Patent Application
20160194274
The present invention refers to phenanthrene derivatives for use as medicaments, mainly in the prevention and/or treatment of human diseases involving presence of toxic RNAs comprising: Myotonic Dystrophy Type 1 (DM1), Huntington's Disease Like 2 (HDL2), Spinocerebellar Ataxia Type 8 (SCA8), Myotonic Dystrophy Type 2 (DM2), Spinocerebellar Ataxia Type 3 (SCA3), Fragile-X-Associated Tremor/Ataxia Syndrome (FXTAS), Frontotemporal Degeneration/Amyotrophic Lateral Sclerosis (FTD/ALS), and Spinocerebellar Ataxia Type 31 (SCA31). In a preferred embodiment, said phenanthrene derivatives are used as antimyotonic agents. Therefore, this invention may be included either in the whole pharmaceutical or medical field.
DM1, also called Dystrophia Myotonica or Steinert's disease, was described more than 100 years ago. DM1 displays an autosomal dominant mode of inheritance and is now recognized as one of the most common forms of muscular dystrophy in adults with a prevalence of 1:8,000-20,000 individuals worldwide (Harper P S. Major Problems in Neurology: Myotonic Dystrophy. London, UK: WB Saunders (2001)). The name myotonic dystrophy refers to two main muscle symptoms identified in this disease: myotonia refers to a state of delay in muscle relaxation, while dystrophy indicates the progressive deterioration of muscle. In addition to muscular dystrophy and myotonia, DM1 includes a consistent group of apparently unrelated and rare clinical symptoms, like: cardiac conduction defects, insulin resistance, ocular cataracts, cognitive abnormalities (developmental delays and learning problems), daytime sleepiness and testicular atrophy. Due to said diversity in symptoms, this disease is perhaps best classified as a multisystemic neuromuscular disease. It is important to emphasize that not all the above cited problems occur in a single patient because DM1 is not only highly variable in the type of symptoms, but also severity and age of onset strongly differ.
In 1992, the genetic mutation underlying DM1 (OMIM #160900) was identified as an expanding (CTG)n repeat within the DM1 locus in the 3′ untranslated region of the DMPK (Dystrophy Myotonic Protein Kinase) gene on chromosome 19q13.3 (Brook J. et al. Cell, 68:799-808 (1992)). Manifestation of disease generally correlates with (CTG)n repeat length, as determined in blood DNA. Number of repeats in the DMPK gene tends to increase from generation to generation, accounting for the genetic anticipation (i.e. the earlier onset of disease manifestation in successive generations) in DM1 families. Next to intergenerational CTG-triplet repeat instability, somatic instability in tissues is also observed during adulthood and ageing. Four categories of clinical manifestation of DM1 can be distinguished based on the number of CTG repeats: (i) normal or unaffected individuals carry a CTG repeat between 5 and 37 triplets; (ii) mild or late onset of disease is seen in patients with 50 to 150 triplets, most of these patients only have cataracts; (iii) classic disease manifestation is associated with repeat lengths of 100 to 1000 CTG triplets; and (iv) DM1 patients with congenital or juvenile onset disease carry several thousands of CTG triplets in their blood DNA. Molecular mechanism underlying DM1 pathogenesis implicates several cellular pathways and is not yet well established. However, the most widely accepted pathogenic pathway is a toxic RNA gain of function where repeat containing mRNAs form toxic dsRNA hairpins that are retained in nuclear foci. These hairpins sequester several nuclear factors, including the splicing factor Muscleblind-like 1 (MBNL1) (Jiang H et al. Hum. Mol. Genet. 13(24):3079-88 (2004)). Sequestration of MBNL1 has a key role in DM1 pathogenesis, since MBNL1 knockout (KO) mice showed much of the pathogenesis seen in CTG mouse models (Kanadia R N et al. Science, 302:1978-80 (2003)), and over-expression of MBNL1 in a DM1 mouse model is able to reduce disease symptoms (Kanadia R N et al. Proc. Natl. Acad. Sci. USA, 103:11748-53 (2006)). Importantly, several mRNAs whose splicing is regulated by MBNL1 showed aberrant splicing in DM1 (Du H et al. Nat. Struct. Mol. Biol. 17(2):187-93 (2010)). Thus, DM1 has been defined as a spliceopathy, a disease mainly caused by splicing defects, and some of the most relevant symptoms in this disease have been related to splicing defects: aberrant exclusion of insulin receptor exon 11 originates a lower signaling receptor that probably causes insulin resistance; unusual inclusion of ClC-1 7a exon generates a STOP codon which triggers this mRNA to the Non Sense Mediated Decay pathway (Ranum L P and Cooper T A. Annu. Rev. Neurosci. 29:259-77(2006)); and recently, muscle degeneration has been linked to aberrant splicing of bridging integrator-1 (BIN1) (Fugier C et al. Nat. Med. 17(6):720-5. (2011)).
