WO2016099394A1

WO2016099394A1 - Novel selective delta-opioid receptor agonists useful for the treatment of pain, anxiety and depression.

Info

Publication number: WO2016099394A1
Application number: PCT/SE2015/051364
Authority: WO
Inventors: Bengt Von Mentzer; Ingemar Starke; Peter Brandt
Original assignee: Pharmnovo Ab
Priority date: 2014-12-19
Filing date: 2015-12-17
Publication date: 2016-06-23

Abstract

A compound of formula I, For use in pain therapy and a pharmaceutical composition comprising said compound.

Description

Novel selective delta-opioid receptor agonists useful for the treatment of pain, anxiety and depression.

Field of Invention

The present invention is directed to novel compounds, to a process for their preparation, their use and pharmaceutical compositions comprising the novel compounds. The novel selective δ-opioid receptor agonists are useful in therapy, and in particular for the treatment of pain, anxiety and depression.

Background of Invention

Delta opioid receptors (DORs) have been considered as a potential target to relieve pain conditions and depression and anxiety disorders. Physical pain is a typical sensory experience that may be described as the unpleasant awareness of a noxious stimulus or bodily harm. Individuals experience pain by various daily hurts and aches, and sometimes through more serious injuries or illnesses. For scientific and clinical purposes, pain is defined by the International Association for the Study of Pain (IASP) as "an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage".

Pain is a common reason for physician consultation. It is a major symptom in many medical conditions, significantly interfering with a person's quality of life and general functioning. Diagnosis is based on characterizing pain in various ways, according to duration, intensity, type (dull, burning, throbbing or stabbing), source, or location in body. Usually pain stops without treatment or responds to simple measures such as resting or taking an analgesic, and it is then called 'acute' pain. But it may also become intractable and develop into a condition called chronic pain, in which pain is no longer considered a symptom but an illness by itself.

Pain is the most common symptom for which patients seek medical advice and treatment. Pain can be acute or chronic. While acute pain is usually self-limited, chronic pain can persist for 3 months or longer and lead to significant changes in a patient's personality, lifestyle, functional ability or overall quality of life (K. M. Foley, Pain, in Cecil Textbook of Medicine 100-107, J. C. Bennett and F. Plum eds., 20th ed. 1996).

Current analgesic therapies are only poorly effective and are associated with significant side effects, especially concerning chronic conditions.

The impact of chronic pain can vary from mild discomfort to the worst pain

imaginable, having a devastating effect on the sufferers and their families. The types of pain are many and varied and include lower back pain, arthritis (especially osteoarthritis), headache (including migraine), fibromyalgia, nerve damage

(neuropathy), neurological disease (e.g. multiple sclerosis), and post-viral illness (e.g. shingles). l Other CNS-related diseases such as Alzheimer's and Parkinson's have a high prevalence of associated pain. The incidence of chronic pain increases with advancing age and is associated with, and potentiated by depressive illness and loss of sleep.

DORs have multiple roles in the central nervous system disorders beside pain, such as depression, anxiety, epilepsy, and stress; gastrointestinal disorders such as diarrhea, postoperative ileus, ulceration, and irritable bowel syndrome and related inflammatory disorders such as osteoarthritis and rheumatoid arthritis, and others including respiratory, alcoholism and obesity/binge eating. Thus, the current compounds can be used in the cure of many diseases.

The identification of at least three different populations of opioid receptors (δ), (μ) and (κ) receptors is now well established and all three are apparent in both central and peripheral nervous systems of many species including man.

A recent survey of more than 45,000 people in 16 countries revealed that almost 1 in 5 of the European population suffers moderate or severe pain persisting for more than 6 months. Chronic pain, therefore, constitutes an enormous burden on national economies and patients' quality of life. Given the number of different chronic pain states, the patient population is extremely varied, although it is very likely to be skewed towards the upper age range with neuropathies and joint pain being particularly prominent. In institutionalized elderly patients for example, up to 80% report a current pain problem although a significant proportion receive inadequate analgesic therapy. There is, therefore, an urgent need for novel pain therapies that are effective over prolonged periods with low side-effect risk.

