO DE LA INVENCIÓN FIELD OF THE INVENTION
La presente invención se relaciona con la industria química y un producto para uso en el área farmacéutica. En particular, la presente invención se relaciona con un método para obtener una síntesis escalable del compuesto desmetilxestospongina B (dmXeB), mediante una estrategia que permite la preparación de xestoesponginas con estereoquímica variable y oxidación a C9/9'. Además, se estableció que dmXeB es un inhibidor efectivo de la transferencia constitutiva de calcio desde el retículo endoplasmático (ER) a mitocondrias, y posteriormente se confirmó su capacidad para inducir la muerte selectiva de células cancerosas en una variedad de líneas celulares, incluido el cáncer metastásico, mientras que las células normales casi no se ven afectadas. The present invention relates to the chemical industry and a product for use in the pharmaceutical area. In particular, the present invention relates to a method for obtaining a scalable synthesis of the compound desmethylxestospongin B (dmXeB), by means of a strategy that allows the preparation of xestospongins with variable stereochemistry and oxidation to C9/9'. Furthermore, dmXeB was established to be an effective inhibitor of constitutive calcium transfer from the endoplasmic reticulum (ER) to mitochondria, and its ability to induce selective cancer cell death in a variety of cell lines, including cancer, was subsequently confirmed. metastatic, while normal cells are hardly affected.
ESTADO DEL ARTE STATE OF THE ART
Las Xestosponginas son un grupo de compuestos naturales aislados de la esponga marina Xestospongia sp. Xestospongina B (XeB) que inhibe al receptor a IP3R en forma específica, alterando la función mitocondrial, causando la muerte selectiva de células de origen tumoral. La disponibilidad de XeB natural es escasa por lo que la síntesis total de este compuesto aparece actualmente como una necesidad. Mediante la presente invención se soluciona dicho problema de la técnica ya que se sintetiza el producto natural desmetilxestospongina B (dmXeB), asumiendo que el grupo metilo no afecta la inhibición a receptores de IP3R y por lo tanto, seguirá teniendo un efecto antitumoral. La síntesis está basada en la aplicación de la reorganización de Ireland-Claisen, macrolactamización y en la parte final, la instalación de una unidad de oxaquinolizina a través de una reducción de lactam. Xestospongins are a group of natural compounds isolated from the marine sponge Xestospongia sp. Xestospongin B (XeB) that specifically inhibits the IP3R receptor, altering mitochondrial function, causing the selective death of cells of tumor origin. The availability of natural XeB is scarce, so the total synthesis of this compound currently appears to be a necessity. By means of the present invention, said problem of the technique is solved since the natural product desmethylxestospongin B (dmXeB) is synthesized, assuming that the methyl group does not affect the inhibition of IP3R receptors and therefore, it will continue to have an antitumor effect. The synthesis is based on the application of the Ireland-Claisen rearrangement, macrolactamization and, in the final part, the installation of an oxaquinolizine unit through a lactam reduction.
Las xestoesponginas, junto con las aragusponginas, son un grupo de productos naturales aislados de esponjas marinas de Xestospongia sp. Estructuralmente, estos compuestos están formados por dos unidades de oxaquinolizidina atadas por cadenas saturadas de alquildieno para formar un núcleo macrocíclico, y tienen oxidación variable y estereoquímica en C9/C9' como se muestra a continuación.
Figure imgf000003_0001
Figure imgf000003_0002
Xestospongins, together with araguspongins, are a group of natural products isolated from marine sponges of Xestospongia sp. Structurally, these compounds are made up of two oxaquinolizidine units linked by saturated alkyldiene chains to form a macrocyclic nucleus, and they have variable oxidation and stereochemistry at C9/C9' as shown below.
Figure imgf000003_0001
Figure imgf000003_0002
Si bien se informó un rango de actividad biológica para las xestoesponjas, a los investigadores de la presente invención les intrigó la creciente evidencia de la capacidad única de la xestoespongina B para influir en el metabolismo mitocondrial mediante la modulación de la señalización de calcio entre el retículo endoplásmico (ER) y las mitocondñas a través de la inhibición de los receptores de inositol-1 ,4,5-trifosfato (IP3Rs). La actividad constitutiva de los IP3R es esencial para la bioenergética celular en una variedad de tipos de células. Su inhibición interrumpe la transferencia constitutiva de calcio de ER a mitocondria provocando una caída en la respiración mitocondrial que genera una crisis bioenergética caracterizada por la activación de AMPK y autofagia. Se ha demostrado que la interrupción de esta comunicación conduce a una muerte selectiva y sustancial de las células cancerosas que no afecta a las células normales. Although a range of biological activity has been reported for xestosponges, investigators of the present invention were intrigued by the growing evidence for xestospongin B's unique ability to influence mitochondrial metabolism by modulating calcium signaling between the reticulum. endoplasmic (ER) and mitochondria through inhibition of inositol-1,4,5-triphosphate receptors (IP3Rs). The constitutive activity of IP3Rs is essential for cellular bioenergetics in a variety of cell types. Its inhibition interrupts the constitutive transfer of calcium from the ER to the mitochondria, causing a drop in mitochondrial respiration that generates a bioenergetic crisis characterized by AMPK activation and autophagy. Disruption of this communication has been shown to lead to selective and substantial death of cancer cells that spares normal cells.
Uno de los objetivos de la presente solicitud de patente fue evaluar el efecto de la desmetilxestoespongina B en la respiración mitocondrial en líneas celulares de cáncer de mama, y la selectividad resultante en la inducción de la muerte de las células cancerosas dejando a las células normales casi sin afectar. Mientras que la xestoespongina B (XeB) ya no está disponible en las fuentes naturales o presenta gran dificultad para ser obtenido desde fuentes naturales, la síntesis artificial proporciona una fuente factible de material. Los investigadores de la presente invención consideraron que la desmetilxestoespongina B (dmXeB), es un producto natural dentro de esta familia de metabolites, basándose en la premisa de que el grupo 3'-metilo no tiene ningún efecto sobre la actividad inhibitoria de IP3R, y obviar la necesidad de instalarlo simplificaría el desarrollo de una síntesis escalable. Si bien Xe C y Ar B son más accesibles, muestran una menor especificidad con efectos secundarios notables sobre la homeostasis del calcio, y no hay datos disponibles para Ar C. One of the objectives of the present patent application was to evaluate the effect of desmethylxestospongin B on mitochondrial respiration in breast cancer cell lines, and the resulting selectivity in inducing cancer cell death while leaving normal cells nearly unaffected. without affecting. While xestospongin B (XeB) is no longer available from natural sources or presents great difficulty in obtaining it from natural sources, artificial synthesis provides a feasible source of material. Investigators of the present invention considered desmethylxestospongin B (dmXeB) to be a natural product within this family of metabolites, based on the premise that the 3'-methyl group has no effect on IP3R inhibitory activity, and obviating the need to install it would simplify the development of a scalable synthesis. Although Xe C and Ar B are more accessible, they show lower specificity with notable side effects on calcium homeostasis, and no data are available for Ar C.
