BR112016014193B1 - COMPOUND, COMPOSITION CONTAINING THE SAME, USE OF SAID COMPOUND, PROCESS FOR PREPARING SAID COMPOUND AND INTERMEDIATE COMPOUND - Google Patents

Abstract

GAMMA-AMINOBUTYRIC ACID (GABA) ANALOGS FOR THE TREATMENT OF PAIN AND OTHER DISORDERS. A compound of Formula (1) wherein R1 represents hydrogen, halo, a C1-C4 alkyl group, a C1-C4 alkyl halide group, a C2-C4, C1-C4 alkoxy-alkyl group, a C2-C4 alkenyl group , a C2-C4 alkynyl group or a C3-C7 cycloalkyl group; R2 represents Formula (1)' or Formula (1)'' a tautomer thereof; and R3 represents hydrogen, a C1-C4 alkyl group, a C1-C4 alkoxy-C2-C4 alkyl group or a C7-cycloalkyl group; or a pharmaceutically acceptable salt or solvate thereof. Processes for preparing said compounds and new intermediates are also claimed. This compound finds use in the treatment of neuropathic pain and disorders of the central nervous system.

Description

[1] The present invention relates to new therapeutic agents, in particular to y-aminobutyric acid derivatives, their pharmaceutical compositions, processes for their preparation and their therapeutic activity in the treatment of pain.

Introduction

[2] Voltage-gated calcium channels are formed by combinations of the pore-forming subunit a1 and auxiliary proteins a2d, ß and Y (Caterall (2000) Annu. Rev. Cell Dev. Biol. 16:521-555). The a2d protein is known to regulate calcium channel density and the voltage-dependent kinetics of these channels (Felix et al (1997) J. Neuroscience 17: 6884-6891; Klugbauer et al (1999) J. Neuroscience 19:684- 691; Hobom et al (2000) Eur. J. Neuroscience 12: 1217-1226; and Qin et al (2002) Mol. Pharmacol. 62:485-496).

[3] Gabapentin (GBP) is an antiepileptic, antihyperalgesic and anxiolytic drug that binds with high affinity to the two subtypes of a2d1 and a2d2 subunits of the a2d calcium channels. GBP was originally developed for epilepsy and has also found application in the treatment of pain and anxiety (Taylor et al (1998) Epilepsy Res. 29:223-249). The mechanism underlying the action of GBP is still poorly understood. GBP was originally designed as an analogue of lipophilic Y-amino butyric acid (GABA), but was subsequently shown to not interact with any of the enzymes in the GABA metabolic pathway, nor does it directly interact with the GABAA or GABAB receptors. However, it is able to effectively cross the blood-brain barrier via an L-system amino acid transporter.

[4] Pregabalin (PGB) is a second-generation, more potent GBP successor for the treatment of the same conditions as those listed above. GBP (Structure G, below) and PGB (Structure P, below) bind to the a2d-1 subunit with IC50 values of 140 and 80 nM, respectively (Dolphin (2013) Biochim Biophys Acta 1828: 1541-1549).

[5] GBP shows few, if any, toxic side effects at clinically relevant doses. However, it has a relatively short half-life and is excreted unchanged, possibly due to its very high water solubility and apparent lack of protein binding in vivo. Mild sedation, ataxia and dizziness are the main dose-limiting side effects and these are believed to be centrally mediated.

[6] GBP and PGB, unlike many other centrally acting drugs, are hydrophilic and doubly charged at neutral pH, which makes them insoluble in lipids such as cell membranes. However, both compounds appear to cross gut membrane barriers, blood-brain barrier and cell membranes via a specialized transporter system (L system) that also transports endogenous amino acids such as L-leucine, L-isoleucine and L- valine (Su et al (2005) J. Pharmacol. Exp. Ther. 313, 1-10).

[7] In mammals, there are four related subtypes of the a2d protein, each encoded by a different gene. Each protein subtype has a molecular weight of about 150kD and consists of 997-1150 amino acid residues. Only a2d subtypes 1 and 2 bind PGB with high affinity; subtypes 3 and 4 are devoid of significant drug binding (Fink et al (2002), Neuropharmacology, 42, 229-236). The binding affinity of PGB is similar for type 1 and type 2 a2d recombinant proteins, demonstrating that PGB is not subtype selective (Piechan et al (2004) Soc. Neuroscience Abstr., (program No 115)).

[8] European patent application 2192109A relates to bicyclic Y-amino acid compounds of Formula A wherein R1, R2, R2', and R4-R8 and R8' are each independently a hydrogen atom, a halogen atom, or a C1-C6 alkyl group, or R2 and R2' together with the atom carbon to which they are bonded forming a C3-C7 cycloalkyl group; and R3 is a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-C6 alkyl halide group, a hydroxy-C1-C6 alkyl group, a sulfanyl-C1-C6 alkyl group, a C1 group -C6 alkoxy-C1-C6 alkyl, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, a C1-C6 alkylsulfanyl group, a C1-C6-alkylsulfanyl-C1-C6 alkyl group, a C2-C7 acylthio-C1-C6 alkyl group, a C2-C7 acyloxy-C1-C6 alkyl group, or a C3-C7 cycloalkyl group.

[9] Said derivatives are described as having activity as a2d ligands and as being effective in the treatment of pain or disorders in the central nervous system. US 2012071685 discloses preparative methods for producing certain of said bicyclic y-amino acid derivatives. Summary of the Invention In one aspect, the present invention provides compounds of Formula 1 wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group; R2 represents or a tautomer thereof; and R3 represents hydrogen, a C1-4 alkyl group, a (C1-4 alkoxy) (C2-4 alkyl) or a C3-7 cycloalkyl group; or a pharmaceutically acceptable salt or solvate thereof.

[10] In another aspect, the present invention also provides a method of treating neuropathic pain in a subject, comprising administering a therapeutically effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof or solvate thereof to said subject.

[11] In a further aspect, the present invention provides a method of treating a central nervous system disorder in a subject, comprising administering a therapeutically effective amount of a compound of Formula 1 or a pharmaceutically acceptable salt thereof or solvate thereof to the said subject.

[12] The present invention provides compounds that bind with high affinity to the a2d-1 subunit of voltage-dependent calcium channels.

[13] In a still further aspect, the present invention provides a pharmaceutical composition comprising the compounds described herein together with a suitable excipient or a suitable lotion. The pharmaceutical composition may be in any form convenient for administration to a subject, for example, in the form of a capsule, a tablet, a lozenge, or in the form of a topical ointment.

Detailed Description of the Invention

[14] The present invention relates to compounds of Formula 1 wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group; R2 represents or or a tautomer thereof; and R3 represents hydrogen, a C1-4 alkyl group, a (C1-4 alkoxy) (C2-4 alkyl) or a C3-7 cycloalkyl group; or a pharmaceutically acceptable salt or solvate thereof.

[15] The following terms shall be understood to have the following meanings:

[16] The term "C1-4 alkyl" means a straight or branched aliphatic hydrocarbon chain having 1 to 4 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl .

[17] The term "C1-4 alkyl halide" means a C1-4 alkyl group substituted by one or more halo atoms.

[18] The term "(C1-4 alkoxy (C2-4 alkyl)" means a C2-4 alkyl group substituted with one or more C1-4 alkoxy groups.

[19] The term "C2-4 alkenyl" means a branched aliphatic hydrocarbon chain having 2 to 4 carbon atoms and containing a carbon-carbon double bond, for example, ethenyl, propenyl, n-butenyl and isobutenyl or linear.

[20] The term "C2-4 alkynyl" means a linear or branched aliphatic hydrocarbon chain having 2 to 4 carbon atoms and containing a carbon-carbon triple bond, for example ethynyl, propynyl, n-butynyl and isobutynyl.

[21] The term "C3-7 cycloalkyl" means a non-aromatic mono- or multi-cyclic ring system of 3-7 carbon atoms, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

[22] The term "halo" means fluorine, chlorine, bromine or iodine.

