AU2001244982A1 - Quinolinyl-piperidin-4-ylidene-methyl-benzamide derivatives for the treatment of pain - Google Patents

Description

QUINOLINYL-PIPERroiN-4-YLjDENE-METHYL-BENZAMIDE DERIVATIVES FOR THE TREATMENT OF PALN

Field of the 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 compounds are useful in therapy, and in particular for the treatment of pain.

Background and prior art

The δ receptor has been identified as having a role in many bodily functions such as circulatory and pain systems. Ligands for the δ receptor may therefore find potential use as analgesics, and/or as antihypertensive agents. Ligands for the δ receptor have also been shown to possess immunomodulatory activities.

The identification of at least three different populations of opioid receptors (μ, δ and K) is now well established and all three are apparent in both central and peripheral nervous systems of many species including man. Analgesia has been observed in various animal models when one or more of these receptors has been activated.

With few exceptions, currently available selective opioid δ ligands are peptidic in nature and are unsuitable for administration by systemic routes. One example of a non-peptidic δ-agonist is SNC80 (Bilsky E.J. et al., Journal of Pharmacology and Experimental Therapeutics, 273(1), pp. 359-366 (1995)). There is however still a need for selective δ-agonists having not only improved selectivity, but also an improved side-effect profile.

Thus, the problem underlying the present invention was to find new analgesics having improved analgesic effects, but also with an improved side-effect profile over current μ agonists, as well as having improved systemic efficacy. Analgesics that have been identified and are existing in the pπor art have many disadvantages in that they suffer from poor pharmacokinetics and are not analgesic when administered by systemic routes. Also, it has been documented that preferred δ agonist compounds, descπbed within the pπor art, show significant convulsive effects when administered systemically.

We have now found that certain compounds not specifically disclosed by, but included within the scope of WO 98/28275, exhibit surpπsingly improved δ-agonist properties and in vivo potency

Outline of the invention

The novel compounds according to the present invention are defined by the formula I

wherein

R is selected from any one of

(I) phenyl;

20

(n) pyπdinyl

(in) thienyl

(iv) furanyl

(v) lmidazolyl / s. i J N H

(vi) tπazolyl

where each R phenyl πng and R heteroaromatic πng may optionally and independently be further substituted by 1, 2 or 3 substituents selected from straight and branched

Ci-Cβ alkyl, NO2, CF3, Ci-Cβ alkoxy, chloro, fluoro, bromo, and lodo The substitutions on the phenyl πng and on the heteroaromatic πng may take place in any position on said πng systems,

A preferred embodiment of the present invention is a compound according to figure I wwhheerreeiinn RR iiss aass ddeeffiinneedd aabboovvee aanndd eeaacchh RR pphheennyyll ππnng and R heteroaromatic πng may independently be further substituted by a methyl group

A more preferred embodiment of the present invention is a compound according to figure I wherein R is pyπdinyl, thienyl or furanyl

Within the scope of the invention are also salts and enantiomers of the compounds of the formula I, including enantiomers of salts When the phenyl ring and the heteroaromatic ring(s) are substituted, the preferred substituents are selected from anyone of CF3, methyl, iodo, bromo, fluoro and chloro.

Reaction step g in Scheme 1, vide infra, is performed by reacting an intermediate compound of the general formula II

wherein PG is a urethane or benzyl-like protecting group, such as Boc. with 8-quinolinyl boronic acid, using a palladium catalyst, e.g. Pd(PPh3)4, in the presence of a base. e.g. Na2C03, to give the compounds of general formula III,

which is thereafter deprotected, under standard conditions and alkylated under reductive conditions with a compound of the general formula R -CHO to give compounds of the general formula I. Suitable palladium catalysts include, but is not limited to, PdCb (with a phosphine), Pd(OAc)₂ (with a phosphine), Pd(dba)₂, PdCl₂(dppf) CH₂C1₂, Pd(PPh₃)₄, and Pd/C.

Suitable bases include, but is not limited to, tnethylamine, sodium and potassium carbonate

Suitable reducing agents to be used includes, but is not limited to, sodium cyanoborohydπde and sodium tπacetoxyborohydπde.

The novel compounds of the present invention are useful in therapy, especially for the treatment of vaπous pain conditions such as chronic pain, neuropathic pain, acute pain, cancer pain, pain caused by rheumatoid arthπtis, migraine, visceral pain etc. This list should however not be interpreted as exhaustive.

Compounds of the invention are useful as lmmunomodulators, especially for autoimmune diseases, such as arthπtis, for skin grafts, organ transplants and similar surgical needs, for collagen diseases, vaπous allergies, for use as anti-tumour agents and anti viral agents.

Compounds of the invention are useful in disease states where degeneration or dysfunction of opioid receptors is present or implicated m that paradigm. This may involve the use of isotopically labelled 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 for the treatment of diarrhoea, depression, anxiety, uπnary incontinence, vaπous mental illnesses, cough, lung oedema, vaπous gastrointestinal disorders, spinal injury and drug addiction, 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 an analgesic agent for use dunng general anaesthesia and monitored anaesthesia care Combinations of agents with different propei ties are often used to achieve a balance of effects needed to maintain the anaesthetic state (e g amnesia, analgesia, muscle relaxation and sedation) Included in this combination are inhaled anaesthetics, hypnotics, anxiolytics, neuromuscular blockers and opioids

Also within the scope of the invention is the use of any of the compounds according to the formula I above, for the manufacture of a medicament for the treatment of any of the conditions discussed above

A further aspect of the invention is a method for the treatment of a subject suffeπng from any of the conditions discussed above, whereby an effective amount of a compound according to the formula I above, is administered to a patient in need of such treatment

A further aspect of the present invention is intermediates of the general formula II,

wherein PG is a urethane or benzyl-like protecting group, such as Boc,

Methods of preparation

The compounds according to the present invention may be prepared by following the known procedures described m e g "Advanced Organic Chemistry" third edition by Jerry March, John Wiley and Sons Inc , New York (1985) Step (a) p848, Step (b) p848, Step (c): p657; Step (d): p875; Step (e): p371-373; Step (f): p364-366; Step (g): N. Miyaura and A. Suzuki, Chem. Rev., 95, 2457-2483(1995); Step (h): "Protective Groups in Organic synthesis" p 327-329, by Theodora W. Greene and Peter G.M. Wuts, Second Edition, John Wiley and Sons Inc.; New York (1991). These references are hereby incorporated in full.

