EA045021B1 - COMBINATIONS CONTAINING POSITIVE ALLOSTERIC MODULATORS OF METABOTROPIC GLUTAMATERGIC RECEPTOR SUBTYPE 2 AND THEIR APPLICATION - Google Patents

Description

Field of invention

The present invention relates to combinations containing a positive allosteric modulator (PAM) of metabotropic glutamatergic receptor subtype 2 (mGluR2) or a pharmaceutically acceptable salt or solvate thereof, and a synaptic vesicle protein 2A (SV2A) ligand.

BACKGROUND OF THE INVENTION

The term epilepsy describes a condition in which an individual experiences recurrent epileptic seizures caused by a chronic process in the deep structures of the brain. Epilepsy refers to a clinical symptom rather than a separate nosological form, since there are a large number of forms and causes of epilepsy. Using the definition of epilepsy as two or more unprovoked epileptic seizures, the incidence of epilepsy is estimated to be approximately 0.3-0.5 percent in various populations worldwide, with the prevalence of epilepsy estimated at 5-10 people per 1000.

An important step in the evaluation and management of a patient with an epileptic seizure is to determine the type of epileptic seizure that has occurred. The main characteristic that allows different categories of epileptic seizures to be distinguished is to determine whether the seizure activity is partial (synonymous with focal) or generalized.

Partial seizures include those in which seizure activity is limited to certain areas of the cerebral cortex. If consciousness is fully maintained during an epileptic seizure, then the clinical manifestations are considered to be relatively simple, and the epileptic seizure is called a simple partial seizure. If consciousness is impaired, the epileptic seizure is called a complex partial seizure. An important additional subgroup includes epileptic seizures that begin with a partial seizure and then spread diffusely throughout the cerebral cortex, which are known as partial seizures with secondary generalization.

Generalized epileptic seizures simultaneously involve diffuse brain regions in a bilaterally symmetrical manner. Petite seizures, or mild forms of epileptic seizures, are characterized by sudden, short-term lapses in consciousness without loss of control over body position. Atypical petit mal seizures typically include longer duration of unconsciousness, less violent onset and offset, and more severe motor signs, which may include focal or lateralizing signs. Generalized tonic-clonic or grand mal seizures, which are the main type of generalized epileptic seizures, are characterized by a violent onset without warning. In the initial phase of an epileptic seizure, there is usually a tonic muscle spasm, impaired breathing, and a significant increase in sympathetic tone, leading to an increase in heart rate, blood pressure and pupil size. After 10-20 s, the tonic phase of an epileptic seizure usually passes into the clonic phase, which consists of alternating periods of muscle relaxation and tonic muscle contraction. Relaxation periods gradually increase until the end of the ictal phase, which usually lasts no more than 1 minute. The postictal phase is characterized by unresponsiveness, muscle relaxation and increased salivation, which can cause wheezing and partial airway obstruction. Atonic seizures are characterized by a sudden loss of postural muscle tone, lasting 1-2 seconds. Consciousness is briefly impaired but is not usually accompanied by postictal confusion. Myoclonic seizures are characterized by sudden and brief muscle contractions that may involve part of the body or the entire body.

Synaptic vesicle protein 2A (SV2A) has been identified as a broad-spectrum anticonvulsant target in models of partial and generalized epilepsy. Results from studies in animal models and human tissues suggest that changes in SV2A expression result in the development of epilepsy (for a review, see, for example: (a) Mendoza-Torreblanca et al. Synaptic vesicle protein 2A: basic facts and role in synaptic function European Journal of Neuroscience 2013, pp. 1-11 (b) Kaminski RM, et al. Targeting SV2A for Discovery of Antiepileptic Drugs. B: Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies [Internet]. 4th edition. Bethesda (MD): National Center for Biotechnology Information (US); 2012. Available at http://www.ncbi.nlm.nih.gov/books/NBK98183/) .

The exact role of SV2A remains unclear, but research suggests that changes in SV2A expression affect synaptic function (Nowack et al. Levetiracetam reverses synaptic deficits produced by overexpression of SV2A PLoS One 2011, Volume 6 (12), e29560). It has also been suggested that SV2A is a key player in exocytosis and is involved in the transmission of nerve impulses (Crowder et al. Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A) Proc Nat Acad Sci USA 1999, 96, pp. 15268-15273) , and results from studies in knockout mice suggest that SV2A deficiency results in an imbalance between glutamatergic and GABAergic nerve impulse transmission (Venkatesan et al. Altered balance between exci

- 1 045021 tatory and inhibitory inputs onto CA pyramidal neurons from SV2A-deficient but not SV2B-deficient mice J Neurosci Res 2012, 90, pp. 2317-2327). Decreased expression of SV2A may cause seizure activity and may be involved in the progression of epilepsy (van Vliet et al. Decreased expression of synaptic vesicle protein 2A, the binding site for levetiracetam, during epileptogenesis and chronic epilepsy Epilepsia 2009, 50, pp. 422-433 ; Feng et al. Down-regulation of synaptic vesicle protein 2A in the anterior temporal neocortex of patients with intractable epilepsy J Mol Neurosci 2009, 39, pp. 354-359; Toering et al. Expression patterns of synaptic vesicle protein 2A in focal cortical dysplasia and TSC-cortical tubers Epilepsia 2009, 50, pp. 1409-1418) and epileptogenesis in patients with brain tumors (de Groot et al. Expression of synaptic vesicle protein 2A in epilepsy-associated brain tumors and in the peritumoral cortex Neuro- Oncology 2010, 12, pp. 265-273).

SV2A ligands include levetiracetam (Lynch et al. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam Proc. Natl. Acad. Sci. USA 2004, Vol. 101, pp. 9861-9866), brivaracetam and seletracetam (Kaminski RM, et al. Targeting SV2A for Discovery of Antiepileptic Drugs. B: Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies [Internet]. 4th edition. Bethesda (MD): National Center for Biotechnology Information (US), 2012. Available at http://www.ncbi.nlm.nih.gov/books/NBK98183/; Nowack et al. Levetiracetam reverses synaptic deficits produced by overexpression of SV2A PLoSone December 2011, Vol. 6 (12), e29560).

Levetiracetam, (-)-(S)-α-ethyl-2-oxo-1-pyrrolidinacetamide or (S)-2-(2-oxopyrrolidin-1yl)butanamide,

is an antiepileptic drug. Levetiracetam has not demonstrated activity in traditional acute models (maximal shock and pentylenetetrazole seizure tests), but has been found to be effective in models of chronic epilepsy and in genetic models of generalized epilepsy. A high safety margin has been shown compared to other antiepileptic drugs (Klitgaard Levetiracetam: the preclinical profile of a new class of antiepileptic drugs Epilepsia 2001, 42 (Supplement 4), pp. 13-18). It is marketed under the brand name Keppra® and is available in tablet form, as an oral solution, and as a concentrate for infusion. Keppra® has been approved in Europe as monotherapy in patients over 16 years of age with newly diagnosed epilepsy, for the treatment of partial seizures (convulsions) with or without secondary generalization, and as adjunctive therapy for use with other antiepileptic medicinal products in the treatment of partial seizures with or without secondary generalization. generalization or without it in patients over 1 month of age; myoclonic seizures in patients over 12 years of age with juvenile myoclonic epilepsy; and primary generalized tonic-clonic seizures in patients over 12 years of age with idiopathic generalized epilepsy (www.ema.europa.eu). Keppra® has also been approved in the US as adjunctive therapy for the treatment of partial-onset seizures in patients 1 month of age and older; myoclonic seizures in patients 12 years of age and older with juvenile myoclonic epilepsy; and primary generalized tonic-clonic seizures in patients 6 years of age and older with idiopathic generalized epilepsy. Keppra XR®, available as sustained-release tablets, has been approved in the United States for the adjunctive treatment of partial-onset seizures in patients 16 years of age and older with epilepsy (http://www.accessdata.fda.gov/scripts/cder/drugsatfda/index .with fm).

Brivaracetam, 4-p-propyl analogue of levetiracetam, (2S)-2-[(4R)-oxo-4-πpropyl-πpyrrolidin-1yl]butanamide,

is in clinical trials and is being investigated as monotherapy for partial-onset seizures and postherpetic neuralgia, and as adjunctive therapy for refractory partial-onset seizures, Unferricht-Lundborg disease in adolescents and adults, and photogenic epilepsy (www.clinicaltrials.gov).

Seletracetam, (2S)-2-[(4S)-4-(2,2,-difluorovinyl)-2-oxo-pyrrolidin-1-yl]butanamide,

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has been tested in clinical trials.

Methods for preparing these three compounds are known from the literature. For example, methods for producing levetiracetam are disclosed, for example, in patent documents EP 0162036 and GB 2225322. A method for producing brivaracetam is disclosed, for example, in patent document WO 01/6272 6. A method for producing seletracetam is known, for example, in patent document WO 2005/121082 . Alternative methods for preparing these three compounds are disclosed in patent document EP 1806339.

The use of antiepileptic drugs in neurological and mental disorders, including neuropathic pain, migraine, essential tremor, and anxiety, schizophrenia and bipolar disorder has been established (Landmarck Antiepileptic drugs in non-epileptic disorders. Relations between mechanisms of action and clinical efficacy CNS Drugs 2008, Vol. 22(1), pp. 27-47; Calabresi et al. Antiepileptic drugs in migraine: from clinical aspects to cellular mechanisms Trends in Pharmacological Sciences 2007, Vol. 28(4), pp. 188-195; Rogawski and Loscher The neurobiology of antiepileptic drugs for the treatment of nonepileptic conditions Nat Med 2004, Vol. 10, pp. 685-692).

Levetiracetam has been found to be effective or potentially effective in a wide range of neuropsychiatric disorders, including mood disorders (Muralidharan and Bhagwagar Potential of levetiracetam in mood disorders: a preliminary review CNS Drugs 2006, Vol. 20, pp. 969-979; Mula et al. The role of anticonvulsant drugs in anxiety disorders: a critical review of the evidence J Clin Pshycopharmacol 2007, Vol. 27, pp. 263-272), anxiety disorders (Kinrys et al. Levetiracetam as adjunctive therapy for refractory anxiety disorders J Clin Psychiatry 2007 , Vol. 68, pp. 1010-1013; Zhang et al. Levetiracetam in social phobia: a placebo controlled pilot study J Psychopharmacol 2005, Vol. 19, pp. 551553; Kinrys et al. Levetiracetam for treatment-refractory posttraumatic stress disorder J Clin Psychiatry 2006, Vol. 67, pp. 211-214), pain (Enggaard et al. Specific effect of levetiracetam in experimental human pain models Eur J Pain 2006, Vol. 10, pp. 193-198; Dunteman Levetiracetam as an adjunctive analgesic in neoplastic plexopathies: case series and commentary J Pain Palliative Care Pharmacother 2005, Vol. 19, pp. 35-43; Price Levetiracetam in the treatment of neuropathic pain: three case studies Clin J Pain 2004, Vol. 20, pp. 33-36), movement disorders (Bushara et al. The effect of levetiracetam on essential tremor Neurology 2005, Vol. 64, pp. 1078-1080; McGavin et al Levetiracetam as a treatment for tardive dyskinesia: a case report Neurology 2003, Vol 61, pp. 419; Woods et al. Effects of levetiracetam on tardive dyskinesia: a randomized, double-blind, placebo-controlled study J Clin Psychiatry 2008, Vol. 69, pp. 546-554; Zivkovic et al. Treatment of tardive dyskinesia with levetiracetam in a transplant patient Acta Neurol Scand 2008, Vol. 117, pp. 351-353; Striano et al. Dramatic response to levetiracetam in post-ischemic Holmes' tremor J Neurol Neurosurg Psychiatry 2007, Vol. 78, pp. 438-439), and is believed to have potentially beneficial effects on cognitive function (Piazzini et al. Levetiracetam: An improvement of attention and of oral fluency in patients with partial epilepsy Epilepsy Research 2006, Vol. 68, pp. 181- 188; de Groot et al. Levetiracetam improves verbal memory in high-grade glioma patients Neuro-oncology 2013, Vol. 15(2), pp. 216-223; Bakker et al. Reduction of hippocampal hyperactivity improves cognition in amnestic mild cognitive impairment Neuron 2012, Vol. 74, pp. 467-474; for review: Eddy et al. The cognitive impact of antiepileptic drugs Ther Adv Neurol Disord 2011, Vol. 4(6), pp. 385-407 and references cited herein; Wheless Levetiracetam in the treatment of childhood epilepsy Neuropsychiatric Disease and Treatment 2007, Vol. 3(4), pp. 409-421) and behavioral symptoms in dementia (Dolder and Nealy The efficacy and safety of newer anticonvulsants in patients with dementia Drugs Aging 2012, Vol. 29(8), pp. 627-637). Animal data and some results from preliminary clinical trials suggest that levetiracetam may have the potential to curb post-traumatic epilepsy, such as epilepsies precipitated by status epilepticus, traumatic brain injury and ischemic stroke, and appears to have neuroprotective effects. The potential of levetiracetam to attenuate epileptogenesis or cognitive dysfunction remains to be determined through definitive clinical and animal studies (for review: Loscher and Brandt Prevention or modification of epileptogenesis after brain insults: experimental approaches and translational research Pharmacol Rev 2010, Vol. 62, 668-700 Shetty Prospects of levetiracetam as a neuroprotective drug against status epilepticus, traumatic brain injury and stroke Front. Neur.2013, 4:172. Doi: 10.3389/fneur.2013.00172), since it showed antiepileptogenic activity in the kindling model in mice and rats. It has also been suggested that levetiracetam inhibits glutamate release (Lee et al. Levetiracetam inhibits glutamate transmission through presynaptic P/Q-type calcium channels on the granule cells of the dentate gyrus British Journal of Pharmacology 2009, Vol. 158, pp. 17531762) .

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Seletracetam and brivaracetam were found to reduce the severity of dystonia in a dt ^sz mutant hamster model and may be useful for some patients suffering from dyskinetic and dystonic movement disorder (Hamann et al. Brivaracetam and seletracetam, two new SV2A ligands, improve paroxysmal dystonia in the dt ^sz mutant hamster European Journal of Pharmacology 2008, Vol. 601, pp. 99-102).

Positive allosteric modulators of mGluR2 have recently been discovered as promising new therapeutic approaches for the treatment of a number of CNS diseases, including epilepsy, and several mGluR2 PAMs are currently in clinical trials for the treatment of schizophrenia and depression associated with feelings of inferiority (www.clinicaltrials.gov, see for example: JNJ-40411813/ADX71149 by Addex Therapeutics and Janssen Pharmaceuticals, Inc.). The initial assumption that drugs that attenuate glutamatergic nerve impulse transmission may be effective for the treatment of epilepsy was advanced by the results of non-clinical studies of immediate effects with mixed mGlu2/3 receptor agonists (Moldrich et al. Glutamate metabotropic receptors as targets for drug therapy in epilepsy Eur J Pharmacol. 2003, Vol. 476, pp. 3-16). LY379268 and LY389795, two mGlu2/3 receptor agonists, were found to be ineffective in blocking MES seizures up to doses that caused motor impairment, but were found to be effective in a dose-dependent b-Hz stimulation model (Barton et al. Comparison of the effect of glutamate receptor modulators in the 6 Hz and maximal electroshock seizure models Epilepsy Research 2003, Vol. 56, pp. 17-26). Chronic administration of an mGlu2/3 agonist paradoxically induced seizure activity in long-term toxicology studies (Dunayevich et al. Efficacy and tolerability of an mGlu2/3 agonist in the treatment of generalized anxiety disorder Neuropsychopharmacology. 2008, Vol. 33(7), pp. 1603 -10). This paradoxical effect may be associated with agonist-induced changes in the sensitivity of the receptor system (tachyphylaxis), but, nevertheless, it was not noted in preclinical studies of epilepsy in model objects. In contrast, positive allosteric modulators modulate the continuous transmission of nerve impulses, but do not have a direct stimulatory effect, thereby reducing the risk of developing tachyphylaxis.

Before seizure activity, extracellular glutamate levels in the human hippocampus are measured to increase, and this increase persists during epileptogenic activity (During and Spencer Extracellular hippocampal glutamate and spontaneous seizure in the conscious human brain Lancet 1993, Vol. 341(8861), pp. 1607-10), thus providing support for the idea that reducing glutamate levels may have a beneficial effect in the treatment of epilepsy. In fact, during seizure activity, glutamate levels increase to potentially non-irrotoxic levels. Seizure activity results in progressive structural damage to the human brain, inducing additional abnormalities in glutamate metabolism (Petroff et al. Glutamate-glutamine cycling in the epileptic human hippocampus Epilepsia 2002, Vol. 43(7), pp. 703-10) . Thus, it is hypothesized that a positive allosteric modulator of mGluR2 or an orthosteric agonist of mGluR2 may protect against seizure-induced neuronal damage.

Patent documents WO 2009/033704 and WO 2010/130424 disclose positive allosteric modulators of mGluR2, their applications and methods for synthesizing these compounds. Patent documents WO 1997/18199 and WO 2003/104217 disclose motor amino acid receptor modulator compounds that were later shown to have orthosteric mGlu2/3 agonist activity (see, for example, Rorick-Kehn et al. (2007) The Journal of Pharmacology and Experimental Therapeutics Vol. 321, No. 1, pp. 308-317), additional examples of compounds having orthosteric mGlu2/3 agonist activity are disclosed in the scientific and patent literature, and patent document WO 2008/150233 discloses compounds with allosteric activating activity against mGluR2.

Currently available antiepileptic drugs do not act exclusively on glutamatergic nerve impulse transmission. Their mechanism of action is usually explained by a change in the balance between excitatory (glutamate-mediated) and inhibitory (GABA-mediated) nerve impulse transmission (Johannessen Landmark Antiepileptic drugs in non-epileptic disorders: relations between mechanisms of action and clinical efficacy CNS Drugs 2008, Vol. 22(1 ), pp. 27-47).

A significant limiting factor in the use of SV2A ligands is tolerability and side effect profile. For example, the effective dose of levetiracetam for partial seizures is 1000, 2000 and 3000 mg, administered twice daily. Side effects reported for levetiracetam include aggressive or restless behavior, anxiety, personality changes, fever, cough or hoarseness, crying, depersonalization, diarrhea, xerostomia, euphoria, fever, a general feeling of discomfort or soreness, headache, hyperventilation, irregular heartbeat, irritability, joint pain, loss of appetite, low back or side pain, depression, muscle aches and pains, nausea, painful or difficult urination, paranoia, overreacting or being overly emotional, rapidly changing moods, restlessness

- 4 045021 in, shaking paralysis, tremors, shortness of breath, drowsiness or unusual lethargy, sore throat, stuffy or runny nose, sweating, sleep disturbance, unusual fatigue or weakness and vomiting. Thus, there remains a need to provide effective treatment with a low effective dose of levetiracetam and a more favorable side effect profile for the treatment of epilepsy and related disorders, not only for adults, but also for pediatric patients.

Brief description of graphic materials

Fig. 1: dose-dependent effect for stimulated 6 Hz 44 mA determination of ED ₅₀ for conn. No. 2 and LEV separately and in combination.

Fig. 2: isobolographic analysis for combination of conn. No. 1 with levetiracetam (LEV) in a 6 Hz (44 mA) assay. Initial ED ₅₀ values (shown below) were determined as for conn. No. 1, and for LEV (points on the graph along the x and y axes; shaded diamonds). The theoretical additivity line connects the calculated ED ₅₀ values for the two compounds (solid black line). Theoretical ED50 (+ SEM) for three combinations with fixed dose ratios (LEV: compound no. 1) are plotted: 1:3 - hatched squares/solid black line, 1:1 - hatched, upward-pointing triangles/solid black line, and 3:1 - shaded, downward-facing triangles/solid black line. Doses for experimental treatments were initially derived from theoretical values and were adjusted according to observed effects. Also shown are the experimentally determined ED ₅₀ (+ SEM) values for each combination with fixed dose ratios: 1:3' open squares/dashed line, 1:1' open up-pointing triangles/dashed line, and 3:1' open, downward triangles/dashed line. Comparisons between theoretical and experimentally determined ED ₅₀ values were made using the t test (***P<0.001). N=8 per group. In fig. 2 ratio LEV and conn. No. 1 is displayed as follows:_____________________________________________

LEV ratio: conn. No. 1 1:3 ▲ 1:1 W 3:1 —H— 1:3’ --A-- 1:1’ —V— 3:1’ ED ₅₀ (LEV) = 345 mg/kg (211-485) (ip, ip) ED ₅₀ (compound no. 1) = 10.2 mg/kg (3.1-12.4) (subcutaneous, sc)

Fig. 3: combination studies for conn. No. 25-a with levetiracetam (LEV) in an assay with stimulation at 6 Hz (44 mA). At a dose of 10 mg/kg, sc, conn. No. 25-a increases the efficiency of LEV, resulting in an approximately 70-fold shift in ED ₅₀ . This indicates a positive pharmacodynamic relationship.

Fig. 4: combination studies for conn. No. 2-a with levetiracetam (LEV) in the analysis with stimulation at 6 Hz (44 mA). At a dose of 10 mg/kg, sc, conn. No. 2-a increases the efficiency of LEV, resulting in approximately a 35-fold shift in ED ₅₀ . This indicates a positive pharmacodynamic relationship.

Fig. 5: Combination studies for conn. No. 6-b with levetiracetam (LEV) in an assay with stimulation at 6 Hz (44 mA). At a dose of 10 mg/kg, p.o., conn. No. 6-b increases the efficiency of LEV, resulting in an approximately 100-fold shift in ED ₅₀ . This indicates a positive pharmacodynamic relationship.

Fig. 6: Combination studies for LY-404039 with levetiracetam (LEV) in a 6 Hz (44 mA) stimulation assay. At a dose of 5 mg/kg, sc, LY-404039 increases the efficacy of LEV, resulting in an approximately 27-fold shift in ED ₅₀ . This indicates a positive pharmacodynamic relationship.

Description of the invention

The present invention relates to a combination for the treatment or prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, and bipolar and related disorders, comprising (a) a synaptic vesicle protein 2A (SV2A) ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) a positive allosteric modulator of metabotropic glutamatergic receptor subtype 2 (mGluR2 PAM), which is a compound of formula (I-B)

- 5 045021

or its stereochemically isomeric form, where

R ¹ is selected from the group consisting of C _1-6 alkyl, (C _3-8 cycloalkyl) C _1-3 alkyl and (C _1-3 alkyloxy) C _1-3 alkyl;

each R ² is independently selected from F, Cl, C _1-3 alkyl, C _1-3 alkyloxy, mono- or polyhalo-C _1-3 alkyl, and mono- or polyhalo-C _1-3 alkyloxy;

n is an integer selected from 1, 2 and 3;

or a pharmaceutically acceptable salt or solvate thereof.

In a further embodiment, the present invention relates to a combination described herein for use as a drug.

A further embodiment of the present invention relates to the use of a combination described herein for the manufacture of a medicament or pharmaceutical product for the treatment or prevention of epilepsy and related disorders; neuropathic pain; migraine or persistent headache and bipolar and related disorders.

A further embodiment of the present invention relates to the use of a combination described herein for the manufacture of a medicament or pharmaceutical product for neuroprotection.

A further embodiment of the present invention relates to the use of a combination described herein for the manufacture of a medicament or pharmaceutical product for the prevention of epileptogenesis.

