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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61K31/00—Medicinal preparations containing organic active ingredients
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  • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
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• • A—HUMAN NECESSITIES
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  • C07—ORGANIC CHEMISTRY
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  • C07C59/40—Unsaturated compounds
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  • C07C59/40—Unsaturated compounds
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  • C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
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  • C07C69/74—Esters of carboxylic acids having an esterified carboxyl group bound to a carbon atom of a ring other than a six-membered aromatic ring
  • C07C69/757—Esters of carboxylic acids having an esterified carboxyl group bound to a carbon atom of a ring other than a six-membered aromatic ring having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
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  • C07C2602/14—All rings being cycloaliphatic
  • C07C2602/32—All rings being cycloaliphatic the ring system containing at least eleven carbon atoms

Definitions

• the present invention relates to novel compounds that are suitable for use in the treat ment of acute brain injury.
• the invention also relates to use of already known com pounds such as compound A, B, C and D for use in the treatment of acute brain injury.
• Injuries to the brain or spinal cord (termed Central Nervous System, CNS) from epi sodes of reduced blood flow such as stroke, trauma or neurodegenerative disorders produce loss of behavioural function and limited recovery.
• the loss of function gener ally occurs in two ways. First, the injury causes complete damage at the center of the insult, resulting in damage to neural circuits that control a bodily function, such as movement, sensation, memory or language. Second, the injury causes partial damage to neural circuits that are adjacent to the injury site (termed peri-infarct cortex), and dis ables the function of these circuits.
• Stroke which can be either ischemic or haemorrhagic in nature, is a major worldwide health issue, often resulting in long-term disability. As the population ages, the number of stroke patients will increase, contributing to a significant social and economic burden on society. To date, the only medical therapy available is administration of tissue plas minogen activator (tPA), i.e. thrombolysis, which must be given within a 4.5-hr time window after the onset of stroke. Thus, there is a large, unmet need for neuroprotective compounds that can halt neuronal death in the early phase of brain ischemia.
• tPA tissue plas minogen activator
• tPA works by dissolving blood clots and opening brain blood vessels.
• tPA is thus a“neuroprotective” therapy because it works by restoring blood flow and preventing the expansion of ischemic cell death in the brain following acute injury.
• tPA confers a modest behavioral benefit in functional recovery, but has a very narrow therapeutic window.
• tPA must be delivered within 4.5 hrs from the time the patient suffers a stroke, otherwise the risks associated with tPA administration outweigh any potential benefit. At present this time window precludes administration of tPA to approximately 90% of all stroke patients. Furthermore, this treatment is effective only in a subset of stroke patients.
• tPA is not an ap limbate treatment option following haemorrhagic stroke, as the“clot bursting” features of the drug can exacerbate the bleeding in this type of stroke. Therefore, having a com pound that extends the narrow therapeutic window for treatment and facilitates an im provement in neurological function is of great need.
• the shortcoming of the various tPA treatments include the rather limited window of treatment, which is within 4.5 hrs after stroke and the possibility of haemorrhagic malformation. Due to the typical delay between the occurrence of an ischemic stroke and medical attention, this type of treatment is often contraindicated. Thus, other compounds are needed in order to halt and prevent neuronal death after an ischemic stroke within a clinically relevant time frame.
• GHB g-hydroxybutyric acid
• GABA g-aminobutyric acid me tabolite and a neuromodulator that is present in micromolar concentrations in the mam malian brain.
• GHB sodium oxybate
• GHB displays both low affinity (milli- molar) binding to GABA B receptors and high affinity (nanomolar to micromolar) binding to a specific protein in neurons.
• GABA B receptors one well-established pharmacological effect of GHB is a lowering of body temperature (Kaupmann et al. , 2003).
• the neuro-physiological and -pharmacological effects related to the high-affinity binding site are still unknown, because the precise molecular identity of this binding site has been elusive.
• the present invention provides compounds having the following general formula (for mula I): (formula I) wherein, when R 5 is H, and R 1 and R 2 form a ring system, then said compound is se lected from the following compounds of formula II or formula IV
• n 0 or 1 ;
• X is selected from O or NH
• Y is NH, O, S, CH 2
• R 3 is selected from H, linear or branched CrC 6 -alkyl including -Me, -Et, -Pr, -iPr, -Bu, - iBu, -tBu, pentyl, neopentyl, hexyl, branched hexyl; -benzyl, polyethylenglycolyl (PEG), or a group such as
• Rg and Rio independently of each other are selected from linear or branched CrC 6 -alkyl including-Me, -Et, -Pr, -iPr, -Bu, -iBu, -tBu, pentyl, neopentyl, or hexyl; no tably Rio is selected from H, -Me, -Et, -iPr;
• Rn and R I2 independently of each other are selected from linear or branched CrC 6 -alkyl including-Me, -Et, -Pr, -iPr, -Bu, -iBu, -tBu, pentyl, neopentyl, or hexyl; no tably R I2 is selected from H, -Me, -Et, -iPr, -iBu;
• n 0 or 1 ;
• X is O or NH
• n 0 or 1 ;
• R 3 is selected from H, linear or branched CrC 6 -alkyl including -Me, -Et, -Pr, -iPr, -Bu, - iBu, -tBu, pentyl, neopentyl, hexyl, branched hexyl; benzyl, polyethylenglycolyl (PEG), or a group such as wherein Rg and R10 independently of each other are selected from linear or branched CrC 6 -alkyl including-Me, -Et, -Pr, -iPr, -Bu, -iBu, -tBu, pentyl, neopentyl, or hexyl; nota bly R10 is selected from H, -Me, -Et, -iPr;
• Rn and R12 independently of each other are selected from linear or branched CrC 6 -alkyl including-Me, -Et, -Pr, -iPr, -Bu, -iBu, -tBu, pentyl, neopentyl, or hexyl; nota bly R12 is selected from H, -Me, -Et, -iPr;
• R’ is COOH, R” is H and R’” is OCH 3 , or
• R’ is COOH, R” is CH 3 and R’” is OH.
• linear or branched CrC6-alkyl includes methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert. butyl, pentyl, neopentyl and other branched pentyl hexyl and branched hexyl.
• the therm linear or branched CrCs-alkyl included linear or branched CrC6-alkyl as mentioned above and heptyl, branched heptyl, octyl and branched octyl.
• xvv) the compound has one of the following structures
• the present invention also relates to a compound of formula (I) for use in medicine.
• the invention relates to a compound of formula (I) for use in the treat ment of acute brain injury as defined herein.