The commonly used opioids, such as, codeine, dihydrocodeine (for mild to moderate pain) and oxycodone, tramadol etc. (for severe pain) are either non-selective, acting on all three opioid receptor sub-types or somewhat μ receptor-biased and they produce the full range of both beneficial and unwanted effects.

The widespread distribution of opioid receptor subtypes in the CNS and peripheral tissues results in the associated side-effects of nausea, constipation, itching and danger of life-threatening respiratory depression, produced by mixed opioid ligands. The development of tolerance additionally limits their clinical efficacy.

The development of tolerance additionally limits their clinical efficacy. Meta-analysis- derived evidence regarding the clinical efficacy of traditional opioids and other commonly used analgesics in chronic pain states is weak or negative; amitriptyline (a first line analgesic for neuropathic pain) benefits less than 40% of patients and, only about one third of patients treated with gabapentin obtain significant relief. The currently available voltage-activated sodium channel blockers also have very limited efficacy and significant CNS and cardio toxicity risks. This commonality offers the possibility that modifying the activity of individual targets, such as the δ-opioid receptor (DOR) will provide benefit in pain treatment.

Outline of Invention so Compounds of this invention show selective high potency for the δ-opioid receptor (DOR). Compounds of the invention deliver a widened therapeutic window, and have the potential, in contrast to existing analgesics delivering only moderate pain relief, to produce maintained analgesia in pain states with less risk of unwanted effects including respiratory depression and constipation.

85 Compounds of this invention will significantly expand treatment options for clinicians and increase the quality of life for a huge number of patients.

The present invention provides a compound of formula (I)

90 (I)

Wherein:

R is selected from anyone of

According to an aspect of the invention, a compound of formula I is

la

According to another aspect of the invention, a compound of formula I is

lb

According to another aspect of the invention, a compound of formula I is

According to another aspect of the invention, a compound of formula I is

Id According to an aspect of the invention the compounds of formula I are used in pain therapy, such as treating acute pain, and chronic pain.

no Compounds of the invention are highly potent and will retain analgesic potency on repeated administration. Compounds of the invention will be useful in therapy, especially for the treatment of various pain conditions such as chronic pain, neuropathic pain, acute pain, cancer pain, pain caused by rheumatoid arthritis, osteoarthritis, fibromyalgia, migraine, visceral pain, diabetic pain, etc.

lis Compounds of the invention are equally useful for the treatment of depression and anxiety as for various types of pain conditions, as mentioned above.

Compounds of the invention are useful for the treatment of urinary incontinence, various mental illnesses, cough, lung edema, various gastro-intestinal disorders (irritable bowel syndrome, irritable bowel disease), spinal injury and drug addiction, 120 including the treatment of alcohol, nicotine, opioid and other drug abuse and for disorders of the sympathetic nervous system for example hypertension.

Compounds of the invention are useful as immunomodulators, especially for autoimmune diseases, such as arthritis, for skin grafts and organ transplants.

Compounds of the invention are useful in disease states where degeneration or 125 dysfunction of opioid receptors is present or implicated in that paradigm. This may involve the use of isotopically labeled versions of the compounds of the invention in diagnostic techniques and imaging applications such as positron emission

tomography (PET).

Compounds of the invention are useful as an analgesic agent for use during general 130 anesthesia and monitored anesthesia care. Combinations of agents with different properties are often used to achieve a balance of effects needed to maintain the anesthetic state (e.g. amnesia, analgesia, muscle relaxation and sedation). Included in this combination are inhaled anesthetics, hypnotics, anxiolytics, neuromuscular blockers, neuropeptide receptor blockers and other opioids.

135 Compounds of the invention can be used in combination therapy with other pain

effective compounds.

Within the scope of the invention is the use of the compound of formula I above, for the manufacture of a medicament for the treatment of any of the conditions discussed above.

140 A further aspect of the invention is a method for the treatment of a subject suffering from any of the conditions discussed above, whereby an effective amount of compound according to the formula I above, is administered to a patient in need of such treatment. According to another aspect of the invention there is provided for a pharmaceutical 145 composition comprising at least a compound of the formula I as an active

ingredient, or a physiologically acceptable salt thereof together with a

pharmaceutically acceptable carrier.