A further embodiment relates to treating or preventing epilepsy and related disorders in a subject; neuropathic pain; migraine or persistent headache; and bipolar and related disorders, comprising simultaneous or sequential administration to a subject in need thereof of a synaptic vesicle protein 2A (SV2A) ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and a positive allosteric modulator (PAM) of metabotropic glutamatergic receptor subtype 2 (mGluR2), which is a compound of formula (I-B)

or its stereochemically isomeric form, where

R ¹ is selected from the group consisting of C _1-6 alkyl, (C ₃ - ₈ cycloalkyl) C ₁ - ₃ alkyl and (C ₁ - ₃ alkoxy) C ₁ - ₃ alkyl;

each R ² is independently selected from F, Cl, C ₁ _ ₃ alkyl, C ₁ _ ₃ alkyloxy, mono- or polyhalo-C _1-3 alkyl and mono- or polyhalo-C ₁ _ ₃ alkyloxy;

n is an integer selected from 1, 2 and 3;

or a pharmaceutically acceptable salt or solvate thereof, in amounts that will be therapeutically effective when administered together with the SV2A ligand and the mGluR2 compound.

A further embodiment relates to a combination described herein for neuroprotection; or to a combination described herein for use in neuroprotection.

A further embodiment relates to a combination described herein for the prevention of epileptogenesis; or a combination described herein for use in the prevention of epileptogenesis.

In a further embodiment, the present invention relates to a method for treating or preventing epilepsy and related disorders in patients; neuropathic pain; migraine or persistent headache; bipolar and related disorders, comprising the administration of a fixed dose combination of (a) a synaptic vesicle protein 2A (SV2A) ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) a positive allosteric modulator (PAM) of metabotropic glutamatergic receptor subtype 2 (mGluR2), which is a compound of formula (I-B)

- 6 045021

or its stereochemically isomeric form, where

R ¹ is selected from the group consisting of C ₁ - ₆ alkyl, (C ₃ - ₈ cycloalkyl) C ₁ - ₃ alkyl and (C ₁ - ₃ alkyloxy) C ₁ - ₃ alkyl;

each R ² is independently selected from F, Cl, C ₁ _ ₃ alkyl, C ₁ _ ₃ alkyloxy, mono- or polyhalo-C ₁ - ₃ alkyl and mono- or polyhalo gene-C ₁ _ ₃ alkylkCi;

n is an integer selected from 1, 2 and 3;

or a pharmaceutically acceptable salt or solvate thereof in amounts that may be therapeutically effective when administered together with the SV2A ligand and the mGluR2 compound.

In a further embodiment, the present invention relates to a method of neuroprotection using a combination as defined herein.

In a further embodiment, the present invention relates to a method of antiepileptogenesis using a combination as defined herein.

A further embodiment relates to a method of treating or preventing epilepsy and related disorders; neuropathic pain; migraine or persistent headache; bipolar and related disorders, the method comprising administering a therapeutically effective amount of a combination or combination product that contains (a) a synaptic vesicle protein 2A (SV2A) ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) a positive allosteric modulator (PAM) of metabotropic glutamatergic receptor subtype 2 (mGluR2), which is a compound of formula (I-B)

or its stereochemically isomeric form, where

R ¹ is selected from the group consisting of C _1-6 alkyl, (C ₃ - ₈ cycloalkyl) C _1-3 alkyl and (C _1-3 alkyloxy) C _1-3 alkyl;

each R ² is independently selected from F, Cl, C _1-3 alkyl, C _1-3 alkyloxy, mono- or polyhalo-C _1-3 alkyl, and mono- or polyhalo-C _1-3 alkyloxy;

n is an integer selected from 1, 2 and 3;

or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof, such as a warm-blooded animal, in particular a human.

A further embodiment provides a method of neuroprotection, the method comprising administering a therapeutically effective amount of a combination or combination product that contains (a) a synaptic vesicle protein 2A (SV2A) ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) a positive allosteric modulator (PAM) of metabotropic glutamatergic receptor subtype 2 (mGluR2), which is a compound of formula (I-B)

or its stereochemically isomeric form, where

R ¹ is selected from the group consisting of C ₁ - ₆ alkyl, (C ₃ - ₈ cycloalkyl) C ₁ - ₃ alkyl and (C ₁ - ₃ alkyloxy) C ₁ - ₃ alkyl;

each R ² is independently selected from F, Cl, C ₁ _ ₃ alkyl, C ₁ _ ₃ alkyloxy, mono- or polyhalo-C _1-3 alkyl and mono- or polyhalo-C ₁ _ ₃ alkyloxy;

n is an integer selected from 1, 2 and 3; or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof, such as a warm-blooded animal, in particular a human.

A further embodiment provides a method of antiepileptogenesis, the method comprising administering a therapeutically effective amount of a combination or combination product that contains (a) a synaptic vesicle protein 2A (SV2A) ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) a positive allosteric modulator (PAM) of the metabotropic glutamatergic response

- 7 045021 receptor subtype 2 (mGluR2), which is a compound of formula (I-B)

or its stereochemically isomeric form, where

R ¹ is selected from the group consisting of C _1-6 alkyl, (C ₃ - ₈ cycloalkyl) C ₁ - ₃ alkyl and (C ₁ - ₃ alkyloxy) C ₁ - ₃ alkyl;

each R ² is independently selected from F, Cl, C ₁ _ ₃ alkyl _, C ₁ _ ₃ alkyloxy, mono- or polyhalo-C ₁ - ₃ alkyl, and mono- or polyhalo gen-C _{1_3} alkyloxy;

n is an integer selected from 1, 2 and 3; or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof, such as a warm-blooded animal, in particular a human.

In a further embodiment, the present invention relates to a pharmaceutical product or commercial package that contains a combination in accordance with the present invention described herein, in particular together with instructions, for simultaneous, separate or sequential use thereof in the treatment or prevention of epilepsy and related disorders; neuropathic pain; migraine or persistent headache, bipolar and related disorders.

In a further embodiment, the present invention relates to a pharmaceutical product or commercial packaging that contains a combination in accordance with the present invention described herein, in particular together with instructions, for simultaneous, separate or sequential use thereof in neuroprotection.

In a further embodiment, the present invention relates to a pharmaceutical product or commercial package that contains a combination in accordance with the present invention described herein, in particular together with instructions for simultaneous, separate or sequential use thereof for the purpose of antiepileptogenesis.

In a further embodiment, the present invention provides a combination comprising an amount that is collectively therapeutically effective for epilepsy and related disorders; neuropathic pain; migraine or persistent headache; bipolar and related disorders, (a) synaptic vesicle protein 2A (SV2A) ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) a positive allosteric modulator (PAM) of metabotropic glutamatergic receptor subtype 2 (mGluR2), which is a compound of formula (I-B)

or its stereochemically isomeric form, where

R ¹ is selected from the group consisting of C ₁ - ₆ alkyl, (C _3-8 cycloalkyl) C _1-3 alkyl and (C _1-3 alkyloxy) C _1-3 alkyl;

each R ² is independently selected from F, Cl, C _{1_3} alkyl, C _1-3 alkyloxy, mono- or polyhalo _- C _1-3 alkyl, _and mono- or polyhalo-C _{1_3} alkyloxy;

n is an integer selected from 1, 2 and 3;

or a pharmaceutically acceptable salt or solvate thereof and at least one pharmaceutically acceptable carrier.

In a further embodiment, the present invention provides a combination containing an amount that is collectively therapeutically effective as a neuroprotector of (a) a synaptic vesicle protein 2A (SV2A) ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) a positive allosteric modulator (PAM) of metabotropic glutamatergic receptor subtype 2 (mGluR2), which is a compound of formula (I-B)

or its stereochemically isomeric form, where

R ¹ is selected from the group consisting of C _1-6 alkyl, (C ₃ - ₈ cycloalkyl) C ₁ - ₃ alkyl and (C _1-3 αlkyloxy) C _1-3 alkyl;

- 8 045021 each R ² is independently selected from F, Cl, C _1-3 alkyl, C 1-zalkyloxy, mono- or polyhalo-C _1-3 alkyl and mono- or polyhalo gene-C _1-3 alkyloxy;

n is an integer selected from 1, 2 and 3;

or a pharmaceutically acceptable salt or solvate thereof and at least one pharmaceutically acceptable carrier.

In a further embodiment, the present invention provides a combination comprising an amount that is collectively therapeutically effective in preventing epileptogenesis of (a) a synaptic vesicle protein 2A (SV2A) ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) a positive allosteric modulator (PAM) of metabotropic glutamatergic receptor subtype 2 (mGluR2), which is a compound of formula (I-B)

(I-B) or its stereochemically isomeric form, where

R ¹ is selected from the group consisting of C ₁ - ₆ alkyl, (C ₃ - ₈ cycloalkyl) C ₁ - ₃ alkyl and (C ₁ - ₃ alkyloxy) C ₁ - ₃ alkyl;

each R ² is independently selected from F, Cl, C ₁ _ ₃ alkyl, C ₁ _ ₃ alkyloxy, mono- or polyhalo-C1_ ₃ alkyl, and mono- or polyhalo-C _{1_ 3} alkyloxy;

n is an integer selected from 1, 2 and 3;

or a pharmaceutically acceptable salt or solvate thereof and at least one pharmaceutically acceptable carrier.

In a further embodiment, the present invention relates to the use of (a) a synaptic vesicle protein 2A (SV2A) ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) a positive allosteric modulator (PAM) of metabotropic glutamatergic receptor subtype 2 (mGluR2), which is a compound of formula (I-B)

(I-B) or its stereochemically isomeric form, where

R ¹ is selected _{from the} group consisting of _C1-6 alkyl _, ( _C3-8 cycloalkyl) _{C1_3} alkyl and ( _{C1_3} alkyloxy) _{C1_3 alkyl} ;

each ^R2 _is independently selected from F, Cl, _{C1_3alkyl} , _{C1_3alkyloxy} , mono- or polyhalo- _{C1_3alkyl} , and mono- or _polyhalo - _{C1_3alkyloxy} ;

n is an integer selected from 1, 2 and 3;

or a pharmaceutically acceptable salt or solvate thereof to obtain a combination product in accordance with the present invention.

Components (b) of the combinations of the present invention are generally referred to herein as mGluR2 compounds, or mGluR2 PAM/mGluR2 agonist compounds, or mGluR2 positive allosteric modulator/mGluR2 orthosteric agonist compound, which means that the compounds primarily have metabotropic activity. glutamatergic receptor subtype 2 and, in particular, are selected from positive allosteric modulators (PAMs) of the metabotropic glutamatergic receptor subtype 2 and orthosteric agonists of the metabotropic glutamatergic receptor subtype 2. One skilled in the art will be aware of the high homology between mGluR2 and mGluR3 such that some mGluR2 orthosteric agonists also exhibit activity similar to mGluR3 orthosteric agonists. This is the case, for example, with (-)(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]-hexane-4,6-dicarboxylic acid (also known as LY-404,039 [CAS 635318-11-5]) with Ki = 149 nM (mGlu2 receptor) and Ki = 92 nM (mGlu3 receptor), having 100-fold selectivity for mGlu2 and mGlu3 compared to mGlu4a, -6, -7a and -8a, and it lacks activity against mGlula and mGlu5a (Rorick-Kehn et al. (2007) The Journal of Pharmacology and Experimental Therapeutics Vol. 321, No. 1, pp. 308-317). Thus, the term mGluR2 compounds, or mGluR2 PAM/mGluR2 agonist compounds, or mGluR2 positive allosteric modulator/mGluR2 orthosteric agonist compound does not exclude compounds that exhibit some additional minor activity in vitro or in vivo.

The PAM mGluR2 combination compounds of the present invention are in particular selected from those disclosed in patent document WO 2010/130424. A specific subset of these compounds disclosed in patent document WO 2010/130424 can be characterized by the following formula (I):

- 9 045021

or a stereoisomeric form thereof, wherein R ¹ is selected from the group consisting of (C ₃ _ ₇ cycloalkyl)C ₁ _ ₃ αalkyl-, mono- or polyhalo-C _1-4 alkyl and (C _1-4 alkyl)-O- ( C ₁ _ ₄ alkyl);

R ² represents halogen or polyhalo-C _1-4 alkyl; A represents a covalent bond or -CH ₂ -; L is selected from radicals (a), (b) and (c):

where R ^3a is selected from unsubstituted phenyl or phenyl substituted with 1 or 2 halogen substituents;

R ^4a is selected from the group of hydrogen, C ₁ _ ₃ alkyl and halogen;

or R ^3a -CR ^4a together represent a radical of formula (a-1)

where R ^5a is hydrogen or halogen;

R ^3b is selected from the group of phenyl substituted with 1 or 2 halogen substituents, pyridinyl substituted with 1 or 2 halogen substituents, unsubstituted pyrimidinyl and pyrimidinyl substituted with 1 or 2 C ₁ - ₃ alkyloxy substituents;

or a pharmaceutically acceptable salt or solvate thereof.

Thus, according to a specific embodiment of the present invention, the metabotropic glutamatergic receptor subtype 2 (mGluR2) positive allosteric modulator (PAM) compound is a compound of formula (I-B) as defined herein.

The compounds of formula (I) are disclosed in patent document WO2010/130424 and can be prepared in accordance with the methods described herein, which are incorporated herein by reference in their entirety.

Specific compounds of formula (I) include

- 10 045021

The compounds of formula (I-A) are disclosed in patent document WO 2009/033704 and can be prepared in accordance with the methods described herein, which are incorporated herein by reference in their entirety.

Specific compounds of formula (I-A) include

- 11 045021

- 12 045021

- 13 045021

as well as their pharmaceutically acceptable salts and solvates.

The PAM mGluR2 combination compounds of the present invention are also particularly selected from the compounds disclosed in PCT/EP2014/068676. These compounds disclosed in PCT/EP2014/068676 can be defined by the following formula (I-B):

(IB) and stereochemically isomeric forms thereof, wherein R ¹ is selected from the group consisting of C _1-6 alkyl, (C _3-8 cycloalkyl) C _1-3 alkyl and (C _1-3 alkyloxy) C ₁ - ₃ alkyl;

_each R ² is independently selected from F, Cl, C _1-3 alkyl, C _{1_3} αlkyloxy, mono- or polyhalo _- C _1-3 alkyl, and mono- or polyhalogen-C _{1_3} alkyloxy;

n is an integer selected from 1, 2 and 3;

as well as their pharmaceutically acceptable salts and solvates.

The PAM mGluR2 combination compounds of the present invention are particularly selected from compounds of formula (IB) as defined herein above, and stereoisomeric forms thereof, wherein R ¹ is selected from the group consisting of CH ₃ CH ₂ , CH ₃ CH ₂ CH ₂ , (cyclopropyl)methyl, (cyclobutyl)methyl, ethyloxymethyl and methyloxymethyl; and the remaining variables are as defined herein; and pharmaceutically acceptable salts and solvates thereof.

In a further embodiment, the PAM mGluR2 combination compounds of the present invention are particularly selected from compounds of formula (IB) as defined herein above, and stereoisomeric forms thereof, wherein R ¹ is selected from the group consisting of CH ₃ CH ₂ , (cyclopropyl )methyl, (cyclobutyl)methyl and methyloxymethyl; and the remaining variables are as defined herein; and pharmaceutically acceptable salts and solvates thereof.

In a further embodiment, the PAM mGluR2 combination compounds of the present invention are particularly selected from compounds of formula (IB) as defined herein above, and stereoisomeric forms thereof, wherein R ¹ is selected from the group consisting of CH ₃ CH ₂ , (cyclopropyl )methyl, (cyclobutyl)methyl and ethyloxymethyl; and the remaining variables are as defined herein; and pharmaceutically acceptable salts and solvates thereof.

Thus, in accordance with a specific embodiment of the present invention, the compound that is a positive allosteric modulator (PAM) of metabotropic glutamatergic receptor subtype 2 (mGluR2) is a compound of formula (I-B) as defined herein.

In a further embodiment, the compounds of formula (IB) are compounds as defined herein, wherein each R ² is independently selected from F, Cl, CH3, CH3O and CF3; as well as their pharmaceutically acceptable salts and solvates.

In a further embodiment, compounds of formula (I-B) are compounds that are defined herein as having formula (I-Ba)

(1-Ba) wherein the variables are defined in formula (I-B) herein, as well as their pharmaceutically acceptable salts and solvates.

In a further embodiment, compounds of formula (I-B) are compounds that are defined herein as having formula (I-Bb)

(1-Bb) wherein the variables are defined in formula (I-B) herein, as well as their pharmaceutically acceptable salts and solvates.

Specific compounds of formula (I-B) include

3-(cyclopropylmethyl)-7-[1-(4-fluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclopropylmethyl)-7-[(1*R)-1-(4-fluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclopropylmethyl)-7-[(1*S)-1-(4-fluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclopropylmethyl)-7-[(IS)-1-(2,4-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclopropylmethyl)-7-[(1R)-1-(2,4-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclopropylmethyl)-7-[1-(2,4-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclopropylmethyl)-7-[(IS)-1-(3,5-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclopropylmethyl)-7-[(IS)-1-(3,4-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclopropylmethyl)-7-[(IS)-1-(2,3-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclopropylmethyl)-7-[(IS)-1-(2,5-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclopropylmethyl)-7-[(IS)-1-(2,6-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclopropylmethyl)-7-[(IS)-1-(4-fluoro-2-methoxyphenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclobutylmethyl)-7-[1-(2,4-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

7-[(IS)-1-(2-chloro-4-methylphenoxy)ethyl]-3-(cyclopropylmethyl)-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

- 15 045021

3-(cyclopropylmethyl)-7-[(IS)-1-(4-fluoro-2-methylphenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine; 3-(cyclopropylmethyl)-8-(trifluoromethyl)-7-[(13)-1-(2,4,6trifluorophenoxy)ethyl][1,2,4]triazolo[4,3-a]pyridine; 7-[1-(2,4-difluorophenoxy)ethyl]-3-(ethoxymethyl)-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-ethyl-8-(trifluoromethyl)-7-[1-(2,4,6trifluorophenoxy)ethyl][1,2,4]triazolo[4,3-a]pyridine;

7-[1-(2,4-difluorophenoxy)ethyl]-3-ethyl-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclobutylmethyl)-7-[(1*R)-1-(2,4-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(cyclobutylmethyl)-7-[(1*S)-1-(2,4-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-(ethoxymethyl)-8-(trifluoromethyl)-7-[(1*R)-1-(2,4,6trifluorophenoxy)ethyl][1,2,4]triazolo[4,3-a]pyridine;

3-(ethoxymethyl)-8-(trifluoromethyl)-7-[(l*S)-l-(2,4,6trifluorophenoxy)ethyl][1,2,4]triazolo[4,3-a]pyridine;

7-[(1*S)-1-(2,4-difluorophenoxy)ethyl]-3-(ethoxymethyl)-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

7-[(1*R)-1-(2,4-difluorophenoxy)ethyl]-3-(ethoxymethyl)-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

7-[(1*R)-1-(2,4-difluorophenoxy)ethyl]-3-ethyl-8(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

7-[(1*S)-1-(2,4-difluorophenoxy)ethyl]-3-ethyl-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

7-[1-(2,4-difluorophenoxy)ethyl]-3-propyl-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine;

3-ethyl-8-(trifluoromethyl)-7-[(1*R)-1-(2,4,6-trifluorophenoxy)ethyl][1,2,4]triazolo[4,3-a]pyridine;

3-ethyl-8-(trifluoromethyl)-7-[(1* S)-1-(2,4,6-trifluorophenoxy)ethyl][1,2,4]triazolo[4,3-a]pyridine;

7-[(1*R)-(2,4-difluorophenoxy)ethyl]-3-propyl-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine and

7-[(1*S)-(2,4-difluorophenoxy)ethyl]-3-propyl-8-(trifluoromethyl)[1,2,4]triazolo[4,3-a]pyridine.

The scope of this list includes their stereoisomeric forms, pharmaceutically acceptable salts and solvates.

In a further embodiment, the compound may be selected from the hydrochloride salt of 3-(cyclopropylmethyl)-7-[(1S)-1-(2,4-difluorophenoxy)ethyl]-8(trifluoromethyl)[1,2,4]triazolo[4 ,3-a]pyridine.

The orthosteric mGluR2/mGluR2/3 agonist combinations of the present invention include, but are not limited to, LY-404039; LY-2969822; LY-2934747; LY-379268; DCG-IV; LY-354740; LY314582; LY-544344; LY-2140023;

LY-181837; LY-389795; LY-446433; LY-450477; LY-395756; LY-566332; LY-541850; LY-2300559; LY-404040; LY-281223; LY-2979165; talaglumetad; MGS008; MGS0022; MGS0028; MGS0039;

(-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate; (+)-4-amino-2sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid; (+) -2-amino-4-fluorobicyclo-[3.1.0]hexane2,6-dicarboxylic acid; 1 S,2R,5S,6S-2-amino-6-fluoro-4-oxobicyclo-[3.1.0]hexane-2,6-dicarboxylic acid;

- 16 045021

S,2R,4S,5S,6S-2-amino-6-fluoro-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylic acid;

1S,2R,3R,5S,6S-2-amino-3-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid;

S,2R,3S,5S,6S-2-amino-6-fluoro-3-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylic acid; (+)-4amino-2-sulfonylbicyclo-[3.1.0]hexane-4,6-dicarboxylic acid; (+)-2-amino-4fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid;

1S,2R,5S,6S-2-amino-6-fluoro-4-oxobicyclo[3.1.0]hexane-2,6-dicarboxylic acid;

1S,2R,4S,5S,6S-2-amino-6-fluoro-4-hydroxybicyclo[3.1.0]hexane-2,6-dicarboxylic acid;

1S,2R,3R,5S,6S-2-amino-3-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid or 1S,2R,3S,5S,6S-2-amino-6-fluoro-3 -hydroxybicyclo-[3.1.0]hexane-2,6-dicarboxylic acid.

A specific group of mGluR2 agonists include LY-37 9268; DCG-IV; LY-354740; LY-404039; LY2969822; LY-2934747; LY-544344 and LY-2140023.

The orthosteric agonists of the metabotropic glutamatergic receptor subtype 2 combinations of the present invention are, in particular, additionally selected agonists disclosed in WO 1997/18199 and WO 2003/104217, included herein in their entirety. The specific compounds disclosed herein are (-)-(1R,4S,5S,6S)-4-amino-2sulfonylbicyclo[3.1.0]-hexane-4,6-dicarboxylic acid (also known as LY-404039)

or a salt or solvate thereof and 2,2-dioxide (1R,4S,5S,6S)-4-[[(2S)-2-amino-4-(methylthio)-1oxobutyl]amino]-2-thiabicyclo[3.1. 0]hexane-4,6-dicarboxylic acid (also known as LY2140023 [CAS 635318-55-7])

or a salt or solvate thereof, for example a monohydrate thereof.

The names of the compounds of the present invention were compiled according to the rules of nomenclature adopted by the Chemical Abstract Service (C.A.S.) using Advanced Chemical Development, Inc. software. (ACD/Name product version 10.01.0.14105, October 2006). In the case of tautomeric forms, the name of the represented tautomeric form of the structure was compiled. However, it should be understood that other tautomeric form not shown is also included within the scope of the present invention.

As used herein, the terminology C _{1_3} alkyl, _{C1_4} alkyl, or C _{1_6} alkyl as a group or part of _{a group} indicates a saturated, straight or branched hydrocarbon radical having from 1 to 3, or from 1 to 4, or from 1 to 6 carbon atoms, such as methyl, ethyl, 1-propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methyl-1-propyl, 1,1-dimethylethyl, 3-methyl-1-butyl, 1-pentyl, 1hexyl, etc.

The terminology C ₃ - ₇ cycloalkyl or C ₃ - ₈ cycloalkyl as a group or part of a group indicates a saturated, cyclic hydrocarbon radical having from 3 to 7 or 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

As used herein, the terminology halide or halogen as a group or part of a group refers to fluorine, chlorine, bromine or iodine, with fluorine or chlorine being preferred.

The terminology mono- and polyhalo- _C1-3 alkyl or _mono- and polyhalo-C1-3 alkyl will respectively indicate _{C1_3} alkyl or _C1-4 alkyl as previously defined, substituted with 1, 2, 3 or, where possible, a large number of halogen atoms as previously defined.