Since the discovery that RNAs with CUG expansions may be toxic for cells, a whole series of scientific evidence supports the hypothesis that these expansions, or similar expansions, in other RNAs, may cause hereditary genetic pathologies similar to DM1. For example, in DM2, expansions of the CCUG tetranucleotide in the first intron of the RNAs transcribed from the CNBP (zinc-30 finger protein 9, Entrez #7555) gene trigger a pathology that is very similar to DM1 (OMIM #602668). In fact, in the state of the art, genes related to the nervous function present altered levels in both DM1 and DM2 patients, which supports the idea of a pathogenesis mechanism common to both diseases. This list is growing and includes diseases as some spinocerebellar ataxias (SCA8, OMIM #603680; SCA3 OMIM #109150, SCA10 OMIM #603516, SCA31 OMIM #117210, SCA36 OMIM #614153), Fragile X-associated tremor/ataxia syndrome (FXTAS, OMIM #300623), Huntington's disease-like 2 (HDL2, OMIM #606438) (reviewed in Dickson A M and Wilusz C J. WIREs RNA 1:173-192 (2010)), and recently frontotemporal dementia and/or amyotrophic lateral sclerosis (FTD/ALS #105550) (DeJesus-Hernandez M et al. Neuron. 72(2):245-56 (2011)).
Although DM1 was first described in 1909, there is still not an effective therapy available. The mechanism of pathogenesis of the DM1 is nowadays reasonable well understood and several experimental therapies are being investigated in animal models of the disease. This is the case of compounds described in the international application WO2009105691 that provides compounds, which are small organic molecules, such as diamide or a derivative thereof, which may be used in methods of treatment of myotonic dystrophy or other toxic RNA diseases. However, although all the treatments applied are palliative and contribute to mitigate the development of symptoms, in no case prevent the onset thereof or treat the disease in a definitive manner. For example, there is no compound capable of reverting lack of chloride conductance so as to reduce myotonia. Some compounds, such as mexiletine, quinidine, phenytoin, procainamide or carbamazepine, which inhibit the sodium entry necessary for the initiation and propagation of impulses, are administered to patients in order to treat this symptom. Types of potentially new anti-myotonia compounds are described in Talon S. et al., (Talon S. et al. Br J Pharmacol. 2001; 134(7):1523-31) that discloses new derivatives of tocainide as antimyotonic agents, in which the chiral carbon atom is constrained in a rigid alpha-proline or pyrrolo-imidazolic cycle in order to improve the potency and the stereoselectivity of Na(+) channel blockers. Also in Desaphy J F. et al (Desaphy J F. et al. Neuropharmacology. 2013; 65:21-7), that evaluated in vivo the antimyotonic activity of β-adrenergic drugs, such as β2-agonist clenbuterol, β-antagonist propranolol, (β2-agonist salbutamol and the β-antagonist nadolol, compared to that of mexiletine. The results demonstrate that both clenbuterol and propranolol show a time-dependent antimyotonic effect in the dose range of 5-40 mg/kg, quite similar to that of mexiletine. In contrast, either the β2-agonist salbutamol or the β-antagonist nadolol, both unable to block sodium channels, did not show any significant antimyotonic effect. Occasionally, said molecules are used, in turn, as a treatment for cardiac arrhythmias; consequently, given the risk that they entail due to their effect on the cardiac function, it is preferable to avoid using them as anti-myotonic agents. Moreover, many of these treatments reduce muscular strength.
