Optionally 18f-labeled 1,4-dihydropyridines
Patent Information
- Application Number
- PCT/EP2026/058838
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-03-27
- Filing Date
- 2026-03-26
- Publication Date
- 2026-10-01
Smart Images
Figure EP2026058838_01102026_PF_FP_ABST
Abstract
Description
[0001] 18F-LABELED 1,4-DIHYDROPYRIDINES AND OTHER COMPOUNDS FIELD OF THE INVENTION
[0002] The present invention relates to18F-labeled compounds , it also relates to system and method for making such compounds and uses thereof, in particular as a compound or a library of novel PET tracers.
[0003] A more specific aspect, the present invention provides these18F-labeled compounds whereby the C4 stereogenic center dihydropyridine ring is enantioenriched (being either S or R" refers to a specific feature of a molecule, particularly its chirality or handedness at a specific carbon atom (C4) in a ring structure. Hereby the carbon atom 4 (C4) in the18F-labeled compound is a stereogenic center (chiral center), attached to four different groups, a configuration allowing the carbon to exist in two possible mirror-image forms (enantiomers), either the S or R form. The C4 stereogenic center is part of a cyclic (dihydropyridine ring) structure in the18F -lab eled and non-labelled compounds.
[0004] The invention provides a way to the design and synthesis of a library consisting of homochiral [18F]1,4-DHP derivatives, hereby named [18F]radipines. The invention also concerns the use of such compounds in pharmacological assessment of disorders related to dysfunction of L-type voltage-gated calcium cannels and in particular their use as neuroimaging agents for positron emission tomography. In vivo pharmacokinetic assessment and radiometabolite studies, demonstrated [18F]radipinel to be a promising PET tracer for imaging of neurodegeneration related to disfunction of voltage-gated calcium channels. In vivo preblocking experiments including selfblock (with the non-labeled version) and heteroblock (with isradipine) carried out were able to decrease the radioactive signal by 30% compared to baseline signal in brain regions brain regions known to express L-type calcium channels (LTCCs or CaVl.l-CaV1.4). Additionally, preblocking experiments in presence of amlodipine as a competitor did not decrease baseline signal, as expected, because amlodipine does not cross the blood-brain barrier. In vivo blocking studies were confirmed via in citro autoradiography as well. In addition, [18F]radipine2 has an outstanding profile as a radioligand for ex-vivo brain autoradiography applications.
[0005] BACKGROUND ART
[0006] L-type calcium channels (LTCCs or Cavl.l-Cavl.4) are protein complexes responsible for various cellular functions including neurotransmitter release, hormone secretion, gene expression, and Ca2+ homeostasis. (Caulfield, M. E. and Manfredsson, F. P.; Steece-Collier, K., ed., Vol. 279; Springer Berlin Heidelberg: Berlin, Heidelberg, 2023, pp 1-31)Such LTCCs have traditionally been recognized for their role in the pathophysiology of cardiovascular diseases, such as hypertension and cardiac arrhythmias. (Shah, K.; et al; Cells 2022, 11 (6), 943.)
[0007] However, more recently, they have been linked to neurodegenerative (Caulfield, M. E. and Manfredsson, F. P.; Steece-Collier, K., ed., Vol. 279; Springer Berlin Heidelberg: Berlin, Heidelberg, 2023, pp 1-31, Sandoval, A. et al. Int. J. Neurosci. 2022, 1-10; bCrossley, C. A.; Rajani, V. and Yuan, Q., Comput. Struct. Biotechnol. J. 2023, 21, 11-20) and neuropsychiatric diseases (Andrade, A.; et al. Int. J. Mol. Sci. 2019, 20 (14), 3537; Harrison, P. J. et al; Neuropharmacology 2022, 220, 109262; cColbourne, L.; Harrison, P. J., Mol. Psychiatry 2022, 27 (9), 3904-3912) , as well as cancer. ( Martinez-Delgado, G.; Felix, R., Oncology 2017, 93 (1), 1-10).
[0008] In light of these findings, researchers have envisioned novel therapeutic applications for LTCC antagonists known as calcium channel blockers (CCBs), which have long been used in the treatment of cardiovascular diseases. Unfortunately, their cardiovascular effects pose a significant limitation when attempting to repurpose these drugs for the treatment of other diseases. And there is a need to the art to have carefully studied.
[0009] Present invention solves this problem by providing novel compounds and in particular such 18F-labeled compounds with LTCC antagonists activity and this with nano-molar potency, and improved blood-brain barrier penetration. The labelled compounds of present invention are particular useful for crafting chemical probes therewith tailored for LTCC imaging applications.
[0010] SUMMARY OF THE INVENTION
[0011] The present invention solves the problems of the related art by providing L-type calcium channel (LTCCs or Cavl .1 Cavl .4) antagonist with high binding potency, increased blood brain barrier (BBB) penetration and metabolic stability for neuroimaging as labelled compounds or neurodegenerative disorder treatment.
[0012] In accordance with the purpose of the invention, as embodied and broadly described herein, the invention is broadly drawn to A compound having the general structure of Formula I
[0013]
[0014] wherein RF is Trifluorom ethyl (CF3), Difluoromethyl (CF2H), Fluoromethyl (CFH2), Trifluorom ethoxy (OCF3), Difluoromethoxy (OCF2H), Fluoromethoxy (OCFH2), Pentafluorosulfanyl (SF5) or fluorosulfate (OSO2F) and all relative18F isotopologues thereof and wherein R1is Methyl (CH3), Tri deuteri omethyl (CD3), Ethyl (C2H5), Pentadeuterioethyl (C2D5), isopropyl ((CH3)2CH-), Propyl (C3H7) or Heptadeuteri opropyl (C2D7) and wherein R2is Tosyl (OTs), Mesyl (OMs), Bromo (Br), Chloro (Cl), fluorine-18 ([18F]F), wherein each X is independently chosen from Hydrogen (H) or Deuterium (D) and wherein R3and R4are independently chosen from the dihydropyridine ring consisting of Methyl (CH3), Ethyl (C2H5), Propyl (C3H), Butyl (C4H9) and Pentyl (C5H11), CH2O(CH2CH2O)2NH2, CH2OCH2CH2NH2, CH2(OCH2CH2)3NH2. Another aspect of the invention in such compound wherein R2is fluorine-18 ([18F]F), for use as diagnostic agents in vivo targeting brain LTCCs and in particular as diagnostic agents for neurodegerative and neuroinflammatory disorders including Alzheimer's Disease (AD), Parkinson's Disease (PD), Stroke or Ischemic Brain Injury.
[0015] Another aspect of the invention in such [18F]-labeled compound wherein R2is Tosyl (OTs), Mesyl (OMs), Bromo (Br) or Chloro (Cl), for use as diagnostic agents in vivo targeting brain LTCCs and in particular as diagnostic agents for neurodegerative and neuroinflammatory disorders including Alzheimer's Disease (AD), Parkinson's Disease (PD), Stroke or Ischemic Brain Injury.
[0016] In still another aspect of the invention, such as the non-labeled derivatives for use as neuroprotective agents for treating neurodegenerative disorders related to L-type calcium channel disfunctions such as as Alzheimer's Disease (AD), Parkinson's Disease (PD). In addition, it extends to their application as antihypertensives, therapeutic agents in stroke or ischemic brain injury as well as antiarrhythmics class-IV. In addition, increased intracellular calcium concentrations relate directly to pain and calcium channels blockers such as these can find applications in treating different pain conditions.
[0017] BRIEF DESCRIPTION OF THE DRAWINGSThe present invention will become more fully understood from the detailed description given herein below and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein:
[0018] Figure 1 is a schematic view showing two lead structures that were initially taken into consideration for designing the novel PET ligands targeting L-type calcium channels, named Rad-DiHyP-Bl and Rad-DiHyPB2.