[23] As used herein, the term "compounds of formula 1" includes pharmaceutically acceptable salts and solvates. References to intermediate compounds include salts and solvates. Pharmaceutically acceptable salts of the compounds of the present invention may include base addition salts of the compound. Such salts may be formed with an inorganic base that provides a pharmaceutically acceptable cation, for example, an alkali metal salt, such as a sodium or potassium salt, or an alkaline earth metal salt, such as a calcium salt or of magnesium. The pharmaceutically acceptable salts of the present invention may also include acid addition salts. Such salts may be formed with an inorganic or organic acid that provides a pharmaceutically acceptable anion, for example, a hydrohalide salt, such as a chloride or bromide salt, a sulfate or phosphate salt, or an organic acid salt. , for example a salt with acetate, fumarate, maleate, tartrate, lactate, citrate, pyruvate, succinate, oxalate, methanesulfonate or p-toluenesulfonate. The term "solvate" refers to a compound of Formula 1 in the solid state, in which molecules of a suitable solvent are incorporated into the crystal lattice. A solvent suitable for therapeutic administration is physiologically acceptable at the dosage administered. Examples of suitable solvents for therapeutic administration are ethanol and water. When water is the solvent, the solvate is referred to as a hydrate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions. Typical solvates include hydrates such as monohydrate, dihydrate or trihydrate.

[24] When the compounds produced according to the invention include a chiral center, the compounds exist in two enantiomeric forms, according to which the present compounds include the racemate, individual enantiomers or mixtures thereof. Enantiomers can be resolved by methods known to those skilled in the art, for example chiral high performance liquid chromatography (HPLC), for example silica with a chiral ligand attached. Enantiomers can also be resolved by methods involving the formation of salts with chiral acids or bases to form diastereomeric salts, followed by crystallization. The compounds produced in accordance with the invention may also include the individual diastereoisomers or mixtures thereof. Diastereomers can be separated by methods known to those skilled in the art, for example, by formation of salts with acids or bases, followed by crystallization.

[25] All tautomeric forms are also encompassed here.

[26] The term "therapeutically effective amount" describes the amount of a compound according to the present invention that should be used by human subjects in order to achieve a desired therapeutic effect. This amount may vary per individual and depends on parameters such as age, weight, height, physical condition and medical history. The compound according to the present invention may be effective for reducing, inhibiting or ameliorating undesirable symptoms in a patient.

[27] In one embodiment of the compounds according to the present invention, R1 represents hydrogen, a C1-4 alkyl or halo group, preferably hydrogen or a C1-4 alkyl group, more preferably a C1-4 alkyl group, e.g. example, methyl, ethyl or propyl. In other preferred compounds of Formula 1, R1 represents ethyl.

[28] In one embodiment of the compounds according to the present invention, R3 represents hydrogen or a C1-C4 alkyl group, for example, methyl, ethyl or propyl. In the preferred compounds of Formula 1, R3 represents hydrogen or ethyl. In other preferred compounds of Formula 1, R3 represents hydrogen.

[29] In one embodiment, the invention is a compound of Formula 2: wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group; or a pharmaceutically acceptable salt or solvate thereof.

[30] The compound of Formula 2 may have specific stereochemistry as shown in a compound of formula 2a

[31] In another embodiment, the invention is a compound of Formula 3: wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group; or a pharmaceutically acceptable salt or solvate thereof.

[32] The compound of Formula 3 may have specific stereochemistry as shown in a compound of Formula 3a

[33] In another embodiment, the invention is a compound of Formula 4:

[34] The compound of Formula 4 may have specific stereochemistry as shown in a compound of Formula 4a

[35] In another embodiment, the invention is a compound of Formula 5:

[36] The compound of Formula 5 may have specific stereochemistry as shown in a compound of formula 5a

[37] The specific compounds of Formula 1 are: 3-(((1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl)methyl)- 1,2,4-oxadiazol-5(4H)-one 3-(((1S,5R,6R)-6-(aminomethyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl) methyl)-1,2,4-oxadiazol-5(4H)-one 3-(((1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo [3.2.0]hept-3-en-6 -yl)methyl)-1,2,4- oxadiazol-5(4H)-racemic one ((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2 .0]hept-3-en-6-yl)methanamine ((1S,5R,6R)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3- en-6-yl)methanamine ((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl)methanamine racemic N-(((lR,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl)methyl)ethanamine N -(((1S,5R,6R)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl)methyl)ethanamine N-( ((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo[3.2.0]hept-3-en-6-yl)methyl)racemic ethanamine

[38] Those skilled in the art will understand that chiral molecules can be rotated but maintain the same relative stereochemical arrangement. For example, compounds of general formulas 4a and 5a can be represented as follows.

[39] The present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of a compound of Formula 1, together with a pharmaceutically acceptable carrier. The compound of Formula 1 is used in an effective amount to treat, reduce or ameliorate neuropathic pain in a subject, especially a human subject suffering from a painful condition. Such pain treatment may or may not be associated with a disorder of the central nervous system (CNS) or peripheral nervous system (PNS). The Formula 1 compound is also effective for treating, reducing or improving any other non-painful CNS-related disorders. The compositions of the present invention comprise a therapeutically effective amount of the compound of Formula 1, which is generally in the range of 0.1-95% w/w of the compound of Formula 1, but is dependent on the exact nature of the active and the mode of administration. administration. Typically, the dose of active ingredient is in the range of 0.1 to 500 mg as single or divided doses, depending on the precise nature of the active ingredient and the mode of administration.

[40] In therapeutic use, compounds of Formula 1 can be administered orally, rectally, parenterally or topically. The pharmaceutical compositions according to the present invention may take the form of any rectal, oral, parenteral or topical composition known to those skilled in the art, using carriers well known in the art of pharmacy. Such compositions are generally prepared in unit dosage form. Compositions for oral administration may include solid dosage forms, such as tablets, capsules or pills, or liquid dosage forms, such as aqueous or oily syrups and suspensions. Solid dosage forms such as tablets and capsules can be prepared by mixing a compound of Formula 1 with an inert diluent, in the presence of disintegrating agents and other formulation aids such as lubricants. Capsules can be in the form of hard capsules, for example, hard gelatin capsules or soft capsules that are prepared by conventional processes, in which the active ingredient is incorporated into a carrier and encapsulated. Optionally, these dosages may include an enteric coating prepared according to conventional procedures that can be used to modify the rate of release, or an excipient that delays release to provide a delayed release or a sustained release composition. Liquid dosage forms can be prepared by dissolving the active substance in a suitable liquid carrier such as water or an oily excipient, optionally in the presence of one or more dissolving agents, surfactant agents and/or suspending aids. Compositions for rectal administration are known pharmaceutical forms for such administration, for example, suppositories with a polyethylene glycol or wax base. Compositions for parenteral administration are also known pharmaceutical forms for such administration, for example sterile solutions or suspensions in a suitable solvent system. Compositions for topical administration may include creams, lotions, ointments, gels, or other such dosages which may be administered by applying the composition directly to the affected area, or by incorporating the composition into a vehicle, such as a transdermal patch or a composition contained within of a permeable membrane for application to a painful area. Conventional aqueous and non-aqueous carriers, such as mineral oils and waxes, can be used alone or in combination to prepare creams, lotions or ointments. Gels can be prepared by mixing the compound of Formula 1 with a topical vehicle comprising a gelling agent, for example carbomer, in the presence of water. Optionally other formulation aids such as transdermal accelerators, thickening agents can also be incorporated.

[41] In another embodiment, the compound of the invention can be used in combination with a suitable pharmaceutical excipient for the topical treatment of back pain. The combination of the compound and the pharmaceutical excipient may be in the form of a gel, the gel molded and adapted for placement on the skin of a subject in pain. In another embodiment, the combination of the compound and the pharmaceutical excipient may be incorporated into the fabric of a patch, the patch molded and adapted for placement on and/or adhering to the skin of a distressed subject. In a more preferred embodiment, the compound is released at a slow rate from the pharmaceutical excipient into the adhesive tissue.