Methods of Preparation

150 The compounds of formula (I) prepared as outlined in scheme 1 .

Wherein R'CH=0 is:

155

Scheme 1 Example 1 d has been made by 1 ) de-protection of the 1 -nitrogen of compound III to 160 form compound VII, 2) reductive amination with 3-pyridinaldehvde to form compound VIII, and 3) coupling reaction with 3-aminocarbonylphenyl boronic acid.

The following abbreviations have been used:

Dppf = (diphenylphosphino)ferrocene

165 HATU = 1 -[Bis(dimethylamino)methylene]-1 /-/-1 ,2,3-triazolo[4,5-Jb]pyridinium 3-oxid hexafluorophosphate

Examples

170 The Invention is illustrated by the following examples;

la

la. 3-({4-[(2-Hydroxyethyl)(methyl)carbamoyl]phenyl}(1 -{[6-(trifluoromethyl) 180 pyridin-3-yl]methyl}piperidin-4-ylidene)methyl)benzamide

3-({4-[(2-Hydroxyethyl)(methyl)carbamoyl]phenyl}(piperidin-4-ylidene)methyl) benzamide (79.0 mg, 0.20 mmol) was dissolved in 1 ,2-dichloroethane (2 ml_) and acetic acid (57 μΙ_, 1 .0 mmol) was added followed by 6-(trifluoromethyl)

185 nicotinaldehyde (70.0 mg, 0.40 mmol) and NaBH(OAc)₃ (169 mg, 0.8 mmol). The reaction was stirred at 50°C for 48 h. The crude was purified by flash

chromatography (ethyl acetate and ethyl acetate/methanol 75:25) to give the title compound as a crude material. This material was then purified by preparative HPLC (X-bridge column, 50 mM NH4HCO3 as mobile phase, CH3CN from 10 to 40) to give

190 12 mg of the title compound. ¹H NMR (CD3OD): δ 2.37 (m, 2H), 2.42 (m, 2H), 2.57 (m, 4H), 3.07 (d, 3H), 3.33 (s, 1 H), 3.41 (m, 1 H), 3.62 (m, 2H), 3.69 (s, 2H), 3.79 (m, 1 H), 7.23 (m, 2H), 7.31 (m, 1 H), 7.39 (m, 3H), 7.66 (m, 1 H), 7.72 (dt, 1 H), 7.78 (d, 1 H), 8.03 (m, 1 H), 8.68 (m, 1 H). MS m/z 553 [M+H]⁺.

lb lb. 3-({4-[(2-Hydroxyethyl)(methyl)carbamoyl]phenyl}({l-[(4-methyl-lH-imidazol-5- 205 yl)methyl]piperidin-4-ylidene})methyl)benzamide

3-({4-[(2-Hydroxyethyl)(methyl)carbam

benzamide (79.0 mg, 0.2 mmol) was dissolved in 1 ,2-dichloroethane (2 mL) and acetic acid (57 μΙ_, 1 .0 mmol) was added followed by 5-methylimidazole-4-

210 carboxaldehyde (44.0 mg, 0.4 mmol) and NaBH(OAc)3 (169 mg, 0.8 mmol). The reaction was stirred at 50°C for 48 h. The crude was purified by flash

chromatography (ethyl acetate, ethyl acetate/methanol 75:25) to give the title compound as a crude material. This material was then purified by preparative HPLC (X-bridge column, 50 mM NH4HCO3 as mobile phase, CH3CN from 10 to 40) to give

215 5 mg of the title compound. MS m/z 488 [M+H]⁺.

Ic

Ic. 3-({4-[(2-Hydroxyethyl)(methyl)carbamoyl]phenyl}[1 -(1 ,3-thiazol-5- ylmethyl)piperidin-4-ylidene]methyl)benzamide

225

3-({4-[(2-Hydroxyethyl)(methyl)carbamoyl]phenyl}(piperidin-4-ylidene)methyl) benzamide (79.0 mg, 0.20 mmol) was dissolved in 1 ,2-dichloroethane (3 mL) and acetic acid ( 57 μΙ_, 1 .0 mmol) was added followed by 5-thiazolecarboxaldehyde (45.3 mg, 0.4 mmol) and NaBH(OAc)3 (169 mg, 0.8 mmol), The reaction was stirred at

230 room temperature overnight. The reaction was then headed to 50°C for 24 h.