En el estado del arte existe la solicitud de patente WO9704783 que divulga “Alcaloides de bis-1 -oxaquinolizidina de una esponja marina con actividad antitumoraf’ , este documento describe compuestos de origen natural, marino y sus usos como agente anti-tumores. Excluye explícitamente Desmethylxestospongin B.
Figure imgf000004_0001
In the state of the art there is patent application WO9704783 that discloses "Bis-1 -oxaquinolizidine alkaloids from a marine sponge with antitumor activity", this document describes compounds of natural marine origin and their uses as antitumor agents. Explicitly excludes Desmethylxestospongin B.
Figure imgf000004_0001
Por otro lado, en el estado del arte hay documentos que mencionan la síntesis para Xestospongins y Araguspongines como por ejemplo E. Baldwin, A. Melman, V. Lee, C. R. Firkin, R. C. Whitehead, J. Am. Chem. Soc. 1998, 120, 8559-8560, así como en el documento T. R. Hoye, J. T. North, L. J. Yao, J. Am. Chem. Soc. 1994, 1 16, 2617-2618. b) T. R. Hoye, Z. Ye, L. J. Yao, J. T. North, J. Am. Chem. Soc. 1996, 1 18, 12074-12081 .Hoye (1994, 1996). On the other hand, in the state of the art there are documents that mention the synthesis for Xestospongins and Araguspongines, such as E. Baldwin, A. Melman, V. Lee, C. R. Firkin, R. C. Whitehead, J. Am. Chem. Soc. 1998, 120, 8559-8560, as well as in T.R. Hoye, J.T. North, L.J. Yao, J.Am. Chem. Soc. 1994, 1 16, 2617-2618. b) T. R. Hoye, Z. Ye, L. J. Yao, J. T. North, J. Am. Chem. Soc. 1996, 1 18, 12074-12081. Hoye (1994, 1996).
Sin embargo, ninguno de ellos interfiere con la presente invención ya que la distinción clave entre los enfoques es la capacidad de controlar la estereoquímica y la sustitución en las posiciones C9/9' que constituyen las diferencias en varios miembros de esta familia de productos naturales. Los enfoques de Baldwin y Hoye no permiten el control en las posiciones C9/9', mientras que la síntesis de Zakarian (Podunavac) permite el control total de la estereoquímica y la sustitución en estas posiciones con una amplia gama de sustituyentes. However, none of them interfere with the present invention as the key distinction between the approaches is the ability to control the stereochemistry and substitution at the C9/9' positions that constitute the differences in various members of this family of natural products. The Baldwin and Hoye approaches do not allow control at the C9/9' positions, while the Zakarian (Podunavac) synthesis allows full control of the stereochemistry and substitution at these positions with a wide range of substituents.
Es así que la síntesis de Baldwin se basa en la dimehzación macrocíclica de 1 ,3- alcanoles alifáticos de cadena larga derivados de piridina, seguida de la reducción del núcleo de piridina. Aunque es breve y eficiente, la sustitución en C9/9' se controla termodinámicamente proporcionando mezclas de estereoisómeros en C9/9' (mezclas de Xe C, Xe A y Ar B). No se ha informado de ningún medio de introducir la sustitución de hidroxilo en C9/9', o cualquier otra sustitución en estas posiciones. Por lo tanto, este enfoque no ha proporcionado acceso a XeB, dmXeB y otros miembros de este grupo de productos naturales en estados de oxidación más altos. Thus, the Baldwin synthesis is based on the macrocyclic dimehzation of long-chain aliphatic 1,3-alkanols derived from pyridine, followed by reduction of the pyridine nucleus. Although brief and efficient, substitution at C9/9' is controlled thermodynamically by providing mixtures of stereoisomers at C9/9' (mixtures of Xe C, Xe A and Ar B). No means of introducing the C9/9' hydroxyl substitution, or any other substitution at these positions, has been reported. Therefore, this approach has not provided access to XeB, dmXeB and other members of this group of natural products in higher oxidation states.
Por otro lado, los aspectos más destacados de la síntesis de Hoye son la dimehzación de aldehidos unidos a 1 ,3-aminoalcanoles mediante un enlazador de tiofeno. Al igual que en la síntesis de Baldwin, la estereoquímica de C9/9' se controla termodinámicamente, proporcionando una mezcla de Xe A y Xe C. No se han informado medios de introducir ninguna sustitución, incluida la sustitución OH. Nuevamente, este enfoque no tiene capacidad demostrable para proporcionar acceso a XeB, dmXeB y otros miembros de este grupo de productos naturales en estados de oxidación más altos. On the other hand, the highlights of the Hoye synthesis are the dimehzation of aldehydes attached to 1,3-aminoalkanols via a thiophene linker. As in the Baldwin synthesis, the C9/9' stereochemistry is controlled thermodynamically, providing a mixture of Xe A and Xe C. No means of introducing any substitution, including OH substitution, have been reported. Again, this approach has no demonstrable ability to provide access to XeB, dmXeB, and other members of this group of natural products in higher oxidation states.
A diferencia de la síntesis de Hoye, el documento Masa Podunavac, Artur K. Mailyan, Jeffrey J. Jackson, Alenka Lovy, Paula Farias, Hernán Huerta, Jordi Molgó, César Cárdenas, Armen Zakarian, WILEY-VCH,” Scalable Total Synthesis, IP3R Inhibitory Activity of Desmethylxestospongin B, and Effect on Mitochondrial Function and Cancer Cell Survival’, Angew. Chem. Int. Ed. 2021 , 60, 1-6, (divulgación inocua de la presente solicitud de patente), describe una estrategia de macrociclización basada en el acoplamiento unitario por formación de amida, seguido de una reacción de macrociclización separada. Esto permite la síntesis simple de miembros asimétricamente sustituidos de esta clase de productos naturales, como XeB, dmXeB, Ar C y todos los demás miembros. El reordenamiento de Ireland-Claisen establece la estereoquímica y la sustitución en C9/9' con alta selectividad, que es otra distinción clave de las síntesis Baldwin y Hoye. Este enfoque ha demostrado un control total de la estereoquímica y la sustitución en estas posiciones cruciales, incluido el acceso a análogos no naturales, como los compuestos fluoro-sustituidos de esta clase. Unlike Hoye's synthesis, the paper Masa Podunavac, Artur K. Mailyan, Jeffrey J. Jackson, Alenka Lovy, Paula Farias, Hernán Huerta, Jordi Molgó, César Cárdenas, Armen Zakarian, WILEY-VCH," Scalable Total Synthesis, IP3R Inhibitory Activity of Desmethylxestospongin B, and Effect on Mitochondrial Function and Cancer Cell Survival', Angew. Chem. Int. Ed. 2021 , 60, 1-6, (innocuous disclosure of the present patent application), describes a macrocyclization strategy based on unitary coupling by amide formation, followed by a separate macrocyclization reaction. This allows for the simple synthesis of asymmetrically substituted members of this class of natural products, such as XeB, dmXeB, Ar C and all other members. The Ireland-Claisen rearrangement establishes stereochemistry and substitution at C9/9' with high selectivity, which is another key distinction from the Baldwin and Hoye syntheses. This approach has demonstrated full control of stereochemistry and substitution at these crucial positions, including access to unnatural analogues, such as fluoro-substituted compounds of this class.