The above mentioned documents disclose compounds for use as antymiotonic agent or for use in the treatment of myotonic dystrophy. The present invention is focused on providing alternative chemical compounds or drugs, involving phenanthrene derivatives, acting as effective antimyotonic agents and also being effective in the treatment of human diseases involving the presence of toxic RNA comprising CTG (DM1, HDL2 and SCA8), CCTG (DM2), CAG (SCA3), CGG (FXTAS), GGGGCC (FTD/ALS), and TGGAA (SCA31) repeats. Up to date, only, patent application US2007149543 discloses phenanthrene derivatives for therapeutic use. Specifically, this patent application discloses phenanthrene derivatives to be used in the inhibition of neuronal cell death, for example in the context of neurodegenerative disorders such as Huntington's disease (HD) caused by polyQ protein expansion.
Such as it is explained above, the present invention refers to phenanthrene derivatives for use as medicaments, mainly in the prevention and/or treatment of human diseases with presence of pathologic toxic RNAs comprising DM1, HDL2, SCA8, DM2, SCA3, FXTAS, FTD/ALS and SCA31. In a preferred embodiment, phenanthrene derivatives of the invention are used as antimyotonic agents.
The compounds of the present invention are primarily based on the massive in vivo screening (in vivo High Throughput Screening (HTS)) of chemical libraries containing thousands of small molecules by using transgenic Drosophila models for DM1 disease as reading tools (Garcia-Lopez et al, 2008 and developed in house stocks). Results after primary screening of 15,000 compounds allowed describing several (more than 20) novel chemical entities (hits) with potential anti-DM1 candidates. The list of compounds of the present invention is built on further development of two specific hits (VLT002 and VLT015) identified with close chemical backbone. The present invention shows that these molecules display anti-DM1 activity after measurement on well-established DM1 phenotypes in different disease models (see figures and examples set below). At the same time, an interesting mechanism of action is suggested since some of the compounds have shown the ability to bind to the toxic double-stranded CUG-RNA hairpin and modify the structure preventing the aberrant binding of MBNL1 and potentially any other molecules whose binding might cause or intensify the pathological phenotype.
Thus, several compounds of Formula I (see below) has been identified in the present invention having therapeutic potential to prevent or treat DM1 which is caused by the presence of toxic RNA repeat transcripts that comprise a CUG motif. For this purpose, it was initially performed in vivo screening of chemical libraries in a Drosophila model of the disease. It was observed that the Drosophila model described in (Garcia-Lopez et al., 2008), which expresses 480 CTG repeats (CTG(480)), displayed important DM1 disease phenotypes that respond to levels of Muscleblind and is susceptible to chemical modification, which makes this model suitable for the systematic search of compounds. Based on this, and developing novel in house Drosophila stocks, it was generated an automated minigene-based drug screening assay by measuring luciferase reporter in adult flies in order to accurately measure potential disease phenotype recovering after the treatment with the compounds of the invention. As explained above, the present invention begins with the screening of several chemical libraries (15,000 small molecules), identification of more than 20 primary hits, and final selection and further development of two interesting ones (VLT002 and VLT015), which give rise to other interesting compounds supported throughout the present invention in terms of activity and mechanism of action. This approach ended with the recognition of a specific Markush formula (see Formula I below), and the assessment of the anti-DM1 activity of the compounds of the invention comprised in said Formula I and also of their capacity to improve or recover DM1 phenotypes characterized by disease hallmarks (precisely the aberrant aggregation of ribonuclear foci). The molecules comprised in the Formula I were further tested and validated in additional disease phenotypes in different models as Drosophila, human cells and mice. The mechanism of action suggested for the compounds of the present invention is linked to their ability to bind CUG repeat toxic structures and switching them to a non-toxic conformation, thereby preventing the aberrant binding or sequestration of MBNL and any other molecules whose binding to CUG hairpin loops could also cause or intensify the pathological phenotype.
On the other hand, the size of the molecules to be used in the present invention as therapeutic agents is an important issue to be considered, since a molecular weight that is too high limits the absorption of the compound inside the cell. The optimization of molecules to produce more active compounds is usually accompanied by an increase in the final size thereof. However, a molecular weight greater than 1,000 Daltons reduces the molecules' therapeutic potential, by reducing the bioavailability thereof, which makes it necessary to start from small compounds. Thus, 80% of the drugs currently commercialized have a molecular weight of less than 450 Daltons. All the specific molecules here identified have a molecular weight of 250 to 500 Daltons.