• the present invention discloses that 3-hydroxycyclopent-1-enecarboxylic acid (A), (Wellendorph et al., 2005), an in-house developed analogue of GHB, possesses neuro- protective properties against acute brain injury. It is contemplated that the compounds of formula (I) have similar neuroprotective properties. However, in contrast to GHB, A does not display any affinity for the GABA B binding site (Klein et al. , 2016; Wellendorph et al., 2005), and so does not have the sedative effect that is problematic for GHB. A binds only to the‘elusive’ high-affinity site.
• This binding site has been probed with a ra- diolabelled version of (£,f?S)-6,7,8,9-tetrahydro-5-hydroxy-5/-/-benzocyclohept-6-yli- dene acetic acid (NCS-382, B), termed 3 H-B. More recently, the inventors have devel oped a radiolabelled version of A ( 3 H-A) (Vogensen et al, 2013), and using this, char acterized and shown 3 H-A binding to be restricted primarily to cortical and hippocampal brain regions in mice, rats and pigs (Klein et al., 2016).
• the inventors have shown that the sodium salt of A readily enters the brain after peripheral administration (Thiesen et al., 2015), and binds with specificity to the high-affinity target in vivo, with no apparent observations of acute toxicity or sedation in rodents after administration of doses as high as 500 mg/kg.
• the present invention further discloses that (£)-2-(5-hydroxy-2-phenyl-5,7,8,9-tetrahy- dro-6/-/-benzo[7]annulen-6-ylidene)acetic acid (2), an in-house developed novel ana logue of B, possesses neuroprotective properties against acute brain injury.
• the inven tors have further shown that 2 and related analogues 1 , 3-10 bind to the‘elusive’ high- affinity site probed by 3 H-A and 3 H-B and passes cellular membranes.
• A affords a significant effect on minimizing the extent of cellular damage and improving functional recovery following stroke. Specifically, ad ministration of A caused a decrease in infarct size and a dose-dependent improve ment in motor function 3-7 days after the induction of the infarct. In the examples herein, A was administered 30 min, 3, 6 or 12 hrs after the induction of a focal stroke to the motor cortex. Surprisingly, and of crucial importance to the clinical use of A in the treatment of acute brain injury, A has a significant effect on both infarct size and motor performance even when given up to 12 hrs after the induction of the infarct. Similarly, 2 affords significant neuroprotection when administered peripherally as the sodium salt 3 or 6 hrs after induction of the injury.
• GHB neuroprotective potential
• A does not bind to the GABA B receptor (Wellendorph et al., 2005). Consistent with this, the inventors show that A does not lower body temperature in mice at a high dose, highlighting that hypothermia is not involved in the neuroprotective activity of A. On the other hand, a decrease in the global central metabolic rate of glucose appears to be a non-GABA B -mediated action. Compound 2 is contemplated to act by a similar mecha nism given the ability to bind to the same high-affinity binding site.
• a well-studied approach in drug development programs targeting stroke has been the use of immunomodulatory compounds. Accumulating evidence supports a linkage be tween activation of the proinflammatory response and infarct size. Likewise, a disrup tion of the blood-brain barrier (BBB) has been found to worsen the outcome following an ischemic stroke, possibly due to increased access for infiltrating lymphocytes and neutrophils. To investigate whether A could reduce these deleterious consequences of brain ischemia, the inventors looked at expression levels of selected inflammatory markers as well as the BBB disruptor, the extracellular proteinase MMP9, a well-known drug target in stroke research.
• BBB blood-brain barrier
• CAMK2A calcium/calmodulin dependent protein kinase II alpha
• CAMK2A protein expression is upregulated in the peri-infarct area following an ischemic stroke.
• CAMK2A is known to phosphorylate both a-amino-3-hydroxy-5-methylisoxazole-4-pro- pionic acid (AMPA) and NMDA receptors, and given that it is implicated in ischemic stroke, CAMK2A is rendered a promising drug target (Coultrap et al. , 2011). Modulating CAMK2A activity directly in pre-clinical models of stroke and global cerebral ischemia, resulted in pronounced neuroprotective effects.
• AMPA a-amino-3-hydroxy-5-methylisoxazole-4-pro- pionic acid
• NMDA receptors NMDA receptors
• CAMK2A The modulation of CAMK2A using the inhibitory peptide Tat-CN21 has shown promise in post-stroke treatment (Vest et al., 2007) and CN21 has been shown to prevent ischemia-induced increases in autophos phorylation of the Thr286 (pThr286) residue of the kinase (Ahmed et al. 2017). How ever, in addition to potential issues with stability and cell penetrance as well as bioa vailability related to the general use of peptides, Tat-peptides are further known to cause hypertension. It is evident, that the modulation of CAMK2A as a target in brain ischemia is not trivial (Coultrap et al., 2011).
• CN21 Whilst CN21 is known to inter act directly with the T-site of CAMK2A (Vest et al., 2007), compound A and related GHB analogues are contemplated to bind to either the kinase domain, the regulatory domain or the hub domain of CAMK2A. Given that no CAMK2A selective, small-mole cule, non-peptide ligands exist, the presented compounds may also represent a novel mechanism of action.
• the inventors herein demonstrate that A and the novel analogue 2 display remarkable neuroprotective properties, and thus hold promise as candidates for diminishing neu ronal death after acute brain injury.
• Acute brain injury refers to a primary cerebral or ischemic in sult that damages brain tissue in an acute manner, but also initiates cascades of dev astating neurotoxic effects. Examples include traumatic brain injury, stroke, subarach noid haemorrhage, neonatal hypoxia-ischemia encephalopathy or associated in utero complications, haemodynamic shock with cardiac arrest, and global hypoperfusion dur ing surgery or as a result from heart failure.
• GHB-related analogue refers to the compounds of the pre sent invention that share a common GHB-related structure (formula I) and bind to a unique site in CAMK2A, for example A, B, C, 1 , 2, or other compounds of the present invention.
• autophosphorylation refers to the phosphorylation of CAMK2A on residues Thr286, Thr305 and Thr306.
• ⁇ AMK2A refers to Calcium/calmodulin-dependent protein kinase type II alpha.
• CMRglc refers to alterations in the cerebral glucose metabo lism as measured by 14 C-2-deoxyglucose autoradiography.
• haemorrhagic refers to a stroke involving bleeding in or around the brain.
• ischemia refers to a disruption of blood supply to the tissue which limits delivery of oxygen and glucose.
• nerve cell damage such as that induced by acute injury to the brain.
• peripheral ischemia refers to the region surrounding the infarct. Permanent middle cerebral artery occlusion (pMCAO) focal ischemia
• pMCAO refers to a permanent disruption of arterial blood flow to a region defined by the area supplied by the middle cerebral artery.
• photoaffinity labelling refers to a UV-light-induced activation of a chemical probe that covalently binds to its target upon such light stimulation.
• photothrombotic focal ischemia refers to method of introduc- ing a cortical infarction through a photochemical reaction with a light-sensitive dye de livered by i.p. injection.