Whenever the term substituted is used in the present invention, it means, unless otherwise stated or is clear from the context, one or more hydrogen atoms, preferably 1 to 3 hydrogen atoms, more preferably 1 to 2 hydrogen atoms, more preferably 1 a hydrogen atom, on an atom or radical designated using the expression substituted, is substituted by a member selected from the group indicated, provided that the normal valence is not exceeded and that the substitution results in a chemically stable compound, i.e., a compound that is sufficiently stable, to withstand isolation from the reaction mixture to a suitable degree of purity and formulation into a therapeutic agent.

As used herein, unless otherwise indicated, the term antiepileptic drug and the abbreviation AED will be used interchangeably with the term anticonvulsant and, in the context of the present invention, refers to an agent that can treat, suppress or prevent seizure activity or ictogenesis when an agent is administered to a subject or patient.

As used herein, unless otherwise noted, the term synaptic vesicle protein 2A ligand and the abbreviation SV2A ligand will be used interchangeably. Examples of SV2A ligands include, but are not limited to, those included in GB 1039113, GB 1309692, EP 1262036, EP 1806339, WO 2001/062726, US 2002/094787, WO 2004/087658, WO 2005/121082, W O 2005/054188, WO 2006 /128692, WO 2006/128693, WO 2007/065595, WO 2008/132139 and WO 2008/132142, WO 2011/047860, WO 2012/143116 and WO 2012/143117. Suitable specific examples of SV2A ligands include, but are not limited to, levetiracetam, brivaracetam, and seletracetam.

Thus, in an embodiment of the present invention, the SV2A ligand is selected from levetiracetam, brivaracetam and seletracetam.

In a specific embodiment, the SV2A ligand is levetiracetam.

In a specific embodiment, the SV2A ligand is brivaracetam.

Methods for preparing the above SV2A ligands are known from the literature and are disclosed, for example, in EP 1806339; in EP 0162036 and in GB 2225322 (levetiracetam); in WO 01/62726 (brivaracetam); and in WO 2005/121082 (seletracetam); such methods are hereby incorporated by reference in their entirety.

In a further embodiment, the combination in accordance with the present invention contains (a) a pharmaceutically effective amount of levetiracetam or brivaracetam; and (b) a pharmaceutically effective amount

or a pharmaceutically acceptable salt thereof, in particular a hydrochloride salt thereof, or a solvate thereof.

In a further embodiment, the pharmaceutical composition in accordance with the present invention contains (a) a pharmaceutically effective amount of levetiracetam or brivaracetam; and (b) a pharmaceutically effective amount

or a pharmaceutically acceptable salt thereof, in particular a hydrochloride salt thereof, or a solvate thereof.

In a further embodiment, the combination according to the present invention contains (a) a pharmaceutically effective amount of levetiracetam or brivaracetam and (b) a pharmaceutically effective amount of LY-404039 or a pharmaceutically acceptable salt thereof, in particular a hydrochloride salt thereof, or a solvate thereof.

In a further embodiment, the pharmaceutical composition in accordance with the present invention contains (a) a pharmaceutically effective amount of levetiracetam or brivaracetam and (b) a pharmaceutically effective amount of LY-404039 or a pharmaceutically acceptable salt thereof, in particular a hydrochloride salt thereof, or a solvate thereof.

In a further embodiment, the combination according to the present invention contains (a) a pharmaceutically effective amount of levetiracetam or brivaracetam and (b) a pharmaceutically effective amount of LY-214 0023, or a pharmaceutically acceptable salt or solvate thereof, particularly a monohydrate thereof.

In a further embodiment, the pharmaceutical composition in accordance with the present invention contains (a) a pharmaceutically effective amount of levetiracetam or brivaracetam and (b) a pharmaceutically effective amount of LY-214 0023, or a pharmaceutically acceptable salt or solvate thereof, particularly a monohydrate thereof.

The combination product of the present invention, in particular the pharmaceutical composition of the present invention, is primarily intended for the treatment of epilepsy and related disorders.

- 18 045021

It should be understood that some mGluR2 compounds, particularly the mGluR2 PAM/mGluR2 agonist compounds of the present invention and their pharmaceutically acceptable addition salts and solvates thereof, may contain one or more chiral centers and exist as stereoisomeric forms.

As used herein, the expression compounds of the present invention is understood to include mGluR2 PAM compounds, in particular compounds of formula (I-B) and mGluR2 agonist compounds as disclosed herein, and their salts and solvates.

In the context of the present invention, any chemical formula with bonds shown only as solid lines rather than as solid wedge or dotted wedge bonds, or otherwise shown as having a specific configuration (e.g., R, S) around one or more atoms, provides each possible stereoisomer or mixture of two or more stereoisomers.

Hereinbefore and below, the terms mGluR2 compound and mGluR2 PAM/mGluR2 agonist compound are understood to include their stereoisomers and their tautomeric forms. Above and below herein, the terms stereoisomers, stereoisomeric forms, or stereochemically isomeric forms are used interchangeably. The present invention includes all stereoisomers of the compounds of the present invention, either as a pure stereoisomer or as a mixture of two or more stereoisomers. Enantiomers are stereoisomers that have non-overlapping mirror images of each other. A 1:1 mixture of a pair of enantiomers is a racemate or racemic mixture. Diastereomers (or diastereoisomers) are stereoisomers that are not enantiomers, i.e. they are not related as mirror images. If the compound contains a double bond, the substituents may be in the E- or Z-configuration. Substituents on divalent cyclic (partially) saturated radicals can be in either the cis or trans configuration; for example, if the compound contains a disubstituted cycloalkyl group, the substituents may be in the cis or trans configuration. Thus, the present invention includes enantiomers, diastereomers, racemates, E-isomers, Z-isomers, cis-isomers, trans-isomers and mixtures thereof whenever chemically possible. The meanings of all these terms, i.e. enantiomers, diastereomers, racemates, E-isomers, Z-isomers, cis-isomers, trans-isomers and mixtures thereof are known to one skilled in the art. The absolute configuration is determined according to the Cahn-Ingold-Prelog system. The configuration for an asymmetric atom is determined by either R or S. Isolated stereoisomers, the absolute configuration of which is unknown, can be designated as (+) or (-) depending on the direction in which they rotate plane-polarized light. For example, isolated enantiomers whose absolute configuration is unknown may be designated as (+) or (-) depending on the direction in which they rotate plane-polarized light.

If a specific stereoisomer is specified, this means that the specified stereoisomer contains substantially no other isomers, i.e. associated with less than 50%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, in particular less than 2% and most preferably less than 1% of other isomers. Thus, if a mGluR2 compound, for example, is listed as (R), this means that the compound contains substantially no (S) isomer; if the compound mGluR2, for example, is indicated as E, then this means that the compound contains essentially no Z isomer; if the compound mGluR2, for example, is listed as cis, this means that the compound contains essentially no trans isomer.

Some mGluR2 compounds can also exist in their tautomeric forms. It is intended that such forms, inasmuch as they may exist although not explicitly shown by the above formula, are included within the scope of the present invention.

It follows that one compound can exist in both stereoisomeric and tautomeric forms.

For medical use, salts of the compounds of the present invention refer to non-toxic pharmaceutically acceptable salts (salts of the compounds of the present invention where the counterion is pharmaceutically acceptable). However, other salts may be useful in the preparation or purification of the compounds of the present invention or pharmaceutically acceptable salts thereof, and may include acids and bases that are not pharmaceutically acceptable. All salts, whether pharmaceutically acceptable or not, are included within the scope of the present invention.

The pharmaceutically acceptable acid and base addition salts mentioned above or below herein are intended to include the therapeutically active, non-toxic acid and base addition salt forms that the compounds of the present invention can form. Suitable pharmaceutically acceptable salts of such compounds include acid addition salts, which can be, for example, formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid, such as, for example, inorganic acids, such as hydrohalic acids, for example, hydrochloric or hydrobromic acid, sulfuric, nitric, phosphoric and other acids; or organic acids, such as, for example, acetic, propionic, hydroxyacetic, lactic, pyruvic, oxalic (i.e. ethanedic acid), malonic, succinic (i.e. butanedicic acid), maleic, fumaric, malic, tartaric , citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, paminosalicylic, pamic and other acids. Conversely, these salt forms can be converted by treatment with an appropriate base into the free base form. In addition, if the compounds of the present invention bear an acid moiety, then suitable pharmaceutically acceptable salts thereof may include organic and inorganic bases.

Suitable forms of basic salts include, for example, ammonium salts, alkali and alkaline earth metal salts, for example, lithium, sodium, potassium, magnesium, calcium, etc. salts, salts of organic bases, for example, primary, secondary and tertiary aliphatic and aromatic amines such as methylamine, ethylamine, propylamine, isopropylamine, four isomers of butylamine, dimethylamine, diethylamine, diethanolamine, dipropylamine, diisopropylamine, di-n-butylamine, pyrrolidine, piperidine, morpholine, trimethylamine, triethylamine, tripropylamine, quinuclidine, pyridine, quinoline and isoquinoline ; salts of benzathine, N-methyl-D-glucamine, hydrabamine, and salts of amino acids such as, for example, arginine, lysine, etc. Conversely, the salt form can be converted by treatment with an acid into the free acid form.

The term solvate includes solvent addition forms, as well as their salts, which can form compounds of formula (I). Examples of such solvent addition forms are, for example, hydrates, alcoholates, and the like.

Preparation of compounds of formula (I-B).

The compounds of formula (I-B) according to the present invention can generally be prepared by a sequence of steps, each of which is known to one skilled in the art. In particular, the compounds can be prepared according to the following synthetic methods.

The compounds of formula (I-B) can be synthesized as racemic mixtures of enantiomers, which can be separated from each other according to separation procedures known in the art. Racemic compounds of formula (I-B) can be converted into the corresponding diastereomeric salt forms by reaction with a suitable chiral acid. These diastereomeric salt forms are then separated, for example by selective or fractional crystallization, and the enantiomers are isolated from there using alkali. An alternative method for separating the enantiomeric forms of compounds of formula (I-B) involves liquid chromatography or supercritical fluid chromatography (SFC) using a chiral stationary phase. Said pure stereochemically isomeric forms can also be prepared from the corresponding pure stereochemically isomeric forms of the corresponding starting materials, provided that the reaction proceeds stereospecifically.

A. Preparation of the final compounds of formula (I-B).

The final compounds of formula (I-B) can be prepared by reacting an intermediate of formula (II) with a compound of formula (III) according to reaction scheme (1), this reaction being carried out under the classical Mitsunobu reaction conditions. The reaction is preferably carried out with phosphine and an azodicarboxylic acid ester or amide in tetrahydrofuran, 1,4-dioxane, diethyl ether, toluene, benzene, dichloromethane or mixtures thereof at -30 to 150° C. under thermal heating or microwave irradiation. Commonly used phosphines are triphenylphosphine and tributylphosphine, which are usually combined with dimethyl azodicarboxylate, diethyl azodicarboxylate, diisopropyl azodicarboxylate, di-(4-chlorobenzyl) azodicarboxylate, dibenzyl azodicarboxylate, di-tert-butyl azodicarboxylate, bis-(dimethyl) azodicarboxylic acid, dipiperidide azodicarboxylic acid or azodicarboxylic acid dimorpholide . In reaction diagram (1), all variables are those defined in formula (I-B).

Reaction scheme 1

B. Preparation of intermediate compounds.

Experimental procedure 2.

Intermediates of formula (II) can be prepared by subjecting the intermediate of formula (IV) to conditions known to those skilled in the art. This is illustrated in reaction diagram (2), where all variables are defined as mentioned above herein. Methods for carrying out these transformations are well known to those skilled in the art. Treatment of the aldehyde of formula (IV) with an organometallic compound such as methyllithium or methylmagnesium bromide gives the compound of formula (II). A suitable solvent for this reaction is an ether such as tetrahydrofuran, and the reaction is usually carried out at a temperature of -78 to 40°C. In reaction scheme 2, all variables are those defined in formula (I-B). Reaction scheme 2

Experimental procedure 3.

Intermediates of formula (IV) can be prepared by reacting an intermediate of formula (V) under dihydroxylation and oxidative cleavage conditions known to those skilled in the art and can be accomplished, for example, using oxon, osmium tetroxide. The process can optionally be carried out in a solvent such as 1,4-dioxane, water, and typically at temperatures from about -100°C to about 100°C. A brief description of such methods is given in Comprehensive Organic Transformations, VCH Publishers, (1989), R.C.Larock, pp.595-596. This is illustrated in reaction diagram (3), where all variables are defined as mentioned above herein.

Reaction scheme 3

Experimental procedure 4.

Intermediates of formula (V) can be prepared by coupling reactions, such as Stille or Suzuki reactions, of an intermediate of formula (VI) with a compound of formula (VII) under conditions known to those skilled in the art. The process can optionally be carried out in a solvent such as 1,4-dioxane, water, and typically at temperatures from about room temperature. to approximately 200°C in the presence of a base. This is illustrated in reaction scheme (4), where all variables are defined as mentioned above herein, where M is a trialkyltin, boronic acid or boronate ester, and a palladium catalyst is present and the halogen is chlorine, bromine or iodine.

Reaction scheme 4

Experimental procedure 5.

Intermediates of formula (VI) can be prepared according to procedures known in the art by cyclizing an intermediate of formula (VIII) in the presence of a halogenating agent such as, for example, phosphorus (V) oxychloride (POCl ₃ ) in a suitable solvent such as , for example, dichloroethane, when stirred under microwave radiation for a suitable period of time to allow completion of the reaction, for example, 5 minutes. at a temperature of 140-200°C. In reaction scheme (5), R ¹ is defined as in formula (IB), and the halogen is chlorine, bromine or iodine.

Reaction scheme 5

Experimental procedure 6.

Intermediates of formula (VIII) can be prepared according to procedures known in the art by reacting a hydrazine intermediate of formula (IX) with acid halides of formula (X). The reaction can be carried out using an inert solvent,

- 21 045021 such as, for example, DCM, in the presence of a base, such as, for example, triethylamine, for example, at room temperature. for a suitable period of time to allow the reaction to complete, for example 20 minutes.

In reaction scheme (6), R ¹ is defined as in formula (IB).

Reaction scheme 6

Experimental procedure 7.

Intermediates of formula (IX) can be prepared by reacting an intermediate of formula (XI) with hydrazine according to reaction scheme (7), this reaction carried out in a suitable reaction-inert solvent such as, for example, ethanol, THF or 1,4-dioxane, under such temperature conditions as, for example, heating the reaction mixture, for example, at 160°C under microwave radiation for 30 minutes. or by classical thermal heating at 70°C for 16 hours. In reaction scheme (7), the halogen is chlorine, bromine or iodine.

Reaction scheme 7

Experimental procedure 8.

Intermediates of formula (XI) can be prepared by reacting an intermediate of formula (XII) with benzyl alcohol according to reaction scheme (8), this reaction carried out in a suitable reaction-inert solvent, such as, for example, N,N-dimethylformamide, in the presence of a suitable base, such as, for example, sodium hydride, at room temperature. for a suitable period of time to allow completion of the reaction, such as 1 hour. In reaction scheme (8), the halogen is chlorine, bromine or iodine.

Reaction scheme 8

Experimental procedure 9.

Intermediates of formula (XII) can be prepared by reacting an intermediate of formula (XIII) with a suitable trifluoromethylating agent, such as, for example, fluorosulfonyl(difluoroacetic acid) methyl ester according to reaction scheme (9). This reaction is carried out in a suitable reaction-inert solvent, such as, for example, N,Ndimethylformamide, in the presence of a suitable coupling agent, such as, for example, copper(I) iodide, under temperature conditions such as heating the reaction mixture, for example, at 160°C under microwave radiation for 45 minutes. In reaction scheme (9), the halogen is chlorine, bromine or iodine.

Reaction scheme 9

The starting materials for the compounds of formulas (II), (VII), (X) or (XIII) are compounds that are commercially available or can be prepared according to conventional reaction procedures well known to those skilled in the art.

As used herein, the expression composition is intended to cover a product containing certain ingredients in certain amounts, as well as any product that is obtained, directly or indirectly, by combining certain ingredients in

- 22 045021 certain quantities.

As used herein, subject refers to an animal, preferably a mammal, most preferably an adult, child or infant, that is or has been the subject of treatment, examination or experiment.

As used herein, the expression "therapeutically effective amount" means that amount of an active compound or pharmaceutical agent that produces a biological or medical effect in a tissue system in an animal or human that is desired by a researcher, veterinarian, physician, or other clinician, which includes alleviation of one or more symptoms of a disease. or disorders being treated; and/or reducing the severity of one or more symptoms of the disease being treated.

A combination of compounds of (a) an SV2A ligand and (b) a positive allosteric modulator (PAM) of metabotropic glutamatergic receptor subtype 2 (mGluR2) or a pharmaceutically acceptable salt or solvate thereof, or an orthosteric agonist of metabotropic glutamatergic receptor subtype 2 or a pharmaceutically acceptable salt or solvate thereof, whatever whether compounds (a) and (b) are administered simultaneously, separately, or sequentially may produce a more beneficial effect compared to the effect of compounds (a) or (b) administered separately. In particular, at least one beneficial effect may be observed, for example, a mutual enhancement of the effect of compounds (a) and (b), an effect exceeding an additive, in particular a synergistic effect; additional beneficial effects include, for example, a significant reduction in the effective dose for the combination of (a) and (b); an additional therapeutic effect not observed for either compound (a) or (b) individually, a more favorable side effect profile, or a combined therapeutic effect where the dosage of either (a) or (b) or both is ineffective connections.

In the context of the present invention, the expression is a fixed 1:1 dose ratio of (a) synaptic vesicle protein 2A ligand and (n) compound of formula (IB), calculated from the ED ₅₀ values of the individual compounds (a) and (b) refers to compositions containing compounds (a) and (b) at a dose corresponding to 50% of the relative dose ED5 ₀ for compounds (a) and (b) individually or a multiple of such fixed dose ratio. The expression fixed dose ratio of (a) synaptic vesicle protein 2A ligand: (b) compounds of formula (IB), 3:1, calculated from the ED ₅₀ values for individual compounds (a) and (b) refers to compositions containing (b) a compound of formula (IB) at a dose corresponding to 75% of the relative ED ₅₀ dose, and compound (a) at a dose corresponding to 25% of the relative ED ₅₀ dose of compound (a), or a multiple of such fixed dose ratio, and etc.

Thus, in another embodiment of the present invention, (a) the SV2A ligand and (b) the compound of formula (IB) are present in the pharmaceutical composition at a fixed dosage ratio of (a):(b) from about 1:10 to about 10:1, preferably from about 1:5 to about 5:1, more preferably from about 1:3 to about 3:1, in another embodiment from about 1:1 to about 3:1; in an alternative embodiment 1:3; in yet another embodiment, 1:1; in an additional embodiment, 3:1; where the fixed dose ratio is calculated from the ED ₅₀ values for individual compounds (a) and (b).

Moreover, the present invention is directed to complex or combination therapy involving the administration of (a) a ligand representing synaptic vesicle protein 2A (SV2A); and (b) an mGluR2 PAM/mGluR2 agonist compound, in particular a compound of formula (I-B), as defined herein, wherein a pharmaceutically or therapeutically effective amount shall mean that amount of the combination of agents taken together such that the combined effect produces the desired biological or medical reaction. For example, a therapeutically effective amount in a combination therapy comprising the administration of (a) an SV2A ligand as defined herein and (b) an mGluR2 PAM/mGluR2 agonist compound, particularly a compound of formula (I-B) as defined herein , may be an amount of (a) an SV2A ligand as defined herein, and an amount (b) of an mGluR2 PAM/mGluR2 agonist compound, particularly a compound of formula (I-B), which, when administered together or sequentially, produces a combined effect that is therapeutically effective. Additionally, one skilled in the art will recognize that in the case of combination therapy with a therapeutically effective amount, as in the example described above, the amount of mGluR2 PAM/mGluR2 agonist compound, particularly a compound of formula (I-B), and/or the amount of a suitable SV2A ligand alone may be or may not be therapeutically effective.

The present invention provides methods of prevention or treatment comprising administering to a subject in need thereof a combination therapy of a therapeutically effective amount of an SV2A ligand and a therapeutically effective amount of an mGluR2 PAM/mGluR2 agonist compound, particularly a compound of formula (I-B) as described herein. To achieve this purpose, the compounds or compositions of the present invention must be administered in the correct therapeutically effective amount or dosage as described below.

The optimal dosages and treatment regimens to be administered can be readily determined by those skilled in the art and will vary depending on the specific compound used, the route of administration, the potency of the drug, and the progression of the disease state. In addition, factors associated with the individual patient being treated, including age, weight, diet, and timing of administration, will necessitate dosage adjustments.

One skilled in the art will appreciate that a therapeutically effective dosage of the compounds of the present invention may include repeat dosing as part of an extended treatment regimen that will provide clinically significant results.

The amounts of mGluR2 PAM/mGluR2 agonist compound, particularly the compound of formula (I-B), in the combinations of the present invention that are administered daily can range from about 0.01 mg to about 2000 mg. Examples of daily amounts of the compound of formula (I-B) are 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50 ,0, 100, 150, 200, 250, 300, 400, 500, 750, and 1000 milligrams for symptomatic dosage adjustments for the patient being treated. An effective amount of drug is typically administered at a dosage level of from about 0.01 mg/kg to about 150.0 mg/kg body weight per day or any range within this range. Preferably, the range is from about 0.1 to about 100.0 mg/kg body weight per day, more preferably from about 0.5 mg/kg to about 50 mg/kg, more preferably from about 1.0 to about 25.0 mg/kg body weight per day. The compounds can be administered in a 1, 2, 3 or 4 times daily regimen. The amounts of SV2A ligand that are administered daily can range from about 0.01 to about 7000 mg, preferably will be from 250 to 5000 mg, or more preferably will be from 500 to 3000 mg. Examples of daily amounts of SV2A ligand are 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250, 500, 750, 1000, 1500 and 3000 milligrams for symptomatic adjusting the dosage in the patient being treated. An effective amount of drug is typically administered at a dosage level of from about 0.01 mg/kg to about 150.0 mg/kg body weight per day or any range within this range. Preferably, the range is from about 0.1 to about 100.0 mg/kg body weight per day, more preferably from about 0.5 mg/kg to about 50 mg/kg, more preferably from about 1.0 to about 25.0 mg/kg body weight per day. The compounds can be administered in a 1, 2, 3 or 4 times daily regimen. All quantities mentioned in this and the following paragraphs refer to the free form (i.e., non-salt form). The above values represent free form equivalents, i.e. values corresponding to the expected introduction of the free form. When introducing salts, amounts should be calculated depending on the ratio of molecular weights of the salt form and the free form.

The above daily doses are calculated for an average body weight of approximately 70 kg and should be recalculated in the case of pediatric use or in the case of use in patients with a significant deviation in body weight.

Dosages may be presented in the form of one, two, three or four or more dosage units administered at regular intervals throughout the day. The dosage used preferably corresponds to the daily amount of mGluR2 PAM/mGluR2 agonist compound, in particular the compound of formula (I-B), or the SV2A ligand mentioned above, or a portion of the dose thereof, such as 1/2, 1/3, 1/4 thereof. The dosage form may contain mGluR2 PAM/mGluR2 agonist compound, in particular compound (I-B), or SV2A ligand, or both in an amount equal to the ranges or amounts mentioned in the previous paragraphs, for example, the dosage form may contain 10, 25, 50 , 100, 150 or 200 mg of PAM/mGluR2 agonist compound, in particular compound (I-B), 10, 25, 50, 100 or 250 mg of SV2A ligand, either in separate formulations or in combination formulations. In one embodiment, mGluR2 PAM/mGluR2 agonist compound, in particular a compound of formula (I-B), is administered once daily (q.d.), in particular as one dose per day, and the SV2A ligand is administered once daily or twice daily day (q.d. or b.i.d.), in particular in the form of one or two doses per day. In the case where both compounds are to be administered once daily, this can be accomplished by administering two separate doses, one with an mGluR2 PAM/mGluR2 agonist compound, in particular a compound of formula (I-B), one with an SV2A ligand, or by administering a combination a dose containing an mGluR2 PAM/mGluR2 agonist compound, in particular a compound of formula (I-B), and an SV2A ligand.

