8 r-hydroxyepoxyboetirane a: synthesis method and uses of same

Abstract

The invention relates to the compound (2S,3S,4R,5R,6S,8R,9S,11R,15R)-3,5,15- triacetoxy-6(17)-epoxy-8-hydroxylathyrane-12E-en-14-one, also called (8R)-8-hydroxyepoxyboetirane A, as well as to a method for obtaining same and the use thereof to promote neuronal differentiation from neural precursors or neural stem cells in culture and in brain lesions. The invention also relates to the use of this compound to prepare a pharmaceutical composition that can be used to treat central nervous system lesions associated with neuron loss.

Description

[0001] 8R-HYDROXYEPOXYBOETHYRANE A: METHOD OF SYNTHESIS AND USES THEREOF

[0002] FIELD OF TECHNOLOGY

[0003] This invention relates to the compound (2S,3S,4R,5R,6S,8R,9S,11R,15R)-3,5,15-triacetoxy-6(17)-epoxy-8-hydroxylatyr-12E-en-14-one, also known as (8R)-8-hydroxyepoxyethiran A, as well as its preparation and use to promote neuronal differentiation from neural precursors or neural stem cells in culture and in brain lesions. Additionally, it also relates to the use of this compound in the preparation of a pharmaceutical composition useful in the treatment of central nervous system lesions involving neuronal loss.

[0004] BACKGROUND OF THE INVENTION

[0005] The frequency of diseases caused by brain damage due to various factors, including traumatic injuries, strokes, and neurodegenerative diseases, has increased in recent years within the global population. These pathologies cause irreversible disorders in cognitive function, the motor and somatosensory systems, and even personality. A common characteristic of all these diseases is the irreversible loss of neurons, which is usually responsible for the symptoms (Blennow et al. 2012. The neuropathology and neurobiology of traumatic brain injury. Neuron, 76: 886-899; Xiong et al. 2013. Animal models of traumatic brain injury. Nat Rev Neurosci, 14: 128-142). Currently, there is no effective curative treatment for brain damage. For this reason, the search for new therapeutic options is one of the challenges of current neuroscience (Grade and Gotz 2017).Neuronal replacement therapy: previous achievements and challenges ahead. NPJ Regen Med, 2: 29). Many advances in this field have been made, thanks to the discovery of neuronal replacement from neural stem cells (NSCs) in the adult central nervous system (CNS). This discovery has led to a new approach in the development of therapies to repair brain damage.

[0006] Neurogenesis is the process of generating neurons from neural stem cells. This process has been extensively studied in rodents. These studies have established that, in rodents under physiological conditions, neurogenesis occurs in specific brain regions of the adult brain called neurogenic niches. The best-studied to date are the dentate gyrus (DG) of the hippocampus, which generates neurons that integrate into hippocampal circuits, and the subventricular zone (SVZ) (Gage et al. 1995. Isolation, characterization, and use of stem cells from the CNS. Annu Rev Neurosci, 18: 159-192; Doetsch et al. 1997. Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci, 17: 5046-5061), where neuroblasts are produced that migrate to the olfactory bulb.In the human brain, the precursors of the adult SVZ also migrate to the striatum, giving rise to mature neurons in this region (Dayere et al. 2005. New GABAergic interneurons in the adult neocortex and striatum are generated from different precursors. J Cell Biol, 168: 415-427; Ernst et al. 2014. Neurogenesis in the striatum of the adult human brain. Cell, 156: 1072-1083; Luzzati et al. 2014. Quiescent neuronal progenitors are activated in the juvenile guinea pig lateral striatum and give rise to transient neurons. Development, 141: 4065-4075). These homeostatic mechanisms, which are harmoniously orchestrated, allow the continuous generation of neurons that will replace existing ones or integrate into a circuit, participating in functions such as learning, memory, or olfactory learning (Anacker and Hen 2017. Adult hippocampal neurogenesis and cognitive flexibility - linking memory and mood. Nat Rev Neurosci, 18: 335-346; Avaro et al. 2022).Scent of stem cells: How can neurogenesis make us smell better? Front Neurosci, 16: 964395).

[0007] Given the existence of these mechanisms, it seems logical to think that brain tissue repair is possible in the face of injury. However, brain lesions disrupt the homeostasis of neurogenic niches, activate neural stem cells in the vicinity of the lesion (Uorens-Bobadilla et al. 2015. Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury. Cell Stem Cell, 17: 329-340), and modify the migratory pathways of their progeny (reviewed in (Grade and Gotz 2017. Neuronal replacement therapy: previous achievements and challenges ahead. NPJ Regen Med, 2: 29)). Additionally, following injury, cells with NSC characteristics appear in the immediate vicinity of the lesion, possessing the potential to generate neurons in response to the injury. Thus, in addition to this mobilization of cells from neurogenic niches, there is also local activation of NSCs at the lesion site.These mechanisms could be a tissue response to generate new neurons (Ohira et al. 2010. Ischemia-induced neurogenesis of neocortical layer 1 progenitor cells. Nat Neurosci, 13: 173-179).