[0019] Figure 2 is a schematic diagram showing the synthetic route of Rad-DiHyP-B 1 and Rad-DiHyP-B2 (Example 1). Both compounds were obtained as racemates via a one-pot two-step mechanism based on a Knoevenagel condensation / Michael addition sequence.
[0020] Figure 3 is a front view showing the preliminary results for both lead compounds, which were initially taken into consideration to develop PET ligands for targeting LTCCs, obtained via automated patch-clamp experiments using the automated SyncroPatch 384i platform. Compounds were provided as lOmM stock concentrations in DMSO and the required concentrations (n=6 concentrations per compound) were freshly prepared in aqueous solution and experiments were carried out in antagonist mode in a HEK293 cell stably expressing the CavE2 complex (aic + 01261 +P2). Nifedipine was used as positive control and DMSO as negative control. The experiment was performed by testing six concentrations per compound (0.1 nM-10 pM ) and measuring a minimum of 2 cells per concentration. The voltage protocol generation (peak pulse protocol starting from -90 mV holding potential to 0 mV over 200 ms and repeated every 20 s) and data collection were performed by SB Drug Discovery using PatchController384 VI.6.6 and Data Controller VI.8. The protocol consists of three steps: i) stabilization involving three consecutive additions of extracellular physiological solution (8-9 min), ii) addition of one concentration of measured compound (3 min) and iii) addition of 10 pM nifedipine as reference compound (2 min). The inhibition was calculated using the following equation and building a concentration-response curve (results shown in Example 1).
[0021] Figure 4 is a schematic diagram describing the preparative route of the chiral auxiliary 9 that will be later used as a building block for the synthesis of Enantioenriched 1,4-DHPs (Example 2). The reaction has been successfully scaled up to 0.1 mol.
[0022] Figure 5 is a schematic diagram depicting the preparation diastereoisomeric 1,4-DHPs via the chiral auxiliary 9 prepared as described in Figure 4. The separation of all diastereoisomers have been achieved via flash chromatography and the purity is defined via 'H and13C nuclear magnetic resonance (NMR) spectroscopy, given the different behaviour of diastereoisomers to the magnetic field. The synthetic procedure has been described in Example 2.Figure 6 is a schematic diagram describing the preparation of enantiopure 1,4-DHP carboxylic acids via beta-hydrolysis, by employing DBU as a non-nucleophilic base.
[0023] Figure 7 is a schematic description for the general synthesis of 1,4-dihydropyri dines including precursors that undergo radiolabeling and cold radipines that served as precursors.
[0024] Figure 8 is a front view of selected enantiomeric pairs that have been tested in vitro as well as in vivo. The length of the ester chains influences the lipophilicity as well as the metabolic stability of the selected candidates as shown in example 3.
[0025] Figure 9 is a front diagram describing Inhibition of KCl-induced cytosolic [Ca2+] increases by radipinel-8 in Cal520-loaded SH-SY5Y cells using FDSS / pCELL reader as described in example 4. A) Concentration-dependent inhibition of KCl-induced [Ca2+] increases obtained using six concentrations (0.1 nM-10 pM). Data reported as (FKci-Fo) / (FionomycinFo) and normalized between 0 and 100%. B) Data reported as percentage of nifedipine-sensitive response to determine the IC50. Data points in panels A and B represent mean ± S.D. of biological triplicates, including three technical replicates each. Where not seen, error bars are within the symbol. C) IC50 of nifedipine and radipinel-8. IC50 values have been extrapolated expressing the data as percentage of nifedipine-sensitive response.
[0026] Figure 10 is a front diagram depicting the preparation of deuterated radipines to evaluate in vivo PET imaging. Deuterium incorporation was achieved via deuterium insertion onto building blocks and deuterated radipines were achieved via a multicomponent reaction as it has been described in example 3.
[0027] Figure 11 is a schematic view of a pool of PET ligands that have been radiolabeled with [18F]F via a HalEx reaction as described in example 5. Radiochemical yields (RCY) range between 7-12 %. Optimization of the reaction conditions are reported in Table 1. Radiolabeling routes for all PET ligand candidates are fully automated and easily translated into as GMP setting.
[0028] Figure 12 is a front radioHPLC-chromatogram including the prepapative radio-HPLC chromatogram used to isolate18F-radipines, where as an example in this figure has been shown [18F] -radipine 1 by using a fully automated GE Tracerlab FXFN fully automated module as described in example 5. The radio-HPLC chromatograms below describe the identity of the radiotracer upon co-injection with the cold reference radipinel and the purity of the isolated radiotracer that is above 98% of purity. The radioligand production has been repeated more than 40 times with consistent results for all 8 molecules.
[0029] Figure 13 is a front diagram depicting in vivo biodistribution studies performed on healthy SD female rats (n = 3). A) Biodistribution of [18F]radipinel in various organs. B) Biodistributionof [18F]radipine2 in organs of interest. C) TAC of brain, myocardium, and lungs highlighting the brain-to-lungs and heart-to-lungs ratios for [18F]radipinel. D) TAC of brain, myocardium, and lungs highlighting the brain-to-lungs and heart-to-lungs ratios for [18F]radipine2. Imaging was carried out with SuperArgus PET / CT (SEDECAL) and reconstruction performed using a 2D-OSEM reconstruction technique. Data are represented as ID% / cc.
[0030] Figure 14 is a front graphical representation of the data obtained via in vivo metabolism of [18F]radipinel-8 in SD female rats as described in the example 6. A) Blood plasma samples at 5, 15, and 30 min extracted with CEECN, with the exception of [18F]radipinel collected at 5, 10, 20, and 40 min and [18F]radipine2 collected at 20 and 40 min. Samples were centrifuged and plasma were collected and centrifuged in presence of CEECN. The organic phase was separated and injected in radio-HPLC. B) Brain homogenate extracted with CH3CN / H2O 1:1, and the organic phase was injected and evaluated via radio-HPLC. Results expressed as ratios calculating the percentage of intact radioligand [Rx] over total radioactivity of the sample [Rtot],
[0031] Figure 15 is a graphical representation of the data obtained from the radioactivity quantification of the PET images obtained at baseline and under preblocking conditions on rodents previously injected with a vehicle or with a known competitor for LTCCs as described in the example 7. Preblocking PET imaging studies performed on healthy SD female rats. Baseline = animal treated with [18F]radipinel (n = 6). Self-block = pretreatment with radipinel (4mg / kg) followed by [18F]radipinel (n = 3). Amlodipine-block = pretreatment with amlodipine (1 mg / kg) followed by [18F]radipinel (n = 3). Isradipine-block = pretreatment with Isradipine (0.5 mg / kg) followed by [18F]radipinel (n = 3) A) Whole brain TAC and quantification. Data analyzed with a one-way ANOVA with multiple comparisons. **** = P < 0.0001, *** = P < 0.001, n.s. = no significant difference (P value > 0.05). AUC4-15min = area under the curve. Imaging was carried out with P-cube (MOLECUBES) and reconstruction performed using a 3D-OSEM reconstruction technique. Panel A of this figure 15, demonstrates an example of the brain timeactivity curves. This is a way to represent the concentration of radioactivity (and hence of the radiotracer) in a volume of interest relative to the total injected dose. The lower part of this figure 15 shows that self-blocking and heterologous blocking with isradipine decreases the baseline signal to a value of approximately 70%, whereas amlodipine, given its poor brain penetration, does not decrease the baseline signal.