Las síntesis anteriores en esta área por Baldwin y Hoye se caracterizan por una notable brevedad y elegancia estratégica, apuntando a C2-simétricos, congéneres no hidroxilados como la araguspongina B (Ar B), y Xe C y A, y resolviendo la controversia sobre la configuración absoluta de estos compuestos. Sin embargo, la simetría simplificadora de C2 se rompe en XeB y dmXeBiológicamente por la presencia de 9-OH y, en el primer caso, los grupos 3’-CH3. Previous syntheses in this area by Baldwin and Hoye are characterized by remarkable brevity and strategic elegance, targeting C2-symmetric, non-hydroxylated congeners such as araguspongin B (Ar B), and Xe C and A, and resolving the controversy over the absolute configuration of these compounds. However, the simplifying symmetry of C2 is broken in XeB and dmXe Biologically by the presence of 9-OH and, in the former case, 3'-CH 3 groups.
En resumen, las síntesis previas de Baldwin (1998) y Hoye (1994, 1996) no entregan, producen ni generan el compuesto desmetilxestospongina B (dmXeB) tal como se divulga en la presente invención. In summary, previous syntheses by Baldwin (1998) and Hoye (1994, 1996) do not deliver, produce, or generate the compound desmethylxestospongin B (dmXeB) as disclosed herein.
DESCRIPCIÓN DE LAS FIGURAS DESCRIPTION OF THE FIGURES
Figuras 1 a y 1 b: Estas figuras muestran el aumento de calcio celular mediado por IP3Rs en respuesta a 100 pM de ATP en células MDA-MB-231 tratadas durante 1 h con etanol (control) versus tratadas con 5 pM XeB (figura 1a), y versus dmXeB (figura 1 b). Media ± SEM de 3 experimentos independientes. En cada experimento se analizaron 100 células. Se observa que las células control, responden a ATP aumentando el calcio celular de 0 a 50 (figura 1 a), y de 0 a 80 (figura 1 b), en cambio las células sometidas a XeB (figura 1 a) y dmXeB (figura 1 b), no muestran incrementos en calcio en respuesta a ATP, permaneciendo en 0. Esto indica que XeB y dmXeB han bloqueado en forma efectiva los IP3Rs.Figures 1 a and 1 b: These figures show the increase in cellular calcium mediated by IP3Rs in response to 100 pM ATP in MDA-MB-231 cells treated for 1 h with ethanol (control) versus treated with 5 pM XeB (Figure 1a). , and versus dmXeB (Figure 1b). Mean ± SEM of 3 independent experiments. In each experiment, 100 cells were analyzed. It is observed that control cells respond to ATP by increasing cell calcium from 0 to 50 (figure 1 a), and from 0 to 80 (figure 1 b), while cells subjected to XeB (figure 1 a) and dmXeB ( figure 1 b), do not show increases in calcium in response to ATP, remaining at 0. This indicates that XeB and dmXeB have effectively blocked the IP3Rs.
En la figura AF/F = Cambio de intensidad / Intensidad original antes de la estimulación y AU = Unidades Arbitrarias. Figuras 2a y 2b: Estas figuras muestran la Tasa de consumo basal de oxígeno (OCR) de las células MDA-MB-231 (figura 2 a) y MCF10A (figura 2 b) incubadas durante 24 horas con una concentración creciente de XeB (gris claro) o dmXeB (gris oscuro). La barra negra representa las células básales OCR sin tratamiento (control). Promedio ± SEM (Error Promedio Estándar) de 3 experimentos independientes con 10 réplicas cada uno. *p < 0,05, **p < 0,01 , ***p < 0,001 en comparación con el control respectivo. Se observa que al aumentar la concentración de XeB y dmXeB, la Tasa de consumo de Oxígeno (OCR) disminuye, indicando una reducción del metabolismo celular, versus el control en donde no se observa disminución. In the figure AF/F = Change in intensity / Original intensity before stimulation and AU = Arbitrary Units. Figures 2a and 2b: These figures show the Basal Oxygen Consumption Rate (OCR) of MDA-MB-231 (Figure 2 a) and MCF10A (Figure 2 b) cells incubated for 24 hours with increasing concentration of XeB (gray light) or dmXeB (dark gray). The black bar represents OCR basal cells without treatment (control). Mean ± SEM (Mean Standard Error) of 3 independent experiments with 10 replicates each. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the respective control. It is observed that by increasing the concentration of XeB and dmXeB, the Oxygen Consumption Rate (OCR) decreases, indicating a reduction in cell metabolism, versus the control where no decrease is observed.
Figuras 3a y 3b: Estas figuras muestran ambas líneas celulares, MDA-MB-231 y MCF10A (control). Ambas fueron tratadas con concentraciones crecientes de XeB (figura 3a) y dmXeB (figura 3b) durante 24 h. La muerte celular se determinó mediante la incorporación de yoduro de propidio por citometría de flujo. Media ± SEM de 3 experimentos independientes, cada uno por triplicado. *p < 0,05, **p < 0,01 , ***p < 0,001 , en comparación con el control respectivo. Se observa que en la medida que aumenta la concentración tanto de XeB (figura 3a) y dmXeB (figura 3b) la muerte celular aumenta en forma significativa, especialmente en las células cancerígenas MDA-MB-231 . Figures 3a and 3b: These figures show both cell lines, MDA-MB-231 and MCF10A (control). Both were treated with increasing concentrations of XeB (Figure 3a) and dmXeB (Figure 3b) for 24 h. Cell death was determined by propidium iodide incorporation by flow cytometry. Mean ± SEM of 3 independent experiments, each in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, compared to the respective control. It is observed that as the concentration of both XeB (Figure 3a) and dmXeB (Figure 3b) increases, cell death increases significantly, especially in MDA-MB-231 cancer cells.
Figuras 4a y 4b: Estas figuras muestran 4 líneas celulares, MCF10A (control), BT-549, MCF-7, ZR-75-1 . Todas fueron tratadas con concentraciones crecientes de XeB (figura 4a) y dmXeB (figura 4b) durante 24 h. La muerte celular se determinó mediante la incorporación de yoduro de propidio por citometría de flujo. Media ± SEM de 3 experimentos independientes, cada uno por triplicado. *p < 0,05, **p < 0,01 , ***p < 0,001 , en comparación con el control respectivo. Se observa en ambos casos, una muerte selectiva de las líneas cancerosas BT -549, MCF-7 y ZR-75-1 versus MCF10A (control sin cáncer). Figures 4a and 4b: These figures show 4 cell lines, MCF10A (control), BT-549, MCF-7, ZR-75-1. All were treated with increasing concentrations of XeB (Figure 4a) and dmXeB (Figure 4b) for 24 h. Cell death was determined by propidium iodide incorporation by flow cytometry. Mean ± SEM of 3 independent experiments, each in triplicate. *p < 0.05, **p < 0.01, ***p < 0.001, compared to the respective control. In both cases, a selective death of the cancer lines BT -549, MCF-7 and ZR-75-1 versus MCF10A (control without cancer) is observed.