[0008] Despite the existence of these mechanisms, the CNS's ability to generate neurons in damaged regions is very limited, and although some possible treatments have emerged that enhance this activity, no regeneration of damaged nerve tissue is observed after an injury. (Parent et al. 1997. Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci, 17: 3727-3738; Liu et al. 1998. Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils. J Neurosci, 18: 7768-7778;Jin et al. 2001. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc Natl Acad Sci USA, 98: 4710-4715;Romero-Grimaldi et al. 2011. ADAM-17 / tumor necrosis factor-alpha-converting enzyme inhibits neurogenesis and promotes gliogenesis. neural stem cells. Stem Cells, 29: 1628-1639; Saha et al. 2013.Cortical lesion stimulates adult subventricular zone neural progenitor cell proliferation and migration to the site of injury. Stem Cell Res, 11: 965-977; Moraga et al. 2014. Toll-like receptor 4 modulates cell migration and cortical neurogenesis after focal cerebral ischemia. FASEB J, 28: 4710-4718). Several factors influence the failure of tissue repair mechanisms in the nervous system. On the one hand, the damage stimulates the onset of an inflammatory process, which promotes the activation of glial cells, forming a glial scar. This appears to hinder the arrival of neuronal precursors or neuroblasts migrating from neurogenic regions. On the other hand, the activated glial cells themselves and microglia cells of the immune system secrete substances that modify the microenvironment of the lesion.The secretion of glyogenic / non-neurogenic molecules in the lesion environment prevents stem cells activated after injury from producing neuroblasts that differentiate into mature neurons (Dominguez-Garcia et al. 2020. A novel PKC activating molecule promotes neuroblast differentiation and delivery of newborn neurons in brain injuries. Cell Death Dis, 11: 262; Gomez-Oliva et al. 2023. Targeting epidermal growth factor receptor to recruit newly generated neuroblasts in cortical brain injuries. J Transí Med, 21: 867). Thus, when promoting the repair of damaged nerve tissue by generating new neurons, it is necessary to use molecules that counteract the non-neurogenic microenvironment and facilitate the differentiation of stem cells into neurons, as well as increase the survival of neuroblasts and newly generated neurons (Grade and Gotz 2017. Neuronal replacement therapy: previous achievements and challenges ahead).NPJ Regen Med, 2: 29).

[0009] The repair capacity of the adult brain has so far been described as very limited. Although most studies have focused on ischemic lesions, other types of lesions have also been studied, yielding neuronal replacement rates of between 0.2 and 10%, depending on the affected area and the type of lesion (Parent et al. 1997. Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus. J Neurosci, 17: 3727-3738; Liu et al. 1998. Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils. J Neurosci, 18: 7768-7778; Jin et al. 2001. Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc Natl Acad Sci USA, 98: 4710-4715; Romero-Grimaldi et al. 2011. ADAM-17 / tumor necrosis factor-alpha-converting enzyme inhibits neurogenesis and promotes gliogenesis from neural stem cells.Stem Cells, 29: 1628-1639; Saha et al. 2013. Cortical lesion stimulates adult subventricular zone neural progenitor cell proliferation and migration to the site of injury. Stem Cell Res, 11: 965-977; Moraga et al. 2014. Toll-like receptor 4 modulates cell migration and cortical neurogenesis after focal cerebral ischemia. FASEB J, 28: 4710-4718). However, the fact that the adult brain has the homeostatic capacity to respond to injury suggests that the formation of new neurons is an effective mechanism in repairing small lesions, which remain silent precisely because the neurons that undergo apoptosis are replaced, at least in part, by newly formed neurons. For example, it has been observed that after head trauma in rodents, neurogenesis increases in the dentate gyrus of the hippocampus, and these animals subsequently recover their ability to perform spatial memory tasks.However, if the stem cells that begin to divide in the hippocampus as a consequence of trauma-induced damage are selectively eliminated in these animals, they cannot recover the ability to perform spatial memory tasks (Blaiss et al. 2011. Temporally specified genetic ablation of neurogenesis impairs cognitive recovery after traumatic brain injury. J Neurosci, 31: 4906-4916), thus demonstrating that neuronal regeneration in these regions has an effect on memory recovery after head trauma. The emergence of neurogenesis in brain lesions has also been demonstrated in humans. In brain samples from patients undergoing surgery after a traumatic brain injury, the presence of proliferating neural stem cells and neuroblasts has been observed in the area of ​​the cerebral cortex surrounding the lesion (Zheng et al. 2013. Neurogenesis in adult human brain after traumatic brain injury).J Neurotrauma, 30: 1872-1880).

[0010] The problem is that, in cases of severe injuries with significant neuronal loss, repair does not appear to be a viable solution unless effective therapeutic measures are established to significantly enhance the neurogenic process. In addition to potentiating endogenous neurogenesis, one alternative that could help facilitate neuron generation and address the clinical challenges posed by diseases involving neuronal loss is the transplantation of stem cells that can subsequently generate new neurons. Several laboratories have attempted this strategy in animal models using embryonic stem cells (Cao et al. Pluripotent stem cells engrafted into the normal or lesioned adult rat spinal cord are restricted to a glial lineage. Exp Neurol. 167:48-58, 2001) and neural stem cells from adult animals (Pluchino, S. et al.).Injection of adult neurospheres induces recovery in a chronic model of multiple sclerosis. Nature 422: 688-694, 2003), induced pluripotent stem cells, or cells derived from stem cells subjected to different degrees of in vitro differentiation. The most commonly observed phenomenon is that stem cells implanted in neurogenic areas of the brain (e.g., the subventricular zone or the olfactory bulb in rodents) integrate into the neurogenic process and eventually differentiate into neuroblasts and mature neurons, but this same phenomenon does not occur in non-neurogenic areas of the brain. (Herrera, DG et al. Adult-derived neural precursors transplanted into multiple regions in the adult brain. Ann Neurol. 46:867-77, 1999; Mligiliche et al. Survival of neural progenitor cells from the subventricular zone of the adult rat after transplantation into the host spinal cord of the same strain of adult rat. Anat Sci I nt 80:229-34, 2005).