• recombinant refers to DNA sequences that have been trans- fected into and expressed as proteins in HEK293T cells.
• traumatic brain injury refers to an acute damage to the brain which may lead to a disruption of the normal function of the brain.
• FIG. 1 The GHB-related analogues A, B, C, 1-10 bind with nanomolar affinity to high-affinity forebrain binding sites using either A) 3 H-B or B, C, D) 3 H-A for radioactive labelling.
• the binding site does not recognize the known peptide CN21.
• Figure 2 Compound A does not bind to GABA B receptors whereas GHB does.
• FIG. 3 Compound A does not produce GABA B receptor-mediated hypothermia in mice whereas GBL (GHB) does.
• Figure 4 The GHB prodrug GBL (200 mg/kg) produces a reduction in the cerebral glu cose utilization not mediated by GABA B receptors.
• Figure 5 Compound A (175 mg/kg) significantly reduces infarct size when adminis tered (i.p.) 30 min to mice after photothrombotic focal ischemia induced 3 days earlier.
• Figure 6 Compound A (17.5 or 175 mg/kg) significantly improves motor performance in affected limbs in A) grid-walking or B) cylinder tasks when administered 30 min (i.p.) after photothrombotic focal ischemia induced 3 days earlier.
• Figure 7 Compound A (175 mg/kg) significantly reduces infarct size when adminis tered (i.p.) to mice, either A) 3, 6 or 12 hrs after a photothrombotic focal insult produced 3 days earlier. B) A dose of 90 mg/kg is similarly neuroprotective.
• Figure 8 Compound A (175 mg/kg) significantly improves motor performance in af fected limbs in A) grid-walking or B) when administered 3, 6 or 12 hrs (i.p.) after a pho tothrombotic focal ischemia insult produced 3 days earlier.
• Figure 9 Compound 2 significantly reduces infarct size when A) administered to mice (i.p.) 3 or 6 hrs (175 mg/kg) after a photothrombotic focal ischemia insult produced 7 days earlier, and B, C) significantly improves motor performance in both grid-walking and cylinder tasks measured at day 7 post-injury. D) Similarly at 50 mg/kg at 3 hrs, compound 2 reduces infarct size, and E, F) improves motor performance in both grid walking and cylinder tasks measured at day 7 post-injury.
• Figure 10 Compound A significantly reduces the expression of the molecular markers CD14 and MMP9 when measured A, B, C) 3 days or D) 12 hrs after a photothrombotic focal insult.
• Figure 11 Compound A significantly reduces plasma expression levels of the pro-in flammatory cytokine IL-6 when measured 4 hours after a photothrombotic focal insult.
• Figure 12 Compound A (175 g/kg) significantly reduces infarct size when adminis tered (i.p.) to mice 30 min after a pMCAO focal lesion produced 3 days earlier.
• Figure 13 Compound A (175 g/kg) improves sensory-motor impairment when admin istered 30 min after a pMCAO focal lesion produced 2-3 days earlier. Effects were seen in A, B) rotarod, C) grip strength and D) Hargreaves tests.
• Figure 14 3 H-A radioligand specific binding to forebrain regions confirms brain pene trance and target engagement of A. Mice were injected (i.p.) with radioligand (5 MBq per mouse) 30 min before the brain was dissected and subjected to autoradiography.
• CAMK2A is the high-affinity binding site for GHB in the mammalian brain, identified by A) photoaffinity labelling and proteomics, and validated by B) by Western blot and C, D) radioligand binding studies.
• FIG. 16 GHB as well as the analogues A, B, C, 1 and 2 bind directly to recombinant CAMK2A expressed transiently in HEK293T cells.
• Figure 17 The cellular uptake of compounds A and 2 is mediated by their substrate ac tivity at proton-coupled transporters endogenously present in tsA201 cells.
• Figure 18 Ex vivo pThr286 autophosphorylation assay on tissues from mice subjected to photothrombosis shows that compound A decreases excessive autophosphorylation.
• membranes were in cubated with increasing concentrations of test compound or 100 mM baclofen (Sigma) for non-specific binding in a 50 mM Tris-HCI buffer (pH 7.4) containing 2.5 mM CaCh and 40 mM isoguvacine (Sigma) for 1 hr at room temperature in 48-well setup.
• the binding reactions were terminated by rapid filtration through GF/C filters (Whatman), soaked in 0.1 % polyethylene imine, using a Brandell 48-well harvester and rapid wash- ing with ice-cold binding buffer.
• the dried filters were added Optifluor scintillation liquid (PerkinElmer) and counts determined on a Tricarb 4910 TR Scintillation counter (Perki- nElmer). Data are presented as % specific binding (of control), and IC50 or K values calculated by means of non-linear regression curve-fitting and the Cheng-Prusoff equa tion, respectively.
• mice were pre-treated with saline injections (0.9% saline) i.p. for 4 days prior to the ex periment to minimize stress on the day of the experiment. Experiments were conducted in a quiet room, in which mice were left undisturbed for at least two hrs prior to the ex- periment. After the i.p. injections, mice were left in their home cages for two hrs, had core body temperature recorded, and were then euthanized. The core body tempera ture was measured rectally by a thermometer (model DM 852; ELLAB Instruments; Co penhagen, Denmark) via a lubricated thermistor probe (model PRA-22002-A, 2.2 mm diameter; ELLAB Instruments). Mice were held at the base of the tail and measured un- til a stable temperature measurement was obtained.
• rCMRglc Regional cerebral metabolism rate of glucose (rCMRglc) was measured in conscious free-moving GABABO ) receptor knock-out mice (Kaupmann et al. , 2003) using a semi- quantitative index of rCMRglc (irCMRglc) which avoids the need to perform blood sam pling throughout the experiment. 10 min following GBL (200 mg/kg) or saline i.p. injec tions, mice were injected i.p. with 5 mq ⁇ of 14 C-2-deoxyglucose (specific activity 54.1 mCi/mmol, Sigma, UK) dissolved in 0.4 ml saline.
• mice were euthanized by cervical dislocation and brains were snap-frozen and stored at -80 °C until section ing.
• Coronal sections of 20 pm were collected at 2.68, 1.34, 0.74, -1.7, -3.08 and -5.68 mm corresponding to bregma, and were thaw-mounted onto glass slides (Fischer Sci entific, Denmark).
• Autoradiographic images were produced by exposing sections to a 14 C-sensitive plate (Science Imaging Scandinavia AB, Nacka, Sweden) in cassettes for five days with 14 C-microscales (Amersham, UK). Finally, the imaging plate was scanned on a BAS-2500 scanner (Fujifilm Europe GmbH, Dusseldorf, Germany). Spe cific and non-specific binding in frontal cortex and hippocampus were calculated by measuring pixel density using ImageJ and converted to nCi using the calibration scale.