EXAMPLES

The invention will now be described in more detail by the following Examples, which are not to be construed as limiting the invention.

Example 1

Preparation of N.N-diethyl-4-(8-quinolinyl-piperidin-4-ylidene-methvπ-benzamide

(compound 7) (i) Preparation of 4-(4-methoxycarbonyl-benzylidene)-piperidine-l-carboxylic acid tert-butyl ester (compound 3)

A mixture of compound 1 (11.2 g, 49 mmol) and tπmethyl phosphite (25 mL) was refluxed under N for 5 hrs. Excess tπmethyl phosphite was removed by co-distillation with toluene to give compound 2 in quantitative yield. Η NMR (CDCh) δ 3 20 (d, 2H, J=22 Hz), 3.68 (d, 3H 10.8 Hz), 3.78 (d, 3H, 11.2 Hz), 3.91 (s, 3H), 7.38 (m, 2H), 8.00 (d, 2H, J=8 Hz)

(ii) To a solution of the above product (compound 2) in dry THF (200 mL) was added dropwise lithium dπsopropylamide (32.7 mL 1.5 M m hexanes, 49 mmol) at -78 °C. The reaction mixture was then allowed to warm to room temperature pπor to addition of N- tert-butoxycarbonyl-4-pιpeπdone (9.76 g, 49 mmol in 100 mL dry THF) After 12 hrs, the reaction mixture was quenched with water (300 mL) and extracted with ethyl acetate (3 x 300 mL). The combined organic phases were dπed over MgSO₄ and evaporated to give a crude product, which was puπfied by flash chromatography to provide compound 3 as a white solid (5.64 g, 35%):

IR (NaCl) 3424, 2974, 2855, 1718, 1 688, 1606, 1427, 1362, 1276 cm^{" 1};

1H NMR (CDC1₃) δ 1.44 (s, 1H), 2.31 (t, J=5.5 Hz, 2H), 2.42 (t, 1=5.5 Hz, 2H), 3.37 (t, J=5.5 Hz, 2H), 3.48 (t, J=5.5 Hz, 2H), 3.87(s, 3H), 6 33 (s, 1H), 7.20 (d J=6.7 Hz, 2H), 7.94 (d, J =6.7 Hz, 2H), ¹³C NMR (CDC1₃) δ 28.3, 29.2, 36.19, 51.9, 123 7, 127.8, 128.7, 129.4, 140.5, 142.1, 154.6, 166.8. (iii) Preparation of 4-bromo-4-rbromo-(4-methoxycarbonyl-phenyl)-methyll- piperidine-1-carboxylic acid tert-butyl ester (compound 4)

To a mixture of compound 3 (5.2 g, 16 mmol) and K₂CO₃ (1.0 g) in dry dichloromethane (200 mL) was added a solution of bromine (2.9 g, 18 mmol) in 30 mL CH₂C1₂ at 0 °C. after 1.5 hrs at room temperature, the solution after filtration of K₂C0₃ was condensed. The residue was then dissolved in ethyl acetate (200 mL), washed with water (200 mL), 0.5 M HCl (200 L) and bπne (200 mL), and dπed over MgS0 . Removal of solvents provided a crude product, which was recrystalhzed from methanol to give compound 4 as a white solid (6.07 g, 78%): IR (NaCl) 3425, 2969, 1725, 1669, 1426, 1365, 1279, 1243 cm ';

Η NMR (CDC1₃) δ 1.28 (s, 9H), 1.75 (m, 2H), 1.90 (m, 2H), 2.1 (m, 4H), 3.08 (br, 4H), 3.90 (s, 3H), 4.08 (br, 4H), 5.14 (s, 1H), 7.57 (d, J=8.4 Hz, 2H) 7.98 (d, J=8.4 Hz, 2H);

¹³C NMR (CDC1₃) δ 28.3, 36.6, 38.3, 40.3, 52.1, 63.2, 72.9, 129.0, 130.3, 130.4, 141.9, 154.4, 166.3.

(iv) Preparation of 4-rbromo-(4-caboxy-phenyl)-methylenel-piperidine-l-carboxylic acid tert-butyl ester (compound 5)

A solution of compound 4 (5.4 g 11 mmol) in methanol (300 mL) and 2.0 M NaOH (100 mL) was heated at 40 °C for 3 hrs. The solid was collected by filtration, and dπed ovemight under vacuum. The dry salt was dissolved in 40% acetonitπle/water, and was adjusted to pH 2 using concentrated HCl. The desired product compound 5 (3.8 g, 87%) was isolated as a white powder by filtration: Η NMR (CDC1₃) δ 1.45 (s, 9H), 2.22 (dd, J=5.5 Hz, 6.1 Hz, 2H), 2.64 (dd, J=5.5 Hz, 6.1 Hz, 2H), 3.34 (dd, J=5.5 Hz, 6.1 Hz, 2H), 3.54 (dd, J=5.5 Hz, 6.1 Hz, 2H), 7.35 (d, J=6.7 Hz, 2H), 8.08 (d, J=6.7 Hz, 2H); ¹³C NMR (CDC1₃) δ 28.3, 31.5, 34.2, 44.0, 115.3, 128.7, 129.4, 130.2, 137.7, 145.2, 154.6, 170.3.