Methylene chloride (10 mL) was added and the mixture was washed with brine (2 x 5 mL), dried over Na2S0₄ and concentrated. The residue was purified by flash chromatography (ethyl acetate/methanol 80:20) to yield 27 mg of the title compound. The product was further purified by preparative HPLC (X-bridge column, 50 mM

235 NH₄HC03 as mobile phase, ChhCN from 10 to 40) to give 9 mg of the title

compound. ¹H NMR (CD₃OD): δ 2.36 (m, 2H), 2.41 (m, 2H), 2.56 (m, 4H), 3.06 (d, 3H), 3.30 (s, 1 H), 3.41 (m, 1 H), 3.62 (m, 2H), 3.80 (m, 1 H), 3.85 (s, 2H), 7.22 (m, 2H), 7.30 (dt, 1 H), 7.39 (m, 3H), 7.65 (m, 1 H), 7.72 (m, 1 H), 7.72 (m, 1 H), 7.75 (s, 1 H), 8.94 (s, 1 H). MS m/z 491 [M+H]⁺.

240

Id

Id. 3-({4-[(2-Hydroxyethyl)(methyl)carbamoyl]phenyl}[1 -(pyridin-3-ylmethyl) piperidin-4-ylidene]methyl)benzamide

250

4-{Bromo[1 -(pyridin-3-ylmethyl)piperidin-4-ylidene]methyl}-N-(2-hydroxyethyl)-N- methylbenzamide (88 mg, 0.20 mmol) and (3-aminocarbonylphenyl)boronic acid (39 mg, 0.24 mmol) were dissolved in dimethoxyethane (1.5 mL) and 2 M K2CO3 (0.10 mL, 0.20 mmol) and PdCl2(dppf) (8 mg, 0.01 mmol) were added under nitrogen and

255 the reaction was heated at 80°C for 3.5 h. The reaction was cooled to room

temperature and left over weekend. Methylene chloride and water were added and the phases separated. The aqueous phase was extracted with methylene chloride (3x). The combined organic phases were washed with brine, dried (Na2S0₄), filtered and concentrated. The crude material was purified by preparatory HPLC (MeCN (15

260 to 65%): 50 mM NH₄HC03), fractions were concentrated in vacuo, extracted with methylene chloride and evaporated in vacuo, to give 14.3 mg (15% ) of the title compound. MS m/z 485 [M+H]⁺. 265

Preparation of intermediates

Example III. ferf-Butyl 4-[bromo({4-[(2-hydroxyethyl)(methyl)carbamoyl]

275 phenyl})methylidene]piperidine-1 -carboxylate

4-[Bromo-(4-carboxy-phenyl)-methylene]-piperidine-1 -carboxylic acid tert-butyl ester (1 .19 g, 3.0 mmol) and 2-(methylamino)-ethanol (0.24 mL, 3.0 mmol) were mixed with methylene chloride (10 mL) at room temperature. After 10 minutes, N,N-

280 diisopropylethylamine (1.03 mL, 9.1 mmol) was added followed by HATU (1 .15 g, 3.0 mmol). The reaction mixture was stirred at room temperature overnight. Water (2 mL) was added followed by methylene chloride (80 mL). The mixture was washed with 30 mL of saturated aqueous NaHC03, 30 mL of 1 M HCI, 2 x 30 mL of water. The organic phase was dried over Na2S0₄, filtered and concentrated. The residue was

285 purified with flash chromatography (methylene chloride/methanol 95:5) to give 1 .234 g of the title compound. MS m/z 453, 455 [M+H]⁺.