[0011] Therefore, one of the problems addressed by the present invention is that, while stem cells implanted in neurogenic areas of the brain (e.g., the subventricular zone or the olfactory bulb in rodents) differentiate into neuroblasts and become mature neurons, when transplantation occurs in other areas, they initiate the glial differentiation pathway. In lesions occurring in non-neurogenic areas, proliferating neural precursors appear that could subsequently differentiate, and the endogenous source of neural precursors in these regions is the parenchymal glia. These glial cells are activated after injury and give rise to neural precursors, most of which differentiate into glial cells and very few into neurons. The use of substances that promote neuronal differentiation can be very useful in defining mechanisms for enhancing neuronal repair.Several molecules involved in neuronal differentiation have been described to date, such as BMP factors, Notch protein, and growth factors that activate ErbB family receptors, such as neuregulin (Dominguez-Garcia et al. 2020. A novel PKC activating molecule promotes neuroblast differentiation and delivery of newborn neurons in brain injuries. Cell Death Dis, 11: 262). Promoting the secretion of these types of factors is a direct way to enhance the differentiation of stem cells into mature neurons and is a good strategy for stimulating the repair of damaged brain tissue.

[0012] Given the current state of the art, it is necessary to find molecules with pharmacological activity that promote the differentiation of neural stem cells into mature neurons without affecting stem cell proliferation. BRIEF DESCRIPTION OF THE FIGURES

[0013] Figure 1. The compound (8R)-8-hydroxyepoxyethiran A promotes the secretion of neuregulin 1 (NRG1), whereas other similar compounds do not have this effect.

[0014] AD Panels

[0015] A. Chemical structure of the compounds whose ability to stimulate NRG1 secretion is analyzed. (1) (8 )-8-hydroxyepoxyboethirano A; (2) (8 )-8-(p-methoxyphenylacetoxy)epoxyboethirano A; (3) (8 )-8-(p-methoxyphenylacetoxy)euphoboethirano A.

[0016] B. To analyze the ability of these compounds to stimulate NRG1 secretion, a cDNA was constructed encoding a fusion protein in which NRG1 was flanked by the red fluorescent protein mCherry in its N-terminal domain and the green fluorescent protein EGFP in its C-terminal domain. This construct was cloned into an expression vector with a promoter for expression in mammalian cells. Subsequently, it was transfected into HEK293T cells.

[0017] C. Diagram showing the NRG1 molecule flanked by the two fluorescent proteins. The NRG1 protein is a transmembrane protein with an N-terminal domain on the extracellular side and a C-terminal domain on the intracellular side. Expression of the fusion protein in the plasma membrane results in a fluorescence intermediate between red and green, which is a result of the combination of mCherry and EGFP. The amino acids that constitute the soluble ligand are located in the N-terminal domain. For this ligand to be released into the extracellular environment, it must be cleaved in a proteolytic reaction catalyzed by the enzyme ADAM17. After cleavage, the ligand is released into the environment, and red fluorescence is lost at the plasma membrane, while green fluorescence is retained.

[0018] D. HEK293T cells were transfected with the mCherry-NRG1-EGFP construct and cultured attached to a substrate for 24 hours. After this time, the cells were used for real-time imaging using a fluorescence microscope. After 5 minutes, either a vehicle (PBS) or various compounds were added to the medium to analyze their ability to stimulate NRG1 secretion. The mCherry / EGFP fluorescence rate was measured in 50 different cells over time in the presence of the vehicle and compound (1, 2, or 3). A culture treated with compound E0F2 was used as a positive control (Dominguez-Garcia et al. 2020. A novel PKC activating molecule promotes neuroblast differentiation and delivery of newborn neurons in brain injuries. Cell Death Dis, 11: 262). Figure 2. Compound (8 / ?)-8-hydroxyepoxyboethiranolase A has no effect on neural stem cell proliferation.

[0019] Disaggregated neurosphere cells were seeded in a medium containing the growth factor bFGF, in the absence (Control) or presence of compounds 1, 2 and 3 (see Figure 1. (1) (8 / ?)-8-hydroxyepoxyboethirano A; (2) (8 / ?)-8-(p-methoxyphenylacetoxy)epoxyboethirano A; (3) (8 / ?)-8-(p-methoxyphenylacetoxy)euphoboethirano A) at a concentration of 1 DM. After 48 h of culture, the neurosphere assay was performed, by which the area and number of new neurospheres generated in the culture were measured.

[0020] A. Representative phase-contrast photomicrographs of neurospheres obtained in negative control cultures (with bFGF growth factor)

[0021] B. Representative phase-contrast photomicrographs of neurospheres obtained in positive control cultures treated with the growth factor bFGF + EGF

[0022] CE. Representative phase-contrast photomicrographs of neurospheres obtained from cultures treated with the growth factor bFGF + compounds 1, 2 and 3 (1 DM).

[0023] F. Effect of 1, 2, and 3 on the size (measured as the total area of ​​each neurosphere) of the neurospheres formed. Each bar represents the mean ± standard error of at least three independent experiments. Statistic: *p<0.001 compared to the control using Student's t-test.

[0024] G. Effect of 1, 2, and 3 on the number of neurospheres formed. Each bar represents the mean ± standard error of at least three independent experiments. Statistic: *p<0.001 compared to the control using Student's t-test.