[42] The compounds of Formula 1 are incorporated into pharmaceutical compositions according to the present invention that are useful under the conditions recited below.

[43] The invention contemplates that compounds of Formula 1 can be used in a clinical setting for the treatment of neuropathic pain. In another embodiment, the compounds can be used to treat pain in the central nervous system (CNS). In another embodiment, the compounds of the invention can be used to treat pain that is not associated with the CNS. In another embodiment, the compounds of the invention can be used to treat pain that is not associated with the PNS. In yet another embodiment, the compounds of the invention can be used to treat a CNS disorder. In one embodiment, the CNS disorder is selected from the group consisting of epilepsy, ischemic cerebrovascular disease, stroke, brain tumors, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders. amyotrophic lateral sclerosis and other neuromotor disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system, prion diseases including kuru, Creutzfeldt-Jakob disease and Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, retardation mental and other pervasive developmental disorders of the central nervous system, including Down syndrome, cerebral palsy, neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases, muscular dystrophy and other neuromuscular diseases, diseases of the peripheral nervous system, dermatomyositis and polymyositis, inherited, metabolic, endocrine and toxic myopathies, myasthenia gravis, periodic paralysis, mental disorders including mood, anxiety and schizophrenic disorders, seasonal affective disorder (SAD), akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, Tourette's syndrome, progressive supranuclear palsy, corticobasal degeneration and familial frontotemporal dementia. In another embodiment, the compounds of the invention can be used in the treatment of pain in the central nervous system, such as, but not limited to, headache and migraine.

[44] In another embodiment, the compounds of the invention can be used in combination with a suitable lotion in a pharmaceutical formulation for the topical treatment of back pain. In another embodiment, the compounds of the invention can be used for the topical treatment of joint pain.

[45] The processes for preparing the compounds of the invention will now be described. These processes form another aspect of the present invention.

[46] Compounds of Formula 1 can be prepared by reduction of compounds of Formula 12 wherein R1 and R2 are as defined above, with a suitable reducing agent.

[47] For example, compounds of Formula 1 in which R1 is as defined herein, R2 represents and R3 represents a C1-4 alkyl group, a (C1-4 alkoxy)(C2-4 alkyl) group or a C3-7 cycloalkyl group, can be prepared by reducing compounds of Formula 12 with a suitable reducing agent, followed by a reductive amination with an appropriate aldehyde or ketone. Suitable reagents include powdered iron with ammonium chloride in a suitable solvent, for example an aqueous alcohol such as ethanol, at a suitable temperature, for example from room temperature to reflux temperatures. Further reductive amination in the presence of a suitable aldehyde or ketone with a suitable reducing agent, for example, includes the use of sodium borohydride in a suitable chlorinated or ethereal solvent such as dichloroethane at room temperature.

[48] Compounds of Formula 1, wherein R1 is as defined herein, R2 represents and R3 represents hydrogen, may be prepared by reducing compounds of Formula 12 with a suitable reducing agent, such as powdered iron with ammonium chloride, in a solvent, such as aqueous ethanol, at a temperature, for example, from room temperature to reflux temperatures. Addition of a protecting group to the crude product to produce the intermediate compound of Formula 13 can be used to aid purification followed by deprotection under acidic conditions. Suitable protecting groups include a tert-butoxycarbonyl group obtained by reaction with di-tert-butyl dicarbonate in the presence of a suitable base and solvent. Examples of a suitable base include ?,?-diisopropylethylamine or triethylamine in a suitable solvent such as an aqueous ethereal solvent, for example, aqueous tetrahydrofuran, at room temperature to reflux temperatures. Deprotection can be carried out with strong acids such as hydrochloric or trifluoroacetic acid in a suitable solvent, for example trifluoroacetic acid in dichloromethane.

[49] Compounds of Formula 1 in which R1 and R3 are as defined herein and R2 represents can be prepared by reacting compounds of Formula 12 with a suitable reducing agent, e.g. sodium borohydride in the presence of nickel(II) chloride hexahydrate, in a suitable solvent, e.g. methanol, at 0°C to room temperature.

[50] Compounds of Formula 1 in which R3 represents alkyl or cycloalkyl can generally be prepared by reductive amination of compounds of Formula 1 in which R3 represents hydrogen, for example by treatment with a suitable aldehyde or ketone compound together with a suitable reducing agent , such as sodium borohydride, sodium cyanoborohydride or sodium triacetoxyborohydride. For example, the use of acetaldehyde produces compounds in which R3 is an ethyl group. Alternatively use of cyclopentanone produces compounds in which R3 is a cyclopentyl group.

[51] Compounds of Formula 1 can also generally be prepared by removing a protecting group from compounds of Formula 1 in which R3 is replaced by a protecting group, for example, COOC(CH3)3, which can be removed by reaction with a suitable acid, for example hydrochloric acid or trifluoroacetic acid.

[52] Enantiomers of compounds of Formula 1 can be prepared by resolving the corresponding racemate or a mixture of diastereoisomers, for example by forming diastereomeric salts with suitable chiral acids or bases. Examples of suitable chiral acids include: mandelic acid, α-methoxyphenylacetic acid, tartaric acid, naproxen or Mosher's acid. Examples of suitable chiral bases include: a-methylbenzylamine, 4-chloro-a-methylbenzylamine or ephedrine. Compounds of Formula 12 wherein R1 is as defined herein and R2 represents can be prepared from Formula 6 wherein R1 is as defined herein, by a ring forming reaction with a suitable azide, for example sodium or trimethylsilyl azide, together with suitable commonly known catalysts such as dibutyltin oxide, pyridine hydrochloride or ammonium chloride in a solvent such as dimethylformamide (DMF), N-methylpyrrolidinone (NPO) or toluene, at elevated temperatures ranging between 60°C to 120°C.

[53] Compounds of Formula 12 in which R2 represents can be prepared by reacting a compound of Formula 6 with appropriate reagents to produce the oxadiazolone moiety in a two-step procedure. For example, the first step may include using hydroxylamine to produce the Formula 9 intermediate. where R1 is as defined here. The second ring formation step may involve reacting intermediate 9 with a suitable cyclization reagent that incorporates a carbonyl group. For example, suitable reagents include carbonyldimidazole or phosgene. Treatment with carbonyl diimidazole can occur in a suitable ethereal solvent such as 1,4-dioxane at reflux temperatures.

[54] Compounds of Formula 9 can be prepared by treating compounds of Formula 6 with hydroxylamine in a suitable solvent such as aqueous ethanol at elevated temperatures, for example, in a 100 watt microwave oven.

[55] Compounds of Formula 6 can be prepared by treating compounds of Formula 7 wherein R1 is as defined herein, with a suitable dehydrating agent, for example a Burgess reagent in a suitable solvent, such as dichloromethane, at room temperature.

[56] Compounds of Formula 7 can be prepared by treating compounds of Formula 8 wherein R1 is as defined herein, with a suitable coupling agent, followed by the addition of 0.88 concentrated aqueous ammonia. A suitable coupling agent may be 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazole[4,5-b]pyridinium-3oxydhexafluorophosphate (HATU) in the presence of a suitable base, for example N, N-diisopropylethylamine (Hunig's base) or triethylamine, and in a solvent such as dimethylformamide (DMF) or N-methylpyrrolidinone (NMP).

[57] Compounds of Formula 8 can be prepared by treating a tert-butyl 2-(3-alkyl-6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl)acetate compound with a suitable strong acid, for example hydrochloric acid or trifluoroacetic acid, in a suitable solvent such as dichloromethane or 1,4-dioxane.

[58] Tert-Butyl 2-(3-alkyl-6-(nitromethyl)bicyclo [3.2.0]hept-3-en-6-yl) acetate compounds can be prepared as described in US 2012/0071685.