Figura 5: Estas figuras muestran un Ensayo de Migración de células MDA-MB- 231. Figure 5: These figures show a MDA-MB-Cell Migration Assay 231.
Esta figura muestra fotos de Placas de seis experimentos con células MDA-MB- 231 tratadas con: 0 pM (Control), 5pM y 7,5 pM de XeB (lado izquierdo) y 5pM y 7,5 pM de dmXeB ((lado derecho). Se observa que la migración de estas células disminuye en forma similar con dmXeB y XeB. This figure shows photos of Plates from six experiments with MDA-MB-231 cells treated with: 0 pM (Control), 5pM and 7.5 pM XeB (left side) and 5pM and 7.5 pM dmXeB ((right side). It is observed that the migration of these cells decreases in a similar way with dmXeB and XeB.
Figura 6: Estas figuras muestran un Ensayo de Migración de células BT-549.Figure 6: These figures show a BT-549 Cell Migration Assay.
Esta figura muestra fotos de Placas de seis experimentos con células BT-549 tratadas con: 0 pM (Control), 5pM y 7,5 pM de XeB (lado izquierdo) y 5pM y 7,5 pM de dmXeB ((lado derecho). Se observa que la migración de estas células disminuye en forma similar con dmXeB y XeB. This figure shows photos of Plates from six experiments with BT-549 cells treated with: 0 pM (Control), 5pM and 7.5 pM XeB (left side), and 5pM and 7.5 pM dmXeB ((right side). It is observed that the migration of these cells decreases in a similar way with dmXeB and XeB.
Figura 7: Estas figuras muestran un ensayo de invasión en una matriz de colágeno. Los grupos de células tumorales (esferoides) MDA-MB 231 fueron embebidos en una matriz de colágeno y luego tratados por 0, 24, 48, 72 y 96h con 5 pM y 7,5 pM dmXeB o XeB. En la situación control se observa que las células tumorales invaden la matriz de colágeno vecina, lo que se observa como un aumento del perímetro del esferoide. Este aumento del perímetro y por tanto de la invasión se ve inhibido por la presencia de dmXeB y XeB. Figure 7: These figures show an invasion assay in a collagen matrix. Groups of MDA-MB 231 tumor cells (spheroids) were embedded in a collagen matrix and then treated for 0, 24, 48, 72 and 96h with 5 pM and 7.5 pM dmXeB or XeB. In the control situation, it is observed that the tumor cells invade the neighboring collagen matrix, which is observed as an increase in the perimeter of the spheroid. This increase in the perimeter and therefore of the invasion is inhibited by the presence of dmXeB and XeB.
GLOSARIO GLOSSARY
Para mayor claridad de la invención, los siguientes términos se deben entender como sigue, además se presenta la traducción al idioma español de términos usados en los esquemas los mismos que están presentados en inglés:
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
For greater clarity of the invention, the following terms should be understood as follows, in addition, the translation into Spanish of terms used in the schemes that are presented in English is presented:
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
DESCRIPCIÓN DE LA INVENCIÓN DESCRIPTION OF THE INVENTION
La presente invención divulga un método para la síntesis del compuesto Desmetilxestospongina B, y su uso como inhibidor del crecimiento tumoral y la metástasis. The present invention discloses a method for the synthesis of the compound Desmethylxestospongin B, and its use as an inhibitor of tumor growth and metastasis.
En adelante, se entenderá en forma general que: Las temperaturas indicadas en cualquier esquema y/o procedimiento son de referencia, permitiendo un rango de temperatura de ± 20 °C, lo que tendrá un impacto mínimo en los resultados de las reacciones. Así mismo se permite utilizar solventes alternativos y seguir siendo eficaces para todas las reacciones. También se puede utilizar una gama de agentes secantes, reactivos cromatográficos y agentes de extinción y tratamiento de reacción. Estos incluyen una variedad de sales anhidras en lugar de sulfato de sodio, alternativas al gel de sílice en varias formas y una serie de ácidos, bases, agentes reductores y oxidantes y agentes complejantes de metales para terminar la reacción. Los ejemplos de aplicación se muestran en los esquemas que se citan a continuación. From now on, it will be understood in a general way that: The temperatures indicated in any scheme and/or procedure are reference, allowing a temperature range of ± 20 °C, which will have a minimal impact on the results of the reactions. Likewise, it is allowed to use alternative solvents and continue to be effective for all reactions. A range of drying agents, chromatographic reagents, and reaction quenching and treatment agents may also be used. These include a variety of anhydrous salts in lieu of sodium sulfate, silica gel alternatives in various forms, and a number of acids, bases, reducing and oxidizing agents, and metal complexing agents to terminate the reaction. Application examples are shown in the diagrams below.
En la presente invención se previo que el ensamblaje final de las unidades de 1 - oxaquinolizidina tuviera lugar mediante N-alquilación intramolecular de los grupos amida en bis (lactama) 1 macrocíclico seguido de semireducción de lactama y formación de hemiaminal. In the present invention it was envisioned that the final assembly of the 1-oxaquinolizidine units would take place by intramolecular N-alkylation of the amide groups on macrocyclic bis(lactam)1 followed by lactam half-reduction and hemiaminal formation.
La presente solicitud de patente provee un método para sintetizar dmXeB (producto final), para ello se realiza una esterificación de azidoalcohol 4 con ácido 2-(bencilox¡) -5-cloropentanoico 5 o ácido 5-cloropentanoico para obtener los intermediarios 2 y 3 a través de la transposición de Irlanda-Claisen para establecer centros estereogénicos en C9' y C9, respectivamente. This patent application provides a method to synthesize dmXeB (final product), for which an esterification of azidoalcohol 4 with 2-(benzyloxy¡) -5-chloropentanoic acid 5 or 5-chloropentanoic acid is performed to obtain intermediates 2 and 3 via Ireland-Claisen rearrangement to establish stereogenic centers at C9' and C9, respectively.
Luego se combinan los precursores de cu-aminoácidos, 2 y 3 mediante formación secuencia! de amidas para obtener bis (lactama) 1. The cu-amino acid precursors, 2 and 3 are then combined by sequence formation! of amides to obtain bis(lactam) 1.
Finalmente, mediante la hidrogenación final de los dobles enlaces de bis (lactama) 1 en acetato de etilo se obtiene el compuesto dmXeB, objeto de la presente invención. El diseño de síntesis se representa en el Esquema 1 .
Figure imgf000013_0001
Finally, through the final hydrogenation of the bis double bonds (lactam) 1 in ethyl acetate the dmXeB compound, object of the present invention, is obtained. The synthesis design is depicted in Scheme 1.
Figure imgf000013_0001
Esquema 1 . Diseño de síntesis para dmXeB. Scheme 1 . Synthesis design for dmXeB.
