WO2008075150A1

WO2008075150A1 - O-substituted-dibenzyl urea-derivatives as trpv1 receptor antagonists

Info

Publication number: WO2008075150A1
Application number: PCT/IB2007/003784
Authority: WO
Inventors: Pier Giovanni Baraldi; Pier Andrea Borea; Pierangelo Geppetti; Francesca Fruttarolo; Maria Giovanna Pavani; Marcello Trevisani
Original assignee: Pharmeste S.R.L.
Priority date: 2006-12-21
Filing date: 2007-12-06
Publication date: 2008-06-26
Also published as: MX2009006582A; CY1110833T1; PT2094653E; CA2673219A1; ES2347492T3; EP2094653B1; CN101563317A; ZA200904278B; PL2094653T3; SI2094653T1; JP2010513455A; US7750049B2; KR20090090347A; UA95986C2; RS51555B; US20100105740A1; DE602007008942D1; HRP20100588T1; CN101563317B; EA015152B1

Abstract

The invention relates to compounds of formula (I) in which R is selected from halogen, alkyl, alkoxy, aryl and heteroaryl; R1 is selected from 2-hydroxyethyl, 2,3-dihydroxypropyl, 3-hydroxypropyl, 2,2-dihydroxyethyl, 3,3-dihydroxypropyl, 1,3-dioxolane- ethyl, 1,3-dioxane-methyl, 1,3-dioxolane-methyl, 1,3-dioxane-ethyl, 3-fluoro- 2-hydroxypropyl, 3-carboxy-2-hydroxy-propyl, 3-chloro-2-hydroxypropyl, 2-hydroxypropyl, 2-hydroxy-propen-2-yl, morpholinoethyl, piperazinoethyl, hydroxymethyl, benzyl, 4-(hydroxymethyl)benzyl, 4-chlorobenzyl, 4-fluorobenzyl, and 4-hydroxybenzyl. R2 is te/t-butyl or trifluoromethyl; R3 is independently selected from hydrogen, carboxy, cyano, alkyl or hydroxyalkyl, The compounds of formula (I) can be used for the preparation of pharmaceutical compositions for the therapy of inflammatory states, such as chronic neuropathic pain, over-active bladder syndrome, tumor pain, haemorrhoids, inflammatory hyperalgesia, post-intervention pain, dental extraction, airway and gastro-intestinal diseases.

Description

0-SUBSTITUTED-DIBENZYL UR[EA-DERiVATIVES AS TRPV1 RECEPTOR ANTAGONISTS

Field of the invention

The present invention relates to vanilloid receptor antagonists, in particular to O-hydroxyalkyl dibenzyl urea-derivatives that antagonize the vanilloid TRPV1 receptor.

State of the art

Recent experimental evidences have demonstrated that the expression of the vanilloid TRPV1 receptor (transient receptor potential channel) increases in the course of inflammatory states. This suggested that vanilloid receptor antagonists could be useful for the treatment of inflammatory pain states, for example chronic neuropathic pain, over-active bladder syndrome, haemorrhoids, inflammatory hyperalgesia, post-intervention pain, dental extraction, airway and gastro-intestinal diseases.

A number of vanilloid receptor antagonists are known; some of them derive from capsaicin and are referred to as capsaicinoid antagonists.

Description of the invention

The present invention relates to compounds of formula (I) wherein:

R is selected from halogen, alkyl, alkoxy, aryl and and heteroaryl;

Ri is selected from 2-hydroxyethyl, 2,3-dihydroxypropyl, 3-hydroxypropyl, 2,2-dihydroxyethyl, 3,3-dihydroxypropyl, 1 ,3-dioxolane- ethyl, 1 ,3-dioxane-methyl, 1 ,3-dioxolane-methyl, 1 ,3-dioxane-ethyl, 3-fluoro- 2-hydroxypropyl, 3-carboxy-2-hydroxy-propyl, 3-chloro-2-hydroxypropyl, 2-hydroxy-propen-2-yl, morpholinoethyl, piperazinoethyl, hydroxymethyl, benzyl, 4-(hydroxymethyl)benzyl, 4-chlorobenzyl, 4-fluorobenzyl, and 4-hydroxybenzyl

R2 is terf-butyl or trifluoromethyl;

R3 is independently selected from hydrogen, carboxy, cyano, alkyl or hydroxyalkyl, including all possible optical isomers and diastereisomers thereof.