(v) Preparation of 4 bromo-(4-diethylcarbamoyl-phenyl)-methylene1-piperidine-l- carboxylic acid tert-butyl ester (compound 6) To a solution of compound 5 (1.0 g, 2.5 mmol) in dry dichloromethane ( 10 mL) at - 20 °C was added isobutylchloroformate (450 mg, 3.3 mmol). After 20 min at -20 °C diethylamme (4 mL) was added and the reaction was allowed to warm to room temperature. After 1.5 hrs the solvents were evaporated and the residue was partitioned between ethyl acetate and water. The organic phase was washed with bπne and dπed over MgS0 . Removal of solvents provided a crude product, which was puπfied by flash chromatography to give compound 6 as white needles (800 mg, 73%): IR (NaCl) 3051, 2975, 1694, 1633, 1416, 1281, 1 168, 1115 cm ¹; Η NMR (CDC1₃) δ 1.13 (br, 3H), 1.22 (br, 3H), 1.44 (s. 9H), 2.22

(t, J=5.5 Hz, 2H), 2.62 (t, J=5 5 Hz, 2H), 3.33 (m, 4H). 3.55 (m, 2H), 7.31 (d, J=8 0 Hz, o 22HH)),, 77..3366 ((dd,, JJ==88 00 HHzz,, 22HH)),, ¹¹³³CC NNMMRR ((CCDDCC11₃₃)) δδ 1122..7711,, 1144 1133,, 28.3, 31.5, 34.2. 39.1, 43.2, 79.7, 115.9, 126.3, 129.3, 136.8, 137.1, 140.6, 154.6, 170

(vi) Preparation of N.N-diethyl-4-(8-quinolinv.-piperidin-4-ylidene-methyl)- benzamide (compound 7). s A mixture of compound 6 (902 mg, 2.0 mmol), 8-quιnohnyl boronic acid

(860 mg, 5.0 mmol), 2M Na₂C0₃ (2.5 mL), and tetrakιs(tπphenyl phosphine) palladιum(O) (20 mg) in toluene (degassed, 5 mL) and ethanol (degassed. 5 mL) was refluxed at 90 °C for 4 hrs under N₂. The reaction mixture was then cooled down to r.t., and extracted with ethyl acetate (2 x 100 mL). The combined organic phases were dπed over MgS0 and o evaporated to give a crude product.

The above product was treated with 4.0 M HCl in dioxane at 50 °C for 2 h. After evaporation, the residue was dissolved in 1 M HCl (100 mL) and impuπties were extracted with diethyl ether (3 x 100 mL). The aqueous phase was basified with NI OH and 5 extracted with dichloromethane (3 x 100 mL). The combined organic phases were washed with bπne, dπed over MgSO₄ and evaporated to give the title compound 7 (729 mg, 91 %).

1H-NMR (400 MHz, CDCI3) δ 1.07 (3 H, br m, CH₃CH,-), 1.20 (3 H, br m, CHsOL-), 2.00 (2 H, m, pipeπdine CH-), 2.46 (IH, s, NH). 2.52 (2 H. m, pipeπdine CH-), 2.75 (IH, m, pipeπdine CH-), 2.92 (2 H, m, pipeπdine CH-), 3.05 (1 H, m, pipeπdine CH-), 3.22 (2 H, m, CF N-), 3.49 (2 H, m, CI N-), 7.23 (2 H, m, ArH), 7.32 (2 H, m, ArH), 7.36 (1 H, m, ArH), 7.49 (2 H, m, ArH), 7.72 (1 H, dd, J = 6.4, 3.2 Hz, ArH), 8.11 (1 H, dd, J = 8.4, 1.6 Hz, ArH), 8.91 (1 H, dd. 7 = 4.0, 1 6 Hz, ArH); Its HCl salt: m p. > 170 °C (Dec); IR (NaCl) 3410, 2973, 1614, 1551, 1436, 1284 cm '.

Example 2

Preparation of N.N-diethyl-4-(8-quinolinyl-N-henzyl-piperidin-4-ylidene-methyl)- benzamide (compound 9)

lo (i) Preparation of N.N-diethyl-4-(bromo-N-benzyl-piperidin-4-ylidene-methv.)- benzamide (compound 8)

Compound 6 prepared in Example l(v) above (2.26 g, 5.0 mmoL), was treated with TFA (25 mL) in dichloromethane (25 mL) at room temperature. After 2 h, the reaction mixture was condensed to give a residue, which was dissolved in acetomtπle (20 mL), and was

15 reacted with benzyl bromide ( 5.0 mmol) at r.t. for 2 h. The reaction mixture was condensed, and then dissolved in ethyl acetate (100 mL) The organic solution were washed with IN NH OH and bπne, dπed over MgS0 . Removal of solvents provided a crude product, which was puπfied by flash chromatography to give compound 8 as an oil (1.0 g, 45%): IR (NaCl) 2971, 1630, 1427, 1287, 1094 cm '; Η NMR (CDC1₃) δ 1.13 (br, 3H), 0 1.23 (br, 3H), 2.28 (m, 2H), 2.37 (m, 2H), 2.55 (m, 2H), 2.69 (m, 2H), 3.27 (m, 2H), 3.53 (br, 4H), 7.31 (m, 4H).

(ii) Preparation of N.N-diethyl-4-(8-quinolinyl-N-benzyl-piperidin-4-ylidene-rnethyl)- benzamide (compound 9)

A mixture of compound 8 prepared in step (1) above (1.32 mg, 3.0 mmol), 5 8-qumolιnylboronιc acid (1.04 mg, 6.0 mmol), 2M Na₂CO (3.0 mL), and tetrakιs(tπphenyl phosphine) palladιum(O) (20 mg) in toluene (degassed, 5 mL) and ethanol (degassed, 5 mL) was refluxed at 90 °C for 2 hrs under N₂. The reaction mixture was then cooled down to r.t., and extracted with ethyl acetate (2 x 100 mL). The combined organic phases were washed with bπne, dπed over MgS0₄. Removal of solvents provided a crude product, o which was purified by flash chromatograph}- to give the desired title compound 9 (832 mg. 57 %): IR (NaCl) 2971, 1625, 1551, 1426, 1287 cm^"1; Η-NMR (400 MHz, CDCI3) δ 1.04 (3 H, br m, CHjCH₂-), 1.17 (3 H, br m, CEbO _.-), 2.03 (2 H, m, piperidine CH-), 2.30 (IH, m, pipeπdine CH), 2.51 (2H, m, piperidine CH), 2.59 (2 H, m, piperidine CH-), 2.68 (IH, m, pipeπdine CH-), 3.19 (2 H, m, CH^N-), 3.49 (2 H, m, CH,N-), 3.52 (2H, s, PhCILN), 7.35(10 H, m, ArH), 7.46 (2 H, m, ArH), 7.71 (1 H, m, ArH), 8.10 (1 H, m, ArH), 8.90 (1 H, m, ArH).