[0032] Figure 16 shows a representative image for the study described in example 7 and represented in figure 17. From the images, we can visually observe a decrease in radioactivity uptake under self-block conditions compared to heterologous blocking in presence of amlodipine, an LTCC blocker known not to cross the blood-brain barrier.Figure 17 is a graphical representation of the preblocking PET imaging experiments as described in example 7 focusing on the radioactivity concentration on eight distinct brain regions: cortex, hippocampus, cerebellum, striatum, hypothalamus, brain stem, amygdala, and thalamus. Baseline = animal treated with [18F]radipinel. Self-block = pretreatment with radipinel followed by [18F]radipinel. Data expressed as AUCmsmin. B) Percentage of blocking calculated as [(AUCR-AUCB) / AUCR]*100. AUCR = AUC radioligand 4-15 min. AUCB = AUC block 4-15 min. MRI template for rat W.Schiffer-T2. Data analyzed with a one-way ANOVA with multiple comparisons. * = P < 0.05, ** = P < 0.01, *** = P value <0.001, **** = p value <0.0001 n.s. = no significant difference (P value > 0.05). Imaging was carried out with P-cube (MOLECUBES) and reconstruction performed using a 3D-OSEM reconstruction technique.
[0033] Figure 18 is a graphical representation of radioligand binding assay data deriving from autoradiography studies carried out over rat brain slices upon incubation with either [18F]radipinel (baseline), or [18F]radipinel in presence of radipinel and isradipine. The graph on the left shows that %blocking calculated as [(IR-IB) / IR]*100 where IR = intensity radioligand and IB = intensity block. On the right three representative images of different brain areas that have been quantified. As can be observed, the %block is quite similar to what we have observed in vivo in example 7 and depicted in figure 15.
[0034] Figure 19 is a graphic with results from example 8. It has a panel A that shows that percentage of blocking calculated as [(IR-IB) / IR]*100. IR = intensity radioligand. IB = intensity block and with panel B) showing brain slices with sagittal cut and brain and cerebellum slices with coronal cut incubated with [18F]radipine2 (baseline) or [18F]radipine2 in the presence of radipine2 (selfblock), isradipine or amlodipine (heterologous block).
[0035] DETAILED DESCRIPTION OF THE INVENTION
[0036] The following detailed description of the invention refers to the accompanying drawings. The same reference numbers in different drawings identify the same or similar elements. Also, the following detailed description does not limit the invention. Instead, the scope of the invention is defined by the appended claims and equivalents thereof.
[0037] “Enantioenriched” is herein in the meaning that the18F-labeled compound has a higher proportion of one enantiomer (either the S or R form) compared to the other. It doesn't necessarily mean that it's pure (100%) in one form, but that. And S or R refers to the two possible configurations (stereoisomers) of the chiral center. "S" and "R" denote the absolute configurations according to the Cahn-Ingold-Prelog priority rules, which determine the 3D orientation of the substituents around the chiral center."Isotopologue" refers herein to a compound which differs in its isotopic composition from its "natural" isotopic composition. "Isotopic composition" refers to the amount of each isotope present for a given atom, and "natural isotopic composition" refers to the naturally occurring isotopic composition or abundance for a given atom. Atoms containing their natural isotopic composition may also be referred to herein as "non-enriched" atoms. Unless otherwise designated, the atoms of the compounds recited herein are meant to represent any stable isotope of that atom. For example, unless otherwise stated, when a position is designated specifically as "H" or "hydrogen," the position is understood to have hydrogen at its natural isotopic composition. The description of compounds herein also includes all Isotopologues, in some embodiments, partially deuterated or perdeuterated analogs of all compounds herein.
[0038] "Isotopically enriched" may also refer to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of that atom. "Isotopic enrichment" refers to the percentage of incorporation of an amount of a specific isotope at a given atom in a molecule in the place of that atom's natural isotopic abundance. For example, deuterium enrichment of 1% at a given position means that 1% of the molecules in a given sample contain deuterium at the specified position. Because the naturally occurring distribution of deuterium is about 0.0156%, deuterium enrichment at any position in a compound synthesized using non-enriched starting materials is about 0.0156%. The isotopic enrichment of the compounds provided herein can be determined using conventional analytical methods known to one of ordinary skill in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy. In some embodiments, the compound comprises a -CD3 group.
[0039] Some embodiments of the invention are set forth in claim format directly below:
[0040] Thus, as mentioned above, compound having the general structure of Formula I
[0041]
[0042] wherein RF is Trifluorom ethyl (CF3), Difluoromethyl (CF2H), Fluoromethyl (CFH2), Trifluorom ethoxy (OCF3), Difluoromethoxy (OCF2H), Fluoromethoxy (OCFH2), Pentafluorosulfanyl (SF5) or fluorosulfate (OSO2F) and a relative 18F isotopologues thereof and wherein R1is Methyl (CH3), Tri deuteri omethyl (CD3), Ethyl (C2H5), Pentadeuterioethyl (C2D5), isopropyl ((CEE^CH-), Propyl (C3H7) or Heptadeuteriopropyl (C2D7) and wherein R2is Tosyl (OTs), Mesyl (OMs), Bromo (Br), Chloro (Cl), 18F-fluoride ([18F]F), whereby n is the number of carbon in the carbon chain and is 2, 3, 4 or 5, wherein each X is independently chosen from Hydrogen (H) or Deuterium (D) and wherein R3and R4are independently chosen from the Aryl group consisting of Methyl (CH3), Ethyl (C2H5), Propyl (C3H), Butyl (C4H9), Pentyl (C5H11), CH2O(CH2CH2O)2NH2, CH2OCH2CH2NH2 and CH2(OCH2CH2)3NH2 is part of the present invention.
[0043] In a particular embodiment, the compound as defined above, is a compound whereby Propyl is selected of the group of n-Propyl and Isopropyl and whereby Butyl is selected of the group consisting of n-Butyl, Isobutyl, sec-Butyl, and tert-Butyl and whereby Pentyl is selected of the group consisting of n-Pentyl, Isopentyl (Isoamyl) and Neopentyl.
[0044] In another particular embodiment, the compound as defined above is characterized in that the compound is of the group consisting of a compoud with thee structure (in this application called [18F]radipinel ) and the enantiopure compound with the structure the structure (in this application called [18F]RaDipine 2)
[0045]
[0046] S- or R-[18F]radipine 1 R- or S-[18F]radipine 2 (enantiopure) (enantiopure)
[0047] In another particular embodiment, the compound as defined in any of the embodiments above is characterized in that the compound is enantioenriched and / or isotopically enriched.
[0048] The following aspects are also part of the invention:
[0049] A compound as defined in any one of the embodiments above, wherein R2is fluorine-18-Labeled ([18F]F) for use in a method of neuroimaging.
[0050] A compound as defined in any one of the embodiments above, wherein R2is fluorine-18-Labeled ([18F]F) for use in a method of diagnosis in vivo of a neurodegenerative disease .
[0051] A compound as defined in any one of the embodiments above, wherein R2is fluorine-18-Labeled ([18F]F) for use in a method of diagnosis in vivo of Alzheimer's Disease (AD), Parkinson's Disease (PD, Stroke or Ischemic Brain Injury in a subject.These previous aspects can alternatively be reworded as follows, a method selected from of neuroimaging, diagnosis in vivo of a neurodegenerative disease, and diagnosis in vivo of Alzheimer's Disease (AD), Parkinson's Disease (PD, Stroke or Ischemic Brain Injury in a subject which comprises using a compound as defined in any one of the embodiments above, wherein R2is fluorine- 18-Labeled ([18F]F). It can also be reworded as use of a compound as defined in any one of the embodiments above, wherein R2is fluorine- 18-Labeled ([18F]F) for the preparation of a diagnostic agent for their use in a method selected from of neuroimaging, diagnosis in vivo of a neurodegenerative disease, and diagnosis in vivo of Alzheimer's Disease (AD), Parkinson's Disease (PD, Stroke or Ischemic Brain Injury in a subject.
[0052] A compound as defined above, characterised that is unlabeled, for use in the treatment of a subject in need of obtaining reduced intracellular calcium levels.
[0053] A compound as defined in any one of the embodiments above, characterised that is unlabeled, for use in the treatment of a subject in need of obtaining lower cytosolic calcium concentrations.
[0054] A compound as defined in any one of the embodiments above, characterised that is unlabeled, for use in the treatment of a subject against a calcium dysregulation disorder.