For the purposes of the present application: the term "halogen" indicates a halogen atom selected from fluorine, chlorine, bromine or iodine; the term "alkyl" indicates a straight or branched (Ci-C-t)alkyl group; the term "alkoxy" indicates a straight or branched (Ci-C₄)alkoxy group; the term "aryl" indicated phenyl, optionally substituted with one or more halogen, alkyl, alkoxy groups as defined heirein before, cyano or amino groups, which can be the same or different from one another; the term "heteroaryl" indicates a 5- or 6-membered heterocycle containing one ore more nitrogen, oxygen or sulphur atoms, which can be the same or different from one another, such as pyrrole, thiofene, furane, imidazole, thiazole, isothiazole, oxazole, pyridine or pyrimidine.

A first preferred group of compounds of formula (I) is that wherein:

R is chlorine or bromine; Ri is 2-hydroxyethyl;

R2 is terf-butyl or trifluoromethyl;

R3 is hydrogen.

A second group of preferred compounds of formula (I) is that wherein: R is chlorine or bromine;

Ri is 2,3-dihydroxypropyl;

R2 is trifluoromethyl;

R3 is hydrogen. A third group of preferred compounds of formula (I) is that wherein:

R is methyl, phenyl, pyridine or 4-(substituted)-phenyl;

Ri is (R)-(-)-2,3-dihydroxypropyl;

R2 is trifluoromethyl;

R3 is hydrogen. A fourth group of preferred compounds of formula (I) is that wherein:

R is chlorine or bromine;

Ri is (/?)-(-)-2,3-dihydroxypropyl;

R2 is trifluoromethyl;

R3 Js hydrogen. Examples of particularly preferred compounds are:

1-[4-(2-hydroxyethoxy)-2-bromo-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea;

1-[4-(2-hydroxyethoxy)-2-chloro-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea; 1-[4-(2-hydroxyethoxy)-2-bromo-5-methoxybenzyl]-3-[4-(terf-butyl)- benzyl] urea;

1-[4-(2-hydroxyethoxy)-2-chloro-5-methoxybenzyl]-3-[4-(te/t-butyl)- benzyl] urea;

1-[4-(2,3-dihydroxypropoxy)-2-chloro-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea;

1-[4-(2,3-dihydroxypropoxy)-2-bromo-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea;

1-[4-((R)-(-)-2,3-dihydroxypropoxy)-2-chloro-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea;

1-[4-((R)-(-)-2,3-dihydroxypropoxy)-2-phenyl-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea;

1-[4-((R)-(-)-2,3-dihydroxypropoxy)-2-(pyridin-3-yl)-5-methoxybenzyl]- 3-[4-(trifluoromethyl)-benzyl] urea;

1-[4-((R)-(-)-2,3-dihydroxypropoxy)-2-(4-chlorophenyl)-5- methoxybenzyl]-3-[4-(trifluoromethyl)-benzyl] urea;

1-[4-((R)-(-)-2,3-dihydroxypropoxy)-2-bromo-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea. The compounds of general formula (I) can be prepared by means of conventional methods, such as the reaction of a compound of formula (II), in which R, Ri and R3 are as defined above,

(H) with a compound of formula (III) in which and R2 and R3 is as defined above:

(III)

The compounds of formula (I), their isomers and salts are able to inhibit the vanilloid TRPV1 receptor and can be used for the preparation of pharmaceutical compositions for the treatment of inflammatory states, chronic neuropathic pain, over-active bladder syndrome, haemorrhoids, inflammatory hyperalgesia, post-intervention pain, dental extraction, airway and gastro-intestinal diseases and tumour pain.

These formulations can be prepared by conventional methods and excipients, such as those disclosed in Remington's Pharmaceutical Sciences Handbook, XVII ed. Mack Pub., N.Y., USA.

The invention is hereinafter illustrated in greater detail in Scheme 1 and in the Examples.