[0025] Figure 3: The compound (8 / ?)-8-hydroxyepoxyboethiran A promotes differentiation to neurons and decreases differentiation to glial cells of NPCs in vitro, whereas (8 / ?)-8-(p-methoxyphenylacetoxy)epoxyboethiran A and (8 / ?)-8-(p-methoxyphenylacetoxy)euphoboethiran A do not exert this effect.

[0026] Disaggregated cells derived from neurospheres were seeded under differentiation conditions, in the absence (Control) and presence of compound 1 at concentrations of 1 and 5 pM for 72 h.

[0027] AC. Representative fluorescence microscopy images of neural precursor cultures processed for immunohistochemical detection of the neuronal marker p-III-tubulin (top panels), glial cell marker GFAP (middle panels), and the marker. Nuclei were counterstained with DAPI. D. Effect of 1 (1 and 5 pM) on the percentage of cells |3-I I-tubulin + with regard to control.

[0028] E. Effect of 1 (1 and 5 pM) on the percentage of GFAP cells + with regard to control.

[0029] DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention solves the problem of generating neurogenic niches in both neurogenic and non-neurogenic areas of the Central Nervous System, based on the use of the compound (8 / ?)-8-hydroxyepoxyethanol A (1).

[0031] (8R)-8-Hydroxyepoxyboetirane A (1)

[0032] This compound promotes the differentiation of neural stem cells into neurons, making it an ideal candidate for treatments to repair damaged nerve tissue.

[0033] This new drug overcomes the non-neurogenic environment that develops in injured regions of the adult brain and promotes the generation of new neurons from neural precursors, whether endogenous or transplanted. Therefore, when regenerating lesions in the non-neurogenic region of the injured area, (8 / ?)-8-hydroxyepoxyboethiran A increases the likelihood that both endogenous and transplanted stem cells will give rise to mature and functional neurons.

[0034] Therefore, a first aspect of the present invention relates to a composition comprising the compound of formula I (hereinafter referred to as compound of formula I): Formula I or any salt, stereoisomer or solvate thereof; where

[0035] Ri, R2, and R3 are each independently selected from the list consisting of a C1-C4 alkyl, optionally branched with one or more C1-C4 alkyls; an aromatic ring, such as phenyl or benzyl, optionally substituted with a halogen, C1-C4 alkyl, or (C1-C4)-alkoxy; or a heterocyclic ring, such as pyridine.

[0036] In a preferred embodiment of the first aspect of the invention, R1, R2, and R3 are each independently selected from the list consisting of a C1-C4 alkyl, preferably CH3. More preferably, the compound of formula I is: or any salt thereof. More preferably, the compound of formula I is:

[0037] (8R)-8-Hydroxyepoxyethirano A (1) or any salt thereof.

[0038] A second aspect of the present invention relates to a method or procedure for obtaining a compound of formula I, wherein the method comprises the biotransformation by Mucorcircinelloides NRRL3631 of epoxiboethiran A obtained from Euphorbia boetica plants.

[0039] It should be noted that in example 1 the obtaining of the compound of formula I is explained in detail. In this sense, briefly, E. boetica plants are collected and their aerial part is extracted following the protocol previously described in the literature (Flores-Giubi, E.; Geribaldi-Doldán, N.; Murillo-Carretero, M.; Castro, C.; Durán-Patrón, R.; Macías-Sánchez, AJ; Hernandez-Galan, R. Lathyrane, Premyrsinane, and Related Diterpenes from Euphorbia boetica'. Effect on in vitro Neural Progenitor Cell Proliferation. J. Nat. Prod. 2019, 82, 2517- 2528). Next, the epoxyboethiran A is obtained following the protocol described in example 1 and its biotransformation is carried out with M. circinelloides NRRL3631 as described in example 1, until the compound of formula I is obtained.

[0040] A third aspect of the present invention relates to the in vitro use of a compound of formula I, as defined in the first aspect of the invention, and optionally in any of its preferred embodiments, to promote differentiation or to differentiate neurons in culture, from stem cells of fetal or adult origin capable of differentiating into neurons, such as induced pluripotent stem cells, neural precursors, or neural stem cells.

[0041] In the context of the present invention, neural precursors or neural stem cells are understood to be those neural stem cells isolated from adult or fetal tissue, which have the capacity to self-replicate, but a limited differentiation capacity, since they can only differentiate into the three cell subtypes of the neural lineage: neurons, astrocytes and oligodendrocytes.

[0042] In a preferred embodiment of the third aspect of the invention, said compound of formula I is the compound:

[0043] (8R)-8-Hydroxyepoxyboetirane A (1)

[0044] A fourth aspect of the present invention relates to an in vitro method for obtaining neurons from fetal or adult stem cells capable of differentiating into neurons, comprising: a. contacting said fetal or adult stem cells capable of differentiating into neurons with a compound of formula I, in a medium suitable for the differentiation of said stem cells; and b. optionally, subsequently harvesting the neurons obtained. In a preferred embodiment of the fourth aspect of the present invention, in step a) the stem cells are contacted with the compound of formula I at a concentration ranging from 1 pmol / L to 40 pmol / L, in a medium suitable for the differentiation of the stem cells.