[0055] A compound as defined in any one of the embodiments above, characterised that is unlabeled, for use in the treatment of a subject with a reduced synaptic platicity disorder.
[0056] A compound as defined in any one of the embodiments above, for use in the treatment of a subject for neuroprotection.
[0057] A compound as defined in any one of the embodiments above, characterised that is unlabeled, for use in the treatment to protect neurons of a subject by reducing calcium overload, oxidative stress, and / or excitotoxic damage.
[0058] A compound as defined in any one of the embodiments above, characterised that is unlabeled, for use in the treatment of a disorder of hypertension, Angina Pectoris or the Raynaud's Phenomenon or for use in the treatment of Alzheimer's Disease (AD), Parkinson's Disease (PD, Stroke or Ischemic Brain Injury in a subject.
[0059] A compound as defined in any one of the embodiments above, characterised that is unlabeled, for use in the treatment of a subject against neurodegeneration.
[0060] These previous aspects can alternatively be reworded as follows, a method selected from the group consisting of the treatment of a subject in need of obtaining reduced intracellular calcium levels, the treatment of a subject in need of obtaining lower cytosolic calcium concentrations,the treatment of a subject against a calcium dysregulation disorder, the treatment of a subject for neuroprotection, the treatment to protect neurons of a subject by reducing calcium overload, oxidative stress, and / or excitotoxic damage, the treatment of a disorder of hypertension, Angina Pectoris or the Raynaud's Phenomenon or for use in the treatment of Alzheimer's Disease (AD), Parkinson's Disease (PD, Stroke or Ischemic Brain Injury in a subject the treatment of a subject against neurodegeneration, and the treatment of a subject against neurodegeneration, which comprises using a compound as defined in any one of the embodiments above, characterised that is unlabeled. It can also be reworded as use of a compound as defined in any one of the embodiments above, characterised that is unlabeled, for the preparation of a medicament for their use in a method selected from the group consisting of the treatment of a subject in need of obtaining reduced intracellular calcium levels, the treatment of a subject in need of obtaining lower cytosolic calcium concentrations, the treatment of a subject against a calcium dysregulation disorder, the treatment of a subject for neuroprotection, the treatment to protect neurons of a subject by reducing calcium overload, oxidative stress, and / or excitotoxic damage, the treatment of a disorder of hypertension, Angina Pectoris or the Raynaud's Phenomenon or for use in the treatment of Alzheimer's Disease (AD), Parkinson's Disease (PD, Stroke or Ischemic Brain Injury in a subject the treatment of a subject against neurodegeneration, and the treatment of a subject against neurodegeneration,
[0061] Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein.
[0062] It is intended that the specification and examples be considered as exemplary only .Each and every claim is incorporated into the specification as an embodiment of the present invention. Thus, the claims are part of the description and are a further description and are in addition to the preferred embodiments of the present invention. Each of the claims set out a particular embodiment of the invention.
[0063] Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word “comprise” encompasses the case of “consisting of’. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.
[0064] EXAMPLES
[0065] Example 1 General approachWe synthesized two racemic compounds (Rad -DiHyP -B 1 and Rad-DiHyP-B2, Figure 1) for potency evaluation, before embarking on the development of novel18F-labeled ligands. The one-step synthesis is based on a variation of the Hantzsch synthesis of 1,4-DHPs, involving a Knoevenagel condensation between acetoacetate 22a and benzaldehyde 23, followed by Michael addition of P-enaminocarbonyl 24a, and subsequent cyclization ( details are shown in Figure 1 & Figure 2).
[0066] The evaluation of the compounds’ potency was performed via automated whole-cell patch clamp in a human embryonic kidney (HEK 293) stable cell line expressing the cardiac Cavl.2 protein complex. Both Rad-DiHyP-Bl and Rad-DiHyP-B2 demonstrated nanomolar potency (14 nM and 78 nM, respectively) in inhibiting IcaL (L-type calcium currents) and intracellular [Ca2+] increase in response to voltage pulses (results are shown in Figure 3).
[0067] Synthesis of racemic 1,4-DHPs
[0068] Synthesis of 3-(3-((tert-butyldimethylsilyl)oxy)propyl) 5-methyl 4-(2,3-dichlorophenyl)-2,6-dimethyl-l,4-dihydropyridine-3,5-dicarboxylate and of 3-(3-((tert-butyldimethylsilyl)oxy)propyl) 5-methyl 4-(2-trifluoromethylphenyl)-2,6- dimethyl-1,4-dihy dropy ri dine-3 , 5 -di carb oxy 1 ate
[0069] Metyl-3-iminobutanoate (1 equiv.) was added to a stirring solution of 3-((tert-butyldimethylsilyl)oxy)propyl 3-oxobutanoate (1 equiv.) and the corresponding benzaldehyde (either dichloro- or o-trifluorobenzaldehyde) (1 equiv.) in methanol (40 ml). The mixture was protected from the light and heated under reflux for 24 hours. Afterwards the mixture was slowly cooled to room temperature and a column chromatography (heptane and ethyl acetate 3:7) was performed in order to purify the product.
[0070] Synthesis of 3-methyl 5-(3(tosyloxy)propyl) 4-(2,3-dichlorophenyl)-2,6-dimethyl-l,4-dihydropyridine-3,5-dicarboxylate and of 3-methyl 5-(3(tosyloxy)propyl) 4-(2-trifluoromethylphenyl)-2,6-dimethyl-l,4-dihydropyridine-3,5-dicarboxylate DBU (0.6 equiv.) was added dropwise to a solution of the corresponding silylated precursor (1 equiv.) and tosyl fluoride (1 equiv.) in acetonitrile. The reaction was protected from the light and left to stir for 24 hours. Subsequently, was the reaction mixture extracted with ethyl acetate (3x) and the organic fractions were dried over sodium sulfate. A column chromatography (heptane and ethyl acetate 7:3) was performed in order to purify the crude product. After removal of the solvent in vacuum was compound obtained as a yellow solid, (yield 43%-50%).
[0071] Synthesis of Rad-DiHyP-B 1 and Rad-DiHyP-B2
[0072] A solution of tetra-w-butylammonium fluoride in THF (1 M, 2 equiv.) was added dropwise to a solution of the corresponding tosylate (1 equiv.) in acetonitrile. The mixture was protected from the light and heated at 50°C for 24 hours. Once the reaction mixture was cooled down to roomtemperature it was extracted with ethyl acetate (3x) and the organic fractions where dried over Na2SO4. The crude product was further purified by column chromatography (heptane and ethyl acetate 4:6). Removal of the solvent under vacuum gave the desired product as yellow solid (yield 82%)
[0073] Example 2 Chiral 1,4-dihydropyridines
[0074] Given the preliminary data based on automated patch clamp experiments as shown in Example 1 Rad-DiHyP-Bl was chosen as lead compound, especially with keeping in mind that one of the two enantiomers will have higher potency. Therefore, the synthesis of the novel library of started with the preparation of a chiral auxiliary that once embedded onto the 1,4-DHP core will provide diastereoisomeric racemates that are separable via flash column chromatography. Initially the chiral auxiliary was prepared starting from the commercial D-threonine (schematic details shown in Figure 5).
[0075] Example for the synthesis of chiral auxiliaries:
[0076] The preparation of the chiral auxiliary 9 follows a modified procedure previously described in literature as reported in Figure 4. Important for obtaining the compound 9 are the following steps: From D-threonine 6 to the ester 7 it is important to run the reaction for 1 hour at 0 °C by using 3 equiv thionyl chloride in MeOH. For the protection of the primary amine DBNC1 1 equiv under basic conditions is added at 0 °C and reaction run for 3 hours. Then temperature increased at 25 °C for 14 hours. The formation of the acetoacetate ius obtained upon reaction of 8 with NSC2931 (1 equiv) for 30 minutes at 150 °C in xylene. Important to run the reaction in open vessel. Reaction can be applied to the preparation of other acetoacetates as well, if the corresponding alcohol has a boiling point higher than 150 °C.