Scheme 1

4-hydroxy-3-niethoxybenzylamine 1 2 hydrochloride in or iv

6a-m Reagents and conditions: (i) Acetic anidride, Pyr; (ii) NBS or NCS, DMF, O⁰C; (iii) aq. 10% HCI, Dioxane; (iv) when R=Br, Pd(PPh3)4, Na2CO3, boronic acid, DME, 90⁰C; (v) hydroxyalkyl halide, K₂CO₃, DMF, 10O⁰C; (vi) HCI 37%, EtOH, Rfx; (vii) Triphosgene, 4-(substituted)benzyl amine, DIEA, CH2CI2, 10 min. Substituents: 6a: R= Cl, Ri=2-hydroxyethyl; R₂= terf-butyl, R₃= H; 6b: R= Br, Ri=2-hydroxyethyl; R₂= terf-butyl, R₃= H; 6c: R= Cl, Ri=2,3-dihydroxypropyl; R₂= trifluoromethyl, R₃= H; 6d: R= Br, Ri=2,3-dihydroxypropyl; R₂= trifluoromethyl, R₃= H; 6e: R= Cl, Ri= 3-hydroxypropyl; R₂= trifluoromethyl, R₃= H; 6f: R= Cl, Ri= 3-hydroxypropyl; R₂= terf-butyl, R₃= H; 6g: R= Cl, Ri= 2-hydroxyethyl; R₂= trifluoromethyl, R₃= H; 6h: R= Br, Ri= 2-hydroxyethyl; R₂= trifluoromethyl, R₃= H; 6i: R= Phenyl, Ri= 2,3-dihydroxypropyl; R₂= trifluoromethyl; R₃= H; 6I: R= Pyridin-3-yl, Ri= 2,3-dihydroxypropyl; R₂= trifluoromethyl; R₃= H; 6m: R= 4-(chloro)-phenyl, Ri= 2,3-dihydroxypropyl; R₂= trifluoromethyl; R₃= H. EXAMPLES:

The reactions were routinely monitored by thin-layer chromatography (TLC) on silica gel (precoated F₂45 Merck plates) and the products were visualized with an iodine or potassium permanganate solution. ¹H NMR spectra were recorded in CDCI₃, CF₃COOD or DMSO-de with a Varian VXR 200 spectrometer. Peak positions are given in parts per million (δ) downfield from tetramethylsilane as internal standard, and J values are given in Hz. IR spectra were recorded on a Pye Unicam SP 300 spectrometer using the KBr Wafer technique. Mass spectra were obtained with a Shimadzu QP5050 Dl 50 spectrometer. The expression "Light petroleum ether" refers to the petroleum fraction boiling at 40-60⁰C. Melting points (M. p.) were determined on a Buchi-Tottoli instrument and are uncorrected. Chromatographies were performed using Merck 60-200 mesh silica gel. The synthesized compounds showed ¹H NMR spectra in agreement with the assigned structures. Elemental analyses were within ±0.4% of the theoretical values for C, H and N. Example 1

1.1. Synthesis of 4-acetoxy-3-methoxy-N-acetyl-benzylamine Acetic anhydride (1 ml, 10.5 mmol) was added to a solution of 4-hydroxy-3-methoxy-benzylamine hydrochloride (0.5 g, 2.63 mmol) in pyridine (5 ml) and the mixture was stirred at room temperature for 6 hours. The solvent was evaporated off under reduced pressure and the residue was suspended in water (100 ml). The aqueous layer was extracted with ethyl acetate (3 x 20 ml) and the combined organic phases were anhydrified (Na2SO4) and evaporated under reduced pressure to afford the title compound as white solid (0.45 g, yield 75%).

^IH-NMR(CDCI₃) δ 2.01 (s, 3H₁ CH₃), 2.31 (s, 3H, CH₃), 3.81 (s, 3H, OCH₃), 4.38 (d, 2H, J=6, CH₂), 5.90 (bs, 1 H, NH), 6.90 (m, 3H, aromatic).

MS: m/z 238.1 (M⁺ C12H15NO4). 1.2. Synthesis of 2-bromo-4-acetoxy-5-methoxy-/V-acetyl benzylamine

Λ/-bromosuccinimide (6.3 mmol, 1.1 g) was added to a solution of 4-acetoxy-3-methoxy-Λ/-acetyl-benzylamine of Example 1.1 (1.5 g, 4.2 mmol) in dry DMF (8 ml) and the mixture was stirred for 30' at 0⁰C and then for 16 hours at room temperature. The formation of a white precipitate was observed when water (40 ml) was added to the reaction.