[59] Compounds of Formula 6 can also be prepared by reacting compounds of Formula 14 wherein R1 is as defined herein, with nitromethane in the presence of a suitable base, for example 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and a solvent such as dichloromethane.

[60] Compounds of Formula 14 can be prepared by treating compounds of Formula 15 wherein R1 is as defined herein, with a suitable double bond forming reagent, for example diethyl cyanomethylphosphate, in the presence of a suitable base, such as potassium tert-butoxide in a suitable solvent, for example tetrahydrofuran, at a temperature in the range of 0°C to room temperature.

[61] Intermediate nitromethyl compounds 6, 7, 8, 9, 10, 11 and 12 may be covered by Formula 16 intermediate wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group; R2a represents R2 represents or a respective tautomer.

[62] Details of the preferred process steps for preparing the specific compounds of Formula 1 are in Schemes 1-3 as follows:

[63] Synthesis of the compound of general formula 4a is carried out using the nitro-nitrile intermediate with Formula 6: (Scheme 1)

[64] Synthesis of the compound of general formula 5 is carried out using the nitro-nitrile intermediate with Formula 6: (Scheme 2) Scheme 2

[65] Synthesis of a compound of Formula 4 (shown as 4b) and its enantiomers can also be carried out by scheme 3

[66] Certain intermediate compounds and stereoisomers of formulas 6, 7, 8, 9, 10, 11, 12, 13, 14 and 16 are believed to be new compounds. The invention also contemplates all stereoisomers of intermediates of compounds comprising the chemical structure as shown in Formula 6, Formula 7, Formula 8, Formula 9, Formula 12, Formula 13, Formula 14 and Formula 16 and the specific compounds identified below.

[67] All new compounds are claimed as another aspect of the present invention.

[68] The invention is illustrated by the following non-limiting Examples. Example 1: Synthesis of the compound of Formula 3 Synthesis of 3-(((1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl)methyl)- Racemic 1,2,4-oxadiazol-5(4H)-one Step 1 (Compound of Formula 8) Racemic 2-((1R,5S,6S)-3-ethyl-6-nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl)acetic acid

[69] To a solution of tert-butyl-2-(3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl) acetate (1.8 g, 6.09 mmol ) [preparation described in patent US2012 / 0071685A1, Example 5-d] in dichloromethane (20 ml) trifluoroacetic acid (20 ml) was added and the mixture was left to stand for 1 hour. The solvent was removed and the residue was redissolved in toluene (100 ml) and evaporated to dryness, this procedure was repeated x5 to give 2-(3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3 acid -en-6-yl)acetic acid (1.4 g, 5.62 mmol, 92% yield) as a colorless oil.

[70] LCMS (Agilent, X-Select, Waters 238(M-H)- (ES-), in 2.21 min, 93% purity @ 215 nm.

[71] 1H NMR (400 MHz, DMSO-d6): d 12.25 (1H, s), 5.26 (1H, d, J = 2.0), 4.87 (2H, d, J = 1.6), 3.13 (1H, br. s), 2.85 (1H, quin, J = 7.5), 2.46 - 2.32 (3H, m), 2.21 (1H, ddd, J = 12.5, 8.8, 2.5), 2.11 (2H, q, J = 7.4), 2.03 (1H, br. d, J = 16.5), 1.46 (1H, dd, J = 11.3, 7.3), 1.04 (3H, t, J = 7.4) ppm. (Toluene also present: 7.26 - 7.23 (0.3H, m), 7.18 - 7.12 (0.45H, m), 2.30 (0.45H, m). Step 2 (Compound of formula 7) 2-((1R,5S,6S )-3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl)racemic acetamide

[72] To a solution of 2-(3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl)acetic acid (2.5 g, 10.45 mmol) in dimethylformamide dry (10 ml) Hunig's base (2.74 ml, 15.67 mmol) was added followed by HATU (4.37 g, 11.49 mmol). The mixture was allowed to stir for 10 minutes at room temperature, and then cooled in ice water. To this stirred cooled solution was added 0.88 aqueous ammonia solution (6.46 mL, 104 mmol). The reaction mixture was then warmed to room temperature and stirred for an additional 1 hour.

[73] The solvent was removed by rotary evaporation and the residue was taken up in ethyl acetate and washed with water, dried over sodium sulfate. The crude product was purified by chromatography on silica (40 g silica column, 20-100% ethenisohexane solvent gradient) to obtain 2-(3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept- 3-en-6-yl)acetamide (2.3 g, 9.46 mmol, 91% yield) as a transparent colorless gum.

[74] LCMS (Agilent, X-Select, Waters z 239(M+H)+ (ES+); 237(M-H)-(ES-), in 1,949 min.

[75] 1H NMR (400 MHz, DMSO-d6): d 7.28 (1H, br. s), 6.77 (1H, br. s), 5.27 (1H, d, J = 2.2), 4.88 (2H, dd, J = 22.3, 12.3), 3.12 (1H, br. s), 2.85 - 2.78 (1H, m), 2.46 - 2.39 (1H, m), 2.24 (1H, br. d, J = 1.4), 2.19 (1H , ddd, J = 12.4, 8.8, 2.6), 2.11 (2H, q, J = 7.5), 2.00 (1H, br. d, J = 16.8), 1.42 (1H, dd, J = 12.5, 7.4), 1.05 (3H, t, J = 7.4) ppm. (DCM also present: 5.87 (0.9 H, s)) Step 3 (compound of Formula 6) 2-((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3 -en-6-yl)acetonitrile

[76] To an ice-cooled solution of 2-((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl)acetamide (0. 8 g, 3.36 mmol) in dry dichloromethane (20 ml) was added in portions over 20 minutes of Burgess reagent (0.880 g, 3.69 mmol) and the mixture was allowed to warm to room temperature and stirred for 3 hours. The reaction mixture was evaporated to half volume and applied to a silica cartridge and purified by chromatography over Companion (40 g column, 0-60% ethenisohexane) to obtain 2-((1R,5S,6S)-3 -ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl)acetonitrile (501 mg, 2.23 mmol, 66.4% yield) as a clear colorless oil.

[77] LCMS (Agilent, X-Select, Waters z 221(M+H)+ (ES+); 219(M-H)- (ES-), at 2.34.

[78] 1H NMR (400 MHz, DMSO-d6): d 5.33 (1H, d, J = 2.0), 4.87 (2H, d, J = 1.9), 3.17 (1H, br. s), 2.93 - 2.85 ( 1H, m), 2.65 (2H, br. s), 2.49 - 2.43 (1H, m), 2.23 (1H, ddd, J = 12.5, 8.8, 2.5), 2.16 - 2.05 (3H, m), 1.56 (1H , dd, J = 12.6, 7.2), 1.07 (3H, t, J = 7.4) ppm. Step 4 (Compound of Formula 9) 2-((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo[3.2.01hept-3-en-6-yl)-N-Hydroxyacetimidamide

[79] A mixture of hydroxylamine in water (50% aqueous solution) (543 µl, 8.85 mmol), 2-((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo [3.2. 01hept-3-en-6-yl)acetonitrile (650 mg, 2.95 mmol) in ethanol (10 ml) was heated to 85 °C in a CEM microwave at 100 watts for 2 hours.

[80] The reaction mixture was evaporated to dryness and the residue was taken up in ethyl acetate and washed with water and dried over sodium sulfate. Filtration and evaporation gave 2-((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl)-N-hydroxyacetimidamide (530 mg, 1.883 mmol, 63.8% yield) as a colorless oil, which was used without further purification.

[81] LCMS (Agilent, X-Select, Waters /z 254 (M+H)+(ES+); at 1.25 min.