The combinations of the present invention can be administered one, two, three, four or, if necessary, many times per day. In one embodiment, the combination is administered once daily. In another embodiment, the combination is administered twice daily or three times daily. Dosing can be done through separate dosage forms, i.e. through dosage forms containing only mGluR2 PAM/mGluR2 agonist compound, in particular a compound of formula (I-B), or only SV2A ligand; or through combination dosage forms containing the active ingredients of mGluR2 PAM/mGluR2 agonist compound, in particular a compound of the formula (I-24 045021

B), and SV2A ligand. A combination of the use of a combined dosage form and the use of separate dosage forms can also be used. Dosage forms that can be administered are described later herein, with oral dosage forms, particularly tablets or capsules, being preferred.

The active ingredients may be formulated into pharmaceutical compositions either individually or as a combination pharmaceutical composition. In the latter case, a pharmaceutical composition is prepared containing a therapeutically effective amount of mGluR2 PAM/mGluR2 agonist compound, in particular a compound of formula (I-B) or a pharmaceutically acceptable salt thereof, and an SV2A ligand, the foregoing being as defined herein and pharmaceutically acceptable medium.

In a further aspect, the present invention relates to a method for preparing the pharmaceutical composition described herein, which comprises intimately mixing a pharmaceutically acceptable carrier with a therapeutically effective amount of an mGluR2 PAM/mGluR2 agonist compound, particularly a compound of formula (I-B) or a pharmaceutically acceptable salt or solvate thereof. , and a therapeutically effective amount of at least one SV2A ligand.

The combinations provided herein may also be formulated as a combination preparation for simultaneous, separate or sequential use in the prevention or treatment of epilepsy and related disorders; neuropathic pain; migraine or persistent headache; bipolar and related disorders; in neuroprotection; or in preventing epileptogenesis. In such a case, the mGluR2 PAM/mGluR2 agonist compound, in particular the compound of formula (I-B), is formulated into a pharmaceutical composition containing other pharmaceutically acceptable excipients, and the SV2A ligand is separately formulated into a pharmaceutical composition containing other pharmaceutically acceptable excipients. Relatively speaking, these individual pharmaceutical compositions can be part of a kit for simultaneous, separate or sequential use.

The individual components of the combination of the present invention can be administered simultaneously or separately at different times during the course of therapy or in parallel in divided or single combination forms.

In view of the above, mGluR2 PAM/mGluR2 agonist compounds, in particular compounds of formula (I-B), and SV2A ligand, alone or in combination, can be formulated into various suitable pharmaceutical compositions for administration. In these compositions, a therapeutically effective amount of a particular compound, or both compounds, is combined with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation required to be administered. Pharmaceutical compositions may be formulated as medicinal preparations for oral, parenteral (including subcutaneous (s.c.), intramuscular (i.m.) and intravenous (i.v.)), rectal, transdermal, buccal or intranasal administration. Pharmaceutical compositions may also be prepared for administration directly into the nervous system by routes including, but not limited to, intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal, and/or perispinal routes via delivery via needles and/or intracranial or intravertebral catheters with with or without pumping devices. Suitable compositions for oral administration include powders, granules, aggregates, tablets, compressed or coated pills, dragees, sachets, hard or gelatin capsules, syrups and suspensions. Suitable compositions for parenteral administration include aqueous or non-aqueous solutions or emulsions, while suitable compositions for rectal administration include hydrophilic or hydrophobic filled suppositories. For topical administration, suitable transdermal delivery systems can be used, and for nasal delivery, suitable aerosol delivery systems can be used.

For example, when preparing compositions for oral administration, any conventional pharmaceutical vehicle may be used, such as, for example, water, glycols, oils, alcohols and the like, in the case of liquid compositions for oral administration, such as suspensions, syrups, elixirs, emulsions and solutions ; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrants and the like, in the case of solid compositions. For compositions for parenteral administration, the carrier will generally comprise sterile water, at least in large part, although other ingredients such as solubilizers, emulsifiers or additional adjuvants may be added to the carrier. Injectable solutions can be prepared in which the carrier contains a saline solution, a glucose solution, or a mixture of both. Injectable suspensions can also be prepared, in which case suitable liquid carriers, suspending agents and the like can be used. Also included are preparations in solid form that are intended to be converted immediately before use into preparations in liquid form, such as powders for dissolution. In compositions suitable for subcutaneous administration, the carrier optionally contains a skin penetration enhancer and/or wetting agent, optionally combined with suitable skin-compatible additives in minor quantities. The mGluR2 PAM compound/mGluR2 agonist compound, in particular the compound of formula (I-B), or the SV2A ligand or combinations thereof can also be administered by oral inhalation or insufflation of formulations suitable for this type of administration, such as solution, suspension or dry powder. Suitable pharmaceutical compositions for administration in the form of aerosols or sprays are, for example, suspensions of mGluR2 PAM/mGluR2 agonist compound, in particular a compound of formula (I-B), or an SV2A ligand, or both, in a pharmaceutically acceptable liquid carrier such as ethanol or water or a mixture of them. If necessary, the composition may also additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers, as well as a nebulizing agent. Such a preparation typically contains the active compound in a concentration of from about 0.1 to 50%, in particular from about 0.3 to 3% by weight.

The pharmaceutical compositions may contain the active ingredient mGluR2 PAM compound/mGluR2 agonist compound, particularly a compound of formula (I-B), or an SV2A ligand, or both in combination, at a concentration of from about 0.1% to about 50%, or from about 1% to about 30%, or about 3% to about 20%, or about 5% to about 20%, all percentages by weight, the total of all components in said pharmaceutical compositions not exceeding 100%. In compositions containing both mGluR2 PAM/mGluR2 agonist compound, particularly a compound of formula (I-B), and an SV2A ligand, mGluR2 PAM/mGluR2 agonist compound, particularly a compound of formula (I-B), is present at a concentration of about 0.1 up to about 50%, or from about 1 to about 30%, or from about 3 to about 20%, or from about 5 to about 20%; and the SV2A ligand is present at a concentration of from about 3% to about 50%, or from about 5 to about 50%, or from about 10 to about 50%, or from about 10 to about 40%, or from about 10 to about 30% , and the total of all components in these pharmaceutical compositions does not exceed 100%.

Pharmaceutical compositions may conveniently be presented in unit dosage form for ease of administration and uniformity of dosage. Examples include tablets (including scored or coated tablets), capsules, pills, suppositories, powder packets, wafers, injectable solutions or suspensions and the like, as well as individual pluralities thereof. Of interest are solid dosage forms for oral administration, such as tablets or capsules.

Solid dosage forms in unit dose form may be packaged in any known packaging, with blister packaging being preferred in particular for tablets and capsules. If the mGluR2 PAM/mGluR2 agonist compound, in particular the compound of formula (I-B), and the SV2A ligand are formulated separately, they may be packaged in separate blisters, but one blister may also contain unit dose forms of the mGluR2 PAM/mGluR2 agonist compound, in particular compounds of formula (I-B), and an SV2A ligand, for example, one row with mGluR2 PAM units/mGluR2 agonist compounds, in particular compounds of formula (I-B), and the other with an SV2A ligand. Other options may also be possible.

The combinations of the present invention can be used for the treatment or prevention of epilepsy and related disorders; neuropathic pain; migraine or persistent headache and bipolar and related disorders; or they can be used as a neuroprotector or to prevent epileptogenesis.

As used herein, treatment is intended to refer to all methods that may involve slowing, disrupting, suppressing or stopping the progression of a disease or alleviating symptoms, but does not necessarily mean complete elimination of all symptoms.

In the context of the present invention, unless otherwise specified, the terms epilepsy and related disorders or epilepsy or related disorder will mean any disorder in which a subject (preferably an adult, child or infant) experiences one or more epileptic seizures and/or attacks of tremor. Suitable examples include, but are not limited to, epilepsy (including without limitation localized forms of epilepsy, generalized forms of epilepsy, forms of epilepsy with both generalized and localized seizures, etc.), partial seizures with or without generalization, myoclonic seizures , primary generalized tonic-clonic seizures, especially in patients with idiopathic generalized epilepsy, seizures associated with Lennox-Gastaut syndrome, seizures as a complication of a disease or condition (such as seizures associated with encephalopathy, phenylketonuria, juvenile Gaucher disease , progressive Lundborg myoclonic epilepsy, stroke, head injury, stress, hormonal changes, drug use or drug withdrawal, alcohol use, or

- 26 045021 alcohol withdrawal, sleep deprivation, fever, infection, etc.), status epilepticus (convulsive or non-convulsive), essential tremor, restless limb syndrome, etc. The disorder is preferably selected from epilepsy (regardless of type, underlying cause or origin), essential tremor or restless limb syndrome. More preferably, the disorder is epilepsy (regardless of type, cause or origin) or essential tremor. A specific example of epilepsy is refractory epilepsy, also called treatment- or therapy-resistant epilepsy. This term is often used when patients have been on three or more antiepileptic drugs (AEDs) without success. Refractory epilepsy also includes refractory partial epilepsy and refractory generalized epilepsy (including idiopathic or symptomatic).

As used herein, the term neuropathic pain includes pain resulting from chronic or debilitating conditions or disorders. Chronic or debilitating conditions or disorders that can lead to neuropathic pain include, but are not limited to, the painful form of diabetic peripheral neuropathy, postherpetic neuralgia, trigeminal neuralgia, post-stroke pain, pain associated with multiple sclerosis, pain associated with neuropathies such as idiopathic or post-traumatic neuropathy and mononeuritis, HIV-associated neuropathic pain, cancer-associated neuropathic pain, carpal tunnel-associated neuropathic pain, spinal cord injury-associated pain, complex regional pain syndrome, fibromyalgia-associated neuropathic pain, lumbar and cervical pain, reflex sympathetic dystrophy, phantom pain syndrome and other pain syndromes associated with chronic and debilitating conditions.

As used herein, the term migraine will mean a chronic, episodic and debilitating clinical condition that is diagnosed by the presence of moderate to severe throbbing unilateral headaches lasting 4 to 72 hours, which includes migraine without aura and migraine with aura. As used herein, the term migraine without aura will indicate at least five attacks that meet the following criteria: (a) a headache attack lasting between 4 and 72 hours with a headache that has at least two of the following features: unilateral, throbbing nature, moderate to severe intensity with a direct impact on activities of daily living and aggravation when climbing stairs or similar activities; and (b) during a headache attack, at least the following occur: nausea and/or vomiting and fear of light and phonophobia. As used herein, the term migraine with aura will indicate at least two attacks accompanied by at least 3 of the 4 following features: (a) one or more fully reversible aura symptoms; (b) at least one aura symptom that develops gradually over more than four minutes, or two or more symptoms that occur sequentially; (c) absence of aura symptom that lasts more than 60 minutes; (d) the headache occurs before, simultaneously with, or after the aura, with an indeterminate interval between the aura and the headache of less than approximately 60 minutes.

As used herein, the term bipolar and related disorders will include bipolar I disorder (eg, single manic episode, recent hypomanic episode, recent manic episode, recent mixed episode, recent depressive episode, and recent indeterminate episode), bipolar II disorder, cyclothymic disorder and bipolar disorder not otherwise specified (as these terms are defined by their diagnostic criteria in the Diagnostic and Statistical manual of Mental Disorders 4th Edition, Text Revision, American Psychiatric Association, 2000 (DSM-IV-TR) or ^5th Edition, Text Revision , American Psychiatric Association, 2013 (DSM-5™). Preferably, the bipolar disorder is characterized by depressive and manic (or hypomanic) phases, the phases being cyclical. Preferably, the bipolar disorder is bipolar I disorder or bipolar II disorder. As used herein the term mania will include mania or a phase of manic tendencies, without regard to the underlying cause. The term bipolar mania, as used herein, is intended to mean mania associated with, characteristic of, or a symptom of bipolar disorder. Thus, the methods of treating bipolar mania of the present invention are directed to methods by which mania and/or the manic phase of bipolar disorders are treated. The term bipolar depression, as used herein, is intended to mean depression associated with, characteristic of, or a symptom of bipolar disorder. Thus, the methods of treating bipolar depression of the present invention are directed to methods by which depression and/or the depressive phase of bipolar disorders are treated. As used herein, unless otherwise noted, the terms periodicity or bipolar periodicity will refer to the change in mood from the depressive to manic phases characteristic of bipolar disorders. Thus, the present invention includes methods for stabilizing said cyclicity, including, without limitation,

- 27 045021 reducing the frequency of periodicity and/or reducing the intensity of manic and/or depressive phases.

Thus, in one embodiment, the pharmaceutical composition of the present invention can be used for mood stabilization, in particular mood stabilization for manic depression.

As used herein, the term epileptogenesis refers to the gradual process of development of epilepsy. This process can occur after brain hemorrhages or a number of conditions, including neurodegenerative diseases, traumatic brain injury, stroke, brain tumors, central nervous system infections and status epilepticus, or it can occur after gene mutations.

As used herein, anxiety refers specifically to generalized anxiety disorder.

The expression used herein approximates its generally accepted meaning. In specific embodiments, when associated with a numerical value, it may be interpreted as a numerical value of ±10, or ±5, or ±2, or ±1, or ±0.5, or ±0.1%. In other embodiments, the exact value is implied, i.e. omitting the word approximately.

And/or means that each or both or all of the components or characteristics listed are options, especially two or more of them, alternatively or together.

As used herein, the singular, plural and similar terms used in the context of the present invention (especially in the context of the claims) should be construed to include both singular and plural forms, unless otherwise specified herein or obviously not refuted by the context.

Examples

The following examples are set forth to assist in understanding the present invention and are not intended or should be construed as limiting in any way the present invention as set forth in the claims that follow.

A. Compounds of formula (i-b) - chemistry and IN VITRO testing.

Some methods for preparing the compounds of formula (I-B) of the present invention are illustrated in the following examples. Unless otherwise stated, all starting materials were obtained from private suppliers and used without further purification.

Further in this document aq. means watery; DCE means 1,2-dichloroethane, DCM means dichloromethane; DIPE means diisopropyl ether; DIPEA means N,Ndiisopropylethylamine; DMF means N,N-dimethylformamide; ES stands for electrospray; Et ₃ N means triethylamine; Et ₂ O means diethyl ether; EtOAc means ethyl acetate; h means hours; 'ΉPLC means high performance liquid chromatography; HRMS stands for high resolution mass spectrometry/spectrometry; l or L means liter; LRMS stands for low resolution spectrometry/mass spectra; MeOH means methanol; min. means minute(s); t.pcs. means melting point; Pd(PPh ₃ ) ₄ means tetrakis(triphenylphosphine)palladium(0); RP means reverse phase; k.t. means room temperature; s means seconds; sat. means saturated; SFC stands for supercritical liquid chromatography; sol. means solution; THF stands for tetrahydrofuran.

Microwave-assisted reactions were performed in a single-mode reactor: Initiator™ Sixty EXP microwave reactor (Biotage AB), or in a multi-mode reactor: MicroSYNTH Labstation (Milestone, Inc.).

Thin layer chromatography (TLC) was performed on silica gel 60 F254 (Merck) plates using high purity solvents. Capillary chromatography was performed on 60 A, 230-400 mesh silica gel (Merck) using standard techniques. Automated flash column chromatography was performed using Merck ready-to-connect irregular silica gel cartridges ranging in size from 15-40 μm (disposable normal phase flash chromatography columns) in an Armen Instrument SPOT or LAFLASH system.

The absolute stereochemical configuration for some compounds was determined using vibrational circular dichroism (VCD). They were measured on a Bruker Equinox 55 equipped with a PMA 37 in a liquid cell with a KBr window using CD ₂ Cl ₂ as solvent (PEM: 1350 cm ^-1 , LIA: 1 mV, resolution: 4 cm ^-1 ). A description of the use of VCD to determine the absolute configuration can be found in Dyatkin A.V. et. al., Chirality, 14:215-219 (2002).

Whenever RS is used herein, it means that the compound is a racemic mixture unless otherwise indicated. The stereochemical configuration for a number of compounds was designated as R or S if the mixture was separated; For some compounds, the stereochemical configuration was designated *R or *S if the absolute stereochemical configuration was not determined, although the compound itself was isolated as a separate stereoisomer and is enantiomerically pure. The enantiomeric excess of the compounds described herein was determined by analyzing the racemic mixture by supercritical liquid chromatography (SFC) followed by comparison of the separated enantiomers during SFC. Preparation of intermediate compounds.

Description 1 - intermediate connection 1

Cyclopropylacetic acid ([CAS 5239-82-7], 50 g, 500 mmol) was dissolved in CH ₂ Cl ₂ (300 ml), and then SOCl ₂ (100 ml) was added. The reaction mixture was stirred at 60°C for 2 hours and then the solvent was evaporated to give intermediate 1 (53 g, 90%), which was used without further purification.

Description 2 - intermediate connection 2

To a solution of 2,4-dichloro-3-iodopyridine ([CAS 343781-36-2], 290 g, 1058 mmol) in DMF (1.7 L) was added methyl 2,2-difluoro-2-(fluorosulfonyl) acetate ([CAS 680-15-9], 403 g, 2098 mmol) and CuI (403 g, 2.13 mol), and then the reaction mixture was heated at 100°C for 5 hours.

The reaction mixture was cooled and filtered. The filtrate was diluted with H ₂ O, and extracted with Et ₂ O, and washed with NH ₃ solution. The organic layer was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give intermediate 2 (160 g), which was used without further purification.

Description 3 - intermediate connection 3

Benzyl alcohol (35 g, 325 mmol) was added to a solution of NaH (60% in oil, 24 g, 600 mmol) in DMF (2 L) at 0°C, and the reaction mixture was stirred for 2 min. Intermediate 2 (160 mg, 741 mmol) was added in one portion and stirred at 0°C for 1 hour. The reaction mixture was diluted by adding H2O and extracted with Et ₂ O. The organic layer was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluent: petroleum ether/EtOAc = 20/1). The purified fractions were collected and the solvent was evaporated to give intermediate 3 (100 g, 38%).

Description 4 - intermediate connection 4

NH2NH2 hydrate (85% solution in water, 300 g, 9.11 mol) was added to a solution of intermediate 3 (100 g, 277 mmol) in 1,4-dioxane (1.5 L) and the reaction mixture was then heated in a sealed test tube at 160°C for 2 hours. The mixture was concentrated in vacuo, dissolved in DCM, washed with NaHCO ₃ . The organic layer was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give intermediate 4 (90 g, 90%), which was used without further purification.

Description 5 - intermediate connection 5

To a solution of intermediate 4 (90 g, 318 mmol) in CH ₂ Cl ₂ (1.5 L) was added triethylamine (64.3 g, 636 mmol), the mixture was cooled to 0° C. and then a solution of intermediate 1 ( 53 g, 449 mmol) in CH ₂ Cl ₂ . The solution was stirred at RT for 1 hour. The reaction mixture was washed with sat. aq. sol. NaHCO ₃ and extracted with CH ₂ Cl ₂ . The organic layer was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo to give intermediate 5 (104.4 g, 90%).

The following intermediate compounds were synthesized according to the synthesis sequence similar to that described in description 5 (D5).

Intermediate connection Acid chloride Conditions O ^F \k ^F HN ^x ^^ Intermediate 6 Propionyl chloride ([CAS 79-03-8]) Additional cycle at RT. ^F ^ ^{F n} /? Intermediate 7 Cyclobutane acetyl chloride ([CAS 59543-383]) Conditions are similar to D5. Intermediate 8 2-ethoxyacetyl chloride ([CAS 14077-58-8]) Conditions are similar to D5. Intermediate connection 25 Butyryl chloride ([CAS 141-75-3]) Conditions are similar to D5.

Description 6 (a) - intermediate 9

To a solution of intermediate 5 (101 g, 277 mmol) in CH3CN (1.2 L) was added phosphorus(V) oxychloride (84.7 g, 553 mmol) and N,N-diisopropylethylamine (71.3 g, 553 mmol) . The reaction mixture was stirred at 90°C for 38 hours. The reaction mixture was then diluted with DCM and washed with Na ₂ CO ₃ solution. The organic layer was dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by silica gel column chromatography (eluent: petroleum ether/EtOAc = 4/1). The purified fractions were collected and the solvent was evaporated to give intermediate 9 (31.39 g, 41%).

(b) Intermediate 10

The reaction was carried out in 4 batches, which were then combined for isolation and purification.

To a solution of intermediate 6 (7 g, 20.6 mmol) in DCE (50 ml) was added N,Ndiisopropylethylamine (3.96 ml, 22.69 mmol) followed by phosphorus oxychloride (2.12 ml, 22.69 mmol ), and the reaction mixture was heated in a microwave oven at 150°C for 5 min. DCM was then added and the organic layer was washed with sat. sol. NaHCO ₃ , dried (Na ₂ SO ₄ ) and concentrated in vacuo to give the title compound, which was purified by column chromatography (gradient elution: 100% DCM to 2% MeOH.NH ₃ in DCM) to give intermediate 10 (2 .5 g, 49%).

The following intermediates were synthesized according to a synthetic sequence similar to that described in description 6(a) or (b).

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Intermediate connection Raw material Conditions ^F Intermediate connection 11 Intermediate 7 The reaction was carried out in the same way as in (a), but in CH ₃ CN. After completion of the reaction, the reaction mixture was poured into ice water and then washed with sat. sol. NaHCO ₃ and extracted with DCM, dried (Na ₂ SO ₄ ), filtered and concentrated. Purification was carried out in Spot (cartridge with Si, eluent DCM/EtOAc up to 10-20%). F Ν'-NM II ^F Intermediate 12 Intermediate 8 The reaction was carried out in the same way as in (b). Purification by flash column chromatography (silica gel; EtOAc in DCM from 0/100 to 40/60). ^F Intermediate connection 26 Intermediate 25 The reaction was carried out in the same way as in (a). Purification by flash column chromatography (silica gel; MeOH in CH ₂ C1 ₂ , 0/100 to 4/96).

Description 7 - intermediate connection 13

Into a stirred solution of intermediate 9 (10 g, 36.28 mmol) and 4,4,5,5-tetramethyl2-vinyl-1,3,2-dioxoborolane ([CAS 75927-49-0], 7.77 ml, 43.53 mmol) in deoxygenated dioxane (30 ml) and deoxygenated saturated NaHCO ₃ solution (30 ml) under nitrogen atmosphere was added (Ph ₃ P) ₄ Pd (2.096 g, 1.81 mmol). The mixture was stirred at 100°C for 18 hours. The mixture was diluted with EtOAc/water and filtered through a pad of diatomaceous earth. The filtrate was treated with brine and extracted with EtOAc. The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and the solvents were evaporated in vacuo. The crude product was purified by flash column chromatography (silica gel; EtOAc in CH ₂ Cl ₂ 0/100 to 5/95). The desired fractions were collected and concentrated in vacuo to give intermediate 13 (6.08, 63%) as a yellow solid.

The following intermediates were synthesized according to a synthesis sequence similar to that outlined in Description 7.

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Intermediate connection Raw material Conditions F N—N +0 Intermediate 14 Intermediate 10 The reaction was carried out at 150°C. Purification by flash column chromatography (silica gel; 7N ammonia in methanol in DCM 0/100 to 1/9). F N—N LE Intermediate 15 Intermediate 11 Extraction with DCM, purification by flash column chromatography (silica gel; MeOH in DCM 4/96). F ⁿ a yr Intermediate 16 Intermediate 12 Purification by flash column chromatography (silica gel; EtOAc in DCM from 0/100 to 10/90). F N-N >-- yy Intermediate connection 2 7 Intermediate 26 The reaction mixture was treated at 150°C in a microwave reactor. Purification by flash column chromatography (silica gel; EtOAc in DCM from 0/100 to 10/90).