[0077] Preparation of diastereoisomeric 1,4-dihydropyridines
[0078] Diastereoisomeric 1,4-dihydropyridines were prepared via a one-pot three component reaction upon reacting the homochiral acetoacetate 9 (called chiral auxiliary from hereafter), the enamine 10 and the corresponding aldehyde 11. Key for this step are the following conditions: The reaction must be carried out in dark under reflux in EtOH from 24 to 72 hours depending on the substrate (Figure 5). Additionally, the purification step is key to obtaining enantiomerically pure compounds. Below we report the detailed description of one compounds. Example for the preparation of 3-((2S,3R)-3-((3,5-dinitrobenzoyl)amino)-4-methoxy-4-oxobutan-2-yl) 5-methyl 2,6-dimethyl-4-(2-(trifluoromethyl)phenyl)-l,4-dihydropyridine-3,5-dicarboxylate (12a and 12b)
[0079] Methyl 3-amino crotonate 10a (1.0 equiv) was added to a round bottom flask containing a solution of 2-(trifluoromethyl) benzaldehyde Ila (1.0 equiv) and the desired acetoacetate 9a (1.0 equiv) in ethanol under vigorous stirring. The reaction mixture was heated to reflux in anoil bath for 48 h. Afterward, the former solution was dried under reduced pressure and purified via column chromatography (DCM:EtOAc 95:5 to 90:10) to give the desired 1,4-DHP derivative 12a and 12b as a yellow solid.
[0080] Preparation enantiopure carboxylic acids under basic conditions
[0081] The use of the non-nucleophilic l,8-diazabicyclo(5.4.0)undec-7-ene allows for a deesterification under mild conditions at room temperature (Figure 6). Below we report a detailed description for compound 13a.
[0082] Example for the preparation of 5-(methoxycarbonyl)-2,6-dimethyl-4-(2-(trifluoromethyl)phenyl)-l,4-dihydropyridine-3 -carboxylic acid (enantiopure but undefined chirality).
[0083] A round bottom flask was charged with 12a (1.0 equiv) in methanol and a stir bar. Then, DBU (3.0 equiv) was added dropwise under vigorous stirring and the reaction was left at room temperature for 4 h. Later, the solvent was removed and the crude dissolved in water and washed with diethyl ether (x3). The water layer was then acidified to pH 2 with a solution of HC1, extracted with EtOAc (x3) and the organic phase dried with MgSO4. Purification via column chromatography yielded the desired product 13a.
[0084] Esterification of chiral carboxylic acids for preparing cold references and precursors for radiolabeling
[0085] The enantiopure carboxylic acids used as starting materials to obtain both cold references as well as precursors for radiolabeling. The latter will lead to the development of a library of PET ligands. The preparation of these compounds is obtained upon reacting the free carboxylic acids with thionyl chloride, and corresponding acyl chloride that is formed in situ is not isolated but directly reacted with the corresponding alcohol to yield final compounds 15 (Figure 7).
[0086] General example for the preparation of 5-(methoxycarbonyl)-2,6-dimethyl-4-(2-(trifluoromethyl)phenyl)-l,4-dihydropyridine-3 -carboxylic acid (enantiopure but undefined chirality).
[0087] A flask was charged with 13 and subjected to nitrogen-vacuum cycles. Then, anhydrous DMF was added, and the resulting solution cooled to 0 °C with an ice bath. Thionyl chloride (2.5 equiv) and EtsN (1.5 equiv) were then added, and the reaction was vigorously stirred at 0°C for 30 min. Next, the corresponding alcohol (3.0 equiv) and EtsN (1.5 equiv) were added to the reaction under vigorous stirring. The reaction proceeded for 3 h at 0 °C, after which it was extracted with CH2Ch-water, dried with Na2SO4, and the solvent was evaporated in vacuo. Finally, the product was purified via column chromatography.
[0088] Example 3To strengthen metabolic soft spots responsible for in vivo radiometabolism, hydrogens have been substituted with deuterated atoms. The carbon-deuterium bond strength is compared to the carbon-hydrogen bond, therefore is expected to increase the metabolic stability. On the other hand, swapping a hydrogen with a deuterium does neither decrease the affinity or potency of drugs or increase the steric bulk. For the synthesis of deuterated radipines we have initially developed the corresponding deuterated building blocks (Figure 10).
[0089] The preparation of the deuteroiminobutanoate 16 was achieved as following:
[0090] Initially we reacted the Meldrum acid (1 equiv) with acetyl chloride (1.2 equiv) in presence of 1.2 equiv of pyridine in dichloromethane. The reaction was conducted for 30 minutes at 0 °C then heated to 25 °C overnight yielding 5-acetyl-2,2-dimethyl-l,3-dioxane-4, 6-dione. The corresponding product was isolated and upon dilution in hot ether, filtered and the filtrate was concentrated. Then, the solid was added to 10 equiv of CD3OH and the solution was heated to reflux for 3 hours. Then, the solvent was removed and the solid purified via flash column chromatography. Then, 1 equiv of deuterated acetoacetate was reacted with 5 equiv NFECOOMe at reflux in dichloromethane for 20 hours and converted into the imine derivative quantitatively.
[0091] The preparation of deuterated benzaldehyde was prepared as following:
[0092] The corresponding benzaldehyde (lequiv) was reacted with D2O (30 equiv) in the presence of DIPEA (5mol%) and NHC catalyst SIPr (5mol%) under strict dry conditions in anhydrous toluene (IM). The reaction vial was run at 50 °C for 16 hours. The crude mixture was added on silica gel and purified via hexanes:Et2O.
[0093] Preparation of deutero 1,4-DHPs.
[0094] The intermediate 16 (1 equiv) was reacted with 1 equiv of the corresponding aldehyde and 1 equiv of acetoacetate 9 in EtOH for 24-72 hours. Then, the diastereoisomers were separated via column chromatography and the hydrolysis via P-elimination in presence of DBU yielded the enantiopure carboxylic acids 17. Then, the deuterated carboxylic acid was reacted with the corresponding alcohol to access both reference compounds as well as precursors required for18F radiolabeling.
[0095] A flask was charged with 17 and subjected to nitrogen-vacuum cycles. Then, anhydrous DMF was added, and the resulting solution cooled to 0 °C with an ice bath. Thionyl chloride (2.5 equiv) and EtsN (1.5 equiv) were then added, and the reaction was vigorously stirred at 0 °C for 30 min. Next, 3.0 equiv of the corresponding alcohol, being fully deuterated or not, and Et3N (1.5 equiv) were added to the reaction under vigorous stirring. The reaction proceeded for 3 h at 0 °C, after which it was extracted with CEECh-water, dried with Na2SC>4, and the solvent was evaporated in vacuo. Finally, the product was purified via column chromatography.Example 4
[0096] In vitro testing of the cold compound
[0097] A library of enantiopure 1,4-DHPs (named radipine 1-8) prepared according to the abovementioned procedures and the schemes cited therein have been tested in calcium imaging assays. The list of compounds reported in Figure 8 tested in SH-SY5Y neuroblastoma cells loaded with the Ca2+indicator Cal520-AM according to an in-house protocol designed to analyze dose-dependent responses by using a FDSS / pCELL imaging system (Hamamatsu Photonics). This experiment defined the potencies of the novel compounds (IC50) via dosedependent assays upon triggering depolarization of the membranes via KC1 as shown in Figure 9. Nifedipine, an LTCC-selective drug, was used as a reference compound given that 30-50 % of the KC1 -triggered Ca2+influx across the plasma membrane is considered to be Nifedipinedependent.