Examples 3-10

Compounds 10-17 of Examples 3-10, were prepared by following the synthetic procedures of Scheme 2 below.

Scheme 2: 1(3)

11

Scheme 2: 2(3)

Scheme 2: 3(3)

17 Example 3

Preparation of N,N-diethyl-4-rri-(2-furylmethyl)-4-piperidinylidenel(8- quinoline)methyllbenzamide (compound 10)

To a room temperature solution of secondary amine (300mg; 0.75mmol) in methanol (8ml) 5 was added 2-furaldehyde (261μl; 3.15mmol), followed by acetic acid (0.5ml). The mixture was stirred for two hours then sodium cyanoborohydπde (198mg; 3.15mmol) was added. The reaction mixture was stirred overnight, then sodium hydroxide 2Ν was added and the mixture extracted with methylene chloπde. Combined methylene chloπde extracts were dπed over anhydrous sodium sulphate, filtered and concentrated under reduced pressure 0 Reverse phase puπfication.

(M+l) calculated: 480.62, (M+l) observed: 480.16

s Anal.: calculated for (C₃₁H₃₃N₃0₂ X 2.20 C₂HO₂F-, X 1.00 H₂0): C:56.81%; H: 5.01%; N:5.61%; found: C:56.84%; H:4.96%; N:5.59%

'HNMR (CD₃OD): 8.86-8.90 (m, IH), 8.38-8.50 (m, IH), 7.86-7.96 (m, IH), 7.48-7.66 (M, 4H), 7.31 (d, 2H, J=7.6Hz), 7.21 (d, 2H, J=7.6Hz), 6.61-6.62 (m, IH), 6.43-6.44 (m, o IH), 4.34 (s, 2H), 2.04-3.62 (m, 12H), 1.07-1.16 (m, 3H), 0.94-1.03 (m, 3H)

Example 4

Preparation of N,N-diethyl-4-{8-quinolinvUl-(2-thienylmethyl)-4- piperidinylidenelmethyllbenzam.de (compound 11) 5 To a room temperature solution of secondary amine (300mg; 0.75mmol) in methanol (8ml) was added thιophene-2-carboxaldehyde (294.4μl; 3.15mmol), followed by acetic acid (0.5ml). The mixture was stirred for two hours then sodium cyanoborohydπde (198mg; 3.15mmol) was added. The reaction mixture was stirred overnight, then sodium hydroxide 2Ν was added and the mixture extracted with methylene chloπde. Combined methylene chloride extracts were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Reverse phase purification.

(M+l) calculated: 496.69, (M+l) observed: 496.09

5

Anal.: calculated for (C₃,H₃₃N₃OS X 2.50 C₂HO₂F₃ X 0.60 H₂0): C:54.63%; H:4.67%; N:5.31%; found: C:54.62%; H:4.64%; N:5.45%

'HNMR (CD₃OD): 8.89-8.90 (m, IH), 8.42-8.56 (m, IH), 7.86-7.98 (m, IH), 7.53 (d, IH, 0 J=5.2Hz), 7.50-7.70 (m, 3H), 7.30 (d, 2H, J=7.6Hz), 7.20-7.24 (m, IH), 7.20 (d, 2H. J=8.0Hz), 7.02-7.06 (m, IH), 4.50 (s, 2H), 2.04-3.66 (m, 12H), 1.06-1.16 (m, 3H), 0.94- 1.02 (m, 3H)

Example 5 s Preparation of N,N-diethyl-4-rri-(3-furylmethyl)-4-piperidinylidenel(8- uinolinvDmethyllbenzamide (compound 12)

To a room temperature solution of secondary amine (300mg; 0.75mmol) in methanol (8ml) was added 3-furaldehyde (272.4μl; 3.15mmol), followed by acetic acid (0.5ml). The mixture was stirred for two hours then sodium cyanoborohydride (198mg; 3.15mmol) was o added. The reaction mixture was stirred overnight, then sodium hydroxide 2Ν was added and the mixture extracted with methylene chloride. Combined methylene chloride extracts were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Reverse phase purification.

5 (M+l) calculated: 480.62, (M+l) observed: 480.33

Anal.: calculated for (C₃ιH₃₃N₃0₂ X 1.80 C₂HO₂F₃ X 0.80 H₂0): C:59.43%; H:5.25%; N:6.01%; found: C:59.38%; H:5.24%; N:5.98% 'HNMR (CD₃OD): 8.80-8.88 (m, IH), 8.30-8.42 (m, IH), 7.80-7.92 (m, IH), 7.44-7.68 (m, 4H), 7.26-7 36 (m, 2H), 7.16-7.24 (m, 3H), 6.50 (s, IH), 4.15 (s, 2H), 2.86-3.62 (m, 9H), 2.48-2.66 (m, IH), 2.06-2.36 (m, 2H), 1.06-1.16 (m, 3H), 0.94-1.04 (m, 3H)

5 Example 6

Preparation of N,N-diethyl-4-|8-quinolinyl[l-(3-thienylmethyl)-4- piperidinylidenelmethvDbenzamide (compound 13)

To a room temperature solution of secondary amine (300mg; 0.75mmol) in methanol (8ml) was added thιophene-3-carboxaldehyde (276μl: 3.15mmol). followed by acetic acid o (0.5ml). The mixture was stirred for two hours then sodium cyanoborohydπde (198mg; 3.15mmol) was added. The reaction mixture was stirred overnight, then sodium hydroxide 2N was added and the mixture extracted with methylene chloπde. Combined methylene chloride extracts were dπed over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Reverse phase puπfication. 5

(M+l) calculated: 496.69, (M+l) observed: 496.11

Anal.: calculated for (C₃₁H₃₃N₃OS X 2.70 C₂H0₂F₃ X 0.70 H₂O): C:53.57%; H:4.58%; o N:5.15%; found: C:53.56%; H:4.54%; N:5.40%