[0045] In another preferred embodiment of the fourth aspect of the present invention, the compound of formula I is the compound:

[0046] (8 / ?)-8-Hydroxyepoxyboethrane A (1)

[0047] To use the compound, as defined in the first aspect of the invention, in an in vitro culture, it is preferable to dissolve it prior to use in a solution that can solubilize both the compound and its salt. Examples of solvents include dimethyl sulfoxide, water, or similar solutions. Additionally, this compound can be dissolved in phosphate-buffered saline (PBS).

[0048] It should be noted that the medium of step a) is considered suitable if it does not impede stem cell differentiation. In this regard, as an example, a Dulbecco's modified Eagle's medium (DMEM) / F-12 (1:1) medium, containing 2% of supplement B27 (Invitrogen), 2 mM L-glutamine, and 2 pg / ml gentamicin without serum and in the absence of growth factors, may preferably be used. In a particular aspect of the invention, to culture stem cells with the compound as defined in the first aspect of the invention, the compound is added at a concentration ranging from 1 pmol / L to 40 pmol / L. The stem cells are cultured attached to a substrate at a density of 20 to 200 x 10⁻⁶ 6 cells / L. The compound is added to a static culture at 37°C for 1 to 14 days in a 5% CO2 atmosphere, changing the medium completely or partially every two days.

[0049] The medium in which the cells are cultured can be any medium that does not impede the differentiation of stem cells; for example, preferably Dulbecco's modified Eagle's medium (DMEM) / F-12 (1:1), containing 2% of supplement B27 (Invitrogen), 2 mM L-glutamine, and 2 pg / ml of gentamicin without serum and in the absence of growth factors, can be used. A fifth aspect of the present invention relates to a culture medium suitable for the differentiation of neurons from stem cells of fetal or adult origin with the capacity to differentiate into neurons, such as induced pluripotent stem cells, neural precursors, or neural stem cells, characterized in that said culture medium comprises a compound of formula I, preferably the compound (8 / ?)-8-hydroxyepoxyethanol A, and / or a salt thereof.In a preferred embodiment of the fifth aspect of the present invention, the compound of formula I is found in the medium in a concentration range of 1 pmol / L to 40 pmol / L.

[0050] A sixth aspect of the present invention relates to the use of the culture medium as defined in the fifth aspect of the invention and, optionally, in any of its preferred embodiments, to promote the differentiation or to differentiate into neurons stem cells of fetal or adult origin with the capacity to differentiate into neurons, such as induced pluripotent stem cells, neural precursors, or neural stem cells.

[0051] A seventh aspect of the present invention relates to a composition comprising or consisting of a population of neurons obtainable by the method of the fourth aspect of the invention and, optionally, by any of its preferred embodiments.

[0052] An eighth aspect of the present invention relates to a composition comprising a compound of formula I, for use in the treatment of diseases or injuries involving neuronal loss. Preferably, the composition is a pharmaceutical composition (hereinafter referred to as the pharmaceutical composition of the present invention).

[0053] In a preferred embodiment of the present invention, the pharmaceutical composition of the present invention further comprises neural precursors or neural stem cells. In a further preferred embodiment of the present invention, the pharmaceutical composition of the present invention comprises at least one pharmaceutically acceptable excipient.

[0054] In another preferred embodiment of the present invention, the diseases or injuries that result in neuronal loss are those selected from the list, which consist of:

[0055] Neurodegenerative diseases: These occur as a consequence of premature neuronal death. The most frequent are Alzheimer's disease, with neuronal loss primarily in the hippocampus and cerebral cortex; Parkinson's disease, with selective neuronal death in the substantia nigra; amyotrophic lateral sclerosis (ALS), with neuronal loss in the spinal cord; vascular dementia; and Lewy body dementia. Traumatic brain injury: This is a traumatic injury to the skull, affecting the brain. The damage can be focal—limited to a single area of ​​the brain—or involve more than one area. In the context of this invention, traumatic brain injury can produce brain damage due to neuronal death, which could be treated with therapies aimed at promoting neuronal regeneration.

[0056] Hypoxic-ischemic injury: Reduction of cerebral blood flow to levels insufficient to maintain the metabolism necessary for normal brain function and structure. In adults, ischemia is primarily caused by strokes, which can be focal (of ischemic, hemorrhagic, or mixed origin) or multiple (as in multi-infarct dementia). In newborns, this hypoxia / ischemia is primarily due to fetal or perinatal distress. In the context of the present invention, hypoxia-ischemia is a condition that causes cellular damage due to a lack of oxygen supply to brain tissue, which in most cases leads to neuronal death.

[0057] Central nervous system (CNS) infections: Brain involvement caused by various infectious agents that result in meningitis, encephalitis, or meningoencephalitis. In the context of this invention, CNS infections cause cellular damage either directly or indirectly through the cerebral edema they produce, potentially leading to neuronal death.

[0058] Epilepsy: A chronic disease characterized by one or more neurological disorders that leave a predisposition to generate recurrent seizures, which often leads to neurobiological, cognitive, and psychological consequences.

[0059] Huntington's disease: Huntington's disease (HD) is a neurodegenerative disorder of the central nervous system characterized by involuntary choreic movements, behavioral and psychiatric disturbances, and dementia. It is caused by an expansion of CAG triplet repeats on the short arm of chromosome 4 (4p16.3) in the huntingtin gene, HTT. The larger the CAG repeat expansion, the earlier the disease appears.