More precisely, the present invention refers to “phenanthrene-1-enthylamine” compounds of Formula I
wherein
R1, R2 and R3 are independently selected from: H, alkyl (ie: CH3, CH2CH3, CH2CH2CH3) carbonyl (i.e.: COCH3) or aryl (ie: -benzyl: C7H7) groups. Alternative, one of R1, R2 or R3 is carbon atom C10 of the molecule (part of ring with subsequent lack of double bond between carbon atoms C9-C10).
R4 is independently selected from: H, carbonyl (CO) or alcohol (OH) groups.
X and Y are independently selected from: alcohol (OH), alkyl (ie: CH3, CH2CH3, CH2CH2CH3) carbonyl (i.e.: COCH3,), halide (ie: Cl), alkyl-halide (ie: C2H4Br), aryl (ie: benzyl: C7H7), urethane (ie: C3H6NO, C7H5NF), aminoacid or aminoacid precursors (ie: Arg) or tiourethane (ie: C7H3NSF3) groups. Alternatively, X, Y as part of the ring, wherein, preferably, X=Y=CH2.
A and B are independently selected from: H, alcohol (OH), halide (ie: —Cl), alkyl-halide (ie: OC2H4Br), O-alkyl (ie: OCH3, OCH2CH3, OCH2CH2CH3,) O-carbonyl (i.e.: OCOCH3), O-aryl (ie: O-benzyl: OC7H7), O-urethane (ie: OC3H6NO, OC7H5NF), O-aminoacid or O-aminoacid precursors (ie: OArg) or O-tiourethane radicals (ie: OC7H3NSF3). Alternatively, A, B as part of the ring, wherein, preferably, A=B.
Preferred A, B, X or Y moieties are selected from:
Intermittent/discontinuous bonds in Formula I mean that the bond may be present or not.
In a still preferred embodiment the present invention refers to the following compounds comprised in the above cited Formula I:
VLT002: R1=R2=CH3; X,Y=CH2; A=B=H
VLT025: R1=R2=R3=CH3; X,Y=CH2; A=B=H
VLT024: R1=R2=R3=CH3; X=Y=CH3; A=B=OCH3
VLT023: R1=R2=CH3; X=Y=CH3; A=B=OCH3
VLT026: R1=CH3; R2=Bn; X=Bn; Y=CH3; A=OCH3; B=OBn
VLT051: R1=R2=R3=CH3; X=Y=CH3; A=B=H
VLT015: R1=CH3; R2=Carbon-10; X=H; Y=CH3; A=OCH3; B=OH
VLT040: R1=H; R2=Carbon-10; X,Y=CH2; A=B=H
VLT042: R1=R2=CH3; R3=Carbon-10; X,Y=CH2; A=B=H
VLT043: R1=CO—CH3; R2=Carbon-10; X,Y=CH2; A=B=H
VLT048: R1=CH3; R2=Carbon-10; X=CO—NH—CH2—CH3; Y=CH3; A=OCH3; B=OCONH—CH2—CH3
VLT049: R1=CH3; R2=Carbon-10; X=Y=CH3; A=B=H
VLT045: R1=R2=Carbon-10; X,Y=CH2; A=B=H; C4=C5; C6=C6a; C7=0
VLT053: R1=R2=R3=CH3; X=Y=H; A=B=H
VLT056: R1=R2=CH3; X=Y=COCH3; A=B=OCOCH3
Consequently, the first embodiment of the present invention refers to compounds of Formula I, derivatives, or pharmaceutically acceptable salts thereof, for use as a medicament. In a preferred embodiment, the present invention refers to compounds of Formula I for use as an antimyotonic agent, preferably in the prevention and/or treatment of human diseases involving the presence of toxic RNA comprising: Myotonic Dystrophy Type 1 (DM1), Myotonic Dystrophy Type 2 (DM2), Huntington's Disease Like 2 (HDL2), Spinocerebellar Ataxia Type 8 (SCA8), Spinocerebellar Ataxia Type 3 (SCA3), Fragile-X-Associated Tremor/Ataxia Syndrome (FXTAS), Frontotemporal Degeneration/Amyotrophic Lateral Sclerosis (FTD/ALS), and Spinocerebellar Ataxia Type 31 (SCA31).
For the purpose of the present invention “antimyotonic agent” refers to any compound able to improve the slow relaxation of the muscles after voluntary contraction or electrical stimulation, a symptom of a small handful of certain neuromuscular disorders.