IV

Example IV. ieri-Butyl 4-[(3-carbamoylphenyl)({4-[(2-hydroxyethyl)(methyl) 290 carbamoyl]phenyl}) methylidene]piperidine-1 -carboxylate fe/f-Butyl 4-[bromo({4-[(2-hydroxyethyl)(methyl) carbamoyl] phenyl})methylidene] piperidine-1 -carboxylate (1 .63 g, 3.0 mmol) was dissolved in a mixture of degassed 1 ,4-dioxane/H20 4/1 (30 ml_). (3-Aminocarbonylphenyl) boronic acid (0.594 g, 3.6 mmol), PdCI₂(dppf) (0.122 g, 0.15 mmol) and potassium carbonate (0.828 g, 6.0 mmol) were added. The reaction mixture was heated to 90°C for 1 h. The reaction was diluted with ethyl acetate (150 ml_), and the organic layer was washed with 3 x 50 ml_ of brine, dried over Na2S0₄, filtered and concentrated to give 1.433 g of the title compound. The residue was taken to the next step without additional purification. MS m/z 438 M+H]⁺ - f-Bu.

V

Example V. 3-({4-[(2-Hydroxyethyl)(methyl)carbamoyl]phenyl}(piperidin-4- ylidene)methyl)benzamide fe/f-Butyl 4-[(3-carbamoylphenyl)({4-[(2-hydroxyethyl (methyl)carbamoyl] phenyl}) methylidene]piperidine-1 -carboxylate (1 .433 g, 2.9 mmol) was dissolved in methylene chloride (35 ml_) and trifluoroacetic acid (7 ml_) was added. The reaction was then stirred at room temperature for 1 .5 h. Methanol (5 ml_) was added and the reaction mixture was concentrated to yield 1 .051 g of the title compound. The residue was taken to the next step without additional purification. MS m/z 394 [M+H]⁺.

Example VII. 4-[Bromo(piperidin-4-ylidene)methyl]-N-(2-hydroxyethyl)-N- methylbenzamide

fe/f-Butyl 4-[bromo({4-[(2-tert-hydroxyethyl)(methyl)carbamoyl]phenyl}) methylidene] piperidine-1 -carboxylate (623 mg, 1 .37 mmol) was dissolved in methylene chloride (15 mL). Trifluoroacetic acid (3 mL) was added drop wise. The reaction mixture was stirred for 120 min. The reaction mixture was evaporated and the residue was redissolved in methylene chloride (25 mL). The organic phase was washed with saturated aqueous K2CO3 (10 mL), dried (Na2S0₄), filtered and concentrated to give 325 503 mg of the title compound as a yellow oil with parts of white crystals. MS m/z 353, 355 [M+H]⁺.

VIII

335

Example VIII. 4-{Bromo[1 -(pyridin-3-ylmethyl)piperidin-4-ylidene]methyl}-N-(2- hydroxyethyl)-N-methylbenzamide

4-[Bromo(piperidin-4-ylidene)methyl]-N-(2-hydroxyethyl)-N-methylbenzamide (433 mg, 1 .22 mmol) and 3-pyridinaldehyde (144 mg, 1 .34 mmol) were dissolved in

340 dichloroethane (10 mL). Then acetic acid (0.07 mL, 1 .22 mmol) was added and the reaction stirred at 10 min before NaBH(OAc)3 (414 mg, 1 .96 mmol) was added. The reaction was stirred at room temperature over weekend. Methylene chloride (25 mL) was added and the mixture was washed with water. The aqueous phase was extracted with methylene chloride (3 x 20 mL). The combined organic phases were

345 dried (Na2S0₄), filtered and concentrated. The crude was purified by preparative HPLC (basic method) to give 196 mg of the title compound as a light yellow semisolid. MS m/z 444, 446 [M+H]⁺.

Pharmaceutical Compositions

350 There is provided a method for treatment of a condition which method comprises administration of a therapeutically effective amount of the compound of formula I to a person suffering from, or susceptible to, such a condition. The compounds of the invention will normally be administered via the oral, topical, parenteral, intravenous, intramuscular, subcutaneous, intra articular injection or in other injectable ways,

355 buccal, sublingual, rectal, vaginal, transdermal and/or nasal route and/or via

inhalation, in the form of pharmaceutical preparations comprising the active ingredient or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.

360 Suitable daily doses of the compounds of the invention in therapeutically treatment of humans are about 0.0001 -100 mg/kg body weight, preferably 0.01 -10 mg/kg body weight.