'HNMR (CD₃OD): 8.82-8.90 (m, IH), 8.34-8.50 (m, IH), 7.82-7.96 (m, IH), 7.42-7.70 (m, 4H), 7.24-7.36 (m, 2H), 7.20 (d, 2H, J=7.2Hz), 7.08-7.14 (m, 2H), 4.28 (s, 2H), 2.04-3.58 (m, 12H), 1.06-1.16 (m, 3H), 0.92-1.03 (m, 3H) 5

Example 7

Preparation of N,N-diethyl-4-rri-(2-pyridinylrnethyl)-4-piperidinylidenel(8- quinolinvPmethyllbenzamide (compound 14) To a room temperature solution of secondary amme (300mg; 0.75mmol) in methanol (8ml) was added 2-pyπdιnecarboxaldehyde (299.6μl; 3.15mmol), followed by acetic acid (0.5ml). The mixture was stirred for two hours then sodium cyanoborohydnde (198mg; 3.15mmol) was added. The reaction mixture was stirred overnight, then sodium hydroxide 2N was added and the mixture extracted with methylene chloπde. Combined methylene chloride extracts were dπed over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Reverse phase puπfication.

(M+l) calculated: 491.65, (M+l) observed: 491.11

Anal.: calculated for (C₃₂H ₄N₄0 X 2.40 C₂H0₂F₃ X 1.00 H₂0): C:56.50%; H:4.95%; N:7.16%; found: C:56.47%; H:4.97%; N:7.27%

¹HNMR (CD₃OD): 8.91 (dd, IH, J=4.4, 1.2Hz), 8.57 (d, IH, J=4.4Hz), 8.47 (dd, IH,

J=8.0, 1.6Hz), 7.91 (dd, IH, J=7.2, 2.8Hz), 7.79 (dt, IH, J=8.0, 1.6Hz), 7.56-7.62 (m, 3H), 7.39 (d, IH, J=7.2Hz), 7.32 (d, 2H, J=8.0Hz), 7.30-7.36 (m, IH), 7.21 (d, 2H, J=8.4Hz), 4.41 (s, 2H), 3.08-3.56 (m, 8H), 2.72-2.88 (m, 2H), 2.18-2.36 (m, 2H), 1.06-1.16 (m, 3H), 0.92-1.02 (m, 3H)

Example 8

Preparation of N,N-diethyl-4-rri-(lH-imidazol-2-ylmethyl)-4-piperidinylidenel(8- quinolinvPmethyllbenzarnide (compound 15)

5 To a room temperature solution of secondary amine (300mg; 0.75mmol) in methanol (8ml) was added 2-imιdazolecarboxaldehyde (302.7mg; 3.15mmol), followed by acetic acid (0.5ml). The mixture was stirred for two hours then sodium cyanoborohydπde (198mg; 3.15mmol) was added. The reaction mixture was stirred overnight, then sodium hydroxide 2Ν was added and the mixture extracted with methylene chloπde. Combined methylene to chloπde extracts were dπed over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Reverse phase puπfication.

(M+l) calculated: 480.63, (M+l) observed: 479.97

15 Anal.: calculated for (C₃₀Η₃₃N₅O X 2.80 C₂HO₂F₃ X 2.20 H₂0): C:50.99%; H:4.83%; N:8.35%; found: C:51.07%; H.4.90%; N:8.10%

'HNMR (CD₃OD): 8.91 (dd, IH, J=4.4, 1.2Hz), 8.60 (d, IH, J=7.6Hz), 7.96 (dd, IH. J=6.4, 3.6Hz), 7.62-7.68 (m, 3H), 7.39 (s, 2H), 7.30 (d, 2H, J=8.0Hz), 7.19 (d, 2H, ₀ J=8.8Hz), 4.09 (s, 2H), 3.34-3.46 (m, 2H), 3.02-3.22 (m, 4H), 2.78-2.88 (m, 2H), 2.60-2.70 (m, 2H), 2.04-2.18 (m, 2H), 1.06-1.16 (m, 3H), 0.92-1.02 (m, 3H)

Example 9 5 Preparation of N.N-diethvi-4-rri-(lH-imidazol-4-ylmethyl)-4-piperidinylidenel(8- quinolinvDmethyllbenzamide (compound 16)

To a room temperature solution of secondary amine (300mg; 0.75mmol) in methanol (8ml) was added 4(5)-ιmιdazolecarboxaldehyde (302.7mg; 3.15mmol), followed by acetic acid (0.5ml). The mixture was stiπ'ed for two hours then sodium cyanoborohydπde (198mg; o 3.15mmol) was added. The reaction mixture was stirred overnight, then sodium hydroxide 2N was added and the mixture extracted with methylene chloride. Combined methylene chloride extracts were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Reverse phase purification.

(M+l) calculated: 480.63, (M+l) observed: 480.15

Anal.: calculated for (C₃₀H₃₃N₅O X 3.70 C₂H0₂F₃ X 1.20 H₂0): C:48.66%; H:4.27%; N:7.59%; found: C:48.68%; H:4.33%; N:7.49%

'HNMR (CD₃OD): 8.90 (dd, IH, J=4.4, 1.2Hz), 8.71-8.74 (m, IH), 8.53 (d, IH. J=8.0Hz). 7.93 (dd, IH, J=6.4, 3.6Hz), 7.58-7.63 (m, 4H), 7.29 (d. 2H. J=8.0Hz). 7.19 (d, 2H, J=8.8Hz), 4.40 (s, 2H), 3.28-3.50 (m, 4H), 3.08-3.24 (m, 4H), 2.68-2.84 (m, 2H), 2.14-2.50 (m, 2H). 1.05-1.14 (m, 3H), 0.92-1.02 (m, 3H)

Example 10

Preparation of N,N-diethyl-4-rri-(4-pyridinylmethyl)-4-piperidinylidenel(8- quinolinvDmethyllbenzamide (compound 17)

To a room temperature solution of secondary amine (300mg; 0.75mmol) in methanol (8ml) was added 4-pyridinecarboxaldehyde (241mg; 2.25mmol), followed by acetic acid (0.5ml). The mixture was stirred for 30 minutes then sodium cyanoborohydride (142mg; 2.25mmol) was added. The reaction mixture was stirred overnight, then sodium hydroxide 2N was added and the mixture extracted with methylene chloride. Combined methylene chloride extracts were dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure. Reverse phase purification.