• mice Under anesthesia with isoflurane (2% to 2.5% in 0 2 ) mice were placed in a stereotactic apparatus, the skull exposed through a midline incision, cleared of connective tissue and dried.
• a cold light source KL1500 LCD, Zeiss, Auckland, New Zealand
• a 40x objective providing a 2-mm diameter illumination was positioned 1.5 mm lateral from bregma.
• 0.2 ml of Rose Bengal Sigma-Aldrich, Auckland, New Zealand; 10 mg/ml in nor mal saline was administered i.p.
• mice After 5 min, the brain was illuminated through the ex posed intact skull for 15 min, while keeping body temperature at 37.0 ⁇ 0.3 °C degrees using a heating pad (Harvard apparatus, Holliston, MA, USA). The skin was glued and animals left in a cage placed on a heating pad during the wake-up phase. Mice were housed under a 12-hr light/dark cycle with ad libitum access to food and water. Further, the mice were monitored and weighed on a daily basis.
• the sodium salt of A was synthesized in-house as described previously (Vogensen et al. , 2013).
• the sodium salt of GHB, the GHB prodrug g-butyrolactone (GBL), NCS-382 (B), diclofenac and 4’-hydroxydiclofenac were purchased from Sigma-Aldrich, whereas C was obtained from Carbosynth or SantaCruz (Berkshire, UK).
• the CN21 peptide was obtained from Genscript.
• Forelimb motor performance was determined using the cylinder and grid-walking tasks as previously described (Clarkson et al., 2010). All animals were tested both in a pre- and post-testing session, 1 week before and 3 days or 7 days after the ischemic insult, respectively. Observers who scored the behaviour were blinded to the treatment groups.
• the permanent middle cerebral artery occlusion (pMCAO) model of focal ischemia The pMCAO study was performed using age-matched, young adult (7-8 weeks), male C57BL/6J mice (Taconic). Mice were housed in separate cages under diurnal lighting and given free access to food (1314 Altromin) and water. Mice were acclimatized for seven days prior to surgery in accordance with guidelines approved by the Danish Ani mal Ethical Committee.
• Focal cerebral ischemia was made by permanent occlusion of the distal part of the left middle cerebral artery (MCA).
• MCA left middle cerebral artery
• Mice were anesthetized by injection of a mixture of Hyp- norm (fentanyl citrate 0.315 mg/ml and fluanisone 10 mg/ml; Jansen-Cilag), Stesolid (5 mg/ml Diazepamum; Dumex) and distilled water (1 :1 :2; 0.20 ml/10 g body weight, s.c.). The mouse was placed on a 37 ⁇ 0.5 °C warm heating pad and a skin incision was made from eye to ear.
• Hyp- norm foreignanyl citrate 0.315 mg/ml and fluanisone 10 mg/ml; Jansen-Cilag
• Stesolid 5 mg/ml Diazepamum; Dumex
• distilled water (1 :1 :2; 0.20 ml/10 g body weight, s.c.
• mice were injected with 1 ml isotonic saline and their eyes were coated with ointment. The mice recovered from the surgery in a recov ery room at 28 °C. For treatment of post-surgical pain, mice were supplied s.c. with 0.15 ml Temgesic diluted 1 :30 (stock: 0.3 mg/ml Buprenorphinum; Reckitt & Colman, UK) three times with an 8 hr interval starting immediately after surgery.
• the rotarod measures motor performance in rodents by assessing the time during which the animal remains on a rotating rod.
• the grip strength meter (BIO-GT-3, BIOSEB) allowed the study of neuromuscular functions in mice by determining the maximum force that is required to make the mouse release its grip.
• the mouse is allowed to grasp a metal grid and then pulled backwards in the horizontal plane. The force applied to the grid is recorded as the peak tension.
• Individual (right and left) and total (both) front paw grip strength was measured before (baseline) and 3 days after pMCAO. Each mouse was tested in five sequential trials and the highest grip strength was recorded as the score.
• Thermal hyperalgesia hind paw withdrawal from a normally innocuous heat source was tested with a Hargreaves test setup. The latency times of five stimuli per hindlimb with at least 2 min break in between were recorded. The lowest and highest reflex latency scores were discarded and the average for left and right was calculated and plotted.
• RNA was extracted using a RNA mini kit (Qiagen) following the instructions from the manufacturer. Extracted RNA was treated with DNAse using Turbo DNA-free kit (Ambion), all according to the manufacturer’s protocol. The reverse transcription was performed using qScriptTM cDNA SuperMix (Quanta Biosciences, Gaithersburg, MD, USA) on a standard PCR machine (25 °C for 5 min, 42 °C for 30 min, 85 °C for 5 min) and cDNA stored at -20 °C until further processing.
• qPCR was performed in 96-well plates (Agilent Technologies, Santa Clara, CA, USA) mixing PerfeCTa SYBR Green FastMix (Quanta Biosciences), nuclease free water (Qi agen, West Wales, UK), and primers (TAG Copenhagen A/S (Copenhagen, Den mark).
• the PCR was performed with an initial denaturation step of 95 °C for 30 s, fol lowed by 40 cycles of 5 s at 95 °C, 60 °C for 15 s and 72 °C for 10 s.
• a dissociation curve analysis was performed consisting of 60 s at 95 °C, 30 s at 55 °C and 30 s at 95 °C.
• the qPCR was performed using the Agilent Mx3005P qPCR system (Agilent Technologies), and the corresponding MxPro software was used to determine the Ct values.
• the ACt values were calculated using 2(Refer- ence Ct - Target Ct).
• MCP-1 monocyte chemotactant protein-1
• IL-6 interleukin-6
• I L-1 a interleukin-1 a
• Biotinylated proteins were enriched using PierceTM High Capacity Streptavidin Agarose (ThermoFisher Scientific). The solubilized membranes were diluted to a final concen tration of 0.01 % SDS and incubated with the resin under rotation for 30 min at room temperature. Enrichment was followed by a rigorous washing procedure (3 x 1 min with 10 CV 1x PBS, 0.01 % Tween and 3x 10 min with 10 CV 1x PBS, 0.01 % Tween). Bioti nylated proteins were eluted by boiling in 1x NuPAGETM LDS sample buffer (Ther- moFisher Scientific) supplemented with 100 mM DTT at 100 °C for 10 min under vigor ous shaking.
• In-gel diups were carried out using 70 ng/band endoproteinase Lys-C (Sigma-Aldrich) over night at 37 °C and 175 ng/band trypsin (Sigma-Aldrich) for 8 h at 37 °C.