Description 8 (a) - intermediate 17

To a suspension of intermediate 13 (6.08 g, 20.02 mmol) in dioxane (192 mL) was added osmium tetroxide (2.5% in t-BuOH, 10.103 mL, 0.781 mmol) followed by sodium periodate (12.53 g, 58.58 mmol) in water (48.5 ml). The mixture was stirred at room temperature for 2 hours.

The mixture was treated with water and EtOAc and filtered through a pad of diatomaceous earth. The filtrate was extracted with EtOAc. The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and the solvents were evaporated in vacuo. The crude product was washed with Et ₂ O and filtered and dried to give intermediate 17 (4.25 g, 79%) as a brown solid.

(b) Intermediate 18

To a stirred solution of osmium tetroxide (2.5% in t-BuOH, 4.06 mL, 0.31 mmol) and intermediate 14 (2.08 g, 7.85 mmol) in dioxane (75 mL) was added a suspension of sodium periodate (5.04 g, 23.54 mmol) in distilled water (19 ml). The mixture was stirred at room temperature for 150 minutes, and then the mixture was treated with sat. NaHCO ₃ and saline and extracted with

- 32 045021 using DCM. The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The product was triturated with Et2O and filtered in vacuo, and finally placed in an oven at 50°C for 18 hours to give intermediate 18 (1.6 g, 80%) as a brown solid.

The following intermediates were synthesized according to a synthetic sequence similar to that outlined in Description 8.

Intermediate connection Raw material Conditions F N—N I I O Intermediate 19 Intermediate 15 Same procedure as in (a). 4 gV ^{th F} J1 J o Intermediate connection 2 0 Intermediate 16 Same procedure as in (a). FN—N p ^F II V/~~ ^F J! J O Intermediate 28 Intermediate 27 Same procedure as in (a), order of addition: osmium tetroxide was added to a stirred solution of intermediate 27 in 1,4dioxane, then a suspension of sodium periodate in water was added and the reaction mixture was stirred for 2 hours at room temperature. Filtration through a pad of diatomaceous earth was not performed.

Description 9 (a) - intermediates 21a, 21b and 21c

he he he

Intermediate Intermediate Intermediate connection 21a connection 21b connection 21c

To a stirred suspension of intermediate 17 (4.25 g, 15.79 mmol) in THF (281.07 ml) at -20°C under N2, methylmagnesium bromide (1.4 M in THF, 12.40 ml) was added dropwise , 17.37 mmol). The mixture was stirred at -20°C for 45 minutes. The crude product was treated with sat. sol. NH4Cl and extracted with EtOAc. The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel; MeOH in DCM 0/100 to 4/96). The desired fractions were collected and concentrated in vacuo to give intermediate 21a (racemic mixture) (2.96 g, 66%). Intermediate 21a (1.82 g) was purified using chiral SFC [stationary phase: CHIRALPAK AD-H (5 μm, 250 x 20 mm), mobile phase: 80% CO ₂ , 20% EtOH] to give 21b (R- 33 045021 enantiomer) (0.453 g, 10%) as a pale gray solid and intermediate 21c (S-enantiomer) (0.439 g, 10%).

(b) Intermediate 22

To a stirred suspension of intermediate 18 (1.23 g, 5.06 mmol) in THF (90 ml) at -20°C under N2, methylmagnesium bromide (1.4 M in THF, 3.97 ml, .56 mmol). The mixture was stirred at -20°C for 45 minutes. The crude product was treated with sat. sol. NH ₄ Cl and extracted with EtOAc. The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel; MeOH in DCM 0/100 to 4/96). The desired fractions were collected and concentrated in vacuo. The residue thus obtained was triturated with Et ₂ O to give intermediate 22 (620 mg, 35%) as a pale yellow solid.

The following intermediates were synthesized according to a synthesis sequence similar to that outlined in Description 9.

Intermediate 24a was further split into intermediate 24b and intermediate 24c:

F N-N / He Intermediate connection 24b F NN _o / ΐό' he Intermediate connection 24s Chiral SFC conditions: stationary phase: Chiralpak AD-H, 5 µm, 250*30 mm; mobile phase: 80% CO ₂ , 15% EtOH.

Preparation of the final compounds of formula (I-B).

Example 1.

(a) Synthesis of compounds 4-b, 6-b and 5-b

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Connection 4-b Connection 6-b Connection 5-b

Into a stirred solution of intermediate 21a (2 g, 7.01 mmol), 2,4-difluorophenol (1.00 ml, 10.52 mmol) and triphenylphosphine (2.76 g, 10.52 mmol) in THF (74, 18 ml) at 0°C and under nitrogen atmosphere, DIAD (2.07 ml, 10.52 mmol) was added dropwise. The mixture was stirred at 100°C for 10 minutes under microwave radiation. The mixture was diluted with EtOAc and washed with sat. sol. _NaHCO3 . The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel; MeOH in DCM 0/100 to 97/3). The desired fractions were collected and concentrated in vacuo. The residue was triturated with DIPE to give compound 4-b (1.46 g, 52%) as a white solid, which was purified using chiral SFC [stationary phase: Chiralpak AD (5 µm, 250*30 mm, mobile phase : 85% CO ₂ , 15% iPrOH)] to give compound 6-b (0.659 g, 24%) and compound 5-b (0.693 g, 25%).

(b) Alternative synthesis of compound 6-b

Into a stirred solution of intermediate 21b (30 mg, 0.11 mmol), 2,4-difluorophenol (15.07 µL, 0.16 mmol) and triphenylphosphine (41.38 mg, 0.16 mmol) in THF (1. 11 ml) at 0°C and under a nitrogen atmosphere, DIAD (31.06 µl, 0.16 mmol) was added dropwise. The mixture was stirred at 100°C for 10 minutes under microwave radiation. The mixture was diluted with EtOAc and washed with sat. sol. _NaHCO3 . The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel; MeOH in DCM 0/100 to 97/3). The desired fractions were collected and concentrated in vacuo. The residue was triturated with DIPE to give compound 6-b (40 mg, 96%) as a white solid.

(c) Synthesis of the hydrochloride salt (HCl) of compound 6-b.

Into a stirred solution of intermediate 21b (200 mg, 0.70 mmol), 2,4difluorophenol (100.45 μL, 1.05 mmol) and triphenylphosphine (275.84 mg, 1.0516 mmol) in THF (4 mL) at 0°C and under nitrogen atmosphere, DIAD (207.06 μL, 1.05 mmol) was added dropwise. The mixture was stirred at 100°C for 15 min under microwave radiation. The mixture was diluted with EtOAc and washed with sat. sol. _NaHCO3 .

The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by RP HPLC (stationary phase: C18 XBridge, 30 x 100 mm, 5 µm, mobile phase: gradient from 60% 0.1% NH4CO3H/NH4OH pH 9 in water, 40% CH3CN to 43% 0. 1% NH4CO3H/NH4OH solution at pH 9 in water, 57% CH3CN) to give a white solid, which was dissolved in Et ₂ O (8 ml) and 1,4-dioxane (0.5 ml). HCl (4 M in dioxane, 200 μl) was added dropwise to the solution thus obtained. The white solid was filtered, washed with Et ₂ O, dried (Na ₂ SO ₄ ) and evaporated under vacuum. The white residue thus obtained was triturated with Et ₂ O to give HCl compound 6-b (110 mg, 36%) as a white solid.

The following compounds were synthesized following a synthetic sequence similar to that described in Example 1(b), starting with intermediate 21b.

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Example 2.

Synthesis of compound 7-b

Procedure(s). Into a stirred solution of intermediate 21b (30 mg, 0.105 mmol), 3,5-difluorophenol (20.52 mg, 0.158 mmol) and triphenylphosphine (41.38 mg, 0.158 mmol) in THF (1.113 ml) at 0°C and DIAD (31.06 μL, 0.158 mmol) was added dropwise under nitrogen atmosphere. The mixture was stirred at 100°C for 10 minutes under microwave radiation. The mixture was diluted with EtOAc and washed with sat. sol. _NaHCO3 . The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel; MeOH in DCM 0/100 to 96/4). The desired fractions were collected and concentrated in vacuo. The residue was triturated with DIPE to give compound 7-b (21 mg, 50%) as a white solid.

Procedure (b). Alternatively, compound 7 was also synthesized according to a synthetic sequence similar to that shown in Example 1(b), starting with intermediate 21b.

Example 3.

Synthesis of compound 8-b

Procedure(s). To a stirred solution of intermediate 21b (30 mg, 0.105 mmol), 3,4-difluorophenol (20.52 mg, 0.158 mmol) and triphenylphosphine (41.38 mg, 0.158 mmol) in THF (1.11 ml) at 0° C and under nitrogen atmosphere, DIAD (31.06 μL, 0.158 mmol) was added dropwise. The mixture was stirred at 100°C for 10 minutes under microwave radiation. The mixture was diluted with EtOAc and washed with sat. sol. _NaHCO3 . The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel; MeOH in DCM 0/100 to 96/4). The desired fractions were collected and concentrated in vacuo. The residue was triturated with DIPE to give compound 8-b (10.6 mg, 25%) as a white solid.

Procedure (b). Alternatively, compound 8-b was also synthesized according to a synthetic sequence similar to that outlined in Example 1(b), starting with intermediate 21b.

Example 4.

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Synthesis of compound 15-b

Procedure(s). In a stirred solution of intermediate 21b (150 mg, 0.526 mmol), 2,4,6-trifluorophenol (116.8 mg, 0.789 mmol) and triphenylphosphine (206.88 mg, 0.789 mmol) in THF (5.56 ml) at 0°C and DIAD (155.3 μL, 0.789 mmol) was added dropwise under nitrogen atmosphere. The mixture was stirred at 100°C for 10 minutes under microwave radiation. The mixture was diluted with DCM and washed with sat. sol. _NaHCO3 . The organic layer was separated, dried (Na ₂ SO ₄ ), filtered and concentrated in vacuo, then purified by flash column chromatography (silica gel; MeOH/NH ₃ 7 N in DCM 0/100 to 90/10). The desired fractions were collected and concentrated in vacuo. Purified using RP HPLC (stationary phase: C18 XBridge, 30 x 100 mm, 5 µm, mobile phase: gradient from 54% 0.1% NH4CO3H/NH4OH pH 9 in water, 46% CH3CN to 64% 0.1 % solution of NH4CO3H/NH4OH at pH 9 in water, 36% CH3CN) to obtain a colorless oil that crystallized upon settling (2 days). The solid was triturated with heptane to give 15-b (129.8 mg, 59%) as a white solid.

Procedure (b). Alternatively, compound 15-b was also synthesized according to a synthetic sequence similar to that outlined in Example 1(b), starting with intermediate 21b.

Example 5

Synthesis of compounds 1-b, 2-b and 3-b

Compounds 1-b, 2-b and 3-b were synthesized according to the procedure outlined in example 1(a). Thus, the reaction resulted in DIAD (500.05 μL, 2.54 mmol), intermediate 21a (483 mg, 1.69 mmol), 4-fluorophenol (227.77 mg, 2.03 mmol) and triphenylphosphine (666 .14 mg, 2.54 mmol) in THF (17.91 ml) described in example 1(a) gave a residue which was purified by flash column chromatography (silica gel; EtOAc in DCM 0/100 to 90/ 10). The desired fractions were collected and concentrated in vacuo. The resulting residue was triturated with DIPE to obtain compound 1-b (320 mg, 50%) as a white solid, which was purified using chiral SFC [stationary phase: Chiralpak AD (5 μm, 250*30 mm, mobile phase: 77% CO ₂ , 23% MeOH)] to give compound 2-b (131 mg, 20%) and compound 3-b (129 mg, 20%) as white solids.

Example 6.

Synthesis of compounds 24-b, 26-b and 27-b

Compounds 24-b, 26-b and 27-b were synthesized according to the procedure described in example 1(a). Thus, the reaction resulted in DIAD (364.57 μL, 1.85 mmol), intermediate 22 (320 mg, 1.23 mmol), 2,4-difluorophenol (176.86 μL, 1.85 mmol) and triphenylphosphine (485.67 mg, 1.85 mmol) in THF (13.06 ml) described in Example 1(a) gave a residue which was purified by flash column chromatography (silica gel; MeOH in DCM 0/100 to 96/4). The desired fractions were collected and concentrated in vacuo to give a colorless oil, which crystallized on DIPE to give compound 24 as a white solid, which was purified by RP HPLC (stationary phase: C18 XBridge, 30 x 100 mm, 5 µm; mobile phase: gradient from 54% 0.1% NH4CO3H/NH4OH pH 9 in water, 46% CH3CN to 64% 0.1% NH4CO3H/NH4OH pH 9 in water, 36% _CH3CN ) to give a colorless oil that crystallized trituration with heptane to give 240 mg (52%) of 24-b as a white solid, which was then purified by chiral SFC (stationary phase: CHIRALPAK AD-H, 5 µm, 250 x 20 mm; mobile phase: 85% CO ₂ , 15% iPOH (0.3% iPrNH ₂ )) to obtain compound 26-b (103 mg, 22%) and compound 27-b (107 mg, 23%).

The following compounds were prepared according to a synthesis sequence similar to that outlined in Example 1(a).

Starting Material: Intermediate 22

Chiral SFC conditions: stationary phase: Chiralpak AD-H, 5 µm, 250 x 20 mm; mobile phase: 85% CO ₂ , 15% EtOH/iPrOH mixture, 50/50 v/v. (+0.3% iPrNH ₂ )

Starting Material: Intermediate 23

Chiral SFC conditions: stationary phase: Chiralpak AD-H (5 µm, 250*30 mm); mobile phase: 80% CO ₂ , 20% MeOH/iPrOH mixture, 50/50 v/v. (+0.3% _iPrNH2 )

The following compounds were synthesized according to the synthesis sequence set out in Example 1(b), starting with the indicated intermediates.

- 38 045021

Table A below lists additional compounds of formula (I-B) that were prepared in analogy with the above examples (Exp. No.).

- 39 045021

Table A

Representative compounds of formula (I-B)

N-N Conn. No. No. EXP. R ¹ Ar Stereochemical configuration AND I 1-b E5 ^# b R.S. 2-b E5 ^# -Π *R s-y E5 ^# -Π φ *S 4-b El ^# . LL b LL R.S. 5-b El ^# ''' -LL b U- R 6-b El (a) and (b) ^# . w b LL S 6- b.HCl El(c) ^# 7-b E2 ^# ''' %Τ7 ABOUT F S 8-b E3 ^# ''' %Τ7 LT S 9-b El(b) ''''^7 ύ S

- 40 045021

N-N Conn. No. No. EXP. R ¹ Ar Stereochemical configuration 10-b El(b) ''' 0 T1' S 11-b El(b) ''' ά: S 12-b El(b) ''' -Π h S 13-b El(b) ''' ·. ABOUT S 14-b El(b) LL S 15-b E4 ^# ''' S 16-b El(a) '' . U- LL R.S. 17-b El(a) . LL LL *R 18-b El(a) '' . LL LL *S

- 41 045021

n~n Conn. No. No. EXP. R ¹ Ar Stereochemical configuration 19-b El(b) -n R.S. 20-b El(b) . LL LL *R 21-b El(b) . LL LL *S 22-b El(b) A *R 23-b El(b) . LL A *S 24-b E6 ^# . LL LL R.S. 25-b El(a) . LL A R.S. 26-b E6 ^# LL *R 27-b E6 ^# . LL LL *S

- 42 045021

N-N Conn. No. No. exp. R ¹ Ar Stereochemical configuration 28-b El (a) A *R 29-b E1(a) .•-X A' LL *S 30-b E1(b) ,ΖχΖ . LL LL R.S. 31-b E1 (b) /\z P · *R 32-b E1 (b) zV s/LL *S

# means that the experimental procedure is described in the examples.

Analytical part. Optical rotations.

Optical rotation angles were measured on a Perkin-Elmer 341 polarimeter with a sodium lamp and designated as follows: [α]° (λ, s g/100 ml, solvent, T°C).

[α] _λ ^T = (100α) / (1 x s): where 1 means the path length in dm, and c means the concentration in g/100 ml for the sample at temperature T (°C) and wavelength λ (in nm) . If the wavelength of light used is 589 nm (sodium D line), then the symbol D can be used instead. The direction of rotation sign (+ or -) should always be given. When using this equation, the concentration and solvent are always given in parentheses after the angle of rotation. The angle of rotation is indicated in degrees, and the units of concentration are not given (it is assumed that they are presented in g/100 ml).

LCMS.

The following methods were used to characterize the compounds of the present invention using (LC)MS.

General procedure

High performance liquid chromatography (HPLC) measurements were performed using an LC pump, diode array detector (DAD) or UV detector and column as defined in the respective methods. If necessary, additional detectors were included (see table of methods below).

The column flow was directed to a mass spectrometer (MS), which was equipped with an atmospheric pressure ionization source. It would be within the purview of one skilled in the art to set custom parameters (eg, scan range, minimum measurement time, etc.) to obtain ions capable of determining the nominal monoisotopic molecular weight (MW) of the compound. Data collection was carried out using appropriate software.

Compounds were described by their experimental retention time (Rt) and ions. Unless otherwise noted, in the data table the molecular ion indicated corresponds to [M+H]+ (protonated molecule) and/or [M-H]- (deprotonated molecule). If the compound was not directly capable of ionization, the type of adduct was indicated (ie [M+NH ₄ ]+, [M+HCOO]-, etc.). For molecules with complex isotopic distributions (Br, Cl, etc.), the described value is the value obtained for the smallest isotope mass. All results are subject to experimental errors that are typically associated with the method used.

As used herein, SQD means Single Quadrupole Detector, Rt means Room Temperature, VEN means Bridged Ethyl Siloxane/Silicon Dioxide, HSS means High Strength Silica Gel, DAD means Diode Array Detector.

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Table B

LCMS method codes (flow expressed in ml/min; column temperature (T) in °C; analysis time in minutes)

Poto Device Column Mobile phase Gradient To -- t colo Analysis time for LCMS Way nki Agilent: From 95% A Waters: Acquity®UPLC® - DAD and SQD Eclipse Plus C18 RRHD (1.8 MKM, 2.1 x 50 A: 95% 6.5 mM ch ₃ coonh ₄ + 5% CH ₃ CN, B: CH ₃ CN up to 5% A in 4.6 min. , holding for 1 50 5 1 mm) 0.4 min. From 95% A Waters: Acquity® UPLC®-DAD and SQD Waters: CSH™ Cl8 (1.7 µm, 2.1 x 50 mm) A: 95% 6.5 mM ch ₃ coonh ₄ + 5% CH ₃ CN, B: CH ₃ CN up to 5% A in 4.6 minutes. , holding for 0.4 min. 1 50 5 2 Waters: Waters: A: 95% 7 From 84.2% 0.34 Acquity VEN C18 mm And in 3 UPLC® - (1.7 ch ₃ coonh ₄ / flow — 6, 2 3 DAD and µm, 2.1 5% CH ₃ CN, 0.49 — Quattro x 100 B: CH ₃ CN min. before 40

- 44 045021

Micro™ mm) 10.5% A in 2.18 min. , holding for 1.94 min. , back to 84.2% And in 0.73 min. , holding for 0.73 min. Waters: Acquity® UPLC® - DAD and SQD Waters: CSH™Cl8 (1.7 µm, 2.1 x 50 mm) A: 95% 6.5 mM ch ₃ coonh ₄ + 5% CH ₃ CN, B: CH ₃ CN From 95% A to 5% A in 7.8 minutes. , holding for 1.2 minutes. 1 50 9 4

Melting temperatures.

Values represent peak values and are subject to experimental uncertainties typically associated with the analytical method.

Mettler FP 81HT/FP90 device.

For a number of compounds, melting points were determined in open capillary tubes using an FP 81HT/FP90 instrument (Mettler-Toledo). Melting points were measured at a temperature gradient of 1, 3, 5 or 10°C/minute. The maximum temperature was 300°C. The melting point was read from a digital display.

- 45 045021

Table C

Physicochemical data for a number of compounds, retention time (Rt) in min, peak corresponding to [M+H]+ (protonated molecule), LCMS method, etc. (melting point in °C) (n.a. = not determined)

Conn. No. T . pl. (°C) Rt (min) [MN ⁺ ] Way LCMS Optical rotation 1-b 156, 3 2.32 380 1 2-b 176, 9 2.93 380 3 -58.5° (589 nM, with 0.53 w/v%, DMF, 20°C) s-y 177.3 2.93 380 3 +59.4 ° (589 nM, with 0.52 w/v%, DMF, 20 °C) 4-b 121.7 2.41 398 1 5-b 142 2.99 398, 3 3 +95.7 ° (589 nM, with 0.69 w/v%, DMF, 20 °C) 6-b 142.4 2.99 398, 2 3 -95.4° (589 nM, s 0.7 w/v%, DMF, 20 °C) 7-b 170.08 2.37 398 2 -55.7 ° (589 nM, with 0.96 w/v%, DMF, 2 0 °C) 8-b n. O. 2.32 398 2 n. O. 9-b n. O. 2.32 398 2 n. O. 10-b n. O. 2.25 398 2 n. O. 11-b n. O. 2.28 398 2 n. O.

- 46 045021

12-b n. O. 2, 16 410 2 n O. 13-b 144 , 1 2, 68 410 2 n O. 14-b 161 , 7 2.51 394 2 n O. 15-b 80 3 2.37 416 2 -167.0° 0.55 wt. 20 (589 /rev. °C) nM, s 1, DMF, 16-b n. O. 2.50 412 2 n O. 17-b n. O. 3, 12 412 3 n O. 18-b n. O. 3, 12 412 3 n O. 19-b n. O. 2.39 402 2 n O. 20-b n. O. 2.3 402 2 n O. 21-b n. O. 3, 36 402 n O. 22-b n. O. 2.35 420 2 n O. 23-b n. O. 2.35 420 2 n O. 24-b 135 , 7 2, 05 372 2 n O. 25-b 138 , 3 2, 13 390 2 n O. 26-b n. O. 2, 80 372 3 -83.9 ° (589 nM, with 0.52 w/v%, DMF, 2 5 °C) 27-b n. O. 2, 80 372 3 +92.1 ° (589 nM, with 0.55 w/v%, DMF, 2 5 °C) 28-b n. O. 2.85 390 3 -129.2° (589 0.5 w/v 25 °C) nM, s 5, DMF, 29-b n. O. 2.85 390 3 +137.3° (589 0.51 wt. /oC DMF, 2 5° nM, s WITH) 30-b 130 , 6 2.29 386 2 n O. 31-b 127 85 2.29 386 2 -67.5° (589 0.83 wt. /oC DMF, 2 0° nM, s 5. %, WITH) 32-b 127 69 2.29 386 2 +89.5° (589 0.83 wt. /oC DMF, 2 0° nM, s WITH)

SFC-MS.

General procedure.

Measurements during SFC were carried out using an analytical system from Berger instrument, containing a hydraulic pump control module for two-component mixtures FCM-1200 for the delivery of carbon dioxide (CO ₂ ) and a modifier, an automatic sampler for liquids CTC Analytics, a temperature control module TCM-2 0000 for heating the column from room temperature to 80°C. An Agilent 1100 UV photodiode array detector was used, equipped with a high pressure flow cell that can withstand pressures up to 400 bar. Flow from column times

- 47 045021 was divided for the MS spectrometer. The MS detector was equipped with an atmospheric pressure ionization source. The ionization parameters for the Waters ZQ mass spectrophotometer are as follows: corona current: 9 μA, source temperature: 140°C, cone voltage: 30 V, probe temperature 450°C, extractor voltage 3 V, desolvation gas flow rate 400 l/h, gas flow rate through the cone 70 l/h. Nitrogen was used as a nebulizer gas. Data collection and processing were carried out using the MassLynx-Openlynx data acquisition and processing system from Waters-Micromass.