(M+l) calculated: 491.28, (M+l) observed: 491.09

Anal.: calculated for (C₃₂H₃₄N₄O X 2.60 C₂HO₂F₃ X 1.50 H₂0): C:54.88%; H:4.90%; N:6.88%; found: C:54.87%: H:4.90%; N:6.75% 'HNMR (CD OD) 8 86-896 (m, IH), 8 62-8.70 (m, IH), 8 42-8.52 (m, IH), 7 88-7.98 (m, IH), 7.50-7.70 (m. 4H), 7.28-7.38 (m, 3H), 7.16-7 26 (m, 3H), 4.36 (s, 2H), 2.90-3.60 (m, 8H), 2.50-2.90 (m, 2H), 2.00-2.40 (m, 2H), 0 82-1.16 (m, 6H)

5 Pharmaceutical compositions

The novel compounds according to the present invention may be administered orally, intramuscularly, subcutaneously, topically, intranasally, mtrapeπtoneally, intrathoracially, intravenously, epidurally, intrathecally, mtracerebroventπcularly and by injection into the 10 joints

A preferred route of administration is orally, intravenously or intramuscularly.

The dosage will depend on the route of administration, the seventy of the disease, age and is weight of the patient and other factors normally considered by the attending physician, when determining the individual regimen and dosage level as the most appropnate for a particular patient.

For prepaπng pharmaceutical compositions from the compounds of this invention, inert, o pharmaceutically acceptable earners can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, cachets, and suppositoπes.

A solid earner can be one or more substances which may also act as diluents, flavoring agents, solubihzers, lubπcants, suspending agents, binders, or tablet disintegrating agents; 5 it can also be an encapsulating mateπal.

In powders, the earner is a finely divided solid which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the earner having the necessary binding properties in suitable proportions and compacted in the shape and size o desired For prepaπng suppository compositions, a low-melting wax such as a mixture of fatty acid glyceπdes and cocoa butter is first melted and the active ingredient is dispersed therein by, for example, stirπng. The molten homogeneous mixture is then poured into convenient sized molds and allowed to cool and solidify.

Suitable earners are magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextπn, starch, tragacanth, methyl cellulose, sodium caiboxymethyl cellulose, a low- meltmg wax, cocoa butter, and the like

Salts include, but are not limited to pharmaceutically acceptable salts Examples of pharmaceutically acceptable salts within the scope of the present invention include: acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium acetate, camsylate, carbonate, chloπde, citrate, dihydrochloπde, edetate, edisylate, estolate, esylate, fumarate, glucaptate, gluconate, glutamate, glycollylarsamlate, hexylresorcinate, hydrabamine, hydrobromide, hydrochlonde, hydroxynaphthoate, lsethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate/diphosphate, polygalacturonate, sahcylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate. Examples of pharmaceutically unacceptable salts within the scope of the present invention include: hydroiodide, perchlorate, and tetrafluoroborate. Preferred pharmaceutically acceptable salts are the hydrochloπdes, sulfates and bitartrates. The hydrochlonde and sulfate salts are particularly preferred.

The term composition is intended to include the formulation of the active component with encapsulating matenal as a earner providing a capsule in which the active component (with or without other earners) is surrounded by a earner which is thus in association with it Similarly, cachets are included Tablets, powders, cachets, and capsules can be used as solid dosage forms suitable foi oral administration.

Liquid from compositions include solutions, suspensions, and emulsions. Steπle water or water-propylene glycol solutions of the active compounds may be mentioned as an example of liquid preparations suitable for parenteral administration. Liquid compositions can also be formulated in solution in aqueous polyethylene glycol solution

Aqueous solutions for oral administration can be prepared by dissolving the active component in water and adding suitable colorants, flavoπng agents, stabilizers, and thickening agents as desired. Aqueous suspensions for oral use can be made by dispersing the finely divided active component in water together with a viscous mateπal such as natural synthetic gums, resins, methyl cellulose, sodium carboxymethyl cellulose, and other suspending agents known to the pharmaceutical formulation art.

Preferably the pharmaceutical compositions is in unit dosage form. In such form, the composition is divided into unit doses containing appropnate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropnate number of any of these packaged forms.

BIOLOGICAL EVALUATION In vitro model Cell culture

A. Human 293S cells expressing cloned human μ, δ, and K receptors and neomycin resistance were grown in suspension at 37°C and 5% C0₂ in shaker flasks containing calcium-free DMEM10% FBS, 5% BCS, 0.1% Pluromc F-68, and 600 μg/ml geneticin.

B. Mouse and rat brains were weighed and rinsed in ice-cold PBS (containing 2.5mM EDTA, pH 7 4). The brains were homogenized with a polytron for 15 sec (mouse) or

30 sec (rat) in ice-cold lysis buffer (50mM Tπs, pH 7.0, 2.5mM EDTA, with phenyimethylsulfonyl fluoπde added just pπor use to 0.5MmM from a 0.5M stock in DMSO.ethanol).