• Peptide ex tracts were loaded onto in-house packed Cis STAGE Tips and eluted into a 96-well mi crotiter plate with with 2 x 20 pi 40% acetonitrile, 0.5% acetic acid in water, followed by removal of organic solvents in a vacuum centrifuge and reconstitution of peptides in 2% acetonitrile, 0.5% acetic acid, 0.1 % TFA in water.
• Spray voltage was set to 2kV, S-lens RF level at 50, and heated capillary temperature at 275 °C. All experi ments were performed in the data-dependent acquisition mode to automatically isolate and fragment Top10 multiply-charged precursors according to their intensities. Former target ions were dynamically for 40 s excluded and all experiments were acquired us ing positive polarity mode. Full scan resolution was set to 60.000 at m/z 200 and the mass range was set to m/z 350-1400. Full scan ion target value was 3E6 allowing a maximum fill time of 100 ms. Higher-energy collisional dissociation (HCD) fragment scans was acquired with optimal setting for parallel acquisition using 1.3 m/z isolation width and normalized collision energy of 28.
• HCD collisional dissociation
• Target value for HCD fragment scans was set to 1 e5 with a maximum fill time of 45 ms and analyzed with 60.000 resolution.
• Raw LC-MS/MS data was processed using the MaxQuant software (v. 1.5.5.1) and searched against the rat and mouse UniProt databases (downloaded 13.03.2017).
• the default contaminant protein database was included and any hits to this were excluded from further analysis.
• Carbamidomethylation of cysteine was specified as a fixed modification; phosphorylation of serine, threonine and tyrosine residues, oxi dation of methionine, pyro-glutamate formation from glutamine and protein N-terminal acetylation were set as variable modifications.
• HEK293T were cultured using standard conditions, using Dulbecco’s modified Eagle Medium with GlutaMax, 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37 °C in a humidified atmosphere of 95% 0 2 and 5% CO2.
• Cells were transfected with rat CAMK2A (Origene construct RR201121) or rat CAMK2B (Origene construct RR200520) using PolyFect (Qiagen, West Wales, UK) according to the manufacturer’s protocol.
• Whole cell homogenates were prepared 48 hr post-transfec- tion by washing the cells with ice-cold 1x PBS and harvesting by scraping.
• Cells were collected and centrifuged for 10 min at 1000 x g. Cell pellets were resuspended in ice- cold 1x PBS and homogenized using 2 x 1 mm zirkonium beads in a bullet blender for 20 s at max speed (NextAdvance, NY, USA). Homogenates were cleared by centrifu gation (10 min, 4 °C, 14.000 x g). Protein concentration was determined using the Bradford protein assay. 150-200 pg protein was incubated with 5 nM 3 H-A and test compound in 1 ml total volume for 1 hr at 0-4 °C. Nonspecific binding was determined with 1-10 mM GHB.
• Proteins were then precipitated by addition of ice-cold acetone (4x of the assay volume), vortexing and incubation at -20 °C for 1 hr. Proteins were filtrated rapidly through GF/C unifilters (Whatman) and washed. The dried filters were added scintillation liquid and radioactivity measured on a Tricarb 2100 Scintillation counter (Packard). MCT-mediated uptake of GHB analogues in tsA201 cells
• mice were treated with either saline or 175 mg/kg A (i.p.) 30 min after the injury.
• mice were sacrificed, brains were dissected out and immediately submerged in ice-cold PBS supplemented with 1 % phosphatase and protease inhibitors (Phospha tase inhibitor cocktail 3 #P0044 (Sigma), Phosphatase inhibitor cocktail 2 #P5726 (Sigma) and complete EDTA protease inhibitors (Roche) for 5 min.
• Cortex tissue from the infarct core region i.e.
• the compounds of the general formula Ilia may be prepared as given below from the appropriate substituted 6,7,8,9-tetrahydro-5/-/-benzo[7]annulen-5-on according to the procedures in Examples 1-3.
• the protection groups may be cleaved by BBr3.
• Step 1 To a solution of NaOH (368 mg, 9.1 mmol) in H 2 0 (4.6 ml) and ethanol (10 ml) was added a mixture of 2-bromo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-one (Mu- rineddu et al., 2005) (400 mg, 1.6 mmol) and glyoxylic acid monohydrate (495 mg, 6.6 mmol) in water (10 ml) at room temperature. The mixture was stirred at room tempera ture until dissolution and then heated at reflux for 4 hr.
• Step 2 Under a nitrogen atmosphere, CeCh 7H 2 0 (231 mg, 0.6 mmol) and (£)- 2-(2- bromo-5-oxo-5,7,8,9-tetrahydro-6/-/-benzo[7]annulen-6-ylidene)acetic acid (183 mg, 0.6 mmol) were dissolved in MeOH (30 ml). NaBH 4 (351 mg, 9.3 mmol) was slowly added to the solution at 0 °C. The reaction was stirred at room temperature for 4 hr and then solvent was evaporated in vacuo. H 2 0 (50 ml) was added to the residue and the pH was adjusted to 1 with HCI.
• Step 1 Phenylboronic acid (101 mg, 0.8 mmol) and K 2 C0 3 (173 mg, 1.2 mmol) were added to a solution of 2-bromo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-one (Mu- rineddu et al., 2005) (100 mg, 0.4 mmol) in DMF (16 ml) and H2O (8 ml). The solution was stirred under nitrogen atmosphere for 10 min, then tetrakis(triphenylphosphine)pal- ladium (96 mg, 0.08 mmol) was added and the mixture was stirred under nitrogen at mosphere for additional 10 min. The reaction was heated at reflux for 24 hours.
• Step 2 Performed as describe in example 1 (step 1) using NaOH (59 mg, 1.4 mmol) in H 2 0 (0.7 ml) and ethanol (10 ml), 2-phenyl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5- one (65 mg, 0.2 mmol) and glyoxylic acid monohydrate (79 mg, 1.0 mmol) in H 2 0 (5 ml).
• Step 3 Performed as described in example 1 (step 2) using CeCh, 7H 2 0 (145 mg, 0.3 mmol), (£)-2-(5-oxo-2-phenyl-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (116 mg, 0.3 mmol), MeOH (20 ml) and NaBFU (145 mg, 5.9 mmol).
• the sodium salt of 2 was prepared by dissolving 2 (85.4 mg, 0.290 mmol) in ethanol (2 ml) and NaOH (aq) (282 mI, 0.296 mmol, 0.5M Tritisol) was added. The solvent was re moved in vacuo to give the product (90 mg, 99%) as white solid.