For the purpose of the present invention, the term “derivative” refers to a compound obtained from a compound according to Formula (I), an analog, tautomeric form, stereoisomer, polymorph, hydrate, pharmaceutically acceptable salt or pharmaceutically acceptable solvate thereof, by a simple chemical process converting one or more functional groups, by means of oxidation, hydrogenation, alkylation, esterification, halogenation and the like. As used herein, the term “analog” refers to a compound having a structure similar to that of another one, but differing from it with respect to a certain component. The compound may differ in one or more atoms, functional groups, or substructures, which may be replaced with other atoms, groups, or substructures. In one aspect, such structures possess at least the same or a similar therapeutic efficacy. The term “tautomer” or “tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton. Valence tautomers include interconversions by reorganization of some of the bonding electrons. The term “stereoisomer” refers to one of a set of isomers whose molecules have the same number and kind of atoms bonded to each other, but which differ in the way these atoms are arranged in space. The term “polymorph” refers to crystallographically distinct forms of a substance. The term “hydrate” as used herein includes, but is not limited to, (i) a substance containing water combined in the molecular form and (ii) a crystalline substance containing one or more molecules of water of crystallization or a crystalline material containing free water.
In a preferred embodiment, the compounds of Formula I for use as a medicament, or as an antimyotonic agent, preferably in the prevention and/or treatment of human diseases involving the presence of toxic RNA comprising: DM1, DM2, SCAs, FXTAS, HDL2 or FTD/ALS, are selected from: VLT002, VLT025, VLT024, VLT023, VLT026, VLT051, VLT015, VLT040, VLT042, VLT043, VLT048, VLT049, VLT045, VLT053 or VLT056. In a preferred embodiment, the compounds of Formula I for use as a medicament, or as an antimyotonic agent, preferably in the prevention and/or treatment of DM1, DM2, SCAs, FXTAS, HDL2 or FTD/ALS, are precisely selected from: VLT002, VLT025 or VLT015. In a still preferred embodiment the present invention refers to compounds of Formula I for use in the treatment of DM1, precisely selected from: VLT002, VLT025 or VLT015.
The second embodiment of the present invention refers to a pharmaceutical composition comprising a compound of Formula I as defined above and pharmaceutically acceptable carriers, for use as a medicament, preferably for use in the treatment of DM1, DM2, SCAs, FXTAS, HDL2 or FTD/ALS. In a preferred embodiment the present invention refers to a pharmaceutical composition comprising a compound of Formula I as defined above and pharmaceutically acceptable carriers for use as an antimyotonic agent, preferably for use in the treatment of DM1, DM2, SCAs, FXTAS, HDL2 or FTD/ALS. The last embodiment of the present invention refers to the compounds of Formula I selected from: VLT026 or VLT048.
As cited above, compounds of Formula I can be administered either alone or formulated in pharmaceutical compositions which combine at least two compounds of Formula I. Alternatively, said composition may comprise at least one compound of Formula I and pharmaceutically acceptable carriers or excipients such as: binders, fillers, disintegrators, lubricants, coaters, sweeteners, flavouring excipients, colouring excipients, transporters, etc. or combinations thereof. Also, the compounds of Formula I can be part of pharmaceutical compositions in combination with other active ingredients (the latter not comprised in the Formula I).
On the other hand, the administration of the compounds of Formula I can be carried out by any means, for example enterally (IP), orally, rectally, topically, by inhalation or by intravenous, intramuscular or subcutaneous injection.
In addition, the administration may be either according to the Formula I above described or in any pharmaceutically acceptable salt, solvates or derivative thereof, such as: esters, ethers, alkyl, acyl, phosphate, sulfate, ethyl, methyl, propyl, salts, complexes, etc.
In a preferred embodiment, the compounds of the invention may be administered in the form of prodrugs as a pharmacological substance that is administered in an inactive (or less than fully active) form, and is subsequently converted to an active pharmacological agent (drug) through normal metabolic processes (bioactivation). These prodrugs are used to improve the absorption, distribution, metabolism and excretion of the active principle. Thus, said prodrugs are often designed to improve bioavailability or their selectively to interacts with the intended target. Particularly, in the present invention, the inclusion of carbamates or urethanes gives rise to prodrugs which, as explained above, improve the bioavailability of the compounds of Formula I.