Membrane preparation

Cells were pelleted and resuspended in lysis buffer (50 mM Tns, pH 7.0, 2.5 mM EDTA, with PMSF added just pπor to use to 0.1 mM from a 0.1 M stock in ethanol), incubated on ice for 15 min, then homogenized with a polytron for 30 sec. The suspension was spun at lOOOg (max) for 10 min at 4°C. The supernatant was saved on ice and the pellets resuspended and spun as before. The supernatants from both spins were combined and spun at 46,000 g(max) for 30 mm. The pellets were resuspended in cold Tns buffer (50 mM Tπs/Cl, pH 7.0) and spun again. The final pellets were resuspended in membrane buffer ( 50 mM Tns, 0.32 M sucrose, pH 7.0). Aliquots (1 ml) in polypropylene tubes were frozen in dry ice/ethanol and stored at -70°C until use. The protein concentrations were determined by a modified Lowry assay with sodium dodecyl sulfate. Binding assays

Membranes were thawed at 37°C, cooled on ice, passed 3 times through a 25-gauge needle, and diluted into binding buffer (50 mM Tns, 3 mM MgCl₂, 1 mg/ml BSA (Sigma A-7888), pH 7 4, which was stored at 4°C after filtration through a 0 22 m filter, and to which had been freshly added 5 μg/ml aprotimn, 10 μM bestatin, 10 μM diprotin A, no DTT) Aliquots of 100 μl were added to iced 12x75 mm polypropylene tubes containing 100 μl of the appropnate radiohgand and 100 μl of test compound at vaπous concentrations Total (TB) and nonspecific (NS) binding were determined in the absence and presence of 10 μM naloxone respectively The tubes were vortexed and incubated at 25°C for 60-75 mm, after which time the contents are rapidly vacuum-filtered and washed with about 12 ml/tube iced wash buffer (50 mM Tns, pH 7 0, 3 mM MgCl₂) through GF B filters (Whatman) presoaked for at least 2h in 0 1% polyethyleneimine The radioactivity (dpm) retained on the filters was measured with a beta counter after soaking the filters for at least 12h in minivials containing 6-7 ml scintillation fluid If the assay is set up in 96- place deep well plates, the filtration is over 96-place PEI-soaked umfilters, which were washed with 3 x 1 ml wash buffer, and dned in an oven at 55°C for 2h The filter plates were counted in a TopCount (Packard) after adding 50 μl MS-20 scintillation fluid/well

Functional Assays

The agonist activity of the compounds is measured by determining the degree to which the compounds receptor complex activates the binding of GTP to G-proteins to which the receptors are coupled. In the GTP binding assay, GTP[γ]³⁵S is combined with test compounds and membranes from HEK-293S cells expressing the cloned human opioid receptors or from homogenised rat and mouse brain Agonists stimulate GTP[γ]^j5S binding in these membranes The EC50 and E_max values of compounds are determined from dose- response curves Right shifts of the dose response curve by the delta antagonist naltπndole are performed to venfy that agonist activity is mediated through delta receptors Data analysis

The specific binding (SB) was calculated as TB-NS, and the SB in the presence of vaπous test compounds was expressed as percentage of control SB. Values of IC50 and Hill coefficient (nj-τ) for ligands in displacing specifically bound radioligand were calculated from logit plots or curve fitting programs such as Ligand, GraphPad Prism, SigmaPlot, or ReceptorFit. Values of K, were calculated from the Cheng-Prussoff equation. Mean ± S.E.M. values of IC50, K_t and n^ were reported for ligands tested in at least three displacement curves Biological data are tabulated on the following pages in Table 1

able 1: Biological data.

Table 1 (continued): Biological data.

Receptor saturation expenments

Radioligand Kδ values were determined by performing the binding assays on cell membranes with the appropnate radioligands at concentrations ranging from 0 2 to 5 times the estimated Kδ (up to 10 times if amounts of radioligand required are feasible) The specific radioligand binding was expressed as pmole/mg membrane protein Values of Kδ and B_max from individual expenments were obtained from nonlinear fits of specifically bound (B) vs nM free (F) radioligand from individual according to a one-site model 0

DETERMINATION OF MECHANO-ALLODYNIA USING VON FREY TESTING

Testing was performed between 08 00 and 16 OOh using the method descπbed by Chaplan et al (1994) Rats were placed m Plexiglas cages on top of a wire mesh bottom which s allowed access to the paw. and were left to habituate for 10-15 mm The area tested was the mid-plantar left hind paw, avoiding the less sensitive foot pads The paw was touched with a senes of 8 Von Frey hairs with logaπthmically incremental stiffness (0 41, 0 69, 1.20, 2 04, 3 63, 5 50, 8 51, and 15 14 grams, Stoelting, 111, USA) The von Frey hair was applied from underneath the mesh floor perpendicular to the plantar surface with sufficient o force to cause a slight buckling against the paw, and held for approximately 6-8 seconds A positive response was noted if the paw was sharply withdrawn Flinching immediately upon removal of the hair was also considered a positive response Ambulation was considered an ambiguous response, and in such cases the stimulus was repeated

₅ TESTING PROTOCOL

The animals were tested on postoperative day 1 for the FCA-treated group The 50% withdrawal threshold was determined using the up-down method of Dixon (1980) Testing was started with the 2 04 g hair, in the middle of the senes Stimuli were always presented in a consecutive way, whether ascending or descending In the absence of a paw withdrawal response to the initially selected hair, a stronger stimulus was presented, in the event of paw withdrawal, the next weaker stimulus was chosen. Optimal threshold calculation by this method requires 6 responses in the immediate vicinity of the 50% threshold, and counting of these 6 responses began when the first change in response occurred, e.g. the threshold was first crossed. In cases where thresholds fell outside the range of stimuli, values of 15.14 (normal sensitivity) or 0.41 (maximally allodynic) were respectively assigned The resulting pattern of positive and negative responses was tabulated using the convention, X = no withdrawal; O = withdrawal, and the 50% withdrawal threshold was interpolated using the formula:

50% g threshold = 10^{(X1 +} V / 10,000

where Xf = value of the last von Frey hair used (log units); k = tabular value (from Chaplan et al. (1994)) for the pattern of positive / negative responses; and δ = mean difference between stimuli (log units). Here δ = 0.224.

Von Frey thresholds were converted to percent of maximum possible effect (% MPE), according to Chaplan et al. 1994. The following equation was used to compute % MPE:

% MPE = Drug treated threshold (g) - allodvma threshold (g) X 100

Control threshold (g) - allodynia threshold (g)

ADMINISTRATION OF TEST SUBSTANCE Rats were injected (subcutaneously, intrapentoneally, intravenously or orally) with a test substance pnor to von Frey testing, the time between administration of test compound and the von Frey test varied depending upon the nature of the test compound. WRITHING TEST

Acetic acid will bπng abdominal contractions when administered intrapentoneally in mice. These will then extend their body in a typical pattern When analgesic drugs are administered, this descπbed movement is less frequently observed and the drug selected as 5 a potential good candidate.