[0098] Nifedipine, that is part of the WHO Model List of Essential Medicines and approved for the treatment of hypertension and other cardiovascular conditions, showed an IC50 = 188 nM according to our assays. On the other hand, all radipines showed higher potency from 2-fold up to 400-fold compared to nifedipine ranging from 0.45 - 90 nM (Figure 9.). Therefore, their higher potency, radipines can substitute nifedipine in treating diverse conditions such as high blood pressure, angina, Raynaud’s phenomenon among others. On the other hand, nifedipine and other dihydropyridines in general are not currently use in thew treatment of diseases related to LTCC dysfunctions in the central nervous system due to poor BBB permeability, a clinical need that can be covered by radipines given their ability to cross the BBB.
[0099] Example 5 Radiochemistry
[0100] Among the indications for developing successful PET imaging agents is the higher binding potency or binding affinity. For ligand-receptor interactions obeying the assumption of a simple 1 : 1 bimolecular binding mechanism the IC50 (binding potency) can be considered very similar in value to the KD (binding affinity). Therefore, given the low IC50 values obtained for the radipines, it is expected that these cores lead to successful diagnostic imaging agents. Initially, we proceeded with optimizing the radiolabeling conditions proceeded to exploring radiolabeling conditions for these compounds manually employing low-amount radioactivity. The fluorine-18 produced in the cyclotron was hereby trapped in a QMA cartridge and immediately eluted with a solution of potassium carbonate and kryptofix-222 in water / acetonitrile. Following azeotropic drying, [18F]KF was dissolved in a small volume of acetonitrile and added around 37 MBq (1 mCi) of this solution to the solution of the desired radiotracer precursor. Unlike general radiolabeling procedures, key to achieving this transformation is employing lower amounts of base for the reaction to successfully proceed. This was discovered serendipitously as initial reaction conditions optimized manually could not be implemented to the automated synthesizer. In addition to this, for the radiolabeling reactionto proceed it is required the use of a polar protic solvent such as a bulky alcohol, even though protic solvents are not commonly used in biomolecular nucleophilic substitution reactions (SN2). In Table 1 are reported some of the most relevant conditions employed to optimize the radiolabeling of 1,4-DHPs. As shown in Table 1, the18F-radipine were from both the bromo as well as the tosyl precursor. Additionally, the reaction has been optimized for chloro and mesyl derivatives.
[0101] Table 1 Optimization of the automated radiolabeling conditions on the homochiral compounds
[0102] - [18F]KF / K222, HCO3- ► 15 min, Solvent, T °C
[0103]
[0104] 15a [18F]radipine 1 Entry EG Solvent T (°C) RCC (%) 1 Br ACN 110 °C 2.3 2 Br TAA 110 28 3aBr TAA 110 31
[0105] TAA-CAN
[0106] 4 Br 110 4-7
[0107] 1 / 1
[0108] TAA-ACN
[0109] 5bBr 110 23
[0110] 2 / 1
[0111] TAA-ACN
[0112] 6 Br 110 17-22
[0113] 3 / 1
[0114] TAA-ACN
[0115] 7 OTs 110 7-18 %
[0116] 3 / 1
[0117] TAA-ACN
[0118] 8 Br 150 80
[0119] 3 / 1
[0120] TAA-ACN
[0121] 9dBr 150 28
[0122] 3 / 1TAA-ACN
[0123] 10eBr 150
[0124]
[0125] 3 / 1
[0126] Cartridge conditioning: 5 mL of KHCO3 lg / 10 ml. Elution: 3.5 mg K2CO3 and
[0127] 15 mg of K222 / ml. Precursor: 2 mg.a30 min.b1 mg of precursor .c1 mg K2CO3 and 20 mg of Wml. EE 63 %.d6.74 mg of / -BuNOMs CH3CN / H2O 1 / 1. EE
[0128] 75 %.eCartridge conditioning: 10 mL of 0.5M K3PO4. Eluting conditions: t- BuNOMs 20 mM solution used as an eluting salt ImL in CH3CN / H2O 1 / 1. EE
[0129] 97 %. EE = elution efficiency. TAA = / -amyl alcohol. RCC = radiochemical conversion.
[0130] Then, the radiolabeling conditions described in Table 1, entry 8 were applied to the synthesis of [18F]radipinel-8 using a TRACERlab FXFN synthesis module (GE Healthcare), with isolated amounts of 0.55-6.29 GBq (15-170 mCi) (Figure 12). The radiosynthesis has been fully automated in compliance with GMP practices including critical steps such as purification and reformulation. The identity of the novel radiolabeled18F-radipines was confirmed via coelution with the corresponding reference compounds radipine 1-8 and the purity was always above (>98 %). A representative example of quality control, confirming compound identity by co-inj ection, is reported for [18F]radipine3 in Figure 12).
[0131] Example 6
[0132] In vivo radiometabolite and biodistribution studies
[0133] In vivo metabolism
[0134] Radiometabolite analysis is very important in PET imaging, as radiometabolites formed in vivo might compete with the parent radioligand of the same binding pocket or even bind elsewhere specifically, hence confounding the results. The formation of metabolites for [18F]-radipines was investigated in SD female rats. Radiometabolite analysis for peripheral radiometabolites was executed as follows: Blood samples were collected from the tail vein at 5 min, 15 min, and 30 min post-injection of the radioligand, centrifuged and plasma was separated. Then, ACN was added to the plasma and the organic phase injected into radio-HPLC as a 1 / 1 mixture with deionized water to define radiometabolite profiling. For brain radiometabolites, animals were terminated and brains extracted after transcardial perfusion at 30 minutes. Then, the homogenized brains were dissolved in ACN and the organic phase injected and analyzed via radio-HPLC. The radiometabolite formation for [18F]radipinel-8 is summarized in Figure 14.
[0135] As shown, all [18F]radipines undergo peripheral metabolism, and these metabolites do not seem to cross the blood-brain barrier given the lack of radiometabolites in the brain for [18F]radipinel and [18F]radipine5. Although not described before for other 1,4-DHPs, quite unexpectedly [18F]radipines seem to undergo brain metabolism. Additionally, we observed that metabolicstability is different for the pair of enantiomers, an aspect that has not been described too much for the rest of 1,4-DHPs.
[0136] Example 7
[0137] PET imaging
[0138] Isradipine is one of the few 1,4-DHPs known to accumulate in the brain and that has been repurposed as a treatment for Parkinson’s Disease, reaching phase 3 clinical trials. However, it failed to show the required efficacy probably due to insufficient target engagement at therapeutic doses. Isradipine has a cLogP = 2.00 according to MarvinSketch, a value not ideal for drugs or imaging agents intended to penetrate passively the blood-brain barrier. On the other hand, our 1,4-DHPs possess higher cLogP values between 2.95-3.78 that is considered a predictor of higher brain permeability compared to isradipine. To evaluate in vivo brain permeability, we carried out PET imaging studies with the most metabolically stable ligand, [18F]radipinel . These experiments are carried out upon tail-vein injection of [18F]radipinel after administering either the vehicle or the cold competitor. This enabled us to compare radioactivity uptake from baseline and preblock studies in presence of ligands competing for the same binding pocket as [18F]radipinel. As competitor we employed radipinel itself, isradipine (a 1,4-DHP prototype known to penetrate the BBB) as well as amlodipine (known not to penetrate the BBB). The study was conducted as follows: Initially, were anesthetized and were injected either the vehicle or the competitor dissolved in the vehicle and after 10 minutes, rats were placed in the PET scanner. Upon [18F]radipine injection, dynamic PET scans of 30 minutes were acquired upon bolus injection of the radiotracer (150-200 pCi). The images were acquired in list-mode, reconstructed then analyzed via PMOD software and time-activity curves were generated (Figure 15 A). Both the radiotracer was well as the 1,4-DHP competitors were tolerated and no change in their vital signs were observed .