• Step 1 Styrene (8.4 ml, 6.0 mmol, 2 eq) and triethylamine (5.5 ml, 39.7 mmol, 19 eq) were added to a solution of 2-bromo-6,7,8,9-tetrahydro-5/-/-benzo[7]annulen-5-one (Murineddu et al., 2005) in CH 3 CN (15 ml). The solution was stirred under nitrogen at mosphere for 5 min, then tetrakis(triphenylphosphine)palladium (725 mg, 0.4 mmol) was added and the mixture was stirred under nitrogen atmosphere for an additional 5 min. The reaction was heated at reflux for 22 hrs sat.
• Step 2 Performed as described in example 1 (step 1) using NaOH (152 mg, 10 mmol) in H2O (1.9 ml) and ethanol (7 ml), (£)-2-styryl-6,7,8,9-tetrahydro-5/-/-benzo[7]annulen- 5-one (100 mg, 0.3 mmol) and glyoxylic acid monohydrate (140 mg, 5 mmol) in H2O (5 ml).
• Step 3 Performed as described in example 1 (step 2) using CeCh, 7H2O (70 mg, 0.1 mmol), (£)-2-(5-oxo-2-((£)-styryl)-5,7,8,9-tetrahydro-6/-/-benzo[7]annulen-6-yli- dene)acetic acid (63 mg, 0.1 mmol), MeOH (20 ml) and NaBhU (74 mg, 1.9 mmol).
• Step 1 Performed as describe in example 1 (step 1) using NaOH (1.48 g, 37.1 mmol) in H2O (18 ml_) and EtOH (40 ml_), 2-chloro-6,7,8,9-tetrahydro-5/-/-benzo[7]annulen-5- one (Murineddu et al., 2005) (1.20 g, 6.18 mmol) and glyoxylic acid monohydrate (2.28 g, 24.7 mmol) in H2O (40 ml_).
• Step 3 Performed as described in example 1 (step 2) using CeCh, 7H2O (735 mg, 3.0 mmol), (£)-2-(5-oxo-2-chloro-5,7,8,9-tetrahydro-6/-/-benzo[7]annulen-6-ylidene)acetic acid (740 mg, 3.0 mmol), MeOH (150 ml_) and NaBhU (1.1 g, 30 mmol).
• Step 1 A mixture of 2-bromo-6,7,8,9-tetrahydro-5/-/-benzo[7]annulen-5-one (Murineddu et al. , 2005) (826 mg, 3.5 mmol), bis(tributyltin) (4.0 g, 6.9 mmol) and tetrakis(tri- phenylphosphine)palladium (0) (400 mg, 0.3 mmol) in dry toluene (35 ml_) was heated at reflux under argon atmosphere for 3 hours. The solvent was evaporated in vacuo to dryness. The crude product was carried on to step 2.
• Step 2 A solution of 2-(tributylstannyl)-6,7,8,9-tetrahydro-5/-/-benzo[7]annulen-5-one (1068 g, 2.4 mmol) in THF (50 ml_) was cooled to 0°C and a solution of iodine (905 g,
• Step 3 Performed as describe in example 1 (step 1) using NaOH (215.0 mg, 5.2 mmol) in H 2 0 (3 ml_) and EtOH (6 ml_) and 2-iodo-6,7,8,9-tetrahydro-5/-/-benzo[7]annulen-5- one (272 mg, 0.95 mmol) and glyoxylic acid monohydrate (354 mg, 3.8 mmol) in H 2 0 (7 ml_).
• Step 4 Performed as described in example 1 (step 2) using CeCh, 7H 2 0 (158 mg, 0.4 mmol), (£)-2-(5-oxo-2-iodo-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (145 mg, 0.4 mmol), MeOH (20 ml_) and NaBH 4 (160 mg, 4.2 mmol).
• Step 1 To 2-(tributylstannyl)-6,7,8,9-tetrahydro-5/-/-benzo[7]annulen-5-one (1017 mg, 2.3 mmol) in acetone (35 ml_) at room temperature were added Ag 2 0 (31 mg, 0.1 mmol), NaHCC> 3 (397 mg, 4.5 mmol) and F-TEDA-BF4 (1204 mg, 3.4 mmol). The reac tion mixture was stirred at reflux for 6 hours. After cooling to room temperature, the re action mixture was filtered on a short pad of celite and evaporated in vacuo. The crude product was carried on to the next step.
• Step 2 Performed as describe in example 1 (step 1) using NaOH (221 mg, 5.5 mmol) in H 2 O (8 ml_) and EtOH (16 ml_) and 2-fluoro-6,7,8,9-tetrahydro-5/-/-benzo[7]annulen- 5-one (179 mg, 1.0 mmol) and glyoxylic acid monohydrate (370 mg, 4.0 mmol) in H 2 O (16 ml_).
• Step 3 Performed as described in example 1 (step 2) using CeCh, 7H 2 0 (235.7 mg,
• Step 1 Performed as describe in example 1 (step 1) using NaOH (6.71 g, 167.7 mmol) in H2O (80 ml_) and EtOH (40 ml_), 2-methyl-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5- one (Murineddu et al. , 2005) (4.84 g, 27.8 mmol) and glyoxylic acid monohydrate (10.28 g, 1 11.7 mmol) in H2O (40 ml_). Purification by column chromatography
• Step 2 Performed as described in example 1 (step 2) using CeC , 7H2O (6.35 g, 17.0 mmol), (£)-2-(2-methyl-5-oxo-5,7,8,9-tetrahydro-6/-/-benzo[7]annulen-6-ylidene)acetic acid (3.56 g, 15.5 mmol), MeOH (150 ml_) and NaBhU (8.79 g, 232.3 mmol).
• Step 1 Performed as describe in example 1 (step 1) using NaOH (194 mg, 4.8 mmol) in H2O (3 ml_) and EtOH (7 ml_), 1-bromo-6,7,8,9-tetrahydro-5H-benzo[7]annulen-5-one (Gruber et al. 1983) (192 mg, 0.8 mmol) and glyoxylic acid monohydrate (299 mg, 3.2 mmol) in H2O (7 ml_).
• Step 2 Performed as described in example 1 (step 2) using CeCh, 7H2O (236 mg, 0.6 mmol), (£)-2-(1-bromo-5-oxo-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (166 mg, 0.6 mmol), MeOH (8 ml_) and NaBhU (214 mg, 5.6 mmol).
• Stepl Performed as describe in example 2 (step 1) using phenylboronic acid (255 mg, 2.1 mmol), K 2 CO 3 (436 mg, 3.1 mmol), 1-bromo-6,7,8,9-tetrahydro-5/-/-benzo[7]annu- len-5-one (Gruber et al. 1983) ( (247 mg, 1.0 mmol), tetrakis(triphenylphosphine)palla- dium (239 mg, 0.2 mmol) in DMF (24 mL) and H2O (16 mL).