[82] 1H NMR (400 MHz, DMSO-d6): d 8.92 (1H, s), 5.36 -5.35 (1H, m), 5.26 (2H, br. s), 4.96 (1H, d, J = 12.8) , 4.83 (1H, d, J = 12.8), 3.13 (1H, br. s), 2.77 (1H, quin, J = 7.5), 2.41 (1H, dd, J = 16.4, 8.2), 2.20 - 2.09 (5H , m), 2.02 (1H, d, J = 16.0), 1.53 (1H, dd, J = 12.4, 7.4), 1.06 (3H, t, J = 7.4) ppm. Step 5 (compound of Formula 11) 3-(((1R,5S,6S)-3-ethfayl-6-(nitromethyl)bicyclo [3.2.0]hept-3-en-6-yl)methyl)-1, 2,4-oxadiazol-5(4H)-one

[83] A mixture of 2-((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl)-N-hydroxyacetimidamide (520 mg, 2.053 mmol) and carbonyl diimidazole (666 mg, 4.11 mmol) in dioxane (30 mL) was heated under reflux for 2 hours.

[84] The reaction mixture was evaporated to dryness and the residue was taken up in water (50 ml) and the solution was carefully acidified with 1N HCL. The aqueous mixture was extracted into ether and dried over sodium sulfate.

[85] The crude product was purified by silica chromatography (12 g column, solvent: ether) to obtain 3-(((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo acid [ 3.2.0]hept-3-en-6-yl)methyl)-1,2,4-oxadiazol-5(4H)-one (195 mg, 0.684 mmol, 33.3% yield) as a colorless gum.

[86] LCMS (Agilent, X-Select, Waters (M-H)- (ES-), in 1.58min, 98% purity @ 215 nm.

[87] 1H NMR (400 MHz, DMSO-d6): d 12.12 (1H, s), 5.35 (1H, q, J = 1.9), 4.88 (2H, q, J = 13.5), 3.16 (1H, br. s), 2.87 91H, quin, J = 7.5), 2.66 (2H, d, J = 1.4), 2.44 (1H, dd, J = 16.6, 7.8), 2.17 - 2.09 (3H, m), 2.06 - 2.02 ( 1H, m), 1.62 (1H, dd, J = 12.5, 7.4), 1.06 (3H, t, J = 7.5). Ether present - overlaps with signal at 1.06 + water signal at 3.4 ppm

[88] 13C NMR (100 MHz, DMSO-d6): d 159.44, 157.15, 150.69, 120.63, 80.09, 52.17, 42.75, 41.80, 35.38, 30.19, 28.46, 23.87, 12.25 ppm Step 6 (formula 3 compound) 3 -(((1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl)methyl)-1,2,4-oxadiazole-5(4H) -ona

[89] To an ice-cooled solution of 3-(((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo [3.2.0]hept-3-en-6-yl)methyl acid) -1,2,4-oxadiazol-5(4H)-one (286 mg, 1.024 mmol) and nickel(II) chloride, 6H2O (24.34 mg, 0.102 mmol) in methanol (40 ml) were added boron. sodium hydride in small portions (387 mg, 10.24 mmol) in portions over 15 minutes. After completion of the addition, the reaction mixture was allowed to warm to room temperature.

[90] The reaction mixture was carefully quenched with the addition of acetic acid (1 ml) and the mixture was evaporated to dryness. The residue was taken up in methanol and passed through a silica plug and the eluent was evaporated to dryness to give an off-white solid. The crude product was purified by preparative HPLC (Waters, acidic (0.1% formic acid), acidic, Waters X-Select Prep-C18, 5 µm, 19x50mm column, 25-70% acetonitrile in water) to obtain obtain after lyophilization 3-(((1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl)methyl)-1,2,4-oxadiazole- 5(4H)-one (8.0 mg, 0.031 mmol, 3.07% yield) as a colorless solid.

[91] LCMS (Agilent, X-Select, Waters /z 250(M+H)+ (ES+); 248(M-H)- (ES-), in 1.143 min, 100% purity @ 215 nm.

[92] 1H NMR (400 MHz, DMSO-d6): d 8.27 (0.5H, s), 5.33 (1H, d, J = 1.5), 3.11 (1H, d, J = 13.1), 3.04 (1H, J = 13.1), 2.97 (1H, br. s), 2.76 (1H, quin, J = 7.5), 2.44 - 2.34 (3H, m), 2.12 (2H, q, J = 7.4), 2.03 - 1.97 (2H, m), 1.31 (1H, dd, J = 12.2, 7.4), 1.05 (3H, t, J = 7.4) ppm.

[93] 13C NMR (100 MHz, DMSO-d6): d 170.67, 165.86, 149.09, 121.20, 51.92, 46.98, 42.62, 41.81, 34.69, 32.98, 30.35, 24.11, 12.64 ppm Example 2: Synthesis of the compound of Formula 2 Synthesis of ((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl)methanamine and enantiomers Step 1 (Compound of Formula 10) 5-(((1R,5S,6S-3-ethyl-6-(nitromethyl) bicyclo [3.2.0]hept-3-en-6-yl-methyl-1H-Tetrazol

[94] To a solution of 2-((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl)acetonitrile (280 mg, 1.271 mmol ) (preparation described in step 3, of the compound of Formula 3) in dry toluene (5 ml) was added azidotrimethylsilane (675 µl, 5.08 mmol) and dibutyltin oxide (63.3 mg, 0.254 mmol) and the mixture was heated in a CEM microwave oven: power 100 watts, temperature 110 degrees for 1 hour.

[95] The above process was repeated eleven times and the crude reaction mixtures combined. The combined reaction mixture was evaporated to dryness and the residue was taken up in 0.1 N sodium hydroxide solution (10 ml) and washed with ether (2x20 ml), the aqueous layer was separated and acidified with 1N hydrochloric acid. The crude product was then re-extracted back into ether and dried over sodium sulfate. Filtration and evaporation gave a solid which was purified by Companion chromatography (4 g column, solvent gradient: 10-70% ethenisohexane) to give 5-(((1R,5S,6S)-3-ethyl -6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl)methyl)-1H-tetrazol (1.81 g, 5.50 mmol, 39% yield) as a colorless solid.

[96] LCMS (Agilent, X-Select, Waters ): m/z 264(M+H)+ (ES+); 262(M-H)- (ES-), in 2.05min, 98% purity @ 210 nm.

[97] 1H NMR (400 MHz, DMSO-d6): d 16.12 (1H, br. s), 5.37 (1H, d, J = 1.0), 4.80 (2H, s), 3.22 (1H, br. s ), 3.02 (2H, s), 2.92 - 2.84 (1H, m), 2.50 - 2.44 (1H, m), 2.18 - 2.05 (4H, m), 1.64 (1H, dd, J = 12.4, 7.4), 1.06 (3H, t, J = 7.4) ppm. Step 2 (Compound of formula 13) Tert-butyl (((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3- en racemic -6-yl)methyl)carbamate

[98] To a solution of 5-(((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo [3.2.0]hept-3-en-6-yl)methyl)-1H-tetrazol (400 mg, 1.519 mmol) in a solvent of ethanol (50 ml) and water (15 ml) was added iron powder (848 mg, 15.19 mmol) and ammonium chloride (488 mg, 9.12 mmol). The mixture was stirred and heated at reflux for 1 hour. The reaction mixture was allowed to cool and filtered through a celite pad and washed well with ethanol and the filtrate was evaporated to dryness.

[99] The above residue was dissolved in a mixture of water (20 ml) and tetrahydrofuran (60 ml) and to this solution was added di-tert-butyl dicarbonate (2.8 g, 12.86 mmol) and triethylamine (1 .79 ml, 12.86 mmol) and then heated and stirred at 40° for 1 hour.

[100] The reaction mixture was evaporated to half volume and the residue was acidified by adding 10% aqueous citric acid solution. The crude product was then extracted with ethyl acetate, the organics separated, washed with water and dried over sodium sulfate. The crude product was purified by silica chromatography (12 g column, solvent gradient 0-70% ethenisohexane) to obtain tert-butyl(((1R,5S,6S)-6-((1H-tetrazol-5 -yl)methyl)- 3-ethylbicyclo[3.2.0]hept-3-en-6-yl)methyl)racemic carbamate (380 mg, 1,140 mmol, 89% yield) as a transparent colorless gum.