Este diseño de síntesis tiene la ventaja de la flexibilidad en el acceso a alcaloides de 1 -oxaquinolizidina con sustitución variable en C9/9' (con o sin grupos OH), así como estereoquímica variable en las mismas posiciones. This synthesis design has the advantage of flexibility in accessing 1-oxaquinolizidine alkaloids with variable C9/9' substitution (with or without OH groups), as well as variable stereochemistry at the same positions.
El 1 -cloro-3-buteno sirvió como punto de partida para la síntesis de 4 (Esquema 2a). Resolución cinética hidrolítica de Jacobsen de óxido de 1 -clorobuteno, obtenido por epoxidación con MCPBA, entregó (S)-6 con un rendimiento del 58% y un exceso enantioméñco (ee) del 92%. El epóxido se distribuyó de forma divergente, esto significa que hay dos líneas de generación de compuestos, una que reacciona y genera 7 y otra que no reacciona y que después se trata con cuprato para acoplar (S)-6 y el compuesto 8 como se menciona más adelante, avanzando parte de él a alcohol alílico 7 con iluro de dimetilmetilenosulfuro. Después de la parametoxibencilación reductora (PMB) en condiciones electrofílicas y el desplazamiento del grupo cloro, se obtuvo el yoduro 8 con un rendimiento global del 71 %. Se requirió el desarrollo de la bencilación electrófila debido a la propensión del 7 a sufrir eterificación intramolecular durante la formación de alcóxido bajo varias condiciones de reacción. El acoplamiento de cuprato con el resto de epóxido de cloro (S) -6 con el reactivo formado a partir de 8 se logró con un rendimiento del 78%. La sustitución posterior de cloruro con azida sódica, O-sililación y eliminación del grupo PMB proporcionó 58,1 gramos de 4 con un rendimiento del 87%.
Figure imgf000014_0001
1 -chloro-3-butene served as the starting point for the synthesis of 4 (Scheme 2a). Jacobsen hydrolytic kinetic resolution of 1-chlorobutene oxide, obtained by epoxidation with MCPBA, gave (S)-6 in 58% yield and 92% enantiochemical excess (ee). The epoxide was distributed in a divergent way, this means that there are two lines of generation of compounds, one that reacts and generates 7 and another that does not react and is then treated with cuprate to couple (S)-6 and compound 8 as shown. mentioned below, advancing part of it to allylic alcohol 7 with dimethylmethylenesulfide ylide. After reductive paramethoxybenzylation (PMB) under electrophilic conditions and displacement of the chlorine group, iodide 8 was obtained in an overall yield of 71%. The development of electrophilic benzylation was required due to the propensity of 7 to undergo intramolecular etherification during alkoxide formation under various reaction conditions. Coupling of cuprate with the chloro(S)-6 epoxide moiety with the reagent formed from 8 was achieved in 78% yield. Subsequent substitution of chloride with sodium azide, O-silylation, and removal of the PMB group provided 58.1 grams of 4 in 87% yield.
Figure imgf000014_0001
Esquema 2a. Preparación del intermediario 4. Scheme 2a. Preparation of the intermediary 4.
De acuerdo a la presente invención figura 2a puede que no todo (S)-6 se transforme en el compuesto 7. En ese caso se utiliza un compuesto de cobre que usa este (S)-6 restante y lo combina con 8 para dar 9. According to the present invention figure 2a it may not all (S)-6 be transformed into compound 7. In that case a copper compound is used that uses this remaining (S)-6 and combines it with 8 to give 9 .
Figure imgf000015_0001
Figure imgf000015_0001
Esquema 2b. Preparación del intermediario 14. Scheme 2b. Preparation of the intermediary 14.
Para acceder a congéneres no simétricos tales como dmXeB que poseen un grupo hidroxi C9, se requirió la síntesis de un cloruro de a-benciloxi acilo 14. To access non-symmetrical congeners such as dmXeB possessing a C9 hydroxy group, the synthesis of an α-benzyloxy acyl 14 chloride was required.
Debido a la inestabilidad térmica de los ésteres enolatos generados a partir de los a-alcoxiésteres, la alquilación del a-benciloxiacetato de t-butilo con yoduro 10 se logró a -95 °C con un rendimiento moderado del 45% en condiciones de reacción optimizadas. Después de la desililación, mesilación y sustitución con LiCI, se obtuvo el éster 13 con un rendimiento del 86%. Due to the thermal instability of the enolate esters generated from the a-alkoxyesters, the alkylation of t-butyl a-benzyloxyacetate with iodide 10 was achieved at -95 °C with a moderate yield of 45% under optimized reaction conditions. . After desilylation, mesylation and substitution with LiCI, ester 13 was obtained in 86% yield.
La escisión del éster t-butílico y la clorodeshidroxilación con cloruro de oxalilo completaron la síntesis del cloruro de acilo 14 (rendimiento del 93%, 10,2 gramos). Cleavage of the t-butyl ester and chlorodehydroxylation with oxalyl chloride completed the synthesis of 14-acyl chloride (93% yield, 10.2 grams).
El intermediario 14, se hace reaccionar con piridina y CH2Cl2(diclorometano) para obtener el éster 15, y luego, se hace reaccionar el éster 15 con KN(S¡Me3)2, MeaSiCI, y PhMe, en reacción específica para a-alcox¡ ésteres (según paper Podunavac, M.; Lachahty, J. J.; Jones, K. E.; Zakahan, A. Org. Lett. 2018, 20, 4867-4870), para obtener el intermediario 2, según el siguiente esquema 3a,
Figure imgf000016_0001
Intermediate 14 is reacted with pyridine and CH2Cl2(dichloromethane) to obtain ester 15, and then ester 15 is reacted with KN(S¡Me3)2, MeaSiCI, and PhMe, in a specific reaction for a-alcox ¡ esters (according to paper Podunavac, M.; Lachahty, JJ; Jones, KE; Zakahan, A. Org. Lett. 2018, 20, 4867-4870), to obtain intermediate 2, according to the following scheme 3a,
Figure imgf000016_0001
Esquema 3a. Unión de fragmentos y síntesis del compuesto 2 Scheme 3a. Union of fragments and synthesis of compound 2
Para obtener el intermediario 3 se hace reaccionar el intermediario 15 con piridina, CH2CI2, y cloruro 5-cloropentoil para obtener 16, luego se hace reaccionar 16 con LDA, TBSCI, HMPA, THF, para obtener 17, luego se hace reaccionar 17, para obtener el intermediario 3, en condiciones según el siguiente esquema,
Figure imgf000016_0002
To obtain intermediate 3, intermediate 15 is reacted with pyridine, CH2CI2, and 5-chloropentoyl chloride to obtain 16, then 16 is reacted with LDA, TBSCI, HMPA, THF, to obtain 17, then 17 is reacted, to obtain obtain intermediary 3, under conditions according to the following scheme,
Figure imgf000016_0002
Esquema 3b. Unión de fragmentos y síntesis del compuesto 3 Scheme 3b. Union of fragments and synthesis of compound 3
Finalmente, para obtener el dmXeB, se hace reaccionar los intermediarios 2 y 3, con EDC, HCI, HOBT, CH2CI2, para obtener el intermediario 18, luego se hace reaccionar el intermediario 18, con SnCh, PhSH, EtsN, CH3CN, para obtener el intermediario 19, luego se hace reaccionar 19, con LiOH, H2O, MeOH, THF, y luego con HATU, Pr2Net, DMF, para obtener el intermediario 1 , luego se hace reaccionar el intermediario 1 , con L¡N(S¡Me3)2, THF, para obtener el intermediario 20, luego se hace reaccionar el intermediario 20, con (n-Bu)4NF, THF, y luego con Li, NH3, THF, para obtener el producto final dmXeB, en condiciones según el siguiente esquema,
Figure imgf000017_0001
Finally, to obtain dmXeB, intermediates 2 and 3 are reacted with EDC, HCI, HOBT, CH2CI2, to obtain intermediate 18, then intermediate 18 is reacted with SnCh, PhSH, EtsN, CH3CN, to obtain intermediate 19, then 19 is reacted with LiOH, H2O, MeOH, THF, and then with HATU, Pr2Net, DMF, to obtain intermediate 1, then intermediate 1 is reacted with L¡N(S¡Me3 )2, THF, to obtain intermediate 20, then intermediate 20 is reacted, with (n-Bu)4NF, THF, and then with Li, NH3, THF, to obtain the final product dmXeB, under conditions according to the following scheme,
Figure imgf000017_0001
Esquema 3c. Unión de fragmentos y finalización de la síntesis. Obtención final de dmXeB. Scheme 3c. Union of fragments and completion of the synthesis. Final get dmXeB.