Method 1: In addition to the general procedure, analytical chiral separation during SFC-MS was performed on a CHIRALPAK AD DAICEL column (10 μm, 4.6 x 250 mm) at 35 °C with a flow rate of 3.0 ml/min. The mobile phase was 85% CO ₂ , 15% iPrOH (+ 0.3% iPrNH ₂ ) held for 7 min in isocratic mode.

Method 2: In addition to the general procedure, analytical chiral separation during SFC-MS was performed on a CHIRALPAK AD DAICEL column (10 μm, 4.6 x 250 mm) at 35 °C with a flow rate of 3.0 ml/min. The mobile phase was 75% CO ₂ , 15% iPrOH (+ 0.3% iPrNH ₂ ) held for 7 min in isocratic mode.

Method 3: In addition to the general procedure, analytical chiral separation during SFC-MS was performed on a CHIRALPAK AD DAICEL column (10 μm, 4.6 x 250 mm) at 35 °C with a flow rate of 3.0 ml/min. The mobile phase was 80% CO ₂ , 10% methanol + 10% iPrOH (+ 0.3% iPrNH ₂ ) held for 7 min in isocratic mode.

Table D

SFC Analytical Data

No. conn. _Rt [M+H] ⁺ % area by UV Way Elution order of isomers* 6-b 4.28 398 100 1 A 5-b 5, 98 398 100 1 IN 2-b 2.13 380 100 2 A 3-b 2.97 380 100 2 IN 17-b 2.46 412 100 3 A 18-b 3, 12 412 100 3 IN 31-b 2.93 386 100 1 A 32-b 3, 81 386 100 1 IN

Rt means retention time (in minutes),

[M+H]+ means the mass of the protonated compound, the method refers to the method used for the analysis of enantiomerically pure compounds using SFC/MS. The measurement results were compared with a mixture, * A means the first eluting isomer, * B means the second eluting isomer.

Nuclear magnetic resonance (NMR).

For a number of compounds, 1H NMR spectra were recorded on Bruker DPX-400 or Bruker AV-500 spectrometers with standard pulse sequences operated at 400 MHz and 500 MHz, respectively. Chemical shifts (5) are reported in parts per million (ppm) for the low-field shift from tetramethylsilane (TMS), which was used as an internal standard.

Conn. No. 6-b: 1H NMR (400 MHz, CDCl ₃ ) δ ppm 0.30-0.38 (m, 2H), 0.59-0.68 (m, 2H), 1.14-1.22 ( m, 1H), 1.72 (d, J=6.5 Hz, 3H), 3.02-3.14 (m, 2H), 5.84 (q, J=6.3 Hz, 1H), 6.67-6.73 (m, 1H), 6.80-6.89 (m, 2H), 7.30 (d, J=7.4 Hz, 1H), 8, 11 (d, J= 7.4 Hz, 1H).

Conn. No. 7-b: ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 0.30-0.39 (m, 2H), 0.59-0.68 (m, 2H), 1.11-1.23 (m, 1H), 1.70 (d, J=6.5 Hz, 3H), 3.01-3.14 (m, 2H), 5.83 (q, J=6.2 Hz, 1H) , 6.35-6.45 (m, 3H), 7.13 (d, J=7.2 Hz, 1H), 8.08 (d, J=7.4 Hz, 1H).

Conn. No. 8-b: 1H NMR (400 MHz, CDCl ₃ ) δ ppm 0.30-0.38 (m, 2H), 0.58-0.68 (m, 2H), 1.11-1.22 ( m, 1H), 1.69 (d, J=6.2 Hz, 3H), 3.01-3.13 (m, 2H), 5.79 (q, J=6.2 Hz, 1H), 6.53 (dtd, J=9.2, 3.1, 3.1, 1.7 Hz, 1H), 6.72 (ddd, J=11.6, 6.5, 3.1 Hz, 1H ), 6.95-7.04 (m, 1H), 7.15 (d, J=7.4 Hz, 1H), 8.07 (d, J=7.4 Hz, 1H).

Conn. No. 15-b: 1H NMR (500 MHz, CDCl ₃ ) δ ppm 0.30 - 0.41 (m, 2H), 0.59 - 0.71 (m, 2H), 1.16 - 1.25 (m, 1 N), 1.70 (d, J=6.4 Hz, 3H), 3.05 - 3.16 (m, 2 N), 5.80 (q, J=6.4 Hz, 1 H), 6.62 - 6.70 (m, 2 H), 7.45 (d, J=7.5 Hz, 1H), 8.16 (d, J=7.2 Hz, 1H).

Conn. No. 13-b: 1H NMR (500 MHz, CDCl ₃ ) δ ppm 0.27 - 0.39 (m, 2H), 0.58-0.67 (m, 2H), 1.12-1.21 (m, 1H), 1.73 (d, J=6.4 Hz, 3 H), 2.22 (s, 3H), 3.06 (qd, J=15.4, 6.6 Hz, 2 H), 5.92 (q, J=6.4 Hz, 1H), 6.71 (d, J=8.4 Hz, 1H), 6.89 (dd, J=8.4, 1.4 Hz, 1H), 7.18 (d, J=1.7 Hz, 1H), 7.32 (d, J=7.2 Hz, 1H), 8.07 (d, J=7.2 Hz, 1 H).

Conn. No. 14-b: 1H NMR (500 MHz, CDCl ₃ ) δ ppm 0.28-0.39 (m, 2H), 0.57-0.69 (m, 2H), 1.12-1.21 ( m,

- 48 045021

1H), 1.70 (d, J=6.6 Hz, 3H), 2.31 (s, 3H), 3.01-3.12 (m, 2H), 5.79 (q, J= 6.6 Hz, 1H), 6.55 (dd, J=9.0, 4.3 Hz,

1H), 6.69 (td, J=8.5, 3.0 Hz, 1H), 6.87 (dd, J=9.0, 2.9 Hz, 1H), 7.17 (d, J =7.5 Hz, 1H), 8.06 (d, J=7.2 Hz, 1H).

Conn. No. 20-b: ¹ H NMR (500 MHz, CDCl ₃ ) δ ppm 1.22 (t, J=7.1 Hz, 3H), 1.72 (d, J=6.4 Hz, 3H), 3 .58(q,

J=7.1 Hz, 2H), 5.03-5.10 (m, 2H), 5.84 (q, J=6.5 Hz, 1H), 6.67-6.74 (m, 1H ), 6.81-6.88 (m, 2H), 7.34 (d, J=7.2

Hz, 1H), 8.40 (d, J=7.5 Hz, 1H).

Conn. No. 22-b: 1H NMR (500 MHz, CDCl·,) δ ppm 1.23 (t, J=6.9 Hz, 3H), 1.70 (d, J=6.4 Hz, 3H), 3 .58 (q, J=7.0 Hz, 2H), 5.05-5.12 (m, 2H), 5.81 (q, J=6.6 Hz, 1H), 6.62-6, 70 (m, 2H), 7.48 (d, J=7.5 Hz, 1H), 8.45 (d, J=7.2 Hz, 1H).

Conn. No. 31-b: 1H NMR (400 MHz, CDCI3) δ ppm 1.07 (t, J=7.40 Hz, 3H) 1.72 (d, J=6.24 Hz, 3H) 1.92 (sxt , J=7.63 Hz, 2H) 2.98-3.14 (m, 2H) 5.84 (q, J=6.47 Hz, 1H) 6.65-6.74 (m, 1H) 6 .78-6.89 (m, 2H) 7.29 (d, J=7.40 Hz, 1H) 8.02 (d, J=7.40 Hz, 1H).

In vitro testing of compounds of formula (I-B).

The compounds of formula (IB) provided in the present invention are positive allosteric modulators of mGluR2. These compounds appear to enhance responses to glutamate by binding to an allosteric site other than the glutamate binding site. In the presence of compounds of formula (IB), the response of mGluR2 to glutamate concentration is enhanced. It is believed that the compounds of formula (IB) exert their effect primarily on mGluR2 due to their ability to enhance receptor function. The effects of positive allosteric modulators tested on mGluR2 using the [ ³⁵ S]GTPyS binding assay method described below, which is suitable for identifying such compounds, and more particularly, compounds of formula (IB), are shown in table. E.

[ ³⁵ S]GTPyS binding assay.

The [ ³⁵ S]GTPyS binding assay is a membrane-based functional assay used to study the function of G protein-coupled receptors (GPCRs) that measures the incorporation of a non-hydrolyzable form of GTP, [ ³⁵ S]GTPyS (guanosine-5'- triphosphate labeled with gamma-emitting ³⁵ S). The α subunit of the G protein catalyzes the exchange of guanosine 5'-diphosphate (GDP) for guanosine triphosphate (GTP), and when GPCR is activated by an agonist, [ ³⁵ S]GTPyS is incorporated and cannot be cleaved off, continuing the exchange cycle (Harper (1998) Current Protocols in Pharmacology 2.6.1-10, John Wiley & Sons, Inc.). The amount of radioactive [ ³⁵ S]GTPyS incorporated is a direct indicator of G protein activity, and therefore agonist activity can be determined. mGlu2 receptors have been shown to be preferentially coupled to the Gai protein, which is the preferred coupling for this method, and hence it is widely used to study receptor activation of mGlu2 receptors in both recombinant cell lines and tissues. Herein, the inventors describe the use of a [ ³⁵ S]GTPyS binding assay using human mGlu2 receptor-transfected cell membranes based on Schaffhauser et al. (Molecular Pharmacology, 2003, 4:798-810) to identify the positive allosteric modulation (PAM) properties of the compounds of the present invention.

Preparation of membranes.

CHO cells were cultured to preconfluence and stimulated with 5 mM butyrate for 24 h. Cells were then collected by scraping in PBS and the cell suspension was centrifuged (10 min at 4000 rpm in a benchtop centrifuge). The supernatant was discarded and the pellet was carefully resuspended in 50 mM Tris-HCl, pH 7.4, by vortexing and pipetting and dispensing. The suspension was centrifuged at 16,000 rpm (Sorvall RC5C Plus with SS-34 rotor) for 10 minutes and the supernatant was discarded. The precipitate was homogenized in 5 mM Tris-HCl, pH 7.4, using an Ultra Turrax homogenizer and centrifuged again (18,000 rpm, 20 min, 4°C). The final pellet was resuspended in 50 mM Tris-HCl, pH 7.4, and stored at -80°C in suitable aliquots until use. Protein concentration was determined by the Bradford method (Bio-Rad, USA) with bovine serum albumin as a standard.

[ ³⁵ S]GTPyS binding assay.

The positive allosteric modulation activity of mGluR2 of the test compounds was measured as follows. Test compounds and glutamate were diluted in assay buffer containing 10 mM HEPES acid, 10 mM HEPES salt, pH 7.4, 100 mM NaCl, 3 mM MgCl ₂ and 10 μM GDP. Membranes containing the human mGlu2 receptor were thawed on ice and diluted with assay buffer supplemented with 14 μg/ml saponin. The membranes were preincubated with the compound alone or together with glutamate at a predetermined (~EC ₂₀ ) concentration (PAM assay) for 30 min. at 30°C. After addition of [ ³⁵ S]GTPyS (final concentration 0.1 nM), assay mixtures were vortexed briefly and further incubated to ensure incorporation of [ ³⁵ S]GTPyS upon activation (30 minutes, 30°C). The final assay mixtures contained 7 μg of membrane protein in 10 mM HEPES acid, 10 mM HEPES salt, pH 7.4, 100 mM NaCl, 3 mM MgCl ₂ , 10 μM GDP, and 2 μg/ml saponin. The total volume of the reaction mixture was 200 μl. Reactions were stopped by rapid filtration through

- 49 045021 Unifilter-96 GF/B plates (Perkin Elmer, Massachusetts, USA) using a universal manifold

FilterMate for 96-well plates. The filters were washed 6 times with an ice-cold mixture of 10 mM NaH2PO4/10 mM Na ₂ HPO ₄ , pH 7.4. The filters were then air dried and 40 μl of liquid scintillation mixture (Microscint-O) was added to each well.

The radioactivity of the membrane-bound complexes was counted in a Perkin Elmer scintillation and luminescence microplate counter.

Data analysis.

Concentration-response curves for exemplary compounds of the present invention produced in the presence of the mGluR2 glutamatergic agonist EC ₂₀ to determine positive allosteric modulation (PAM) were performed using the Lexis software interface (developed by J&J). Data were calculated as % of the control response to glutamate, defined as the maximum response evoked when glutamate was added alone. Sigmoidal concentration-response curves, constructed by plotting these percentage values against the logarithm of the concentration of the test compound, were analyzed using nonlinear regression analysis. The concentration causing the half-maximal effect was then calculated as _EC50 .

The following pEC ₅₀ values were calculated as -log EC ₅₀ when EC ₅₀ was expressed in ME. E _max was defined as the relative maximum effect (ie, the maximum effect in % compared to the control response to glutamate).

Table E below shows the pharmacological data obtained for the compounds of formula (IB) and the current pharmacological data obtained for the compounds of formulas (I) and (I-A).

Table E

Pharmacological data for compounds

- 50 045021

- 51 045021

5-a n.t. b-a 6, 34 255 7-a 6, 64 291 8-a 6, 04 157 9-a 6, 59 222 10-a 6, 88 290 11-a 7.11 249 12-a 7.03 242 13-a 6, 67 212 14-a 6, 92 259 15-a 7 253 16-a 7.12 223 17-a 6, 54 261 18-a n.t. 19-a 6.71 240 20-a 6, 91 243 21-a 6, 25 207 22-a 6, 05 259 23-a 6, 58 203 24-a 6, 91 258 25-a 7.07 261 26-a 6, 5 248 27-a 6.48 284 28-a 6, 96 297 29-a 6, 97 317 30-a n.t. 31-a n.t. 32-a 6, 66 347 33-a 6, 58 362

p.s. means that pEC ₅₀ could not be calculated. Nt means not tested.

pEC ₅₀ values were not calculated in cases where the concentration-response curve did not reach a plateau.

All compounds were tested in the presence of the glutamatergic mGluR2 agonist at a predetermined concentration of EC ₂₀ to determine positive allosteric modulation. .pEC ₅₀ values were calculated from a concentration-response experiment using at least 8 concentrations.

B. Anticonvulsant studies with mGluR2 compounds (orthosteric agonist and compounds of formula (I)/(I-A)/(I-B).

General information.

Preparation of test compounds and solutions.

Test compounds were administered using the optimal ratio of volume of liquid form to volume of body fluids. Test compounds were administered to mice in a volume of 0.01 ml/g body weight (White H. S., et al., General Principles: Experimental selection, quantification, and evaluation of antiepileptic drugs, in Antiepileptic Drugs, Fourth Edition, R. H. Levy, R. H. Mattson, and B. S. Meldrum, Editors. 1995, Raven Press, Ltd.: New York, pp. 99-110). For subcutaneous (s.c.) administration, test compounds were administered into a fold of skin along the animal's back, with the exception of compound 6-b, which was administered orally (p.o.). For each of the tests performed on the test compounds (except for compound 6b), the final concentrations of the compounds were administered as an aqueous solution in 20% Hp-e-CD. For compound 6-b, a 40% Hp-e-CD stock solution was first prepared and used to formulate compound 6b at the required concentrations for testing via oral administration; final concentrations of the compound were administered as a suspension in 20% Hp-p-CD. For the groups receiving the inert substance, a 20% solution of Hp-in-CD was used.

For LY-404039, final concentrations of the compound were administered as sodium chloride solution s.c.

For compound CAS 1092453-15-0, final concentrations of compound were administered in 10% inert Hp-p-CD (+NaCl) after dissolution.

- 52 045021

Final concentrations of levetiracetam were administered in an aqueous solution of 0.5% methylcellulose (MC) administered by intraperitoneal (i.p.) injection.

Important reagents.

a. Solutions of inert substances 0.5% methylcellulose (MC) 40% stock solution of hydroxypropyl-p-cyclodextrin (Hp-p-CD).

b. Other solutions.

Tetracaine (0.5% w/v solution) was applied dropwise from a plastic dropper bottle to the eyes of all animals that were subsequently to receive electrical stimulation using corneal electrodes.

Animals and conditions for keeping animals.

Adult male albino CF No. 1 mice (26–35 g) were obtained from Charles River, Portage, Michigan. Animals were maintained on an appropriate diet (Prolab RMH 3000) and were given free access to food and water except for a short period of time when they were removed from their cage for testing. Newly received animals in the laboratory were given sufficient time to adjust for possible food and water restrictions to which they were subjected during transport before being used for testing. All mice were kept in plastic cages in specially designed chambers with controlled humidity, air exchange and controlled lighting (12 hours on -12 hours off). Animals were housed, fed, and cared for in accordance with the recommendations of the National Council publication Guide for the Care and Use of Laboratory Animals.

Minimal motor impairment (MMI).

Acute MMI was assessed through a combination of direct observations of the animal regarding overt symptoms related to the animal's neurological or muscular function. The rotarod procedure was used in mice to identify minimal muscle or neurological deficits. If a mouse was placed on a rod rotating at 6 rpm, the animal was able to maintain its balance for long periods of time. An animal was considered unhealthy if it fell from this rotating rod three times within a period of 1 min.

Determination of median effective and toxic doses (ED ₅₀ u TP ₅₀ ).

To determine the ED ₅₀ or TD ₅₀ for each test compound, the first dose administered was typically the same dose that was used to successfully determine TPE. If the initial dose used was effective or toxic in more than 50% of the animals, then the next dose was half the original dose; if the initial dose was effective or toxic in less than 50% of the animals, then the next dose was twice the original dose. The third and fourth doses were adjusted to obtain a dose-response line with equal intervals between points. There must be a minimum of 4 points that either include 0 and 100% or are in this range.

Definition of TPE.

Animals in groups of typically four each were administered test compounds, and each group was tested at one of five time points: 0.25, 0.5, 1, 2, or 4 hours after treatment (White et al. 1995) . TPE was determined using an assay with b Hz stimulation (32 mA). The time point (0.25, 0.5, 1, 2, or 4 hours after treatment) at which maximum protection was observed was considered the time of peak effect (TPE).

In the TPE defined for this study or previously defined, compounds were tested in an assay with stimulation at 6 Hz (32 and/or 44 mA) at several doses, including doses that caused little or no protection and up to full protection.

ED ₅₀ and 95% confidence interval (CI) were calculated using probit analysis in a computer program provided by the laboratory (Finney Probit Analysis 34d Ed 1971, London: Cambridge University Press).

Collection of blood serum for pK/pD analysis.

In various tests, animals were sacrificed after testing and blood drained from the torso and/or brain tissue (whole brains) were collected to quantify drug levels. Immediately after testing, animals were decapitated and blood drained from the torso was collected into a BD Vacutainer® tube containing K2EDTA and cooled on ice until centrifugation. After centrifugation (13,000-18,000 rpm, 5-7 min), the blood plasma was removed and transferred to a labeled microcentrifuge tube and stored at -80°C. For brain tissue collection, brains were removed immediately after decapitation and snap frozen. The frozen sample was placed in a labeled centrifuge tube and stored at -80°C.

Psychomotor seizure test at 6 Hz stimulation in mice.

The psychomotor seizure test at 6 Hz stimulation is used as a model of drug-resistant limbic seizures. Seizure at 6 Hz stimulation reflects resistance to phenytoin, carbamazepine, lamotrigine, and topiramate (Barton et al. Pharmacological characterization of

- 53 045021 the 6 Hz psychomotor seizure model of partial epilepsy Epilepsy Research 2001, Vol. 47, pp. 217-222).

Method of conducting a test with psychomotor seizures with stimulation at 6 Hz.

Focal seizures were induced in mice by corneal stimulation (6 Hz, 0.2 ms square wave, 3 s duration; Barton et al. 2001). Mice were tested at 32 mA or 44 mA. Before stimulation, 0.5% tetracaine drops were applied to each eye. Seizures induced by corneal stimulation in this analysis were characterized by a minimal clonic phase followed by stereotypical forms of automatic behavior including stupor, forelimb clonus, whisker twitching, and Straub's tail. Animals that did not exhibit these behaviors were considered protected.

Example 1 - studies using compounds 1 and 2.

1.1. Study of combinations with conn. No. 1, conn. No. 2 and levetiracetam.

Each compound was first tested individually at the dose that demonstrated minimal activity in the 6 Hz 44 mA stimulation test at the TPE of each compound. When mGluR2 PAM and levetiracetam were administered in combination (same dose and time point as for individual tests), almost complete protection was observed in the test with stimulation at 6 Hz 44 mA (Table 2). In addition to recording efficacy and toxicity data for such compounds alone or in combination, both plasma and brain samples were collected from each group for pharmacokinetic/pharmacokinetic analysis. No pharmacokinetic interactions were observed based on levels of compounds in plasma and brain samples (data not shown). It should be noted that compounds 1 and 2 showed a positive pharmacodynamic interaction with levetiracetam in the 6 Hz stimulation model, which did not appear to be caused by a pharmacokinetic interaction, and without increasing motor impairment (Tables 2, 2a, 2b). The effect of 1 dose of compound 2 was also tested in relation to the dose-dependent effect of LEV. As can be seen in table. 3, there was a ~200-fold deviation in ED ₅₀ LEV compared to the results obtained when testing LEV alone. LEV appears to marginally improve the efficiency of the connection. No. 2 (Table 3).

1.2. Isobolographic analysis of interactions between connections. No. 1 and levetiracetam in a model of epileptic seizure with stimulation at 6 Hz.

Isobolographic studies were carried out for the combined administration of the compound. No. 1 with levetiracetam (LEV) in an assay with 6 Hz stimulation (44 mA). The studies were carried out according to previously described methods (Madsen et al. 2011). The initial ED ₅₀ values were determined as for the connection. No. 1 and LEV and were used to calculate theoretical ED ₅₀ values (± standard error of the mean, SEM) for three fixed dose ratio combinations (LEV: Comp. No. 1): 1:3, 1:1 and 3:1. The doses used were proportional to the calculated _ED50 values. For example, the dose ratio used for the 1:1 regimen was based on 0.5 x ED ₅₀ for LEV and 0.5 x ED ₅₀ for cond. No. 1. Similarly, in the case of the 1:3 scheme, 0.25 x ED ₅₀ for LEV and 0.75 x ED ₅₀ for connection were used. No. 1. In the case of a dose ratio of 3:1, 0.75 x ED ₅₀ LEV and 0.25 x ED ₅₀ for connection were used. No. 1. Doses for experimental treatments (see Table 4) were based on theoretical values and were adjusted according to observed effects. The experimentally determined ED ₅₀ values (± SEM) for each fixed dose ratio combination were compared for statistical purposes with theoretical values (t-criterion). The dose ratio was defined as superadditive (synergistic) if the experimentally determined ED ₅₀ value was significantly lower than the theoretical ED ₅₀ . Then experimental combined doses were determined for the same schemes in the test of epileptic seizures with stimulation at 6 Hz (Table 4 below). Results from an isobolographic study with Compound 1 and levetiracetam in a 6 Hz stimulation model demonstrated a significant synergistic pharmacodynamic interaction at all dose ratios assessed and closely matched Compound 1 plasma levels. In addition, no motor deficits were observed at any of the dose ratios assessed, indicating that the synergistic pharmacodynamic interaction did not cause increased motor toxicity.

1.3. Mouse model of corneal kindling and studies with compound 1.

Mice were electrically induced to seize with 3 s, 3 mA, 60 Hz stimuli twice daily via corneal electrodes until the Racine stage 5 criterion of 5 consecutive seizures was achieved (Racine Modification of seizure activity by electrical stimulation II motor seizure Electroenceph Clin Neurophysiol 1972, 32, pp. 281294). Once the mice had reached a steady state of kindling, the test compound or inert substance was administered and, at a predetermined TPE, an electrical stimulus was administered to each animal as above. After stimulation, animals were examined for the presence or absence of seizure activity using the Racine scale (0–5), with 5 indicating the highest degree of falling and rising. One dose of LEV and two doses of comp. No. 1 was tested alone and in combination against epileptic seizures induced by repeated corneal stimulation. The combination of compound 1 with levetiracetam in this model indicated a positive pharmacodynamic interaction (Table 5 below).