[0060] In a further preferred embodiment of the present invention, the diseases or injuries that involve neuronal loss are focal cerebral ischemia, traumatic brain injury with neuronal damage, Parkinson's disease, epilepsy, and amyotrophic lateral sclerosis. A ninth aspect of the present invention relates to the population of neurons or neural precursors obtainable according to the seventh aspect of the present invention, for the preparation of a medicament for use in the treatment of diseases or injuries that involve neuronal loss. Alternatively, the seventh aspect of the present invention relates to such a population for use in the treatment of diseases or injuries that involve neuronal loss.In an even more preferred embodiment of the present invention, the diseases or injuries that involve neuronal loss are focal cerebral ischemia, traumatic brain injury with neuronal damage, Parkinson's disease, epilepsy, and amyotrophic lateral sclerosis.

[0061] A tenth aspect of the present invention relates to the use of the compound of formula I to promote the secretion of the growth factors neuregulin 1 and 2.

[0062] The following examples are merely illustrative of the present invention and should in no way be understood as limiting it.

[0063] EXAMPLES

[0064] EXAMPLE 1 (8R)-8-Hydroxyepoxyboethiran A was obtained by biotransformation of epoxiboethiran A, previously obtained by isolation from Euphorbia boetica plants, using Mucor circinelloides NRRL3631.

[0065] The E. boetica plants were collected in El Pinar del Hierro (Chiclana de la Frontera), Cádiz, Spain, in March 2020, with all the necessary permits from the relevant national authorities (General Directorate of Biodiversity, Forests and Desertification, State Secretariat for the Environment, Ministry for Ecological Transition and Demographic Challenge, Reference No. ESNC64, and Ministry of Agriculture, Livestock, Fisheries and Sustainable Development-Territorial Delegation of Cádiz, Junta de Andalucía, Reference No. 201999901092011).

[0066] The M. circinelloides strain NRRL3631 was kindly provided by Prof. Cerdá-Olmedo of the University of Seville and is deposited in the fungal collection of the Biosynthetic Design of Fungicides research group, of the Department of Organic Chemistry at the University of Cádiz.

[0067] Epoxy-Ethyrene A Insulation

[0068] The aerial parts of E. boetica were extracted following the protocol previously described in the literature (Flores-Giubi, E.; Geribaldi-Doldán, N.; Murillo-Carretero, M.; Castro, C.; Durán-Patrón, R.; Macías-Sánchez, AJ; Hernández-Galán, R. Lathyrane, Premyrsinane, and Related Diterpenes from Euphorbia boetica: Effect on in Vitro Neural Progenitor Cell Proliferation. J. Nat. Prod. 2019, 82, 2517-2528). Three kilograms of the aerial parts of fresh plants were used, which were frozen with liquid nitrogen, ground, and extracted with methanol (MeOH) (2.5 L *3) at room temperature for 24 h. The MeOH was evaporated under reduced pressure, obtaining a crude extract that was resuspended in 1 L of water and extracted with hexane (He) (1.5 L *3). After removing the solvent, the He extract (33.3 g) was purified by silica gel column chromatography (CC), using a He:ethyl acetate (EtOAc) gradient (10-100%) of increasing polarity.Twenty-one fractions were obtained according to thin-layer chromatography (TLC). Fractions 9 and 10 were combined and purified again by silica gel TLC using a CH2Cl2-acetone gradient of increasing polarity (0-2%), yielding 350 mg of epoxiboethiran A.

[0069] Biotransformation of epoxiboethiran A with M. circinelloides NRRL3631

[0070] The fungus was cultivated in 500 mL Erlenmeyer flasks containing 200 mL of rich medium, consisting of 20.0 g of glucose and 1.0 g of yeast extract and 500 mL of distilled water; 2.0 g L-asparagin, 5.0 g KH₂PO₄, and 0.5 g MgSO₄·7H₂O in 480 mL of distilled water; 10 mL of Sutter's solution; and 10 mL of Sutter's calcium solution. Sutter's solution contains 0.01% thiamine, 2.0% citric acid, 0.015% Fe(NO₃)₃·9H₂O, 0.01% ZnSO₄·7H₂O, 0.003% MnSC₄·4H₂O, 0.0005% CuSO₄·5H₂O, and 0.0005% Na₂MoC₄·2H₂O. The Sutter calcium solution also contains 0.28% CaCh. Each flask was inoculated with 0.5 x 10 7Fresh conidia were incubated at 24–26 °C and 200 rpm under white light (daylight lamp). After 3 days, 300 pL of an epoxyboethiranolase A solution in DMSO was added to each flask to a final concentration of 100 ppm, and the culture was incubated for another 5 days. When biotransformation was complete, the culture was filtered, and the medium was extracted with EtOAc (x3). The organic extract was dried over anhydrous Na₂S₄, and the solvent was evaporated under reduced pressure. The resulting residue, which contained biotransformation products as determined by thin-layer chromatography comparison with the respective microorganism and substrate controls, was dissolved in EtOAc and purified by silica gel column chromatography. Mixtures of increasing polarity of He:EtOAc were used as the eluent. The collected fractions were purified again by semi-preparative HPLC yielding vapors of biotransformation products.Semi-preparative HPLC purification (He:EtOAc 65:35, RI = 135 min, flow rate 3.0 mL / min, purity >98%) of 8-hydroxyepoxyboethiran A yielded 267.0 mg (58.8% yield). EXAMPLE 2. Compound 1 stimulates neuregulin 1 secretion in cultured cells. This effect is not observed with other similar compounds.