A complete and typical Wπthing reflex is considered only when the following elements are present: the animal is not in movement; the lower back is slightly depressed; the plantar aspect of both paws is observable. In this assay, compounds of the present invention 10 demonstrate significant inhibition of wnthing responses after oral dosing of l-100μmol/kg

(i) Solutions preparation

Acetic acid (AcOH). 120 μL of Acetic Acid is added to 19.88 ml of distilled water in order is to obtain a final volume of 20 ml with a final concentration of 0.6% AcOH. The solution is then mixed (vortex) and ready for injection.

Compound (drug). Each compound is prepared and dissolved in the most suitable vehicle according to standard procedures. o

(ii) Solutions administration

The compound (drug) is administered orally, intrapentoneally (i.p.) , subcutaneously (s.c.) or intravenously (i.v.)) at 10 ml/kg (consideπng the average mice body weight) 20, 30 or 40 minutes (according to the class of compound and its characteπstics) pπor to testing. When 5 the compound is delivered centrally: Intraventπcularly (i.e. v.) or mtrathecally (l.t.) a volume of 5 μL is administered.

The AcOH is administered intrapentoneally (i.p.) in two sites at 10 ml/kg (considering the average mice body weight) immediately pπor to testing. o (iii) Testing

The animal (mouse) is observed for a period of 20 minutes and the number of occasions (Writhing reflex) noted and compiled at the end of the experiment. Mice are kept in individual "shoe box" cages with contact bedding. A total of 4 mice are usually observed at the same time: one control and three doses of drug.

Claims (18)

Claims

1. A compound of the formula I

wherein

R is selected from any one of

(i) phenyl

(ii) pyridinyl

(iii) thienyl

(iv) furanyl

(v) imidazolyl

(vi) tnazolyl

where each R phenyl πng and R heteroaromatic ring may independently be further substituted by 1, 2 or 3 substituents selected from straight and branched C^-Cg alkyl, NO2, CF3, C]-C(5 alkoxy, chloro, fluoro. bromo. and lodo. as well as salts thereof.

2. A compound according to claim 1, wherein each R phenyl ring and R heteroaromatic πng may optionally and independently be further substituted by 1, 2 or 3 substituents selected from methyl, CF3, chloro, fluoro, bromo, and lodo

3. A compound according to claim 1, wherein each R phenyl πng and R heteroaromatic πng may independently be further substituted by a methyl group

4. A compound according to claim 1, wherein R is pyπdinyl, thienyl or furanyl.

5. A compound according to claim 1, selected from any one of

• N,N-Dιethyl-4-(8-quιnohnyl-N-benzyl-pιpeπdιn-4-yhdene-methyl)-benzamιde,

• N,N-dιethyl-4-[[l-(2-furylmethyl)-4-pιpendιnylιdene](8-quιnohne)methyl]benzamιde;

• N,N-dιethyl-4-{8-quιnolιnyl[l-(2-thιenylmethyl)-4-pιpeπdιnylιdene]- methyl Jbenzamide,

• N,N-dιethyl-4-[[l-(3-furylmethyl)-4-pιpeπdιnyhdene](8-quιnolιnyl)methyl]benzamιde; • N,N-dιethyl-4-{ 8-quιnohnyl[l-(3-thιenylmethyl)-4-pιpeπdιnylιdene]methyl }- benzamide,

• N,N-dιethyl-4-[[l-(2-pyndιnylmethyl)-4-pιpendιnylιdene](8-quιnolιnyl)- methyl]benzamιde,

• N,N-dιethyl-4-[[l-(lH-ιmιdazol-2-ylmethyl)-4-pιpeπdιnylιdene](8-quιnohnyl)- methyl]benzamιde.

• N,N-dιethyl-4-[[l-(lH-ιmιdazol-4-ylmethyl)-4-pιpeπdιnylιdene](8-quιnolιnyl)- methyl]benzamιde, and

• N,N-dιethyl-4-[[l-(4-pyπdιnylmethyl)-4-pιpendιnylιdene](8-quιnohnyl)- methyl]benzamιde

6. A compound according to any of the preceding claims, in form of its hydrochlonde, dihydrochloπde, sulfate, tartrate, ditπfluoroacetate or citrate salts.

7. A process for prepaπng a compound of formula I, compnsing the reaction of, A) reacting a compound of the general formula II

wherein PG is a urethane or benzyl-like protecting group with 8-quιnohnyl boronic acid, using a palladium catalyst in the presence of a base to give the compounds of general formula III,

which is thereafter deprotected, under standard conditions and alkylated under rreedduuccttiivvee ccoonnddiittiioonnss wwiitthh aa ccoommppoouurnd of the general formula R -CHO to give compounds of the general formula I

8. A compound according to claim 1 for use in therapy.

9. A compound according to claim 8, wherein the therapy is pain management.

10. A compound according to claim 8, wherein the therapy is directed towards gastrointestinal disorders.

11. A compound according to claim 8, wherein the therapy is directed towards spinal injuries.

12. A compound according to claim 5, wherein the therapy is directed to disorders of the sympathetic nervous system.

13. Use of a compound according to formula I of claim 1 for the manufacture of a medicament for use in the treatment of pain, gastrointestinal disorders, or spinal injuries.

14. A pharmaceutical composition compnsing a compound of the formula I according to claim 1 as an active ingredient, together with a pharmaceutically acceptable earner.

15. A method for the treatment of pain, whereby an effective amount of a compound of the formula I according to claim 1 is administered to a subject in need of pain management

16. A method for the treatment of gastrointestinal disorders, whereby an effective amount of a compound of the formula I according to claim 1, is administered to a subject suffeπng from said gastrointestinal disorder

17. A method for the tieatment of spinal injuπes, whereby an effective amount of a compound of the formula I according to claim 1, is administered to a subject suffenng from said spinal injury.

18. A compound of the general formula II

wherein PG is a urethane or benzyl-like protecting group.