Another embodiment of the present invention refers to a method for the prevention and/or treatment of human diseases involving presence of toxic RNAs comprising: DM1, HDL2, SCA8, DM2, CAG SCA3, FXTAS, FTD/ALS, and SCA31, which comprises the administration to the patients at least a compound of Formula I or a composition comprising thereof.
Such as it can be seen in this
A strong reduction of the aberrant phenotype is observed even from those compounds without reaching a statistically significant value.
As can be seen, flies treated with VLT024 displayed a significant increase of the muscle area relative to DMSO-treated flies (quantified in graphic) * indicates p-value <0.05. A minimum of 8 flies were analyzed in each case.
Consequently, VLT002, VLT025, VLT045, VLT049 and VLT040 are able to effectively reduce the formation of foci and also to relieve MBNL1 sequestration. * indicates p-value <0.05. A minimum of 100 cells were analyzed in each case.
This
The present invention is defined in more detail by means of the following examples, whose purpose is illustrating the invention without limiting its scope of protection.
After screening identification of the compounds of the invention (see
All the assayed compounds of Formula I give rise to a statistically significant reduction in the number of foci/nuclei (
At the same time human cells were recollected for foci counting. Some (150,000-300,000) were prepared for Muscleblind (MBNL1) detection (see
As shown in
Additional assays involved:
Also, an immunohistochemistry approach was used to measure ClC-1 protein levels (see
Myotonia, as important functional DM1 sign displayed by HSALR mice was measured by electromyography (
Fluorescence Electrophoretic Mobility Shift Assay to test RNA-compound binding. Briefly, an aliquot of carboxyfluorescein (FAM)-CUG10 RNA (DM1, HDL2, SCA8; see
Aminopurine Fluorescence assay to detect hairpin double strand RNA unfolding. An aliquot of 2-aminopurine (2AP)-CUG23 RNA (1 μM in binding buffer) was heated at 90° C. for 3 min, placed on ice, and further diluted to 30 nM in a final volume of 1 mL in binding buffer. The fluorescence emission spectra of the RNA were obtained as described in ((Garcia-Lopez A et al. Proc. Natl. Acad. Sci. USA. 108(29):11866-71 (2011)), monitoring fluorescence emission peaks at 363 nm and 375 nm. All measures were taken in a Shimadzu RF-1501 spectrofluorophotometer. As shown in
R1, R2, R3=H or CH3 or CH2CH3 or CH2CH2CH3 or Benzyl
X, Y=H or CH2 or CH3 or OCOCH3 or C7H7 or C2H4Br or C3H6NO or C7H5NF or C7H3NSF3 or CH2CH3
A, B=H or OCH2O or Cl or OCH3 or OCOCH3 or OC3H7 or OC7H7 or OC2H4Br or OC3H6NO or OC7H5NF or OC7H3NSF3 or OCH2CH3 or OH
Plant Material: The concentrated methanolic extract of the bark of Xylopia spp (Annonaceae), or leaves of Berberis spp (Berberidaceae) (3 g), was dissolved in methanol/HCl 5% 1:9. The mixture was heated and stirred, and then filtered. The aqueous-methanolic extract was partially evaporated and basified with NH4OH until pH=9. Then the solution was washed with CH2Cl2 (15 ml) up to five times. The organic layers were evaporated to dryness (385 mg, 13%) and a column chromatography over silica gel flash was eluted with toluene-EtOAc-MeOH-TEA 6:2.5:1.5:0.1. 60 mg (2%) of VLT040 were obtained. The procedure was repeated as many times as necessary.
General Instrumentation.
Melting points were taken on a Cambridge microscope instruments coupled with a Reichert-Jung. EI and FAB mass spectra were recorded on a VG Auto Spec Fisons spectrometer instruments (Fisons, Manchester, United Kingdom). 1H NMR and 13C NMR spectra were recorded with CDCl3 as solvent on a Bruker AC-300, AC-400 or AC-500. Multiplicities of 13C NMR resonances were assigned by DEPT experiments. NOE DIFF irradiations, COSY, HMQC, HSQC and HMBC correlations were recorded at 400 MHz and 500 MHz (Bruker AC-400 or AC-500). All reactions were monitored by analytical TLC with silicagel 60 F254 (Merck 5554). The residues were purified through silica gel 60 (40-63 μm, Merck 9385) column chromatography. Solvents and reagents were used as purchased from commercial sources. Quoted yields are of purified material. The HCl salts of the synthesised compounds were prepared from the corresponding base with 5% HCl in MeOH.