[0071] To analyze the ability of compounds 1, 2, and 3 (see Figure 1. (1) (8)-8-hydroxyepoxyboethyrane A; (2) (8)-8-(p-methoxyphenylacetoxy)epoxyboethyrane A; (3) (8)-8-(p-methoxyphenylacetoxy)epoxyboethyrane A) to stimulate NRG1 secretion, a cDNA was constructed encoding a fusion protein in which NRG1 was flanked by the red fluorescent protein mCherry in its N-terminal domain and the green fluorescent protein EGFP in its C-terminal domain. This construct was cloned into an expression vector with a promoter for expression in mammalian cells. Subsequently, it was transfected into HEK293T cells. The NRG1 protein is a transmembrane protein with an N-terminal domain on the extracellular face and a C-terminal domain in the intracellular space. The expression of the fusion protein in the plasma membrane results in a fluorescence intermediate between red and green, which is a result of the combination of mCherry + EGFP.The N-terminal domain contains the amino acids that constitute the soluble ligand. For this ligand to be released into the extracellular medium, it must be cleaved in a proteolytic reaction catalyzed by the enzyme ADAM 17. After cleavage, the ligand is released into the medium, and red fluorescence is lost at the plasma membrane, while green fluorescence is retained. To study the ability of different compounds to stimulate NRG1 secretion, HEK293T cells were transfected with the mCherry-NRG1-EGFP construct and cultured attached to a substrate for 24 hours. After this time, the cells were used to obtain real-time images using a fluorescence microscope. After 5 minutes, various compounds were added to the vehicle medium (PBS) to analyze their ability to stimulate NRG1 secretion.The mCherry / EGFP fluorescence rate was measured over time in 50 different cells in the presence of vehicle and compounds 1, 2, and 3. A culture treated with compound EOF2 was used as a positive control (Dominguez-Garcia et al. 2020. A novel PKC activating molecule promotes neuroblast differentiation and delivery of newborn neurons in brain injuries. Cell Death Dis, 11: 262).

[0072] The results showed that only compound 1 was able to facilitate the secretion of NRG1. Since this growth factor facilitates the differentiation of neural precursors into neurons and their migration, this compound becomes a candidate for the development of a drug that facilitates neuron generation both in vivo and in vitro. EXAMPLE 3 Compounds 1, 2, and 3 have no effect on stem cell proliferation, measured as the number of neurospheres formed from cultured cells isolated from the SVZ of adult mice.

[0073] The biological activity of compounds 1, 2 and 3 on neural stem cells extracted from the subventricular zone of 7-day postnatal mice has been investigated.

[0074] For this purpose, the lateral walls of the lateral ventricles of the subventricular zone of P7 mice were extracted and enzymatically dissociated in low Ca2, high Mg2 cerebrospinal fluid (CSF) (5 mM KCl, 124 mM NaCl, 3.2 mM MgCl2, 100 pM CaCl2, 26 mM NaHCO3, and 10 mM glucose) supplemented with 1 mg / ml trypsin and 0.2 mg / ml kynurenic acid, preheated to 37°C for 15 minutes. It was incubated at 37°C in an incubator for 13 minutes. The tissue was centrifuged at 9,000 rpm for 5 minutes and resuspended in normal CSF (5 mM KCl, 124 mM NaCl, 1.3 mM MgCl2, 2 mM CaCl2, 26 mM NaHCO3, and 10 mM glucose). It was incubated in an oven for 5 minutes and centrifuged under the same conditions. Subsequently, the cells were resuspended in Dulbecco's modified Eagle's medium (DMEM) / F-12 (1:1) supplemented with 0.7 mg / ml of ovomucoid and mechanically disaggregated with a Pasteur pipette with the diameter reduced by half.The cells were then centrifuged again and resuspended in 6 ml of defined neurosphere medium (45 ml of (DMEM) / F-12, 900 pL of B2α, 2 mM L-glutamine, and 2 pg / ml gentamicin) supplemented with 6 pL of EGF (20 ng / ml) and bFGF (10 ng / ml), and maintained at 37°C in a 5% CO2 atmosphere. After 1–2 days, the cell aggregates that form are what we call neurospheres. Subcultures were passed every 3–4 days by neurosphere centrifugation and mechanical dissociation of the cells in 1 ml of defined neurosphere medium; the cell suspension was then cultured in new flasks with fresh medium to obtain new neurospheres. Experiments were performed between passages 3 and 5.

[0075] Once the neurospheres were obtained, the effect of compounds 1, 2, and 3 on neural stem cells was evaluated at different concentrations. For this purpose, the neurospheres were centrifuged, and the cells were resuspended and dispersed in a defined neurosphere medium. Twenty cells / pL were seeded, and the growth factors EGF (20 ng / mL) and bFGF (10 ng / mL) were added. Compounds 1, 2, and 3 were added simultaneously at a concentration of 1 pM in each well. The analysis was performed in triplicate. During the experiments, the culture conditions were kept anonymous to the operator responsible for taking images and performing the quantification. The number of newly formed neurospheres was counted 72 h later using an Olympus IX70 inverted phase-contrast microscope. To measure the area of ​​the neurospheres, images of 50 neurospheres per well were obtained and analyzed using the Image J analysis system.

[0076] It was observed that neither the area nor the number of neurospheres were affected by these compounds, indicating that they did not exert a proliterative effect.

[0077] EXAMPLE 4 Effect of compound 1 on the generation of neurons from SVZ isolated neural stem cell cultures in vitro

[0078] To determine whether compound 1 promoted the differentiation of neural stem cells into neurons, cells were dissociated from neurospheres and cultured attached to a PLO substrate in the absence of growth factors to promote their differentiation. These cells were cultured in the presence and absence of compound 1 for 72 h.