The solid was filtered off and washed twice with cold water (2 x 20 ml), then dried over P2O5 to afford the title compound as white solid (1.4 g, 99% yield). 1H NMR (DMSO-de) δ 1.89 (s, 3H), 2.24 (s, 3H), 3.76 (s, 3H,

OCH₃),4.27 (d, 2H, CH₂, J=8), 7.09 (s, 1 H, aromatic), 7.25 (s, 1 H, aromatic), 8.35 (t, 1 H, NH).

Bidimensional NOESY (DMSO-dβ): coupling between the singlet at 2.24 ppm and the singlet at 7.25 ppm confirms that bromine is at the 2-position of the aromatic ring.

MS: m/z 316 (M⁺ Ci₂Hi₄BrNO₄).

Example 2

2. General Procedure for the synthesis of 2-(phenyl/pyridine-3-yl/4- (chloro)phenyl)- 4-hydroxy-5-methoxy-N-acetyl benzylamine

A solution of 2-bromo-4-acetoxy-5-methoxy-Λ/-acetyl benzylamine (600 nrig, 1.9 mmol) in DME (15 ml_) was deoxygenated by passing N2 through the mixture for 5 min. Then was added Pd(PPh3)4 (0.09 mol eq) and a solution of the appropriate boronic acid (1.4 mol eq) in abs. ethanol (3 ml_). The mixture was stirred for 10 min. then a 2M aq. solution of Na2CO3 (9 ml_) was added and the reaction was heated at 90°C for 12 h. The solvent was evaporated at reduced pressure, water was added (60 ml_) and the aqueous phase was extracted with EtOAc (3x30 mL). The recombined organic phased were anhydrified over Na2SO4, evaporated and the residue was purified by chromatography (6:4 EtOAc:petroleum ether) to afford the title compounds as solids.

2.1. 2-phenyl-4-hydroxy-5-methoxy-N-acetyl benzylamine White solid, yield 95%. 1H NMR (CDCI₃) δ 1.89 (s, 3H), 3.92 (s, 3H), 4.33 (d, 2H, J=4.4), 5.41

(bs, 1 H), 5.75 (s, 1 H), 6.84 (s, 1 H), 6.94 (s, 1 H), 7.41-7.29 (m, 5H). MS: m/z 271 (M⁺ Ci₆Hi₇NO₃).

2.2. 2-(pyridine-3-yl)-4-hydroxy-5-methoxy-N-acetyl benzylamine Pale yellow solid, yield 68%. 1H NMR (DMSO-de) δ 1.79 (s, 3H), 3.79 (s, 3H), 4.04 (d, 2H, J=4),

6.65 (s, 1 H), 6.98 (s, 1 H), 7.42 (m, 1 H), 7.71 (m, 1 H), 8.41 (bt, 1 H), 8.54 (m, 1 H), 9.19 (s, 1 H).

MS: m/z 272 (M⁺ Ci₅Hi₆N₂O₃).

2.2. 2-(4-chloro)phenyl-4-hydroxy-5-methoxy-N-acetyl benzylamine Pale yellow solid, yield 78%.

¹H NMR (DMSO-d₆) δ 1.81 (s, 3H), 3.79 (s, 3H), 4.08 (d, 2H, J=4), 6.01 (t, 1 H), 6.79 (s, 1 H), 6.98 (s, 1 H), 7.44 (dd, 4H), 8.15 (s, 1 H). MS: m/z 305 (M⁺ C_{I 6}HI₆CINO₃). Example 3

3.1. Synthesis of 2-bromo-4-hydroxy-5-methoxy-Λ/-acetyl benzylamine

10% Aq. hydrochloric acid (2.5 ml) was added to a solution of 2-bromo-4-acetoxy-5-methoxy-Λ/-acetyl-benzylamine 2 (0.45 g, 1.66 mmol) in dioxane (15 ml) and the mixture was refluxed for 2 hours, then cooled and the solvent was concentrated under vacuum and the residue was basified with 10% aq. NaOH. The resulting solid was collected by filtration, washed with cold water and dried to furnish the title compound as white solid in quantitative yield. 1H NMR (DMSO-de) δ 2.18 (s, 3H, CH₃), 3.87 (s, 3H, OCH₃), 4.00 (d,

2H, CH₂), 6.91 (s, 1 H, aromatic), 7.32 (s, 1 H, aromatic), 8.46 (t, 3H, NH₂), 9.80 (bs, 1 H, OH).