The final data for the compounds tested individually are presented in Table. 1, and additional results of studies conducted in accordance with Example 1 are shown in Tables 2-5 below.

Table 1

Summary data of anticonvulsant effects during acute attack in the model with stimulation at 6 Hz at 32 mA and 44 mA for PAM compounds mGluR2 1, 2, 11, 2-a, 25-a, 6-b and LY404039 after s.c. administration (except for compound 6-b, which was tested p.o.)

Conn. No. TRE (h.) ED ₅₀ ( 95% CI mg/kg, S.C. Epileptic seizure score (dose) 32 mA 4 4 mA Provoking seizures via corneal kindling eleven 0.5 4.77 (3.54 - 6.76) 9.41 (1.53- 15.1) 31.5 (15.1 - 47.3) 2.8 (100 mg/kg) 2 0.25 3.83 (1.62 - 6.71) 5.89 (3.89- 8, 45) 3.4 (40 mg/kg) 1 0.5 2.8 (1.3- 4.3) 10.2 (3.1- 12.4) 3.7 (20 mg/kg) 25-a 1 7.7 (2.3- 1 hour TRE: n.t.

18.4) 25.9 (15.5- 33.7) 0.25 hour TPE: 29.1 (21.6-39.6) 2-a 0.5 44.7 (23.4- 80.5) 20.8 (10.0- 31.7) 12.2 (8.4- 17.4) 50% protection at 100 mg/kg 21 (17,927.4) 4.4 (100 mg/kg) 6-b 0.5 7.2 (4.2- 11.8) 16.1 (13.0- 20.1) n.t. LY-404039 0.5 10.2 (3.62- 12.4) n.t. 3.1 (100 mg/kg)

TPE means time of peak effect, CI means confidence interval, s.c. means subcutaneously, p.o. means orally, n.t. means not tested. TPE was determined in a test with stimulation of 6 Hz, 32 mA. Effects were generally observed at doses that did not cause reduction in the rotarod test. Individual values from replicate experiments are presented for compounds 11 and 2-a. For compound 25a, both 0.25 and 1 h time points were used for studies with 6 Hz (44 mA) stimulation.

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Table 1a

Final results of determining TPE with stimulation at 6 Hz 32 mA for connection. No. 1

Dose (mg/kg, s.s.) Time (hours) 6 Hz, 32 mA Motor impairment 10 4 0/4 0/0 2 0/4 0/0 1 3/4 0/0 0.5 4/4 0/0 0.25 4/4 0/0 5 0.5 8/12 0/0 0.25 8/12 0/0 2.5 0.5 5/8 0/0 0.25 1/8 0/0

(number of mice protected by 6 Hz stimulation or susceptible to toxicity as assessed by rotarod resistance/number of mice tested).

Table 1b

Studies of the dose-dependent effect of the connection. No. 1

Test Dose (mg/kg, s.s.) Number protected/number tested Number of those with motor impairment based on the rotarod test/number of those tested 6Hz 32 mA 20 8/8 1/8 10 7/8 0/8 5 8/12 0/12 2.5 7/16 0/16 0.5 1/8 0/8 ED ₅₀ (95% CI): 2.8 mg/kg (from 1.3 up to 4.3) 6Hz 44 mA 20 8/8 1/8 15 7/8 0/8 10 4/8 0/8 2.5 0/8 0/8 ed _5C ) (95% CI) : 1 0.2 mg/kg (from 3.1 UP TO 12.4)

It was previously determined that TPE for connection. No. 1 is 0.5 h (results are shown above in Table 1a). Some doses of conn. No. 1 was administered at a given TPE and tested in the assay with 6 Hz stimulation at both 32 and 44 mA stimulation intensities.

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table 2

Total interaction data conn. No. 1 and conn. No. 2 with levetiracetam (LEV) in a mouse model of epileptic seizure with stimulation at 6 Hz, 44 mA

Dose Time (h) Number protected/number tested Number with toxic effects on motor function/number tested LEV 10 mg/kg, i.p. 1 1/6 0/6 Conn. No. 2 + LEV 3 mg/kg, s . With. 0.25 5/6 0/6 Conn. No. 2 3 mg/kg, S . With. 0.25 1/6 0/6 LEV 10 mg/kg, i.p. 1 1/8 0/8 Conn. No. 1 + LEV 2.5 mg/kg, S. With. 0.5 6/8 0/8 Conn. No. 1 2.5 mg/kg, S. With. 0.5 0/8 0/8

Results are reported as the number of mice experiencing complete protection/total number of mice tested in each dosage group (for given dosage levels of the test compound or combination).

Table 2a

Connection levels No. 1 in plasma and brain in a combination study with levetiracetam (LEV) BQL means below the quantifiable limit.

Conn. No. 1 LEV Plasma blood (ng/ml) Conn. No. 1 Plasma blood (ng/ml) Protection at 6 Hz stimulation 6 Hz 4 4 mA 10 mg/kg 9350 2.5 mg/kg BQL Yes 8580 244 No 10900 314 Yes 10300 382 Yes 9780 416 Yes 9780 377 Yes 13700 2260* No 10100 607 Yes Average plasma level 10311 657.1 (390) 6/8 Average plasma levels (not combination) 1/8 8254 0/8 438

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The mean plasma level shown in parentheses ( ) was calculated with the statistical criterion removed to identify outliers*.

Table 2b

Connection levels No. 2 in plasma and brain in a combination study with levetiracetam (LEV) AQL means above the quantifiable limit

Conn. No. 2 LEV Blood plasma (ng/m l) Brain (ng/ml) Connect . No. 2 Blood plasma (ng/m l) Brain (ng/ml) Protection against stimulation AI at 6 Hz 6 Hz 44 mA 10 mg/k g 6450 6290 3 mg/k g 1830 1540 Yes 8200 7990 386 1020 Yes 3540 4760 4700 1310 Yes 3850 N.A. 467 N.A. No 7150 6380 AQL (>500 ) 1120 Yes 3890 3960 2080 1140 Yes Average plasma/brain levels 5513 5876 1893 1226 5/6 Average plasma/brain levels (not combination) 1/6 8750 5773 1/6 1295 1113

NA - sample not available for analysis.

Table 3

Treatment ED ₅₀ (95% CI) mg/kg Maximum effect (% defense) Conn. No. 2 separately 6, 97 100% (5, 44 - 8.30) Conn. No. 2 1.35 100% + LEV (10 mg/kg) (0.8 - 1.9) LEV separately - 200 75% LEV 1.0 100% + conn. No. 2 (3 mg/kg) (0.23 - 2.24)

Determination of ED50 in an epileptic seizure model with stimulation at 6 Hz (44 mA) for connection. No. 2 and levetiracetam (LEV) separately and in combination LEV at a dose of 10 mg/kg increased the activity of the compound. No. 2 (~5-fold deviation of ED50). Conn. No. 2 at 3 mg/kg increased both efficacy (up to 100% protection) and LEV activity (~200-fold deviation in ED50). In fig. 1 shows the dose-dependent effect for the results of ED50 determinations with stimulation at 6 Hz, 44 mA, for connection. No. 2 and LEV separately and in combination.

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Table 4

Connection results No. 1 and levetiracetam in an isobolographic study

Group LEV (mg/k g, i.p.) f Conn. No. 1 (mg/k G, s. With . ) f Combined dose (mg/kg) Rotating rod Number with toxic effect 6 Hz (44 mA) Number protected/number LO tested

in relation to motor functions/number of tested s 1:1 scheme 181 0, 5 5, 1 0, 5 93, 1 0/8 8/8 90.5 2, 6 46, 6 0/8 6/8 45, 3 1.3 23.3 0/8 3/8 22, 6 0.6 11, 6 0/8 3/8 ED ₅₀ (95% CI; mg/kg): 22.2 (8.4-35.7) scheme 13 45, 3 .2 5 3, 8 . 7 5 14.2 0/8 8/8 22, 6 1.9 7.1 0/8 4/8 11.3 1.0 3, 6 0/8 2/8 ED ₅₀ (95% CI; mg/kg): 5.9 (3.5-8.7) scheme 3:1 271.5 .7 5 2, 6 .2 5 204.3 0/8 8/8 135, 8 1.3 102.2 0/8 3/8 67.9 0.6 51.1 0/8 3/8 33, 9 0.3 25.5 0/8 0/8 ED ₅₀ (95% CI; mg/kg): 86.3 (56.8-131.4)

The results of isobolographic analysis (Fig. 2) indicate that the combination of conn. No. 1 and levetiracetam results in a significant positive synergistic effect.

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Table 5

Connection results #1 levetiracetam combination study in a mouse model of corneal kindling

Connection(s) Number protected/number LO tested protected s Average rating of epileptic seizure Inert substance (20% HPBCD after 30 min., s.s.; 0.5% MS after 60 min., i.p.) 0/10 0% 4.7 LEV, 3 mg/kg 5/13 38% 3.3 Conn. No. 1, 30 mg/kg 3/12 25% 4.0 LEV, 3 mg/kg, and comp. No. 1, 30 mg/kg 10/10 100% 0.6 Conn. No. 1, 20 mg/kg 5/16 31% 3.7 LEV, 3 mg/kg, and comp. No. 1, 20 mg/kg 7/10 70% 1.9 From 0 to 5 Score 0 = no epileptic convulsive activity about a seizure 5 = maximum Racine scale seizure activity

Example 2 - studies with compounds 25-a and 2-a.

2.1. Study of combinations of conn. No. 25-a and levetiracetam.

Independent dose-response studies were carried out in a test with stimulation at 6 Hz, 44 mA, for both compounds in order to determine the ED ₅₀ values in TPE 1 hour, ip, for levetiracetam, and for 1 hour, sc, for comp. No. 25-a.

ED value ₅₀ for conn. No. 25-a was 25.9 mg/kg, and for levetiracetam the value was estimated to be approximately 345 mg/kg. The dose-dependent effect for levetiracetam was repeated with combined administration of 10 mg/kg comp. No. 25-a (dose of compound No. 25-a, which alone did not protect in the model with stimulation at 6 Hz, 44 mA). Combined administration of 10 mg/kg comp. No. 25-a produced an ED ₅₀ for levetiracetam dose-dependent effect of 4.9 mg/kg (~70-fold reduction compared to levetiracetam alone) and, importantly, provided complete protection in a seizure model when stimulated at 6 Hz, 44 mA . These results suggest a positive pharmacodynamic interaction between the compounds. No. 25-a and levetiracetam in a model of epileptic seizure with stimulation at 6 Hz.

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Table 6

Determination of the time to reach the peak effect for the connection. No. 25-a in analysis with stimulation at 6 Hz (32 mA)

Dose (mg/kg, S. With . ) Time (h) Number protected/number tested Number with motor impairment based on rotarod test/number tested Medium levels conn. No. 25-a (ng/ml) in blood plasma 10 0.25 2/4 0/4 10,983 (2,477) 0.5 1/4 0/4 3,330 1 1/4 1/4 700 2 0/4 0/4 256 4 0/4 0/4 40 20 0.25 4/4 0/4 4,095 0.5 3/4 1/4 2,800 1 4/4 1/4 1.765 2 1/4 0/4 618 4 1/4 1/4 28

s.c. means subcutaneous.

This study used two doses, 10 and 20 mg/kg, at multiple time points (0.25-4 hours). The compound showed the greatest degree of protection in the assay with stimulation at 6 Hz, from 0.25 to 1 hour, which was most pronounced at 20 mg/kg. Plasma levels of the compound were generally consistent with protection against behavioral seizures. TPE 0.25 h was used for trials with stimulation at 6 Hz (32 mA), while time points at both 0.25 and 1 h were used for trials with stimulation at 6 Hz (44 mA).

The mean plasma level shown in parentheses ( ) was calculated with the statistical test removed to identify outliers.

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Table 7

Studies of dose-dependent effects for com. No. 25-a in the analysis with stimulation at 6 Hz (32 mA ^a and 44 mA ^b ) CI means confidence interval

Test Dose (mg/kg, S.s.) Number protected / number tested Number of those with motor impairment based on the rotarod test/number of those tested Medium levels conn. No. 25-a (ng/ml) in blood plasma 6Hz 32 mA 20 8/8 0/8 5,570 15 3/8 0/8 1.201 10 4/8 0/8 6, 113 5 4/8 0/8 2,558 1 1/8 0/8 466 ED ₅₀ । (95% CI): 7.7 mg/kg (2.3 to 18.4) 6Hz 4 4 mA 40 7/8 0/8 6,263 thirty 3/8 0/8 7,220 20 2/8 0/8 3,368 10 0/8 0/8 4.345 (1,526) 5 0/8 1/8 1.428 ED ₅₀ (95% ST): 29.1 mg/kg (21.6 to 39.6)

aTime to reach the peak effect in the analysis with stimulation at 6 Hz, 32 mA, for conn. No. 25-a was determined to correspond to 0.25 hours (see Table 1).

^ъ Time to reach the peak effect in the analysis with stimulation at 6 Hz, 44 mA, for connection. No. 25-a was the same for 0.25 hours and 1 hour; results for 1 hour confirmed ED ₅₀ (95% CI) 25.9 (15.5-33.7) (see tables 1 and 6).

The mean plasma level shown in parentheses ( ) was calculated with the statistical test removed to identify outliers.

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Table 8

Combination studies for conn. No. 25-a with levetiracetam (LEV) in the analysis with stimulation at 6 Hz (44 mA)

Medicinal means Dose (mg/kg, S. With . ) Number protected/number tested Number of those with motor impairment based on the rotarod test/number of those tested LEV 200 2/8 0/8 400 4/9 0/9 800 10/12 0/12 ED ₅₀ (95% CI): 345, 4 mg/kg (from 211.0 up to 485.3) LEV + conn. No. 25-a, 10 mg/kg 200 8/8 1/8 100 7/8 2/8 50 5/8 1/8 10 4/8 0/8 1 4/8 1/8 ED ₅₀ (95% CI) : 4.9 (0.0 - 14.2)

Conn. No. 25-a (s.c), 10 mg/kg, tested in combination with LEV (i.p.) - conn. No. 25-a, 10 mg/kg, not active when administered separately.

2.2. Study of combinations of conn. No. 2-a and levetiracetam.

Studies of the dose-dependent effect were carried out in tests with stimulation at 6 Hz, 32 mA and 4-4 mA, (Table 9 below) and in a test of combinations with levetiracetam (the effect of compound No. 2-a on the dose-dependent effect of LEV in Tables 10a and the effect of LEV on dose-response effect of Compound No. 2-a in Table 10b below) in the same manner as described for studies with Compound No. 2-a. No. 25-a and levetiracetam above.

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Table 9 Studies of dose-dependent effects for comp. No. 2-a in the analysis with stimulation at 6 Hz (32 mA and 44 mA; TPE 0.5 h). Time to peak effect of 0.5 h was determined in the test with stimulation at 6 Hz, 32 mA (s.c.), and was used for studies with stimulation at 6 Hz (32 mA and 44 mA).

Test Dose (mg/kg, s.s.) Number protected/number tested Number with motor impairment based on rotarod test/number tested 6 Hz 32 mA 40 8/8 2/8 20 6/8 3/8 10 4/8 0/8 5 0/8 0/8 2.5 0/8 1/8 ED ₅₀ (95% ST): 12.2 mg/kg (8.4 to 17.4) 6 Hz 44 mA 40 8/8 4/8 20 3/8 3/8 0/8 0/8 15 2/8 1/8 10 0/8 1/8

Test Dose (mg/kg, s.s.) Number protected/number tested Number with motor impairment based on rotarod test/number tested 0/8 0/8 ED ₅₀ (95% CI): 21.0 mg/kg (17.9 to 27.4) TD ₅₀ : > 40 mg/kg ^a

mg/kg - 6 out of a total of 16 (combined 32 mA and 4 4 mA) with insufficiency.

Dose chosen for the study of combinations with LEV at 6 Hz (44 mA): conn. No. 2-a 10 mg/kg.

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Table 10a Combination studies for conn. No. 2-a with levetiracetam (LEV) in the analysis with stimulation at 6 Hz (44 mA). Combination 10 mg/kg conc. No. 2-a with different doses of levetiracetam

Medicinal means Dose (mg/kg) Number protected/number tested Number with motor impairment based on rotarod test/number tested LEV 200 2/8 0/8 400 4/9 0/9 800 10/12 0/12 LEV ED ₅₀ (95% CI): 345.4 mg/kg (211.0 to 485.3) LEV + Conn. No. 2-a 10 mg/kg ^a 200 6/8 1/8 100 6/8 0/8 50 6/8 0/8 25 8/8 0/8 12.5 5/8 0/8 6, 25 4/8 0/8 3, 125 3/8 1/8 1.5625 0/8 0/8 LEV ED ₅₀ (95% CI): 9.6 mg/kg (1.7 - 21.9)

^a Conn. No. 2-a (sc) 10 mg/kg, tested in combination with LEV (ip); conn. No. 2-a, 10 mg/kg, not active when administered separately.

Additional controls for LEV (low dose) were tested at 25 and 6.25 mg/kg (1/8 and 0/6 protected, respectively).

Mice treated with inert (0.5% methylcellulose, i.p., (1 h)/20% HPBCD s.c. (0.5 h)) did not show protection (0/8 protected).

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Table 10b

Combination studies for conn. No. 2-a with levetiracetam (LEV) in the analysis with stimulation at 6 Hz (44 mA). Combination of 350 mg/kg levetiracetam with different doses of comp. No. 2-a

Medicinal means Dose (mg/kg) Number protected/number tested Number of those with motor impairment based on the rotarod test/number of those tested LEV (separately) ^a 350 3/8 0/8 LEV 350 mg/kg + comp. No. 2-а ^b 20 8/8 2/8 10 7/8 1/8 5 7/8 1/8 2.5 5/8 0/8 1.25 4/8 0/8

Preliminary value ED ₅₀ for conn. No. 2-a (95% CI): 21.0 mg/kg (from 17.9 to 27.4)

ED ₅₀ for combination of LEV and conn. No. 2-a (95% CI): 1.5 mg/kg (0.1-2.7)

-14-fold activity deviation ^a ED ₅₀ for LEV (presented separately), previously determined at 6 Hz stimulation (44 mA): 345 mg/kg.

^b Conn. No. 2-a (sc) 10 mg/kg, tested in combination with LEV (ip); conn. No. 2-a, 10 mg/kg, not active when administered separately.

Additional controls for LEV (low dose) were tested at 25 and 6.25 mg/kg (1/8 and 0/6 protected, respectively).

Mice treated with the inert substance (0.5% methylcellulose, i.p., (1 h) / 2 0% HPBCD s.c. (0.5 h) did not show protection (0/8 protected).

At a dose of 10 mg/kg, sc, conn. No. 2-a increased the efficiency of LEV, which led to an approximately 35-fold shift in ED ₅₀ . This indicated a positive pharmacodynamic relationship (Table 10a). At a dose of 350 mg/kg, sc, LEV increased the effectiveness of the compound. No. 2-a, which led to an approximately 14-fold shift in _ED50 . This indicated a positive pharmacodynamic relationship (Table 10b).

Example 3 - studies with compound 6-b.

3.1. Study of combinations with conn. No. 6-b and levetiracetam.

Independent dose-response studies were performed in a 6 Hz, 44 mA stimulation test for both compounds to determine ED ₅₀ values in TPE 1 hour, ip, for levetiracetam, and for 0.5 hours p.o. for connection No. 6-b. ED value ₅₀ for conn. No. 6-b was 16.1 mg/kg, and for levetiracetam the value was estimated to be approximately 345 mg/kg. The dose-dependent effect for levetiracetam was repeated with combined administration of 10 mg/kg comp. No. 6-b (dose of compound No. 6-b, which alone did not protect in the model with stimulation at 6 Hz, 44 mA). Combined administration of 10 mg/kg comp. No. 6-b produced an ED ₅₀ for the dose-dependent effect of levetiracetam of 2.4 mg/kg (~100-fold lower compared to levetiracetam alone) and, importantly, yielded complete protection in the epileptic seizure model when stimulated at 6 Hz, 44 mA. These results suggest a positive pharmacodynamic interaction in the epileptic seizure model with stimulation at 6 Hz between connections. No. 6-b and levetiracetam.

The results of studies carried out on compound 6-b are shown in Tables 11-13 below.

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Table 11

Determination of the time to reach the peak effect for the connection. No. 6-b (p.o.) in the analysis with stimulation at 6 Hz (32 mA)

Dose (mg/kg, p.o.) Time (h) Number protected/number tested Number with motor impairment based on rotarod test/number tested 10 0.25 1/4 0/4 0.5 3/4 0/4 1 0/4 0/4 2 1/4 0/4 4 0/4 0/4 20 0.25 4/4 0/4 0.5 3/4 0/4 1 4/4 0/4 2 0/4 0/4 4 1/4 0/4

The TPE was determined to be 0.5 hours.

Table 12

Study of dose-dependent effects for com. No. 6-b in the analysis with stimulation at 6 Hz (32 mA and 4 4 mA; TPE 0.5 h)

Test Dose (mg/kg, p.o. ) Number protected/number tested Number of those with motor impairment based on the rotarod test/number of those tested 6Hz 32 mA 20 7/8 0/8 10 6/8 0/8 5 2/8 0/8 2.5 1/8 0/8 ED ₅₀ (95% CI): 7.2 mg/kg (4.2 to 11.8) 6Hz 44 mA 40 8/8 0/8 20 6/8 0/8 15 4/8 0/8

10 0/8 0/8 ed ₅₀ (95% CI): 16.1 mg/kg (from 13.0 ds e 20.1)

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Table 13

Combination studies for conn. No. 6-b with LEV in the analysis with stimulation at 6 Hz (44 mA)

Medicinal means Dose (mg/kg) Number Protecting others/number tested Number of having motor impairment based on results test with rotating rod/number tested LEV 200 2/8 0/8 400 4/9 0/9 800 10/12 0/12 ED ₅₀ (95% CI): 345.4 mg/kg (from 211.0 to 485, 3) LEV + conn. No. 6-b, 10 mg/kg 200 8/8 0/8 100 8/8 0/8 50 5/8 0/8 10 5/8 0/8 1 5/8 0/8 ED ₅₀ (95% CI): 2.4 (0.0- 6.4)

Conn. No. 6-b (p.o.), 10 mg/kg, tested in combination with LEV (i.p.).

Conn. No. 6-b, 10 mg/kg, not active when administered alone.

Example 4 - studies with compound LY404039.

3.1. Combination Study with LY404039 and Levetiracetam LY-404039 was tested alone and in combination with levetiracetam according to the procedures already described herein above. The results of studies conducted with compound LY-404039 are shown in Tables 14-15.

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Table 14

Dose-response studies for LY404039 in the 6 Hz stimulation assay (32 mAa and 44 mAb)

Test Dose (mg/kg, s.s.) Number protected/number tested Number of those with motor impairment based on the rotarod test/number of those tested 6Hz 32 mA 40 8/8 1/8 20 6/8 1/8 10 5/8 0/8 5 1/16 1/16 ED ₅₀ (95% CI): 10.9 mg/kg (7.8 to 15.9) 6Hz 4 4 mA 40 7/8 2/8 20 7/8 1/8 10 3/8 1/8 5 0/16 0/16 ED ₅₀ (95% CI): 14.1 mg/kg (10.0 to 20.6) TD ₅₀ : > 40 mg/kg ^a

Time to peak effect of 0.5 h was determined in the test with stimulation at 6 Hz, 32 mA (s.c.), and was used for studies with stimulation at 6 Hz (32 mA and 44 mA).

mg/kg - 3 out of a total of 16 (combined 32 mA and 44 mA) with insufficiency.