Oral formulations are tablets or capsules which may be formulated by methods known to those skilled in the art to provide doses of the active compound in the range 365 of 0.007 mg to 700 mg for example 1 mg, 3 mg, 5 mg, 10 mg, 25 mg, 50 mg, 100 mg , 250 and 500 mg.

The optimum dosage and frequency of administration will depend on the particular condition being treated and its severity; the species of the patient; the age, sex, size and weight, diet, and general physical condition of the particular patient; brain/body 370 weight ratio; other medication the patient may be taking; the route of administration; the formulation; and various other factors known to physicians and others skilled in the art.

According to a further aspect of the invention there is thus provided a pharmaceutical formulation including any of the compounds of the invention, or pharmaceutically

375 acceptable derivatives thereof, in admixture with pharmaceutically acceptable

adjuvants, diluents and/or carriers.

The compounds of the invention may be present in the pharmaceutical formulation in a concentration from 0.1 to 99.5%, such as from 0.5 to 95%, by weight of the total formulation.

380 For oral administration the compound of the invention may be admixed with an

adjuvant or a carrier, for example, lactose, saccharose, or sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the

385 like, and then compressed into tablets. If coated tablets are required, the cores,

prepared as described above, may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatine, talcum and titanium dioxide. Alternatively, the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.

390 For the preparation of soft gelatine capsules, the compound of the invention may be admixed with, for example, a vegetable oil or polyethylene glycol. Hard gelatine capsules may contain granules of the compound using either the above-mentioned excipients for tablets. Also liquid or semisolid formulations of the compound of the invention may be filled into hard gelatine capsules. 395 Liquid preparations for oral application may be in the form of syrups or suspensions, for example, solutions containing the compound of the invention, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol. Optionally such liquid preparations may contain coloring agents, flavoring agents, saccharine and/or carboxymethylcellulose as a thickening agent or other excipients known to those

400 skilled in art.

Biological Evaluation

In vitro

δ-opioid receptors (DOR) are coupled to inhibitory G proteins (Gi/o) which inhibit adenylyl cyclase activity, open K⁺ channels and block Ca²⁺ channels. They can also 405 couple, in an agonist-dependent manner to β-arrestin 2, which lead to

desensitization, and to MAP kinase (ERK) phosphorylation.

In vitro assessment of δ-opioid agonist potency: In vitro assessment of δ-opioid agonist potency: cAMP measurements will be made in δ-opioid-expressing CHO cells exposed to increasing agonist concentrations using DiscoveRx cAMP Hunter™

410 assay kits, β-arrestin recruitment will be measured in the presence of increasing

agonist concentrations using DiscoveRx Pathunter^R kits . Selectivity of mu- and kappa-opioid receptors will be performed using Cerep cAMP evaluation from ref. 1392 and 2071 (Cerep Le Bois I'Eveque, 86600 Celle I'Evescault, France).

415

in Vivo

Chronic Pain, Seltzer model

The Seltzer assessment is used to evaluate chronic pain. Thirty male CD1 mice (29- 37 g) are included in the study. They were obtained from Charles-River Ltd. and kept 420 in the Animal Facility of the Department of Pharmacology and Pharmacotherapy at the University of Pecs at 24-25 °C provided with standard chow and water ad libitum. All experimental procedures are carried out according to the 1998/XXVIII Act of the Hungarian Parliament on Animal Protection and Consideration Decree of Scientific Procedures of Animal Experiments (243/1988) and complied with the

425 recommendations of the Helsinki Declaration. The studies are approved by the Ethics Committee on Animal Research of Pecs University according to the Ethical Codex of Animal Experiments and license was given (license No.: BA 02/2000-1 1 -2006). After a conditioning measurement three reliable control mechano-nociceptive thresholds are determined on three consecutive days for all mice and the operation is performed 430 afterwards. On the 7^th day following the nerve ligation the mechano-nociceptive

thresholds are determined directly before and 15 min after i.p. drug administration to observe the difference between pre-injection and post-injection thresholds. Only mice with a minimum of 30% pre-injection hyperalgesia are included in the study.