[101] LCMS (Agilent, X-Select, Waters ): m/z 334(M+H)+ (ES+); 332(M-H)- (ES"), in 2.34 min, 98% purity @ 210 nm.

[102] LCMS (Agilent, X-Select, Waters X-Bridge C18, 2.5 µm, 4.6x30 mm, basic (0.1% ammonium bicarbonate) 4 min method, 5-95% acetonitrile/water ): m/z 334(M+H)+ (ES+); 332(M-H)- (ES-), in 1.51 min, 98% purity @ 215 nm.

[103] 1H NMR (400 MHz, CD3OD): d 6.96 (1H, t, J = 5.8), 5.43 (1H, br. s), 3.27 - 3.15 (2H, m), 3.11 (1H, br. s) , 2.99 (1H, d, J = 14.9), 2.89 (1H, d, J = 14.9), 2.84 - 2.76 (1H, m), 2.51 (1H, dd, J = 16.4, 7.8), 2.17 (2H, q , J = 7.4), 2.07 (1H, br. d, J = 16.4), 1.90 (1H, ddd, J = 11.8, 8.7, 2.6), 1.56 (1H, dd, J = 12.1, 7.3), 1.46 (7H , s,tBu(larger rotamer)), 1.44 (2H, s, tBu(smaller rotamer)), 1.11 (3H, t, J = 7.5) ppm. (Dichloromethane also present: 5.49 (0.5H, s)) Step 3 (compound of Formula 2) (((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [ 3.2.0]hept-3-en-6- racemic methanamine

[104] To a solution of tert-butyl(((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3-en-6 -yl)methyl)carbamate (1.3 g, 3.90 mmol) in dichloromethane (50 ml) trifluoroacetic acid (30 ml, 389 mmol) was added and the mixture was allowed to stand at room temperature for 30 minutes.

[105] The mixture was evaporated to dryness and the residue was redissolved in toluene (60 ml) and evaporated to dryness, this procedure was repeated three times. The residue was dissolved in a 1:1 mixture of methanol and water (30 m). This solution was applied to a 100-200 mesh (10 g) Dowex® 50WX8 hydrogen form ion exchange resin. The resin was washed and eluted with water until the eluent was neutral. The product was then eluted using a 2N methanolic ammonia solution to give a colorless gum after evaporation. This residue was triturated with acetonitrile (13 ml) to give ((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo[3.2.0]hept-3-en - Racemic 6-yl)methanamine as a colorless solid (365 mg, 1.50 mmol, 38.5%).

[106] LCMS (Agilent, X-Select, Waters /z 234(M+H)+ (ES+); 232(M-H)- (ES-), in 1.01 min.

[107] LCMS (Agilent, X-Select, Waters , m/z 234(M+H)+ (ES+); 232(M-H)- (ES-), in 1.21 min.

[108] 1H NMR (400 MHz, CD3OD): d 5.40 (1H, d, / = 1.8), 3.15 (3H, m), 3.05 (1H, d, / = 15.3), 2.94 (1H, d, /= 15.3), 2.83 (1H, m), 2.52 (1H, br. dd, / = 16.4, 7.8), 2.19 (2H, q, / = 7.8), 2.10 (1H, br. d), 1.93 (1H, ddd , / = 12.4, 8.7, 2.7), 1.64 (1H, dd, / = 12.6, 7.5), 1.13 (3H, t, 7 = 7.4) ppm.

[109] 13C NMR(100 MHz, CD3OD): d 159.86, 151.60, 122.57, 53.69, 47.96, 44.73, 43.03, 36.87, 30.21, 25.40, 12.84 ppm. Step 4 Chiral resolution of enantiomers of ((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl)methanamine racemic

[110] ((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl)methanamine (155 mg) was resolved using a Diacel Chiralpak IC, 5 µm, 20x250 mm, 15 ml/min, 50% ethanol:50% isohexane. After evaporation of the fractions, peak 1 was obtained with a retention time of 9.91 min (32 mg) and peak 2 with a retention time of 18.91 min (28 mg).

[111] Chiral Analytical Chromatography: Diacel Chiralpak IC, 5 µm, 4.6x250 mm, 30 min method, 1.5 mL/min, 30% ethanol: 70% isohexane peak 1 retention time 9, 49 min%, peak 2 retention time of 19.41 min at 215 nm. Example 3: Synthesis of the compound of Formula 2 Synthesis of N-(((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept- 3- racemic en-6-yl)methyl)ethanamine

[112] To a suspension of (((1R,5S,6S)-6-((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl) racemic methyl)ethanamine (compound of Formula 2) (50 mg, 0.214 mmol) in dry dichloroethane (10 ml) acetaldehyde (121 µl, 2.143 mmol) was added and the mixture was stirred for 20 min during which time the solution became clear.

[113] The solvent was removed by rotary evaporation and the residue was taken up in ethanol (10 ml) and to this solution sodium borohydride (81 mg, 2.143 mmol) was added and the mixture was stirred for 20 minutes.

[114] The reaction mixture was acidified to pH 1 by dropwise addition of 1N hydrochloric acid and the resulting mixture evaporated to dryness.

[115] The crude product was purified by preparative HPLC (Waters, basic (0.1% ammonium bicarbonate), Basic, Waters X-Bridge Prep-C18, 5 µm, 19x50 mm, 20-50% acetonitrile in water) to obtain N-(((1R,5S,6S)-6- ((1H-tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3-en-6-yl) methyl)ethanamine (24 mg, 0.090 mmol, 42.0% yield) as a colorless solid.

[116] LCMS (Agilent, basic, Waters X-Bridge C18, 2.5 µm, 4.6x30 mm, Basic (0.1% ammonium bicarbonate) method 4 min, 5-95% acetonitrile/water): m /z 262(M+H)+(ES+); 260(M-H)- (ES-), in 1.32min, 98% purity @ 215 nm.

[117] 1H NMR (400 MHz, CD3OD): d 5.35 (1H, d, J = 1.8), 3.25 (2H, dd, J = 16.0, 13.0), 3.18 -3.12 (3H, m), 3.08 (1H, d, J = 15.5), 2.99 (1H, d, J = 15.5), 2.88 (1H, quin, J = 7.4), 2.52 (1H, dd, J, = 16.4, 7.8), 2.21 - 2.16 (2H, m ), 2.10 (1H, d, J = 16.4), 1.99 (1H, ddd, J = 12.4, 8.7, 2.7), 1.60 (1H, dd, J = 12.4, 7.5), 1.40 (3H, t, J = 7.3 ), 1.12 (3H, t, J = 7.4) ppm.

[118] 13C NMR (100 MHz, CD3OD): d 160.10, 151.80, 122.38, 56.59, 53.89, 45.05, 44.50, 42.99, 37.25, 31.93, 31.08, 25.39, 12.80, 11.5 3 ppm. Example 4: Alternative route for the preparation of Compound 10 Step 1 (Compound of Formula 14) (2E/Z)-2-((1R,5S)-3-ethyl-6-bicyclo [3.2.0]hept-3- enylidene)acetonitrile

[119] To a solution of 1.78 M potassium tert-butoxide in tetrahydrofuran (64 mL, 113.9 mmol) diluted with tetrahydrofuran (45 mL) and cooled to 0 °C was added diethyl cyanomethylphosphate ( 21.16g, 119 mmol). The reaction mixture was stirred at 0°C for 10 minutes and allowed to warm to room temperature and stirred for a further 30 minutes. The mixture was transferred to a pressure equalizing funnel and added dropwise to a solution of racemic (1R,5S)-3-ethylbicyclo[3.2.0]hept-3-en-6-one (14.8 g, 109 mmol) in tetrahydrofuran (140 mL) at 0 °C. The mixture was warmed to room temperature and stirred for 18 hours.