Note: No activity was observed after inert injection at 32 or 44 mA.

Dose selected for combination studies with LEV at 6 Hz (44 mA): LY404039 5 mg/kg.

Table 15 Combination studies for LY404039 with levetiracetam (LEV) in the 6 Hz (44 mA) stimulation assay

Medicinal means Dose (mg/kg) Number Protection of others/number of tested Number of those with motor impairment based on the rotarod test/number of those tested LEV ^a 200 2/8 0/8 400 4/9 0/9 800 10/12 0/12 LEV ED ₅₀ (95% CI): 345.4 mg/kg (211.0 to 485.3) LEV + LY404039, 5 mg/kg ^b 200 8/8 0/8 50 6/8 1/8 20 6/8 2/8 5 2/8 1/8 LEV ED ₅₀ (95% CI): 12.8 mg/kg (2.5 - 25.2)

- 69 045021 ^a LEV was shown separately earlier, confirmatory doses were carried out in combination with conn. No. 2-a (see previous table above).

^b LY404039 (sc), 5 mg/kg, tested in combination with LEV (ip); LY404039, 5 mg/kg, was not active when administered alone.

Additional controls for LEV (low dose) were tested at 25 and 6.25 mg/kg (1/8 and 0/6 protected, respectively).

Mice treated with the inert substance (10% sterile water - NaCl; s.c., 0.5 h TPE and 0.5% MS, i.p., 1 h TPE) showed no protection or impairment when assessing rotarod grip.

At a dose of 5 mg/kg, LY404039 increased the efficacy of LEV, resulting in an approximately 27-fold shift in _ED50 . This indicates a positive pharmacodynamic relationship.

Example 4 - studies with compound CAS 1092453-15-0.

4.1. Study of combinations with 2,3-dihydro-7-methyl-5-[3-(1-piperazinylmethyl)-1,2,4-oxadiazol5-yl]-2-[[4-(trifluoromethoxy)phenyl]methyl]-1H -isoindol-1-one [CAS 1092453-15-0] (described in WO 2008150233, WO 2011084098) and levetiracetam

CAS 1092453-15-0 was tested alone and in combination with levetiracetam according to the procedures already described herein above. The results of Example 5 are shown in tables 1617.

Table 16 Dose response studies for CAS 1092453-15-0 in the 6 Hz (32 mA) stimulation assay

Dose Time Number Number of having (mg/kg, s . s . ) (h) protected/number tested motor impairment based on rotarod test/number tested 20 0.25 1/4 0/4 0.5 0/4 0/4 1 1/4 0/4 2 0/4 0/4 4 0/4 0/4 40 0.25 1/4 0/4 0.5 1/4 0/4 1 1/4 0/4 2 0/4 0/4 4 0/4 0/4 80 0.25 0/4 0/4 0.5 0/4 0/4 1 1/4 1/4

Low activity was observed at the doses and time points tested. The greatest activity is after 0.25-1 hour at the tested doses. Combination studies were carried out using

- 70 045021 mg/kg, s.c., 1 hour TPE in the assay with stimulation at 6 Hz (44 mA).

Table 17

Combination studies for CAS 10 92453-15-0 with levetiracetam (LEV) in an assay with 6 Hz stimulation (44 mA)

Medicinal Dose Number Number of having means LEV (mg/kg) protected/number tested motor impairment based on rotarod test/number tested LEV ^a 200 2/8 0/8 400 4/9 0/9 800 10/12 0/12 LEV ED ₅₀ ( 95% CI): 345.4 mg/kg (211.0 to 485.3) [CAS 1092453- 15-0] (20 mg/kg, alone) 0/8 0/8 LEV + [CAS 1092453-15-0] 20 mg/kg ^b 400 4/8 0/8 200 5/8 0/8 50 3/8 0/8 20 2/8 0/8 5 1/8 1/8 LEV ED ₅₀ (95% CI): 238.9 mg/kg (41.6 - higher than highest dose tested)

Additional controls for LEV (low dose) were tested at 25 and 6.25 mg/kg (1/8 and 0/6 protected, respectively).

^b [CAS 1092453-15-0], 20 mg/kg (sc; 1 h TPE), tested in combination with LEV (ip; 1 h TPE); [CAS 1092453-15-0], 20 mg/kg, showed low activity when administered alone (6 Hz, 32 mA) and was not tested in the 6 Hz (44 mA) stimulation assay. This compound exhibited an in vitro EC ₅₀ = 562 nM (E _max = 197%) when tested in the GTPyS assay described above herein, and no occupancy was observed in ex vivo experiments in rats.

Note: Animals treated with inert (10% HPeCD-NaCl, s.c., 1 hour and 0.5% MS, i.p., 1 hour) did not show protection or motor impairment, N=8.

The current data set indicates that mGlu2 PAM or mGlu2 agonist molecules had anticonvulsant activity in an animal model when stimulated at 6 Hz. Tested mGlu2 PAMs with EC ₅₀ activities <150 nM (as determined in the [ ³⁵ S]GTPyS assay), corresponding PK parameters and ability to penetrate into the brain demonstrated activity in both the 32 mA stimulation regimen and the 44 mA stimulation regimen. mA, 6 Hz. In addition, all molecules tested showed synergistic effects with LEV. In contrast, the molecule CAS 1092453-15-0, which was only marginally active (EC _50,562 nM) in vitro, did not demonstrate activity in any of the 6 Hz stimulation tests, nor did it show synergy with LEV.

It is important to note that the data indicate that, under conditions of comparable PK characteristics and corresponding ability to penetrate into the brain, the most active mGlu2 PAMs, based on in vitro EC ₅₀ values, also showed themselves to be the most active in vivo, indicating that in vitro and in vivo activities may be related. In addition, synergistic effects with LEV were consistently observed at doses of mGlu2 PAM similar to the ED ₅₀ obtained in the 32 mA stimulation model, or at a dose at least 2 times lower than the ED ₅₀ determined in the 44 mA stimulation model ( i.e. the dose was inactive in the 44 mA stimulation test when testing single molecules).

Also regarding LY404039, an mGlu2/3 agonist, activity was observed in both tests with stimulation at 6 Hz, and synergy was observed at a dose 3 times less than the ED ₅₀ determined in the model with stimulation at 44 mA, which was inactive at testing separately.

Based on publicly available preclinical data in a 6 Hz mA stimulation model, it appears that combining active SV2A ligand and active mGlu2 PAM results in a 35- to 100-fold reduction in the median effective dose or ED ₅₀ of an SV2A ligand such as LEV.

Thus, without wishing to be bound by any theory, compounds that are positive allosteric modulators of the metabotropic glutamatergic receptor subtype 2 (PAM mGluR2), in particular PAM mGluR2 compounds, are believed to have an EC ₅₀ activity of <150 nM (as determined in [ ³⁵ S]GTPyS assay), where EC ₅₀ is the concentration giving half-maximal effect in the concentration-response curve obtained in the presence of EC ₂₀ glutamate, and with appropriate PK parameters and brain penetration, results in a synergistic combination with the ligand SV2A, in particular levetiracetam, at ineffective doses of one or both of compound (a) and compound (b) of the combination of the present invention.

Thus, in a further embodiment, the compound that is a positive allosteric modulator of metabotropic glutamatergic receptor subtype 2 (PAM mGluR2), a combination of the present invention as defined herein, is selected from a PAM mGluR2 compound having an EC ₅₀ activity of <150 nM ( as determined in the [ ³⁵ S]GTPyS) assay, where _EC50 is the concentration giving half-maximal effect in the concentration-response curve obtained in the presence of EC20 glutamate.

Examples of possible applications.

A. Dominant-submissive relationships (DSR) in rats in an in vivo assay.

The DSR analysis is divided into two models: reduction of mania in the dominant behavior model (RDBM) and reduction of depression in the submissive behavior model (RSBM). An RDBM model in which dominant animals are treated with a test compound predicts the ability of the test compound to treat mania. The RSBM model, in which subordinate animals were treated with a test compound, predicts the ability of the test compound to treat depression.

Male Sprague-Dawley rats (140 to 160 g) from Charles River Laboratories, Wilmington, MA, were used in this assay. Batches of rats were obtained at two-week intervals. Each batch was subjected to a five-day quarantine, a one-week acclimatization period and a one-week selection process, followed by a five-week treatment of the selected pairs with a drug or inert substance.

The rats were kept in cages of four individuals each. Access to food was limited to one hour per day after testing from Monday to Thursday. After testing on Friday, rats were allowed free access to food until they were fasted again on Sunday. Rats were not deprived of water during all time periods. The periods of food deprivation involved had little effect on weight gain, as the average weight of rats at the end of the study would be approximately 300 g. At the end of the experiment, rats were sacrificed by decapitation, blood drained from the torso, and brains were collected for in vitro experiments and drug concentration measurements. facilities.

The main testing device consisted of two chambers connected only by a tunnel large enough to allow one rat to pass through at a time. At the bottom, in the middle part of the tunnel, a container with sweetened milk was placed. This main device was duplicated so that a total of four pairs of rats could be recorded simultaneously on a video camera. The camera could recognize rats marked with different colors. Thus, the heads of rats were painted for the purpose of video recording, red in one cage and yellow in the other. Only one animal at a time can have free access to the feeder, but both animals can drink milk for a five-minute period each day. During daily five-minute periods, the time spent in the feeder area by each rat was recorded by video capture software and saved to a text file.

The test began by randomly dividing the rats into pairs. Each rat from the pair was placed in the opposite chamber of the test device. The time spent by each animal in the feeding area was recorded. During the first week (five days) of testing, the animals were accustomed to the new habitat. Dominance was assigned to the animal with the highest score during the second week of testing when three criteria were met. First, there must be a significant difference (two-tailed t test, P < 0.05) between the mean daily drinking rates of both animals. Secondly, the dominant animal's score must be at least 25% higher than the subordinate animal's score. As a result, there should be no changes during the week of pair selection in which a hypothetically subordinate rat outperformed its dominant partner in isolated cases. Ideally, during the week of acclimatization, changes will be minimal. Only pairs of animals that met these criteria continued in the study.

Significant differences between the time spent at the feeder by dominant and subordinate rats were determined by ANOVA using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA) followed by a two-tailed t test ( P < 0.05). Comparisons were made between treatment groups using normalized dominance values for animals in pairs. Dominance level was a value that measured the social relationship between subjects in a pair. Dominance level (DL) = FTD - FTS, where FTD is the time spent at the feeder by dominant rats and FTS is the time spent at the feeder by subordinate rats. Normalization was carried out in accordance with the formula:

dominance level (week, n in %) = (dominance level (week, n))/(dominance level (week 2).

The statistical significance of the difference in the level of dominance between the control group (pairs of rats where both dominant and subordinate animals were treated with an inert substance) and the treatment group (subordinate rats were treated with a drug and dominant rats with an inert substance) was determined using ANOVA followed by a t-test. . Time to onset of action at 50% response (AOT-50) and minimum and maximum drug responses based on decreasing dominance values were calculated using nonlinear regression analysis (GraphPad Software, Inc., San Diego, CA). For these calculations, normalized DL values were used, where the DL values for treatment weeks were normalized as the percentage of the second week (pre-treatment) of that pair according to the formula above. With these parameters, the minimum response (DL) indicated positive drug activity, consistent with efficacy, as DL values decreased if the drug response was positive. In case of a negative reaction to the drug (increasing severity of symptoms), the DL values increased. If the drug did not exhibit such activity, then the maximum response did not exceed 100%. Any maximum DL value significantly higher than the control value (approximately 100%) indicated negative drug activity.

Levetiracetam and mGljR2 PAM/mGluR2 agonist compound (eg, compounds 2, 2-a, 25-a, 6-b or LY-404039) were evaluated in a rat model of RDBM according to the procedure described in more detail below.

Groups of dominant rats were treated with p.o. QD levetiracetam 10 mg/kg and mGluR2 PAM compound/mGluR2 agonist compound at varying concentrations from approximately 0.05 mg/kg (n>3), in an amount of 0.5 mg/kg (n>3), in an amount of 2, 5 mg/kg (n>3), in an amount of 5.0 mg/kg (n>3) and in an amount of 50.0 mg/kg (n>3). One control group of dominant rats with an inert substance was treated with 0.5% methylcellulose (n>3), and a second control group of dominant rats was treated with i.p. QD sodium valproate at 30 mg/kg (n>6 after 2 studies n>3 each).

All treatments were carried out approximately 1 hour before testing. All treatments began on the Saturday after the second week of testing (selection week). Levetiracetam and mGluR2 PAM/mGluR2 agonist compound were administered orally (po).

When dominant animals were treated with levetiracetam 10 mg/kg and mGluR2 PAM compound/mGluR2 agonist compound, the difference between dominant and subordinate animals disappeared after the first or second week of treatment, depending on the dosage. Similarly, if dominant animals were treated with sodium valproate, the difference between dominant and subordinate rats also disappeared after the first week of treatment. An increase in tolerance could be observed in dominant rats treated with levetiracetam and mGluR2 PAM/mGluR2 agonist compound or sodium valproate. Thus, treated dominant rats allowed their subordinate partners to increase their time at the feeder.

To compare different drugs and dose effects, data were normalized to reference week baseline values. The most potent effect of the combination of levetiracetam and mGluR2 PAM/mGluR2 agonist compound was observed when there was a significant difference in dominance level (DL) values between inert and combination treated rats starting in the second week and continuing through the treatment period up to 5 weeks. In comparison, animals (30 mg/kg) treated with sodium valproate consistently showed reduced levels of dominance after the second week of treatment, with the effect increasing in subsequent weeks.

To assess time to action (TOA), daily mean time spent at the feeder for dominant and subordinate pairs was plotted and significant differences between the two groups were calculated using a two-tailed t test.

To compare time to onset of action (AOT) between different treatments, time to onset

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Claims (76)

actions were assessed using a nonlinear regression plot. The nonlinear regression model produced a plot for each drug, combination, and daily DL values normalized by dose. The effects of levetiracetam and mGluR2 PAM/mGluR2 agonist compound in RDBM were potentially dose dependent. According to this analysis, the combination of levetiracetam and mGluR2 PAM/mGluR2 agonist compound potentially reduced dominant behavior, indicating that the combination is active as an antimanic agent. B. Oral tablets. In a specific embodiment, oral compositions of 100 mg mGluR2 PAM/mGluR2 agonist compound are formulated with sufficiently fine lactose to produce a total amount of 580 to 590 mg to fill a size O hard gelatin capsule. While the foregoing description has set forth the basic concepts of the present invention, with examples given for illustrative purposes, it is understood that the practice of the present invention covers all conventional variations, adaptations and/or modifications that fall within the scope of the following claims, and their equivalents. CLAIM 1. A combination for the treatment or prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, and bipolar and related disorders, containing (a) a synaptic vesicle protein 2A (SV2A) ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam; and (b) a positive allosteric modulator of metabotropic glutamatergic receptor subtype 2 (mGluR2 PAM), which is a compound of formula (I-B) or its stereochemically isomeric form, where R ¹ _is selected _{from the group consisting of C 1_6 alkyl} , (C _{3_8} cycloalkyl) _{C 1_3} αlkyl and ( _{C 1_3 αlkyloxy} ) C _{1_3} alkyl; each R ² is independently selected from F, Cl, C ₁ _ ₃ alkyl _, C ₁ _ ₃ alkyloxy, mono- or polyhalo-C ₁ _ ₃ alkyl, and mono- or polyhalo-C _{1_3} alkyloxy; n is an integer selected from 1, 2 and 3; or a pharmaceutically acceptable salt or solvate thereof. 2. The combination according to claim 1, wherein the SV2A ligand is levetiracetam or brivaracetam. 3. The combination according to claim 1 or 2, where the positive allosteric modulator of the metabotropic glutamatergic receptor subtype 2 is or a pharmaceutically acceptable salt thereof. 4. The combination according to any one of claims 1-3, where the positive allosteric modulator of the metabotropic glutamatergic receptor subtype 2 is or a pharmaceutically acceptable salt thereof, and the SV2A ligand is levetiracetam. 5. The combination according to any one of claims 1 to 3, wherein the positive allosteric modulator of the metabotropic glutamatergic receptor subtype 2 is - 74 045021 or a pharmaceutically acceptable salt thereof, and the SV2A ligand is brivaracetam. 6. The combination according to any one of claims 1 to 5, wherein the positive allosteric modulator of the metabotropic glutamatergic receptor subtype 2 is 7. The combination according to any one of claims 1 to 6, wherein the disorder is epilepsy or related disorders. 8. A combination according to any one of claims 1 to 6, wherein the disorder is bipolar or related disorders. 9. A pharmaceutical composition containing a combination according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier. 10. A method for producing a pharmaceutical composition according to claim 9, wherein the combination according to any one of claims 1 to 8 is thoroughly mixed with a pharmaceutical carrier. 11. A drug containing a combination of an SV2A ligand and a positive allosteric modulator of the metabotropic glutamatergic receptor subtype 2 according to any one of claims 1 to 6, for simultaneous, separate or sequential use in the treatment or prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, bipolar and related disorders. 12. The drug according to claim 11, where the SV2A ligand and the positive allosteric modulator of the metabotropic glutamatergic receptor subtype 2 are intended for separate use. 13. Use of a combination according to any one of claims 1 to 6 for the preparation of a medicinal product for the treatment or prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, bipolar and related disorders. 14. Use of a combination according to any one of claims 1-6 to obtain a medicinal product for the prevention of epileptogenesis. 15. Use according to claim 13, where the medicinal product is intended for the treatment or prevention of epilepsy and related disorders. 16. Use according to claim 15, wherein the epilepsy and related disorders are selected from focal or partial seizures. 17. Use according to claim 16, wherein epilepsy and related disorders are selected from focal or partial seizures with generalization. 18. Use according to claim 16, wherein epilepsy and related disorders are selected from focal or partial seizures without generalization. 19. Use according to claim 15, wherein epilepsy and related disorders are myoclonic seizures. 20. Use according to claim 15, wherein the epilepsy and related disorders are primary generalized tonic-clonic seizures. 21. Use according to claim 15, wherein the epilepsy and related disorders are treatment-resistant or refractory epilepsy. 22. Use according to claim 20, wherein primary generalized tonic-clonic seizures occur in patients with idiopathic generalized epilepsy. 23. Use according to claim 21, wherein the refractory epilepsy is refractory partial epilepsy or refractory generalized epilepsy. 24. Use according to claim 15 as a neuroprotector. 25. Use according to claim 13, wherein the drug is intended for the treatment or prevention of bipolar or related disorders. 26. The use of claim 25, wherein the bipolar or related disorder is bipolar I disorder. 27. The use of claim 25, wherein the bipolar or related disorder is bipolar II disorder. - 75 045021 28. Use according to claim 25, wherein the bipolar or related disorder is cyclothymic disorder. 29. Use according to claim 25, wherein the bipolar or related disorder is characterized by depressive and manic phases, the phases being cyclical. 30. Use of a pharmaceutical composition as defined in claim 9 as a medicament for the treatment or prevention of epilepsy and related disorders, neuropathic pain, migraine or persistent headache, and bipolar and related disorders. 31. Use according to claim 30 in the treatment or prevention of epilepsy and related disorders. 32. Use according to claim 31, wherein epilepsy and related disorders are selected from focal or partial seizures. 33. Use according to claim 31, wherein epilepsy and related disorders are focal or partial seizures with generalization. 34. Use according to claim 32, wherein epilepsy and related disorders are focal or partial seizures without generalization. 35. Use according to claim 32, wherein the epilepsy and related disorders are myoclonic seizures. 36. Use according to claim 31, wherein the epilepsy and related disorders are primary generalized tonic-clonic seizures. 37. Use according to claim 31, wherein the epilepsy and related disorders are treatment-resistant or refractory epilepsy. 38. Use according to claim 36, wherein primary generalized tonic-clonic seizures occur in patients with idiopathic generalized epilepsy. 39. Use according to claim 37, wherein the refractory epilepsy is refractory partial epilepsy or refractory generalized epilepsy. 40. Use according to claim 30 in the treatment or prevention of bipolar or related disorders. 41. The use of claim 40, wherein the bipolar or related disorder is bipolar I disorder. 42. The use of claim 40, wherein the bipolar or related disorder is bipolar II disorder. 43. Use according to claim 40, wherein the bipolar or related disorder is cyclothymic disorder. 44. Use according to claim 40, wherein the bipolar or related disorder is characterized by depressive and manic phases, the phases being cyclical. 45. Use according to claim 30 as a neuroprotector. 46. The use of a pharmaceutical composition as defined in claim 9 in the prevention of epileptogenesis. 47. A method of treating a patient with epilepsy or related disorders, neuropathic pain, migraine or persistent headache, or bipolar and related disorders, comprising administering an mGluR2 PAM compound and an SV2A ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam , where mGluR2 PAM represents or a pharmaceutically acceptable salt thereof. 48. The method of claim 47, wherein the SV2A ligand is levetiracetam. 49. The method of claim 47, wherein the SV2A ligand is brivaracetam. 50. The method according to any one of claims 47-49, where the positive allosteric modulator of the metabotropic glutamatergic receptor subtype 2 is 51. The method according to claim 50, which is intended for the treatment of epilepsy or related disorders. - 76 045021 52. The method of claim 51, wherein the disorder is epilepsy, in particular treatment-resistant epilepsy. 53. The method of claim 51, wherein the epilepsy or related disorder is focal or partial seizures. 54. The method of claim 53, wherein the epilepsy or related disorder is focal or partial seizures with generalization. 55. The method of claim 53, wherein the epilepsy or related disorder is focal or partial seizures without generalization. 56. The method of claim 51, wherein the epilepsy or related disorder is generalized seizure disorder. 57. The method of claim 51, wherein the epilepsy and related disorders are myoclonic seizures. 58. The method of claim 51, wherein the epilepsy and related disorders are primary generalized tonic-clonic seizures. 59. The method of claim 58, wherein primary generalized tonic-clonic seizures occur in patients with idiopathic generalized epilepsy. 60. The method according to claim 52, wherein the refractory epilepsy is refractory partial epilepsy or refractory generalized epilepsy. 61. The method according to claim 50, where the specified compound is used as a neuroprotector. 62. The method according to claim 50, which is intended for the treatment of bipolar or related disorders. 63. The method of claim 62, wherein the bipolar or related disorder is bipolar I disorder. 64. The method of claim 62, wherein the bipolar or related disorder is bipolar II disorder. 65. The method of claim 62, wherein the bipolar or related disorder is cyclothymic disorder. 66. The method of claim 62, wherein the bipolar or related disorder is characterized by depressive and manic phases, the phases being cyclical. 67. The method according to any one of claims 47 to 66, wherein the mGluR2 PAM compound and the SV2A ligand are administered simultaneously. 68. The method according to any one of claims 47 to 66, wherein the mGluR2 PAM compound and the SV2A ligand are administered simultaneously in separate dosage forms. 69. The method according to any one of claims 47 to 66, wherein the mGluR2 PAM compound and the SV2A ligand are administered separately in separate dosage forms during the course of therapy. 70. Use of a mGluR2 PAM compound for the treatment of a patient with epilepsy or related disorders, neuropathic pain, migraine or persistent headache, or bipolar and related disorders, where the patient also receives an SV2A ligand selected from the group consisting of levetiracetam, brivaracetam and seletracetam, where mGluR2 PAM is or a pharmaceutically acceptable salt thereof. 71. Use according to claim 70, wherein the SV2A ligand is levetiracetam. 72. Use according to claim 70, wherein the SV2A ligand is brivaracetam. 73. Use according to any one of claims 70-72, wherein the positive allosteric modulator of the metabotropic glutamatergic receptor subtype 2 is 74. Use according to any one of claims 70-73, in the treatment of epilepsy or related disorders. 75. Use according to claim 74, wherein the disorder is epilepsy, in particular treatment-resistant or refractory epilepsy. 76. Use according to claim 74, wherein epilepsy or a related disorder is a -