[0139] Whole brain analysis confirmed brain penetration of [18F]radipine 1, peaking at 0.87 ± 0.10 %ID / cc between 60-90 s post-injection (Figure 15 A). Notably, it exhibited higher brain uptake compared to [nC]isradipine (0.39 ± 0.08 %ID / cc) as expected.90
[0140] Pre-blocking studies demonstrated that [18F]radipine indeed binds to the brain LTCCs. Pretreatment with radipinel or isradipine led to a significant reduction in whole brain radioactivity uptake compared to baseline. By calculating the area-under-the-curve (AUC) from 4-15 min post-injection, [18F]radipinel shows comparable binding when cold radipinel and isradipine were employed and the baseline signal was reduced to approximately 70 %. On the other hand, pretreatment with amlodipine did not lead to significant differences between preblock and baseline signal as expected, given that amlodipine ionizes at physiological pH and thus is unable to cross the blood-brain barrier (Figure 16).
[0141] In addition, radioactivity uptake upon injection [18F]radipine 1 of was quantified for distinct brain regions known to express LTCCs. These regions include the cerebral cortex,hippocampus, cerebellum, striatum, hypothalamus, thalamus, brain stem, and amygdala (Figure 17).
[0142] All the analysed regions exhibited good radioligand uptake, which decreased significantly upon pretreatment with radipine 1 or isradipine (Figure 17 A). This indicates a certain degree of specific binding and demonstrates binding to the LTCCs. The highest radioligand uptake was detected in the hippocampus, striatum, hypothalamus, thalamus, and brain stem, whereas the cortex and cerebellum displayed the lowest uptake. Pretreatment with radipine 1 resulted in 20-25% blocking in all brain regions, while the percentage of isradipine-block resulted highly region-dependent (Figure 17 B). In the hippocampus, brain stem, and thalamus, isradipine-block reached almost 20%, while in the striatum, hypothalamus, and amygdala it hovered around 15% (Figure 17 B). In contrast, in the cortex and cerebellum, isradipine-block only reached 10%.
[0143] These findings indicate that [18F]radipinel has good brain penetration and uptake in brain regions expressing the LTCCs. In addition, the preblocking experiments highlight a certain degree of specific binding.
[0144] Example 8
[0145] Autoradiography
[0146] Previous in vivo results were confirmed in vitro via autoradiography experiments in brain slices deriving from healthy rodents. For instance, preincubation of brain slices with either isradipine (heterologous blocker) or cold radipine 1 (homologous blocker) led to a baseline signal decrease analogous to in vivo PET imaging.
[0147] The autoradiography experiments were carried out as following:
[0148] Brain tissue was harvested after transcardiac perfusion with saline (0.9 % NaCl), fast-frozen in isopentane (2 -methylbutane, Sigma-Aldrich) on dry ice and stored at -80 °C. Either coronal or sagittal brain slices of 20 pm were obtained using a cryostat (Leica CM3050S, Germany), mounted on a glass slide (Superfrost Ultra Plus, Thermo Fisher, USA) and stored. The day of the experiment, slides were thawed, dried and pre-incubated for 15 min with incubation buffer (Tris-HCl 50 mM, pH 7.4, supplemented with 1 mM MgCh, 1 mM CaCh, 2 mM KC1 and 1% of bovine serum albumin) at room temperature. Subsequently, slices were dried again and incubated in the incubation buffer solution containing 1.5 pCi / mL of18F -radipine 1 during 15 min at room temperature. For the determination of non-specific binding, successive slices were incubated in the radioligand solution containing either 10 pM of non-labelled radipine 1 (homologous blocking) or 10 pM of either isradipine or amlodipine (heterologous blocking). After incubation, slices were removed from the baths, washed two times in ice-cold buffer (50 mM Tris-HCl, pH 7.4, 4 °C) and dipped once in ice-cold ultrapure water. After drying, slices were exposed to a phosphor screen for 5 minutes and the plate was scanned in an Amersham Typhoon 5 Biomolecular Imager (Cytiva, Massachusetts, US) at the highest possible resolution(10 m). For image quantification, regions of interest were defined in the whole brain slices with specific software (ImageQuantTL, Cytiva, Massachusetts, US) and mean values (photostimulated intensity / area) were used to calculate the percentage of non-specific binding. Then we decided to test [18F]radipine2 in autoradiography binding studies, given the very high potency of this enantiomer. Hence, meanwhile we seek alternatives to increase its metabolic stability, we envisaged to repurpose the radioligand for ex vivo autoradiography, assessing its specificity for LTCCs through preblocking experiments.
[0149] Brain tissue slices mounted on slides were preincubated with a buffer containing bovine serum albumin (BSA) and subsequently exposed to a buffer solution containing 1 pCi / mL of [18F]radipine2. To assess specific binding, the slides were incubated in the radioligand solution containing 10 pM of a LTCC blocker. For self-block and heterologous block evaluations, three non-labeled 1,4-DHPs, radipine2, isradipine, and amlodipine, were selected.
[0150] Representative images of the experiment are depicted in (Figure 19). along with the results of the quantification. Notably, [18F]radipine2 exhibited binding to brain regions known to express the LTCCs (vide supra). In particular, the radioligand highlighted the cortex, hippocampus, striatum, and thalamus as high-affinity binding regions, and the brain stem and cerebellum as low-affinity binding regions, where the signal was about 50% lower. Co-incubation with nonlabeled 1,4-DHPs resulted in substantial blocking effects, ranging from 42 to 55% across the various brain regions. Specifically, 42-47% blocking was observed in the high-affinity binding regions, with blocking reaching 50-55% in the hippocampus. Particularly low blocking (about 5%) was observed in the brain stem when using radipine2 and amlodipine as blockers. Conversely, isradipine-block exhibited a 17% reduction in signal, similar to the observed range in the cerebellum (13-18%).
[0151] Present application provides the design and synthesis of a library of [18F]1,4-DHP derivatives, having the general structure of Formula I indicated below wherein RF is Trifluoromethyl (CF3), Difluoromethyl (CF2H), Fluoromethyl (CFH2), Trifluoromethoxy (OCF3), Difluoromethoxy (OCF2H), Fluoromethoxy (OCFH2), Pentafluorosulfanyl (SF5) OR fluorosulfate (OSO2F) and a relative 18F isotopologues thereof and wherein R1is Methyl (CH3), Trideuteri omethyl (CD3), Ethyl (C2H5), Pentadeuterioethyl (C2D5), isopropyl ((CH3)2CH-), Propyl (C3H7) or Heptadeuteri opropyl (C2D7) and wherein R2is Tosyl (OTs), Mesyl (OMs), Bromo (Br), Chloro (Cl,) 18F-fluoride ([18F]F), and wherein each X is independently chosen from Hydrogen (H) or Deuterium (D) and wherein R3and R4are independently chosen from the Aryl group consisting of methyl (CH3), Ethyl (C2H5), Propyl (C3H), Butyl (C4H9) and Pentyl (C5H11), CH2O(CH2CH2O)2NH2, CH2OCH2CH2NH2, CH2(OCH2CH2)3NH2).
[0152]
[0153] H
[0154] Present invention also relates to system and method for making such compounds and uses thereof, in particular as a compound or a library of novel PET tracers. Furthermore, present invention concerns the use of such compounds for LTCC Inhibition.
[0155] In particular [18F]radipines has been demonstrated to be an LTCC PET tracer. The library was based on the scaffold of Rad-DiHyP-B 1 whose synthesis is shown in Figure 2 and automated patch-clamp results shown in Figure 3, a 1,4-DHP derivative that proved to be almost 3-times more potent than nifedipine in whole-cell patch clamp experiments, and included all homochiral compounds.