The blood and the brain samples are taken 60 min after drug administration under 435 deep ketamin-xylazine anesthesia and sent for further analysis. Blood (0.5-1 .5 ml) is taken by cardiac puncture to EDTA-containing tubes and centrifuged. The plasma samples are stored at -70 ^°C. The whole brain with the cerebellum and the brainstem is dissected, put into Eppendorf tubes, immediately frozen in liquid nitrogen and they are stored at -70 ^°C.

440 Chronic Pain MNX OA model

Rats are anaesthetized using 2.5% Isoflurane (Abbott, Maidenhead, UK) in oxygen with a flow rate of 1 L per minute. The left leg is shaved and surgically prepared. The medial collateral ligament is exposed and a section of it removed to expose the meniscus. The meniscus is cut through its full thickness at the narrowest point. The

445 connective tissue layer and skin are then closed with coated Vicryl 8-0 and 4-0

sutures, respectively (Ethicon, Livingstone, UK). No post-operative analgesic drug is administered as pain behavior is an outcome measure of the experiment. Sham operated animals undergo an identical procedure with the exception that the meniscus is not transected. Effects of treatments on weight distribution through the

450 left (ipsilateral) and right (contralateral) knees are assessed using an incapacitance meter (Linton Instruments UK) . The change in hind paw weight distribution is defined as the difference in the amount of weight between the right contralateral control limb and the left ipsilateral treated limb divided by the sum of the weight right and left limbs x 100. Hind paw withdrawal thresholds to mechanical stimulation are measured

455 using calibrated von Frey monofilaments using the up down method. The animals are habituated on at least two occasions before commencement of the experiments. Baseline behavioral pain measurements are made at day 0 prior to model induction and then at day 14, 28, 35, and 49 days.

Acute Carrageenan-induced inflammatory pain

460 λ-Carrageenan (100 L 2% in saline; Sigma, Poole, UK) is injected into the plantar surface of the rat hind paw. Mechanical stimuli (8-100 g von Frey monofilaments) are applied (in ascending order) to the peripheral receptive field at 10-min intervals, for 180 min, following injection of carrageenan and withdrawal responses recorded. Hind paw circumference is measured using suture looped around the paw at metatarsal level and gently tightened. The thread is then opened out and measured to the nearest millimeter. Measurements are taken prior to carrageenan injection and then at 60-min intervals thereafter.

Behavioral studies

470 Rotarod

Co-ordination (Rotarod); the rotarod assessment is used to evaluate general coordinator behavior. It will be assessed by performance on the rotarod apparatus (Ugo Basile) on which mice will be trained for 3 days, each mouse receiving four training trials per day consisting of placing the mice for 2 min on a rod rotating at a speed of 475 up to 24 rpm, the latency to fall off the rotarod onto foam rubber padding being

measured with the trial terminating after 2 min if the mouse has not fallen off.

Open Field

Open field behavior; the open field assessment is use to evaluate general social behavior. Animals will be placed within a standard Open field arena' and behavior 480 monitored by computer tracking (Ethovision) for up to 2 hours per day. Overall

locomotion will be measured and the time spent in the central part of the arena and in rearing will give an indication of anxiety-like behavior.

Claims

485 CLAIMS

1 . A compound of formula I

(I)

Wherein:

R is selected from

pharmaceutical acceptable salts thereof.

2. The compound according to claim 1 , wherein the compound of formula I is:

500

The compound according to claim 1 , wherein the compound of formula I

The compound according to claim 1 , wherein the compound of formula I

510

The compound according to claim 1 , wherein the compound of formula I

A compound of formula I according to claims 1 -5 for use in pain conditions.

A compound of formula I according to claim 6, wherein the pain conditions are directed towards acute pain.

A compound of formula I according to claim 6, wherein the pain conditions are directed towards chronic pain.

9. A compound of formula I according to claim 8, wherein the conditions are directed towards chronic joint pain, such as pain caused by osteoarthritis or fibromyalgia.

10. A pharmaceutical composition comprising at least a compound of the formula I according to claims 1 -5 as an active ingredient, or a

physiologically acceptable salt thereof together with a pharmaceutically acceptable carrier.