En preparación para el ensamblaje de bis (macrolactama), los sintones de aminoácidos individuales se obtuvieron mediante transposición de Irlanda- Claisen (ICR) de dos ásteres preparados a partir del alcohol alílico 4 (Figura 3a y b). El intermediario 2 se obtuvo a partir del áster 15 (rendimiento del 82%, dr10: 1 ) en condiciones de reacción desarrolladas específicamente para a-alcox¡ ásteres, donde encontramos que el mejor resultado controlado por quelación se logra con la combinación KN (S¡Me3)2/PhMe, mientras que, sorprendentemente, LDA/THF proporciona diastereómero controlado sin quelación con buena selectividad, a pesar de la mayor propensión esperada del litio a coordinarse con bases de Lewis de oxígeno. La ICR del éster 16 dio lugar al ácido carboxílico 17 (87% de rendimiento, dr 6: 1 ), que, después de formar el éster metílico con MeaSiCHIX , se adelantó al aminoéster 3 mediante reducción de azida con SnCh / PhSH (82% de rendimiento). In preparation for bis(macrolactam) assembly, individual amino acid synthons were obtained by Ireland-Claisen rearrangement (ICR) of two asters prepared from allylic alcohol 4 (Figure 3a and b). Intermediate 2 was obtained from aster 15 (yield 82%, dr10: 1) under reaction conditions specifically developed for a-alcox¡ esters, where we found that the best chelation-controlled result is achieved with the KN(S) combination. ¡Me3)2/PhMe, whereas, surprisingly, LDA/THF provides controlled diastereomer without chelation with good selectivity, despite the expected higher propensity of lithium to coordinate with Lewis bases of oxygen. ICR of ester 16 gave carboxylic acid 17 (87% yield, 6:1 dr), which, after forming the methyl ester with MeaSiCHIX, was preempted to amino ester 3 via azide reduction with SnCh/PhSH (82% performance).
La unión de los fragmentos se logró mediante la formación de amida a partir del ácido 2 y la amina 3, seguida de otra reducción de azida en las mismas condiciones, hidrólisis del éster y macrolactamización. En esta etapa, los diastereoisómeros menores formados durante la ICR eran separables y se aisló bis (lactama) 1 macrocíclica con un rendimiento del 35% como un solo diastereoisómero. Fragment ligation was achieved by amide formation from acid 2 and amine 3, followed by another azide reduction under the same conditions, ester hydrolysis, and macrolactamization. At this stage, the minor diastereomers formed during ICR were separable and macrocyclic bis(lactam)1 was isolated in 35% yield as a single diastereomer.
La finalización de la síntesis sigue una estrategia bidireccional, comenzando con la formación de dos restos de valerolactama por N-alquilación intramolecular de 1 (L¡N(S¡Me3)2,THF, 85% de rendimiento). Completion of the synthesis follows a two-way strategy, beginning with the formation of two valerolactam residues by intramolecular N-alkylation of 1 (L¡N(S¡Me3)2,THF, 85% yield).
La eliminación de los grupos sililo precedió a la formación crucial reductora de 1 - oxaquinolizidina con el reactivo Li-NHs, que logró simultáneamente la O- desbencilación. En particular, numerosos reactivos alternativos que intentaron lograr la semirreducción de las lactamas (¡BU2AIH, NaAIH2 (OR)2, IrCI (CO) (PPh3) 2/(HMe2S¡)20), resultaron consistentemente en una reducción completa a piperidina. También se observó a-deshidroxilación parcial que conduce a una cantidad menor de 22. Removal of the silyl groups preceded the crucial reductive formation of 1-oxaquinolizidine with the Li-NHs reagent, which simultaneously achieved O-debenzylation. In particular, numerous alternative reagents that attempted to achieve half-reduction of lactams (¡BU2AIH, NaAIH2 (OR)2, IrCI (CO) (PPh3) 2/(HMe2S¡)20), consistently resulted in complete reduction to piperidine. Partial a-dehydroxylation was also observed leading to an amount less than 22.
La hidrogenación final de los dobles enlaces de los compuestos 21 (dmXeB) y 22 se realizó con Rh/AhO3 en acetato de etilo obteniendo de forma fiable y limpia el compuesto objeto de la presente invención que es el dmXeB (rendimiento del 94%). The final hydrogenation of the double bonds of compounds 21 (dmXeB) and 22 was carried out with Rh/AhO3 in ethyl acetate, reliably and cleanly obtaining the compound object of the present invention, which is dmXeB (94% yield).
Una vez obtenido el producto sintético dmXeB mediante el método de la presente invención, se procedió a evaluar su efecto sobre la liberación de calcio mediada por IP3R, la respiración mitocondhal y la actividad anticancerígena selectiva en líneas celulares de cáncer de mama, y cómo se compara con XeB aislado previamente de una esponja marina. Como se muestra en la Figuras 1 a y 1 b, la incubación de 30 minutos con XeB (figura 1 a) y dmXeB (figura 1 b), anuló por completo la liberación de calcio IP3R inducida por ATP en células MDA-MB-231. Posteriormente, la línea celular normal MCF10A y una línea celular de cáncer MDA-MB-231 se trataron con concentraciones crecientes de dmXeB y XeB durante 24 h, y las tasas de consumo de oxígeno (OCR) se determinaron utilizando el sistema Seahorse. Como se muestra en las Figuras 2a y 2b, ambos compuestos disminuyen el OCR de manera similar dependiente de la dosis. Once the synthetic product dmXeB was obtained by the method of the present invention, its effect on IP3R-mediated calcium release, mitochondal respiration and selective anticancer activity in breast cancer cell lines was evaluated, and how it compares with XeB previously isolated from a marine sponge. As shown in Figures 1a and 1b, 30 min incubation with XeB (Figure 1a) and dmXeB (Figure 1b), completely abrogated ATP-induced IP3R calcium release in MDA-MB-231 cells. Subsequently, the normal cell line MCF10A and a cancer cell line MDA-MB-231 were treated with increasing concentrations of dmXeB and XeB for 24 h, and oxygen consumption rates (OCR) were determined using the Seahorse system. As shown in Figures 2a and 2b, both compounds decrease OCR in a similar dose-dependent manner.