• Step 2 Performed as describe in example 1 (step 1) using NaOH (214 mg, 5.3 mmol) in H2O (3 mL) and EtOH (8 mL), 1 -phenyl-6, 7,8, 9-tetrahydro-5/-/-benzo[7]annulen-5- one (210 mg, 0.9 mmol) and glyoxylic acid monohydrate (329 mg, 3.6 mmol) in H2O (5 mL).
• the crude (£)-2-(5-oxo-1 -phenyl-5, 7,8, 9-tetrahydro-6/-/-benzo[7]annulen-6-yli- dene)acetic acid was used for next step without any purification.
• Step 3 Performed as described in example 1 (step 2) using CeCh, 7H2O (362 mg, 1.0 mmol), (£)-2-(5-oxo-1 -phenyl-5, 7, 8, 9-tetrahydro-6/-/-benzo[7]annulen-6-ylidene)acetic acid (258 mg, 0.9 mmol), MeOH (14 ml_) and NaBhU (338 mg, 8.9 mmol).
• Step 1 Performed as describe in example 1 (step 1) using NaOH (246 mg, 6.0 mmol) in H2O (3 mL) and EtOH (7 mL), 3-bromo-6,7,8,9-tetrahydro-5/-/-benzo[7]annulen-5-one (Gruber et al. 1983) (237 mg, 1.0 mmol) and glyoxylic acid monohydrate (366 mg, 4.0 mmol) in H 2 0 (7 mL). Purification by column chromatography (DCM/MeOH 9.5:0.5 +
• Step 2 Performed as described in example 1 (step 2) using CeCh, 7H2O (356 mg, 1.0 mmol), (£)-2-(3-bromo-5-oxo-5,7,8,9-tetrahydro-6/-/-benzo[7]annulen-6-ylidene)acetic acid (256 mg, 0.9 mmol), MeOH (10 ml_) and NaBhU (332 mg, 8.7 mmol).
• Stepl To a solution of 3-oxocyclopent-1-ene-1-carboxylic acid 4 (126 mg, 1.0 mmol), 4- dimethylaminopyridine (24 mg, 0.2 mmol) and 2,2-dimethylpropan-1-ol (124 mg, 1.4 mmol) in DCM (9 ml_) at 0 °C was added A/-(3-dimethylaminopropyl)-/ ⁇ /'-ethylcar- bodiimide hydrochloride (260 mg, 1.4 mmol) dissolved in DCM (4 ml_). The mixture was then allowed to warm to room temperature and stirred for overnight.
• Step 2 Performed as described in example 1 (step 2) using CeCh, 7H2O (309 mg, 0.8 mmol), neopentyl 3-oxocyclopent-1-ene-1-carboxylate (131 mg, 0.7 mmol), MeOH (7 ml_) and NaBhU (127 mg, 3.4 mmol). Purification by column chromatography (hep- tane/EtOAc 3:1) furnished neopentyl 3-hydroxycyclopent-1-ene-1-carboxylate (57 mg, 43%) as a transparent oil.
• Stepl To a solution of 3-oxocyclopent-1-ene-1-carboxylic acid 4 (209 mg, 1.7 mmol) in DMF (5 ml_), tert- butyl 2,2,2- trichloroacetimidate (3.0 ml_, 16.7 mmol) and boron trifluoride diethyl etherate (0.1 ml_, 0.9 mmol) were added. The mixture was stirred overnight at 50°C. A solution of saturated NaHCC>3 was added and then aqueous phase was extracted with EtOAc. The combined organic phase was dried over anhydrous Na2SC>4, filtered, and evaporated in vacuo to dryness.
• Step 2 Performed as described in example 1 (step 2) using CeC , 7H2O (499 mg, 1.3 mmol), tert- butyl 3-oxocyclopent-1-ene-1-carboxylate (203 mg, 1.1 mmol), MeOH (11 ml_) and NaBhU (213 mg, 5.6 mmol).
• Stepl 3-Hydroxycyclopent-1-ene-1-carboxylic acid (HOCPCA) (Vogensen et al., 2013) (355 mg, 2.8 mmol), 4-dimethylaminopyridine (35 mg, 0.3 mmol) were dissolved in THF (15 ml_), and cooled to 0 °C under argon. Acetic anhydride (0.4 ml_, 4.2 mmol) was added dropwise, and stirred overnight at room temperature. Water was added and the mixture was extracted with EtOAc. The combined organic phase was dried over Na 2 SC>4, filtered, and evaporated in vacuo. Purification by column chromatography (heptane/EtOAc 1 :3 + 1 % of AcOH) furnished 3-acetoxycyclopent-1-ene-1-carboxylic acid (434 mg, 92%) as a white solid.
• HOCPCA 3-Hydroxycyclopent-1-ene-1-carboxylic acid
• Step 2 3-Acetoxvcvclopent-1-ene-1-carboxylic acid (137 mg, 0.8 mmol), A/-(3-dimethyl- aminopropyl)-/ ⁇ /'-ethylcarbodiimide hydrochloride (152 mg, 0.8 mmol), hydroxybenzotri- azole (108 mg, 0.8 mmol), triethylamine (0.22 ml_, 1.6 mmol) were dissolved in THF (3 ml_) and stirred for 10 minutes under argon.
• Step 3 To a solution of methyl (2S)-3-(3-(3-acetoxycyclopent-1-ene-1-carboxam- ido)phenyl)-2-((tert-butoxycarbonyl)amino)propanoate (139 mg, 0.3 mmol) in dioxane (1.2 mL) was added dropwise with 4M HCI/ dioxane (1.2 mL, 4.7 mmol) at 0 °C. The mixture was allowed to stir for 1h at room temperature. A solution of saturated NaHCC was added and then aqueous phase was extracted with EtOAc. The combined organic phase was dried over anhydrous Na2S0 4 , filtered, and evaporated in vacuo to dryness.
• GHB-related analogues bind with high affinity to specific GHB sites in rat brain synaptic membranes at both pH values of 6.0 and 7.4 (fig. 1)
• Novel analogues were tested in established binding assays and compared to reference compounds.
• compounds 1-10 were found to inhibit A) 3 H-B binding (respective i values of 50, 92, 30, 52, 53, 108, 325, 225, 382, 9687 nM) compared to compound B with a K, value of 440 nM.
• B) Compounds 1, 2 and 3 also inhibited 3 H-A binding in a concentration-dependent manner with similar affinities (respective K, values of 56 nM, 144 nM and 50 nM) compared to GHB itself with a , value of 2.75 mM.
• GHB analogues fig. 2
• compounds A and C were tested for affinity for the GABA B receptor in an estab lished binding assay. Whereas GHB at both 0.1 mM and 1 mM could inhibit 3 H-GABA B binding, compound A displayed no ability to compete up to concentrations of 1 mM and compound C showed limited inhibition at a concentration of 1 mM.