[0156] The invention thus also concerns the library of unlabeled [18F] 1,4-DHP derivatives and more particularly L-type calcium channels (LTCCs or Cavl.1-1.4) binding compounds having the general structure of Formula I indicated above wherein RF is Trifluorom ethyl (CF3), Difluoromethyl (CF2H), Fluoromethyl (CFH2), Trifluorom ethoxy (OCF3), Difluoromethoxy (OCF2H), Fluoromethoxy (OCFH2), Pentafluorosulfanyl (SF5) OR fluorosulfate (OSO2F) and a relative 18F isotopologues thereof and wherein R1is Methyl (CH3), Trideuteri omethyl (CD3), Ethyl (C2H5), Pentadeuterioethyl (C2D5), isopropyl ((CH3)2CH-), Propyl (C3H7) or Heptadeuteri opropyl (C2D7) and wherein R2is Tosyl (OTs), Mesyl (OMs), Bromo (Br), Chloro (Cl,) Fluorine-18 ([18F]F), and wherein each X is independently chosen from Hydrogen (H) or Deuterium (D) and wherein R3and R4are independently chosen from the Aryl group consisting of Methyl (CH3), Ethyl (C2H5), Propyl (C3H7), Butyl (C4H9) and Pentyl (C5H11), CH2O(CH2CH2O)2NH2, CH2OCH2CH2NH2, CH2(OCH2CH2)3NH2.
[0157] Present invention also relates to system and method for making such compounds and uses thereof, in particular as a compound or a library of novel PET tracers. Furthermore, the present invention concerns the use of such compounds for targeting L-type calcium channels (LTCCs or Cavl .1-1.4) and track any of their roles responsible for various pathophysiological functions deriving from L-type calcium currents (IcaL) such as impaired neurotransmitter release, hormone secretion, gene expression, and / or Ca2+homeostasis in a subject, in particular amammalian subject. In the examples such compounds have shown high potency for LTCCs and their fluorine-18 radiolabeled Isotopologues have been successfully used as in vivo radioligands.
[0158] This is the result of LTCC inhibition decreases the influx of calcium ions, leading to lower cytosolic calcium concentrations, reduced activation of calcium-dependent signaling pathways, such as calmodulin (CaM)-dependent signalling, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) activity and diminished activation of transcription factors like CREB (cAMP response element-binding protein).
[0159] In neurons such compounds reduced synaptic plasticity. LTCC inhibition can impair processes like long-term potentiation (LTP), which is crucial for memory and learning. On the other hand excessive calcium entry through LTCCs contributes to exci totoxi city and neuronal death, which are implicated in neurodegenerative diseases. Thus inhibiting LTCCs can protect neurons by reducing calcium overload, oxidative stress, and excitotoxic damage. Replacing F by F18 provides an aid in neuroimaging which is useful to investigate these bioactive molecules.
[0160] The pharmacological assessment of the cold ligands, radipine 1-8, via live-cell Ca2+imaging showed nM potency towards the LTCCs for all the analyzed compounds, with an IC50 of 4.7 nM for radipine 2 and <1 nM for radipine 4, the most potent compounds.
[0161] The evaluation of the metabolic stability of the PET tracer candidates [18F]radipinel-8 highlighted rapid formation of radiometabolites in the blood plasma for most compounds, with the most stable compound being [18F]radipinel displaying similar stability to [nC]S12968 (Valette, H.; Dolle, F.; Guenther, I.; Hinnen, F.; Fuseau, C.; Coulon, C.; Peglion, J.-L.; Crouzel, C., J. Nucl. Med. 2001, 42 (6), 932). However, [18F]radipinel and [18F]radipine5 showed high percentage of intact radioligand in the brain (> 97 %) 30 min post-injection, thus being a better neuroimaging agent. In preblocking experiments with [18F]radipinel targeting the CNS, notable blocking effects were observed, resulting in a significant reduction in detected radioactivity upon pretreatment with the blocker across the whole brain and in brain regions known to express LTCCs.
[0162] In addition, despite its limited metabolic stability, the assessment of [18F]radipine2 in ex vivo autoradiography revealed specific binding in key brain regions, such as cortex, thalamus, hippocampus, striatum, and cerebellum. This indicates its potential for ex vivo investigations of LTCCs in the brain. Moreover, its high potency and good specificity demonstrates that improving the metabolic stability of [18F]radipine2 will lead to an excellent PET tracer.
Claims
24Claims1. A compound having the general structure of Formula Iwherein RF is Trifluoromethyl (CF3), Difluoromethyl (CF2H), Fluoromethyl (CFH2), Trifluoromethoxy (OCF3), Difluoromethoxy (OCF2H), Fluoromethoxy (OCFH2), Pentafluorosulfanyl (SF5) or fluorosulfate (OSO2F) and a relative 18F isotopologues thereof and- wherein R1is Methyl (CH3), Tri deuteri omethyl (CD3), Ethyl (C2H5), Pentadeuterioethyl (C2D5), isopropyl ((CEE^CH-), Propyl (C3H7) or Heptadeuteri opropyl (C2D7) andwherein R2is Tosyl (OTs), Mesyl (OMs), Bromo (Br), Chloro (Cl), 18F-fluoride ([18F]F)whereby n is the number of carbon in the carbon chain and is 2, 3, 4 or 5 - wherein each X is independently chosen from Hydrogen (H) or Deuterium (D) and - wherein R3and R4are independently chosen from the Aryl group consisting of Methyl (CH3), Ethyl (C2H5), Propyl (C3H), Butyl (C4H9), Pentyl (C5H11), CH2O(CH2CH2O)2NH2, CH2OCH2CH2NH2 and CH2(OCH2CH2)3NH2.
2. The compound according to claim 1, whereby Propyl is selected of the group of n- Propyl and Isopropyl and whereby Butyl is selected of the group consisting of n-Butyl, Isobutyl, sec-Butyl, and tert-Butyl and whereby Pentyl is selected of the group consisting of n-Pentyl, Isopentyl (Isoamyl) and Neopentyl.
3. The compound according to claim 1, characterized in that the compound is of the group consisting of a compoud with the structure (in this application called [18F]radipinel ) and the enantiopure compound with the structure (in this application called [18F]RaDipine 2)S- or R-[18F]radipine 1 R- or S-[18F]radipine 2 (enantiopure) (enantiopure)4. A compound according to any one of claims 1 to 3, characterized in that the compound is enantioenriched and / or isotopically enriched.
5. A compound according to any one of claims 1 to 4 wherein R2is fluorine- 18-Labeled ([18F]F) for use in a method of neuroimaging.
6. A compound according to any one of claims 1 to 5, wherein R2is fluorine- 18-Labeled ([18F]F) for use in a method of diagnosis in vivo of a neurodegenerative disease.
7. A compound according to any one of claims 1 to 5, wherein R2is fluorine- 18-Labeled ([18F]F) for use in a method of diagnosis in vivo of Alzheimer's Disease (AD), Parkinson's Disease (PD, Stroke or Ischemic Brain Injury in a subject.
8. A compound according to any one of claims 1 to 4, characterised that is unlabeled, for use in the treatment of a subject in need of obtaining reduced intracellular calcium levels.
9. A compound according to any one of claimd 1 to 4, characterised that is unlabeled, for use in the treatment of a subject in need of obtaining lower cytosolic calcium concentrations.
10. A compound according to any one of claims 1 to 4, characterised that is unlabeled, for use in the treatment of a subject against a calcium dysregulation disorder.IL A compound according to any one of claims 1 to 4, characterised that is unlabeled, for use in the treatment of a subject with a reduced synaptic platicity disorder.
12. A compound according to any one of claims 1 to 4, for use in the treatment of a subject for neuroprotection.
13. Acompound according to any one of claims 1 to 4, characterised that is unlabeled, for use in the treatment to protect neurons of a subject by reducing calcium overload, oxidative stress, and / or excitotoxic damage.
14. A compound according to any one of claims 1 to 4, characterised that is unlabeled, for use in the treatment of a disorder of hypertension, Angina Pectoris or the Raynaud's Phenomenon or for use in the treatment of Alzheimer's Disease (AD), Parkinson's Disease (PD, Stroke or Ischemic Brain Injury in a subject.
15. A compound according to any one of claims 1 to 4, characterised that is unlabeled, for use in the treatment of a subject against neurodegeneration.