[120] The mixture was diluted with saturated aqueous ammonium chloride (100 mL) and ethyl acetate (200 mL) and the layers were separated. The aqueous layer was extracted with ethyl acetate (50 ml) and the combined organic layers were washed with brine (50 ml) and dried over magnesium sulfate. The residue after filtration was purified by chromatography on silica (2.5% ethyl acetatedsohexane) to obtain (2E)-2-((1R,5S)-3-ethyl-6-bicyclo[3.2.0]hept-3 -enylidene)acetonitrile racemic as a mixture of E/Z isomers (14.45 g, 84%).

[121] LCMS (Agilent, Waters C18 Sunfire, 4.6x30mm, formic acid, acetonitrile/water): m/z 160.2 (M+H)+ ES+ in 2.88 min.

[122] 1H NMR (400 MHz, CDCl3): ~60:40 mixture of d alkene isomers 5.43 (0.4H, m), 5.23 (0.6H, m), 5.09 (0.6H, m), 4.98 (0.4H , m), 4.12 (0.4H, br s), 3.93 (0.6H, br s), 3.19-2.90 (2H, m), 2.74-2.46 (2H, m), 2.29-2.07 (2H, m), 1.141 .06 (3H, m). Step 2 (compound of formula 6) 2-((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo [3.2.0]hept-3-en-6-yl)racemic acetonitrile

[123] To a solution of (1R,5S)-3-ethylbicyclo [3.2.0]hept-3-en-6-one (9.59 g, 60.3 mmol) in nitromethane (75 mL, 84.6 g, 1.38 mol) under nitrogen was added 1,8-diazabicyclo [5.4.0]undec-7-ene (10 mL, 10.2 g, 66.9 mmol) and the mixture was stirred for 18 hours at temperature environment.

[124] The reaction mixture was poured into a 5% aqueous solution of potassium dihydrogen phosphate orthophosphate (400 mL) and ethyl acetate (300 mL) added. The layers were separated and the aqueous layer was further extracted with ethyl acetate (2 x 150 ml). The combined organic layers were dried over magnesium sulfate and evaporated to obtain a crude product that was combined with the crude product from a previous reaction carried out at half scale. The residue was purified by chromatography on silica (5-10% acetate-sohexane acetate) to obtain 2-((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3- en-6-yl)acetonitrile (16.5 g, 75 mmol, 83% yield) as a ~2:1 mixture of diastereomers. Data for the larger diastereomer.

[125] LCMS (Agilent, Waters C18 Sunfire, 4.6x30mm, Acidic (0.05% formic acid, 6 min method, 3-97% acetonitrile/water): m/z 221 (M+H)+ (ES+ ) in 2.79 min.

[126] 1H NMR (400 MHz, DMSO-d6): d 5.32 (1H, d, J = 2.1), 4.87 (2H, s), 3.16 (1H, br. s), 2.97 -2.82 (1H, m) , 2.65 (2H, s), 2.48 - 2.40 (1H, m), 2.23 (1H, ddd, J = 12.4, 8.8 ,2.5), 2.16 - 2.02 (3H, m), 1.56 (1H, dd, J = 12.5 , 7.2), 1.07 (3H, t, J = 7.5) ppm. Step 3 (compound of Formula 10) 5-(((1R,5S,6S)-3-ethyl-6-(nitromethyl)-6-bicyclo [3.2.0]hept-3-enyl)-methyl)-1H- racemic tetrazole

[127] To a solution of racemic 2-((2R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo [3.2.0] hept-3-en-6-yl)acetonitrile (200 mg, 0.909 mmol) in dry toluene (4 mL) was added azidotrimethylsilane (590 µl, 4.44 mmol) and dibutyltin oxide (113 mg, 0.45 mmol). The container was sealed and heated at 110°C for 18 hours. The mixture was cooled to room temperature and partitioned between water (20 ml) and ethyl acetate (20 ml). The organic layer was treated with 2M sodium hydroxide solution (20 ml) and the aqueous layer was separated and then acidified with cone hydrochloric acid to ~pH1. The acidic aqueous layer was re-extracted with ethyl acetate (2 x 20 mL) and the combined organic layers were dried over magnesium sulfate. Filtration and evaporation gave a crude product purified by chromatography on silica (50% ethenisohexane) to obtain 5-(((1R,5S,6S)-3-ethyl-6-(nitromethyl)-6-bicyclo[3.2. 0] hept-3-enyl)methyl)-1H-tetrazol (10 mg, 0.038 mmol, 4% yield).

[128] LCMS (Agilent, Waters C18 Sunfire, 4.6x30mm, Acidic (0.05% formic acid, 6 min method, 3-97% acetonitrile/water): m/z 264 (M+H)+ (ES+ ); 262 (M-H)- (ES-), in 2.35 min.

[129] 1H NMR (400 MHz, DMSO-d6): d 11.09 (1H, br. s), 5.37 (1H, d, J = 1.2), 4.80 (2H, s), 3.22 (1H, br. s) , 3.01 (2H, s), 2.93 - 2.81 (1H, m), 2.50 - 2.40 (1H, m), 2.19 - 2.05 (4H, m), 1.63 (1H, dd, J = 12.5, 7.5), 1.05 ( 3H, t, J = 7.6) ppm. Alternative conditions for step 3 (Compound of Formula 10) 5-(((1R,5S,6S)-3-ethyl-6-(nitromethyl)-6-bicyclo [3.2.0]hept-3-enyl)-methyl )-1H- racemic tetrazole

[130] To a solution of racemic 2-((1R,5S,6S)-3-ethyl-6-(nitromethyl)bicyclo[3.2.0]hept-3-en-6-yl)acetonitrile (170 mg, 0.772 mmol) in 1-methyl-2-pyrrolidinone (2.7 mL) was added pyridine hydrochloride (180 mg, 1.57 mmol) and sodium azide (263 mg, 4.04 mmol). The flask was heated under nitrogen at 100 °C for 18 hours. The temperature of the flask was then increased to 117-120°C for a further 4 hours after which it was allowed to cool to room temperature. The mixture was poured into water (20 ml) and carefully acidified with 2M aqueous hydrochloric acid. The aqueous layer was extracted with ethyl acetate (2 x 20 mL) and then the organic layer was stirred with a 2M sodium hydroxide solution (1 x 20 mL, 1 x 10 mL). The combined aqueous layers were then acidified with cone hydrochloric acid to ~pHL and re-extracted with ethyl acetate (3 x 20 mL). The combined organic layers were washed with water (10 mL) and dried over magnesium sulfate. Filtration and evaporation gave a crude product purified by silica chromatography (7 g silica, diethyl ether: isohexane: acetic acid 200:300:8) to give 5- (((1R,5S,6S)- 3-ethyl-6-(nitromethyl)-6-bicyclo[3.2.0]hept-3-enyl)methyl)-1H-tetrazol (81 mg, 0.304 mmol, 40% yield).

[131] LCMS (Agilent, Waters C18 Sunfire, 4.6x30mm, Acidic (0.05% formic acid, 6 min method, 3-97% acetonitrile/water): m/z 264 (M+H)+ (ES+ ); 262 (M-H)- (ES-), in 2.35 min 1H NMR (400 MHz, DMSO-d6): d 16.10 (1H, br. s), 5.34 (1H, d, J = 1.4), 4.80 ( 2H, s), 3.22 (1H, br. s), 3.02 (2H, s), 2.94 - 2.81 (1H, m), 2.48 - 2.40 (1H, m), 2.19 - 2.05 (4H, m), 1.64 ( 1H, dd, J = 12.5, 7.4), 1.05 (3H, t, J = 7.4) ppm.

Example 5

[132] The therapeutic activity of the compounds of the present invention was demonstrated by an a2d-1 affinity binding assay. This test was performed as follows. Calcium Channel Subunit a2d-1 Binding Assay

[133] This section describes a scintillation proximity assay (SPA) to measure binding of [H]gabapentin ([3H]GBP) to membranes containing a2d-1 and its use for compound profiling (Calvo et al (2012) J. Biomol. Screen. 17: 1041-1049).