• Compound A does not produce hypothermia in mice, but GBL does (fig. 3)
• GBL induces a reduction in the cerebral metabolic rate of glucose not mediated by GABA B receptors (fig. 4)
• GHB prodrug GBL alters glucose metabolism via a non-GABA B re ceptor-dependent mechanism
• either saline (black bars) or GBL (200 mg/kg) (white bars) were administered (i.p.) to GABA BI receptor knock-out mice.
• 14 C- deoxyglucose was administered (i.p.) to all mice to estimate brain glucose consump tion. This showed a significant GHB-induced lowering of the cerebral metabolic rate of glucose in frontal cortex and hippocampus after 45 min (10.46 vs. 6.78 nCi/ROI/min for frontal cortex and 9.74 vs. 6.45 for hippocampus, *P ⁇ 0.05, error bars depicted as SD).
• Compound A reduces infarct size when administered 30 min after photothrom- botic focal ischemia (fig. 5)
• Compound A improves motor performance in affected limbs when administered 30 min after photothrombotic focal ischemia (fig. 6)
• Compound A reduces infarct size when administered 3, 6 or 12 hrs after photothrombotic focal ischemia (fig. 7)
• compound A was given 3, 6 or 12 hrs after induc tion of the infarct at two different doses.
• Compound A improves motor performance in affected limbs when administered 3, 6 or 12 hrs after photothrombotic focal ischemia (fig. 8)
• asymmetry was measured between left and right forelimb in the grid-walking and cylinder tasks 7 days after the focal ischemia.
• Compound 2 reduces infarct size and improves motor performance in affected limbs when administered 3 or 6 hrs after photothrombotic focal ischemia (fig. 9)
• the novel compound 2 was given (i.p.) 3 or 6 hrs after induction of the infarct at doses of 175 mg/kg (A-C) or 50 mg/kg (D-F).
• Compound 2 administra tion significantly reduced
• mice treated with the lower dose displayed E-F) significantly improved motor performance in both asymmetry tests.
• Compound A treatment reduces the expression of selected molecular markers related to photothrombotic focal ischemia (fig. 10)
• mRNA expression levels of the markers GFAP, CD14 and MMP9 were measured in the brain tissue surrounding the ischemic core 3 days post-stroke.
• A (17.5 mg/kg
• MMP9 mRNA levels were significantly lower than in animals receiving saline.
• Compound A treatment reduces plasma levels of selected pro-inflammatory cytokines in photothrombotic focal ischemia (fig. 11 )
• Compound A reduces infarct size in the permanent middle cerebral artery occlusion (pMCAO) model of focal ischemia (fig. 12)
• Compound A (175 mg/kg) was administered to mice 30 min after induction of a focal stroke via a permanent occlusion of the middle cerebral artery and compared to saline- treated animals. Three days after the induced stroke, an infarct was visible in the left motor cortex. Quantification of infarcts revealed a significant reduction in infarct volume when comparing saline with A (175 mg/kg) after 30 min (16.6 vs. 12.3 mm 3 , *P ⁇ 0.05).
• Compound A improves functional recovery when administered 30 min after a pMCAO focal lesion (fig. 13)
• Compound A (175 mg/kg) was administered to mice 30 min after induction of a focal stroke via a permanent occlusion of the middle cerebral artery and compared to saline- treated animals and both motor- and sensory impairment was investigated using rotarod, grip strength and Hargreaves tests.
• mice were exposed to 4 trials (T1-T4) 48 hrs post-stroke.
• A) The anti-biotin western blot shows the specifi cally labelled band at ⁇ 55 kDa in the presence (first lane) and absence (second lane) of photoligand.
• GHB and the GHB-related analogues A, B, C, 1 and 2 bind directly to recombinant CAMK2A (fig. 16)
• Human/rat CAMK2A expressed in HEK cells was assayed in an in-house established 3 H-A filtration binding assay performed on whole cell lysates of CAMK2A-transfected HEK293T cells. As seen for binding to synaptic membranes from rat brain cortex, com pounds GHB, A, B, C, 1 and 2 were able to concentration-dependently inhibit radiolig and binding.
• Compound A prevents pThr286 CAMK2A autophosphorylation in mice subjected to photothrombotic focal ischemia (fig. 18)
• mice or mice were treated with either saline or 175 mg/kg A (i.p.) 3 hrs after the injury. Thirty (30) min after the injury, mice were sacrificed and cortex tis sue processed. Autophosphorylation was assessed by Western blot analysis and levels of pThr286 CAMK2A normalized to total CAMK2A to detect changes in autophosphory lation. In accordance with other reports on ischemia (Ahmed et al., 2017), focal ische mia induced by photothrombosis significantly increased autophosphorylation ( # P ⁇ 0.05). This response was significantly inhibited by compound A (*P ⁇ 0.05) amounting to a 73% decrease in autophosphorylation compared to the sham condition.
• Tricyclic pyrazoles 3. Synthesis, biological evaluation, and molecular modeling of analogues of the cannabinoid antagonist 8-chloro-1-(2',4'- dichlorophenyl)-N-piperidin-1-yl-1 ,4,5,6-tetrahydrobenzo[6,7]cyclohepta[1 ,2-c]pyrazole- 3-carboxamide. J Med Chem 48, 7351-7362.
• Ri and R 2 form a ring system to obtain a compound selected from
• R3 is selected from H, -Me, -Et, -Pr, -iPr, -Bu, -tBu, -benzyl, polyethylenglycolyl (PEG), or a group such as
• R 9 is selected from -Me, -Et, -Pr, -iPr, -Bu,-iBu, or -tBu, and wherein R10 is se lected from H, -Me, -Et, -iPr;
• Rn is selected from -Me, -Et, -Pr, -iPr, -Bu, iBu, or -tBu, and wherein R 12 is se lected from H, -Me, -Et, -iPr;
• X is N, O, S, CH 2 or a pharmaceutically acceptable salt therof;
• a method for treating a subject suffering from acute brain injury the treatment com prises administering to said subject an effective amount of a compound as defined in any of items 1-13.
• n 0 or 1 ;
• R 3 is selected from H, -Me, -Et, -Pr, -iPr, -Bu, -tBu, -benzyl, polyethylenglycolyl (PEG), or a group such as wherein Rg is selected from -Me, -Et, -Pr, -iPr, -Bu or -tBu, and wherein R10 is selected from H, -Me, -Et, -iPr;
• Rn is selected from -Me, -Et, -Pr, -iPr, -Bu or -tBu, and wherein R 12 is se lected from H, -Me, -Et, -iPr;
• X is N, O, S, CH 2 or a pharmaceutically acceptable salt therof for use in the treatment of acute brain injury.

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