[0079] To analyze the differentiation of neural precursors isolated from the SVZ in vitro, the cells were seeded on glass slides, each with a surface area of ​​0.8 cm² 2The wells were pre-treated with poly-L-ornithine (PLO) (25 pg / ml) in 0.1 M borate buffer, pH 8.4. 300 pg of PLO was added to each well and incubated overnight at 37°C. The following day, the plates were washed with sterile water and allowed to dry for at least two hours under sterile conditions. Once the plates were dry, the neurospheres were disaggregated and seeded in a defined medium in the absence of growth factors at a density of 40,000 cells per well in a final 300 pg. Compound 1 was added at a concentration of 5 pM. The absence of growth factors promotes cell cycle exit and differentiation of neural precursors into neurons, astrocytes, and oligodendrocytes, allowing for the in vitro study of this process.

[0080] Subsequently, the cell phenotypes present in the culture were analyzed using immunocytochemistry. Under these conditions, cells from neurosphere cultures differentiate into different neural phenotypes (glial or neuronal). The percentage of neuroblasts and neurons (pi1 l-tubulin cells) was quantified. + ) and of astrocytes or glial progenitors (GFAP cells) + ). It was observed that in the presence of compound 1 the percentage of cells pi I l-tubulin + was approximately double that in the control cultures (Figure 3 AD). The percentage of GFAP cells was also observed to be +There was no variation from one culture to another, and the differences between them were not statistically significant. These results seemed to indicate that compound 1 is capable of inducing the differentiation of NPCs into neuroblasts at the expense of differentiation into glioblasts and suggested that its use in brain lesions could reverse the glyogenic / non-neurogenic environment of these lesions and transform it into a neurogenic environment where new neurons could be generated. For this reason, compound 1 is postulated as a candidate for the treatment of brain lesions, as a compound capable of modifying the glyogenic niche of the lesion, converting it into a neurogenic niche.

Claims

CLAIMS 1. Composition comprising the compound of formula I: Formula I or any salt, stereoisomer or solvate thereof; where Ri, R2 and R3 are each independently selected from the list consisting of a C1-C4 alkyl, optionally branched with one or more C1-C4 alkyls; an aromatic ring, such as phenyl or benzyl, optionally substituted with a halogen, C1-C4 alkyl, or (C1-C4)-alkoxy; or a heterocyclic ring, such as pyridine.

2. Composition according to claim 1, wherein R1, R2 and R3 are each independently selected from the list consisting of a C1-C4 alkyl, preferably CH3.

3. Composition according to any of claims 1 to 2, wherein the compound of formula I is: or any salt thereof, and where R1, R2 and R3 are preferably CH3.

4. Composition according to any of claims 1 to 2, wherein the compound of formula I is: (8R)-8-Hydroxyepoxyethanol A (1) or any salt thereof.

5. Method or procedure for obtaining a compound of formula I according to any of claims 1 to 4, wherein the method comprises the biotransformation by M. circinelloides NRRL3631 of epoxiboetyran A obtained from E. boetica plants.

6. In vitro use of a compound of formula I, as defined in any of claims 1 to 4, for promoting differentiation or for differentiating neurons, in culture, from stem cells of fetal or adult origin with the capacity to differentiate into neurons, such as induced pluripotent stem cells, neural precursors, or neural stem cells.

7. The use according to claim 6, wherein said compound of formula I is the compound defined in claim 3 or 4.

8. An in vitro method for obtaining neurons from fetal or adult stem cells capable of differentiating into neurons, comprising: a. contacting said fetal or adult stem cells capable of differentiating into neurons, with a compound of formula I as defined in any of claims 1 to 4, in a medium suitable for the differentiation of said stem cells; and b. optionally, subsequently harvesting the neurons obtained.

9. Method according to claim 8, wherein in step a) the stem cells are contacted with a concentration range from 1 pmol / L to 40 pmol / L with the compound of formula I, as defined in any of claims 1 to 4, in a medium suitable for the differentiation of neural stem cells or neural precursors.

10. Method according to claim 8 or 9, wherein the compound of formula I is the compound defined in claim 3 or 4.

11. Culture medium suitable for differentiation to neurons, characterized in that it comprises a compound of formula I, as defined in any of claims 1 to 4, preferably the compound (8 / ?)-8-hydroxyepoxyethanol A, and / or a salt thereof.

12. Culture medium according to claim 11, and wherein the compound of formula I is in the medium in a concentration range of 1 pmol / L to 40 pmol / L.

13. Use of the culture medium as defined in any of claims 11 or 12, to promote the differentiation or to differentiate into neurons stem cells of fetal or adult origin with the capacity to differentiate into neurons, such as induced pluripotent stem cells, neural precursors or neural stem cells.

14. Composition comprising a population of neurons obtainable by the method according to any of claims 8 to 10.

15. Composition comprising a compound of formula I as defined in any of claim 1 or 4, for use in the treatment of diseases or injuries involving neuronal loss selected from the group consisting of: focal cerebral ischemia, traumatic brain injury with neuronal damage, Parkinson's disease, epilepsy, and amyotrophic lateral sclerosis.

16. Composition for use according to claim 15, further comprising one or more pharmaceutically acceptable excipients.

17. Composition for use according to any of claims 15 or 16, further comprising a population of neural precursors, neural stem cells or other stem cells with the ability to differentiate into neurons.

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