VLT040.
1H NMR* (300 MHz, CDCl3): δ=8.00 (d, 1H, J=8, H-11), 7.28-7.11 (m, 3H, H-8, H-9, H-10), 6.48 (s, 1H, H-3), 5.96 and 5.82 (2d, 2H, J=1.2 Hz, OCH2O), 3.90 (m, 1H, H-6a), 3.30 (m, 1H, H-5α), 3.00 (m, 1H, H-4α), 2.95 (m, 1H, H-5β), 2.90 (m, 1H, H-7α), 2.80 (m, 2H, H-4β and H-7β); 13C NMR* (75 MHz, CDCl3): δ=146.8 (C-2), 142.5 (C-1), 135.4 (C-7a), 131.0 (C-11a), 128.7 (C-4a), 128.1 (CH-1b), 127.5 (CH-8), 127.2 (CH-10), 127.1 (CH-9), 127.0 (CH-11), 116.3 (C-1a), 108.0 (CH-3), 100.6 (OCH2O), 53.6 (CH-6a), 43.6 (CH2-5), 37.4 (CH2-7), 29.6 (CH2-4); *The assignments were made by COSY 45, DEPT, HSQC and HMBC.
VTL015.
1H NMR* (300 MHz, CDCl3): δ=7.89 (s, 1H, H-11), □ 6.90 (s, 1H, H-8), 6.60 (s, 1H, H-3), 3.92 (s, 3H, OCH3-10), 3.61 (s, 3H, OCH3-1), 3.2-2.9 (m, 4H, H-6a, H-4α, H-5α, H-7-α), 2.70-2.40 (m, 3H, H-4β, H-5β, H-5β), 2.58 (s, 3H, N—CH3); 13C NMR* (75 MHz, CDCl3): δ=148.1 (C-10), 145.6 (C-1), 145.0 (C-2), 142.2 (C-9), 130.0 (C-7a), 129.3 (CH-4a), 126.2 (C-1a), 126.1 (C-1b), □123.5 (C-11a), 114.4 (CH-8), 113.4 (CH-3), 110.5 (CH-11), 62.4 (CH-6a), 60.2 (OCH3-1), 56.2 (OCH3-10), 53.2 (CH2-5), 43.8 (N—CH3), 33.8 (CH2-7), 28.4 (CH2-4); *The assignments were made by COSY 45, DEPT, HSQC and HMBC.
A. General Process for the Preparation of the General Formula I as Derivatives from Aporphines:
B. General Procedure for N,N-dimethylation of Aporphine.
The appropriate aporphine, e.g., VLT040 (pm=265, 80 mg, 0.3 mmol) was dissolved in 0.3 ml of MeOH and 5.5 ml of Et2O; 1.3 ml of iodomethane was added, and the mixture stirred at room temperature for 6 hours. The reaction mixture was evaporated to dryness and redissolved in 2 ml of MeOH; 30 ml of Et2O were added and the suspension was centrifuged for five minutes at 1500 rpm speed. The supernatant was removed and the pellet redissolved in 2 ml of MeOH. This procedure was repeated as necessary. The pellet was taken and evaporated to dryness to afford 85 mg of N,N-dimethyl-VLT040 (pm=294, 0.28 mmol, 93%) N,N-dimethyl-VLT040 (II.1).
C. General Procedure for Aporphine Hofmann's Elimination: L.1 (VLT002)
Formation of the phenanthrenic alkaloids was carried out under Hofmann's conditions using the quaternary ammonium salt of the corresponding aporphine, e.g., N,N-dimethyl-VLT040, II.1 (85 mg, pm=294, 0.28 mmol), which is dissolved in 20 ml of MeOH; 20 ml of KOH 3N were added. The mixture was stirred at 45° C. for eight hours. After partial evaporation of organic solvent, the aqueous solution is extracted with CH2Cl2. The organic layers were dried with Na2SO4 and concentrated to dryness. The residue obtained was purified by a silica gel flash column (CH2Cl2-MeOH 95:5) to afford 60 mg of phenantrenic alkaloid I.1 (pm=293, 0.20 mmol, 73%).
Stephenanthrine, I.1 (VLT002).