[134] Human Cav1.2/ß3/a2d-1 Calcium Channel Membranes (Chantest) were thawed on ice, aliquoted, and stored at -80 °C for subsequent use. Membranes were diluted to 200 µg/ml (3 µg/final well assay concentration (FAC)) in assay buffer (10 mM HEPES (Sigma), (pH 7.4)). [3H]GBP stock solution (Perkin Elmer) was stored at −20 °C. [3H]GBP was diluted to 40 nM (10 nM FAC) in assay buffer. SPA beads (Perkin Elmer) were resuspended at 100 mg/ml in 10 mM HEPES (pH 7.4). Beads were diluted to 40 mg/ml (0.6 mg/FAC well) in assay buffer. Non-specific binding (NSB) was generated using an excess of pregabalin (Tocris). Pregabalin was solubilized in 10 mM Milli-Q H2O. 10 mM Pregabalin was diluted to 400 µM (100 µM FAC) in assay buffer.

[135] Compounds were diluted to 100 µM and then diluted in half log series. These were then diluted 1:100 in assay buffer to an assay concentration of 4X (a higher FAC dilution of 1 µM).

[136] SPA granules 15 µl; Membranes 15 µl; Pregabalin or assay buffer/test compound 15 µl and [3H]GBP 15 µl were added to a white 96-well isoplate, (Perkin Elmer). The assay plate was sealed and mixed for 10 seconds on a plate shaker, then placed in plate stack and inserted into the reader stacker. The plate was incubated overnight (20 hours) and then read on a 1450 MicroBeta TriLux Microplate Scintillation and Luminescence Counter at room temperature (RT). Data analysis

[137] The NSB values obtained by adding 100 µM pregabalin were subtracted from the values obtained for the compounds to generate a specific binding value. Specific binding in counts per minute (cpm) was plotted against compound concentration (M) and fitted using a 4-parameter logistic equation. Compound IC50 values were calculated, where the compound concentration produces a 50% inhibition of specific binding. The results of the test compounds are shown in Table 1. Table 1: Synthesized compounds and a2d-1 binding affinity data: IC50 values for compounds tested in the [3H] gabapentin binding assay using cell membranes that express a recombinant a2d-1 with the ß3 ancillary protein (Chan test) t: No relevant literature.

Example 6 Method to determine binding kinetic parameters (association and dissociation rates) and affinities (Kd) of GABA analogues for a2d-1 subunits.

[138] It is well known in the art that binding affinity can be expressed in KD, or dissociation equilibrium constant, wherein increasing binding affinity correlates with decreasing KD, which can be calculated from the kinetic constants binding. Binding kinetic analysis of GABA analogues to a2d-1 subunits can be determined by surface plasmon resonance technology applied on Biacore™ SPR instruments (Biacore, GE Healthcare, Uppsala). Kinetic association rates (ka; k-on) and dissociation rates and (kd; k-off) are obtained. Dissociation equilibrium constant (KD; affinity) values are calculated as k-off/k-on.

[139] In the prior art, and in scientific publications, a short region within the full-length voltage-gated calcium channel a2d-1 subunits is referred to as the binding site for gabapentin and pregabalin (Wang et al (1999 ) Biochem. J. 342, 313-320; Field et al (2006) Proc. Natl. Acad. Sci. U.S.A, 14, 103, 17537-17542).

[140] This example reveals that the GABA analogs ((1R,5S,6S)-6 -((1H- tetrazol-5-yl)methyl)-3-ethylbicyclo [3.2.0]hept-3-en-6 -yl)methanamine ("Test Compound": prepared as described in Example 2) and (1R,5S,6S)-6-(aminomethyl)-3-ethylbicyclo[3.2.0]hept-3-en-6- acid yl]acetic acid ("Reference Compound": prepared as described in WO 2010/079668 and US 2012 / 0071685A1) are capable of binding to a recombinant fragment of the human voltage-gated calcium channel subunit (a2d-1, CACNA2D1 ). Furthermore, comparative kinetic binding analysis of these two compounds was performed in order to identify and characterize the potentially different binding properties of these two compounds to their target (CACNA2D1).

[141] Recombinant human CACNA2D1 was covalently immobilized at a high density (26000 RU) to the surface of a Biacore CM5 optical sensor chip as target ligand using thiol coupling chemistry, according to the manufacturer's instructions (Biacore Thiol Coupling Kit, Order Code: BR-10057; GE-Healthcare, Uppsala). Bovine serum albumin was immobilized on a reference flow cell in order to compensate for nonspecific background binding. Increasing concentrations of the test compound and the reference compound, dissolved in Biacore HBS-P buffer (Order code: BR-100368; GE-Healthcare, Uppsala), were passed through the flow cells at a flow rate of 30 µl. /min. The sensor chip surfaces were regenerated after each assay with 10mM hydrochloric acid (flow rate = 30µl/min) in order to remove pre-bound material and reconstitute active compound binding sites. Subtractive sensorgrams (CACNA2D1 recombinant protein minus reference BSA) were then generated.

[142] Kinetic binding analysis was subsequently performed by unique mathematical sensorgram assembly of each subtractive binding sensorgram using a 1:1 Langmuir interaction algorithm as provided by BiaEvaluation 4.0 software.

[143] The experimental results described in the current example revealed that both compounds under study were capable of specifically binding to the recombinant human CACNA2D1 protein. Furthermore, the data disclosed in this example allows us to differentiate the binding properties between the test compound and the reference compound. These differences are predictive of an increase in the pharmacological activity of the test compound over the reference compound.

[144] It will be understood that the above examples are intended as illustrations of the present invention and are not intended to be limiting in any way. The scope of the invention, therefore, shall be determined with reference to the appended claims, together with the full scope of the equivalents to which the claims are directed.

Claims (12)

1. Compound, characterized by having Formula 1 wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group; R2 represents or a tautomer thereof; and R3 represents hydrogen, a C1-4 alkyl group, a (C1-4 alkoxy)(C2-4 alkyl) group or a C3-7 cycloalkyl group, or a pharmaceutically acceptable salt thereof. 2. Compound, according to claim 1, characterized by the fact that it has Formula 2 wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group, or a pharmaceutically acceptable salt thereof. 3. Compound, according to claim 1, characterized by the fact that it has Formula 3 wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group, or a pharmaceutically acceptable salt thereof. 4. Compound according to any one of claims 1 to 3, characterized in that R1 represents ethyl. 5. Compound, according to claim 1, characterized by the fact that it has Formula 4 6. Compound, according to claim 5, characterized by the fact that it has Formula 4a 7. Compound, according to claim 3, characterized by the fact that it has Formula 5 8. Pharmaceutical composition, characterized by the fact that it comprises a compound as defined in any one of claims 1 to 7 and a pharmaceutically acceptable carrier for the same. 9. Use of a compound of Formula 1 or a pharmaceutically acceptable salt thereof, as defined in any one of claims 1 to 7, characterized in that it is for the manufacture of a medicament for treating neuropathic pain, epilepsy, anxiety, pain headache or migraine in a patient in need of the same. 10. Process for preparing a compound of Formula 1, as defined in any one of claims 1 to 7, characterized by the fact that it comprises the step of reducing a compound of Formula 12 wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group; and R2 represents or a tautomer thereof. 11. Process for preparing a compound of Formula 2a, wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group; characterized by the fact that it comprises the step of resolving a mixture of Formula 1 enantiomers wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group; R2 represents or a tautomer thereof; and R3 represents hydrogen. 12. Intermediate compound, characterized by the fact that it has Formula 16 wherein R1 represents hydrogen, halo, a C1-4 alkyl group, a C1-4 alkyhalide group, a (C1-4 alkoxy)(C2-4 alkyl) group, a C2-4 alkenyl group, a C2-4 alkynyl group or a C3-7 cycloalkyl group; R2a represents -R2; and R2 represents or a tautomer thereof.