En las figuras 3a y 3b se observó un efecto similar en dos líneas celulares MCF10A (negro-control)) versus MDA-MB-231 (gris). In Figures 3a and 3b a similar effect was observed in two cell lines MCF10A (black-control)) versus MDA-MB-231 (grey).
También se demostró con la presente invención que dmXeB induce la muerte celular selectiva en líneas celulares de cáncer de mama. Vahas líneas celulares de cáncer de mama que representan la heterogeneidad del cáncer de mama y la línea celular normal MCF10A se trataron con diferentes concentraciones de XeB o dmXeB durante 24 h y la muerte celular se midió mediante la incorporación de yoduro de propidio a través de citometría de flujo. Como se muestra en las Figuras 3a y 3b, 7,5 pM XeB indujo un aumento significativo en la muerte celular en células MDA-MB-231 , sin afectar la línea celular normal MCF10A. A 10 pM, XeB es aún más eficaz en las células MDA-MB-231 , pero algunos efectos en las células MCF10A se vuelven medibles, lo que indica una selectividad reducida. Por otro lado, dmXeB de hecho muestra un cierto aumento de la selectividad, induciendo la muerte celular en MDA-MB-231 a 5 pM, que se mantiene a 10 pM sin ningún efecto sobre las células MCF10 normales (Figuras 3a y 3b). A 20 pM, las células MDA-MB-231 son aún más sensibles, con casi el 100% de la población muerta, mientras que MCF10A muestra un aumento inicial en la muerte celular. It was also shown with the present invention that dmXeB induces selective cell death in breast cancer cell lines. Several breast cancer cell lines representing the heterogeneity of breast cancer and the normal cell line MCF10A were treated with different concentrations of XeB or dmXeB for 24 h and cell death was measured by propidium iodide incorporation via cell cytometry. flow. As shown in Figures 3a and 3b, 7.5 pM XeB induced a significant increase in cell death in MDA-MB-231 cells, without affecting the normal MCF10A cell line. At 10 pM, XeB is still more effective in MDA-MB-231 cells, but some effects in MCF10A cells become measurable, indicating reduced selectivity. On the other hand, dmXeB indeed shows some increased selectivity, inducing cell death in MDA-MB-231 at 5 pM, which is maintained at 10 pM without any effect on normal MCF10 cells (Figures 3a and 3b). At 20 pM, MDA-MB-231 cells are even more sensitive, with almost 100% of the population killed, while MCF10A shows an initial increase in cell death.
Como se muestra en las Figuras 4a y 4b, las líneas celulares representativas de luminal A (MCF-7) y luminal B (ZR-75-1 ) las líneas celulares de cáncer de mama XeB y dmXeB muestran muerte celular selectiva en concentraciones entre 5 y 10 pM. De manera similar a las observaciones con la línea celular MDA-MB-231 , dmXeB a una concentración de 20 pM causó una muerte celular casi completa en las líneas celulares ZR-75-1 y MCF-7, mientras que también afectó a la línea celular normal MCF10A en mucha menor medida. Las células BT-549 son más resistentes a ambos compuestos, mostrando solo selectividad a 7,5 pM. As shown in Figures 4a and 4b, representative cell lines of the luminal A (MCF-7) and luminal B (ZR-75-1) breast cancer cell lines XeB and dmXeB show selective cell death at concentrations between 5 and 10 pM. Similar to observations with the MDA-MB-231 cell line, dmXeB at a concentration of 20 pM caused almost complete cell death. in cell lines ZR-75-1 and MCF-7, while it also affected the normal cell line MCF10A to a much lesser extent. BT-549 cells are more resistant to both compounds, showing only selectivity at 7.5 pM.
Por último, cabe destacar que distintos parámetros particulares de la invención, como las temperaturas indicadas en cualquier esquema y/o procedimiento descritas anteriormente pueden vahar o ser modificadas en función de los requerimientos de operación. En consecuencia, las configuraciones específicas descritas anteriormente no pretenden ser limitantes, y dichas variaciones y/o modificaciones se encuentran dentro del espíritu y alcance de la invención. Finally, it should be noted that different particular parameters of the invention, such as the temperatures indicated in any diagram and/or procedure described above, can vary or be modified depending on the operating requirements. Accordingly, the specific configurations described above are not intended to be limiting, and such variations and/or modifications are within the spirit and scope of the invention.

Claims

REIVINDICACIONES
1 Método para sintetizar el compuesto desmetilxestospongina B (dmXeB), de fórmula
Figure imgf000021_0001
donde R es H, 1 Method for synthesizing the compound desmethylxestospongin B (dmXeB), with the formula
Figure imgf000021_0001
where R is H,
CARACTERIZADO porque se sintetiza como se indica en el esquema siguiente
Figure imgf000021_0002
en donde se realiza una esterificación de azidoalcohol 4 con ácido 2-(bencilox¡) -5- cloropentanoico 5 o ácido 5-cloropentanoico para obtener los intermediarios 2 y 3 a través de la transposición de Irlanda-Claisen para establecer centros estereogénicos en C9' y C9, respectivamente; luego se combinan los precursores de ω -aminoácidos, 2 y 3 mediante formación secuencial de amidas para obtener bis (lactama) 1; y luego, mediante la hidrogenación final de los dobles enlaces de bis (lactama) 1 en acetato de etilo se obtiene el compuesto dmXeB. CHARACTERIZED because it is synthesized as indicated in the following scheme
Figure imgf000021_0002
wherein an esterification of azidoalcohol 4 with 2-(benzyloxy¡) -5-chloropentanoic acid 5 or 5-chloropentanoic acid is performed to obtain intermediates 2 and 3 through the Ireland-Claisen rearrangement to establish stereogenic centers at C9' and C9, respectively; then the ω-amino acid precursors, 2 and 3 are combined by sequential amide formation to obtain bis(lactam) 1; and then, by final hydrogenation of the bis(lactam) 1 double bonds in ethyl acetate, the dmXeB compound is obtained.