1H NMR* (300 MHz, CDCl3): δ=9.06 (d, 1H, H-5, J=8), 7.86 (d, 1H, H-10, J=9), 7.82 (m, 1H, H-8), 7.62-7.42 (m, 3H, H-6, H-7, H-9), 7.17 (s, 1H, H-2), 6.15 (s, 2H, OCH2O), 3.35 (t, 2H, CH2-β), 2.85 (t, 2H, CH2-α), 2.54 (s, 6H, N—(CH3)2); 13C NMR* (75 MHz, CDCl3): δ=145.0 (C-3), 142.2 (C-4), 132.0 (C-8a), 130.8 (C-1), 128.8 (C-4b), 127.6 (CH-8), 127.3 (CH-5), 126.7 (CH-7), 126.2 (CH-6), 126.0 (C-10a), 125.1 (CH-9), 122.7 (C-10), 117.1 (C-4a), 110.5 (CH-2), 101.1 (OCH2O), 61.2 (CH2-α), 45.3 (N(CH3)2), 31.9 (CH2-β); *The assignments were made by COSY 45, DEPT, HSQC and HMBC; ESMS m/z (%): 294 (100) [M+1]+.
D. General Procedure for Aporphine Methylation and Ring Opening: 1.2 (VLT024)
A mixture of VTL015 (pm=327, 150 mg, 0.45 mmol), iodomethane (6 eq, 1.83 mmol, 0.12 ml) and anhydrous K2CO3 (4 eq, 1.83 mmol, 126 mg) in dry acetone (15 ml) was stirred and refluxed for 24 hours. The solution was concentrated to dryness and extracted with CH2Cl2 (20 ml×3). The organic layers were washed with brine and concentrated to dryness to afford 122 mg of the resulting product 1.2 (VLT024) (pm=384, 0.31 mmol, 70%). 1.2 (VLT024). 1H NMR* (300 MHz, DMSO-d6): δ=9.15 (s, 1H, H-5), 7.82 (d, 1H, H-10, J=10), 7.78 (d, 1H, H-9, J=10), 7.50 (s, 1H, H-8), 7.43 (s, 1H, H-2), 4.05, 3.99, 3.93 and 3.86 (4s, 12H, 4-OCH3), 3.75-3.57 (m, 4H, CH2-α, CH2-β), 3.30 (s, 9H, 3-NCH3); 13C NMR* (75 MHz, CDCl3): ESMS m/z (%): 384 (100) [M]+.
E. General Procedure for Aporphine Benzylation and Ring Opening: 1.3 (VLT026)
A mixture of the aporphine, e.g, VTL015 (pm=327, 250 mg, 0.76 mmol), benzyl chloride (3 eq, 2.28 mmol, 0.26 ml) and anhydrous K2CO3 (1.5 eq, 1.14 mmol, 157 mg) in EtOH (10 ml) was refluxed overnight. After being stirred, the reaction mixture was concentrated to dryness and redissolved in 10 ml of CH2Cl2, and then 5% aqueous NaOH (3×10 ml) was added. The organic layer was washed with brine (2×10 ml) and H2O (2×10 ml), dried with anhydrous Na2SO4 and evaporated to dryness. The residue obtained was purified by a silica gel flash column (toluene-EtOAc-MeOH-TEA 8:1:1:0.1) to afford the resulting compound (I.3) (90 mg; pm=597, 0.15 nmol; 20%). I.3 (VLT026). 1H NMR* (300 MHz, CDCl3): δ=9.3 (s, 1H, H-11), 7.8 (d, 1H, H-10), 6.6 (s, 1H, H-3), 7.6-7.2 (m, 16H-OBn, -NBn, H-9), 7.2 (s, 1H, H-8), 5.4 (2H, OCH2Ph), 5.3 (OCH2Ph), 4.1 (s, 3H, —OCH3), 4.0 (s, 3H, —OCH3), 3.6 (s, 2H, NCH2Ph), 3.2 (t, 2H, CH2-α), 2.8 (t, CH2-β), 2.4 ppm (s, 3H, NCH3); 13C NMR* (75 MHz, CDCl3): δ=; ESMS m/z (%): 598 (100) [M+1]+.
| Number | Date | Country | Kind |
|---|---|---|---|
| 12197087.5 | Dec 2012 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/076615 | 12/13/2013 | WO | 00 |