2.- Método para sintetizar el compuesto desmetilxestospongina B (dmXeB) de acuerdo a la reivindicación 1 , CARACTERIZADO porque el intermediario 4, se sintetiza de acuerdo al siguiente esquema: 2.- Method for synthesizing the compound desmethylxestospongin B (dmXeB) according to claim 1, CHARACTERIZED in that intermediate 4 is synthesized according to the following scheme:
Figure imgf000022_0001
se hace reaccionar óxido de 1-clorobuteno con MCPBA con una resolución cinética hidrolítica de Jacobsen y epoxidación para obtener (S)-6, luego una parte del epóxido (S)- 6 reacciona con dimetilmetilenosulfuro para obtener alcohol alílico 7, luego se reacciona 7 en una parametoxibencilación reductora (PMB) en condiciones electrofílicas con un desplazamiento del grupo cloro para obtener 8, la parte de (S)-6 que no reacciona con dimetilmetilenosulfuro se acopla con cuprato y el compuesto 8 para dar origen al compuesto 9, finalmente se hace reaccionar 9 con cloruro y azida sódica en una sustitución posterior de O-sililación y eliminación del grupo PMB, para obtener al intermediario 4.
Figure imgf000022_0001
1-chlorobutene oxide is reacted with MCPBA with Jacobsen hydrolytic kinetic resolution and epoxidation to obtain (S)-6, then a part of the epoxide (S)- 6 reacts with dimethylmethylenesulfide to obtain allyl alcohol 7, then reacts 7 in a reductive paramethoxybenzylation (PMB) under electrophilic conditions with a displacement of the chlorine group to obtain 8, the part of (S)-6 that does not react with dimethylmethylenesulfide is coupled with cuprate and compound 8 to give rise to compound 9, finally it is reacts 9 with chloride and sodium azide in a subsequent O-silylation substitution and elimination of the PMB group, to obtain intermediate 4.
3.- Método para sintetizar el compuesto desmetilxestospongina B (dmXeB) de acuerdo a la reivindicación 1 , CARACTERIZADO porque para obtener el intermediario 2 se prepara primero el intermediario 14 cloruro de a-benciloxi acilo, el cual se sintetiza de acuerdo al siguiente esquema:
Figure imgf000022_0002
la alquilación del a-benclloxlacetato de t-butilo con yoduro 10 producen 11 y 12, luego con la desililación, mesilación y sustitución de 12 con LiCI, se obtiene el éster 13, luego la escisión del éster t-butílico y la clorodeshidroxilación con cloruro de oxalilo se obtiene el cloruro de acilo 14, posteriormente el intermediario 14, se hace reaccionar con piridina y CH2CI2 para obtener el éster 15, y luego, se hace reaccionar el éster 15 con KN(S¡Me3)2, MesSICI, y PhMe, en reacción específica para a-alcox¡ ésteres, para obtener el intermediario 2, según el siguiente esquema,
Figure imgf000023_0001
3.- Method for synthesizing the compound desmethylxestospongin B (dmXeB) according to claim 1, CHARACTERIZED in that to obtain intermediate 2, intermediate 14 a-benzyloxy acyl chloride is first prepared, which is synthesized according to the following scheme:
Figure imgf000022_0002
alkylation of t-butyl a-benzyloxlacetate with iodide 10 yields 11 and 12, then desilylation, mesylation and substitution of 12 with LiCl, yields ester 13, then cleavage of t-butyl ester and chlorodehydroxylation with chloride oxalyl acyl chloride 14 is obtained, then intermediate 14 is reacted with pyridine and CH2CI2 to obtain ester 15, and then ester 15 is reacted with KN(S¡Me3)2, MesSICI, and PhMe , in a specific reaction for a-alkoxy esters, to obtain intermediate 2, according to the following scheme,
Figure imgf000023_0001
4.- Método para sintetizar el compuesto desmetilxestospongina B (dmXeB) de acuerdo a la reivindicación 1 , CARACTERIZADO porque para obtener el intermediario 3 se hace reaccionar el intermediario 15 con piridina, CH2CI2, y cloruro 5-cloropentoil para obtener 16, luego se hace reaccionar 16 con LDA, TBSCI, HMPA, THF, para obtener 17, luego se hace reaccionar 17, para obtener el intermediario 3, en condiciones según el siguiente esquema,
Figure imgf000023_0002
4.- Method for synthesizing the compound desmethylxestospongin B (dmXeB) according to claim 1, CHARACTERIZED in that to obtain intermediate 3, intermediate 15 is reacted with pyridine, CH2CI2, and 5-chloropentoyl chloride to obtain 16, then it is made react 16 with LDA, TBSCI, HMPA, THF, to obtain 17, then 17 is reacted, to obtain intermediate 3, under conditions according to the following scheme,
Figure imgf000023_0002
5.- Método para sintetizar el compuesto desmetilxestospongina B (dmXeB) de acuerdo a la reivindicación 1 , CARACTERIZADO porque para obtener el dmXeB, se hace reaccionar los intermediarios 2 y 3, con EDC, HCI, HOBT, CH2CI2, para obtener el intermediario 18, luego se hace reaccionar el intermediario 18, con SnCh, PhSH, EtsN, CH3CN, para obtener el intermediario 19, luego se hace reaccionar 19, con LiOH, H2O, MeOH, THF, y luego con HATU, Pr2Net, DMF, para obtener el intermediario 1, luego se hace reaccionar el intermediario 1 , con L¡N(S¡Me3)2, THF, para obtener el intermediario 20, luego se hace reaccionar el intermediario 20, con (n-Bu)4NF, THF, y luego con LI, NH3, THF, para obtener el producto final dmXeB, en condiciones según el siguiente esquema,
Figure imgf000024_0001
5.- Method for synthesizing the compound desmethylxestospongin B (dmXeB) according to claim 1, CHARACTERIZED in that to obtain dmXeB, intermediates 2 and 3 are reacted with EDC, HCI, HOBT, CH2CI2, to obtain intermediate 18 , then intermediate 18 is reacted with SnCh, PhSH, EtsN, CH3CN, to obtain intermediate 19, then 19 is reacted with LiOH, H2O, MeOH, THF, and then with HATU, Pr2Net, DMF, to obtain intermediate 1, then intermediate 1 is reacted with L¡N(S¡Me3)2, THF, to obtain intermediate 20, then react intermediate 20, with (n-Bu)4NF, THF, and then with LI, NH3, THF, to obtain the final product dmXeB, under conditions according to the following scheme,
Figure imgf000024_0001
6.- Uso del compuesto sintetizado desmetilxestospongina B (dmXeB) obtenido por el método de las reivindicaciones 1 a 5, CARACTERIZADO porque sirve para preparar un medicamento útil como Inhibidor del crecimiento tumoral y la metástasis. 6.- Use of the synthesized compound desmethylxestospongin B (dmXeB) obtained by the method of claims 1 to 5, CHARACTERIZED in that it serves to prepare a drug useful as an inhibitor of tumor growth and metastasis.
7.- Uso del compuesto sintetizado desmetilxestospongina B (dmXeB) obtenido por el método de las reivindicaciones 1 a 5, CARACTERIZADO porque sirve para preparar un medicamento para tratar el cáncer de mama. 7.- Use of the synthesized compound desmethylxestospongin B (dmXeB) obtained by the method of claims 1 to 5, CHARACTERIZED in that it is used to prepare a drug to treat breast cancer.
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