M. p.: >300°C.

3.2. Synthesis of 2-bromo-4-(2-hydroxyethoxy)-5-methoxy-/V-acetyl benzylamine

To a solution of compound 3 (0.4 g, 1.46 mmol) in dry DMF (15 ml) dry K₂CO₃ (2 mol eq) and 2-iodoethanol (2 mol eq) were added. The mixture was refluxed for 6 hours, then the solvent was evaporated off under reduced pressure. After addition of water the aqueous layer was extracted with EtOAc (3X 25 ml) and the organic phases were anhydrified over Na₂SO₄ and evaporated under reduced pressure to furnish the title compound as pale yellow solid (0.38 g, 81 % yield).

¹H NMR (CDCI₃) δ 2.00 (s, 3H), 3.84 (s, 3H), 3.92 (t, 2H, J=2), 4.09 (t, 2H, J=2.1), 4.44 (d, 2H, J=4), 5.21 (t, 1 H), 5.90 (bs, 1 H), 6.96 (s, 1 H), 7.07 (s, 1 H).

3.3. Synthesis of 2-bromo-4-(2-hydroxyethoxy)-5-methoxy- benzylamine hydrochloride

37% Hydrochloric acid (0.2 ml) was added to a solution of 2-bromo-4- (2-hydroxyethoxy)-5-methoxy-Λ/-acetyl benzylamine 4 (0.1 g, 0.31 mmol) in abs. ethanol (5 ml) and the mixture was refluxed for 12 hours. After cooling, the solvent was evaporated off under reduced pressure and the residue was recrystallized from a methanol/ethyl ether mixture to afford the title compound as pale yellow solid in quantitative yield.

¹H NMR (DMSO-de) δ 3.95 (s, 3H), 4.14 (t, 2H, J=2), 4.19 (m, 2H₁), 5.01 (m, 2H₁), 5.44 (t, 1 H), 7.02 (s, 1 H), 7.27 (s, 1 H), 7.38 (m, 3H).

Example 4

General Procedure for the synthesis of 2-(phenyl/pyridine-3-yl/4- (chloro)phenyl)-4-(2,3-dihydroxypropoxy)-5-methoxy-N-acetyl benzylamine

To a solution of 2-(phenyl/pyridine-3-yl/4-(chloro)phenyl)-4-hydroxy-5- methoxy-N-acetyl benzylamine (1.1 mmol) in dry DMF (10 ml) dry K2CO3 (2 mol eq) and 3-chloro-1 ,2-dihydroxypropane (2 mol eq) were added. The mixture was refluxed for 12 hours, then the solvent was evaporated off under reduced pressure. After addition of water the aqueous layer was extracted with EtOAc (3X 25 ml) and the organic phases were washed with NaOH 3% (20 ml_), anhydrified over Na2SO4 and evaporated off under reduced pressure to furnish the title compound as solids after crystallization from Et₂O. 4.1. 2-phenyl-4-(2,3-dihydroxypropoxy)-5-methoxy-N-acetyl benzylamine

Pale yellow solid, yield 72%.

¹H NMR (DMSO-de) δ 1.83 (s, 1 H), 3.44 (t, 2H), 3.79 (s, 1 H), 3.95 (m, 7H), 4.10 (d, 2H, J=4.2), 4.62 (t, 1 H), 4.92 (d, 1 H), 6.79 (s, 1 H), 6.98 (s, 1 H), 7.36 (m, 5H), 8.16 (t, 1 H).

4.2. 2-(pyridine-3-yl)-4-(2,3-dihydroxypropoxy)-5-methoxy-N-acetyl benzylamine

Pale yellow solid, yield 60%. ¹H NMR (DMSO-de) δ 1.80 (s, 3H), 3.44 (t, 2H), 3.80 (s, 3H), 3.96 (m, 3H), 4.08 (d, 2H), 4.62 (t, 1 H), 4.93 (d, 1 H), 6.84 (s, 1 H), 7.02 (s, 1 H)₁ 7.44 (m, 1 H), 7.77 (m, 1 H), 8.20 (bt, 1 H), 8.56 (m, 2H).

4.3. 2-(4-chloro)-phenyl-4-(2,3-dihydroxypropoxy)-5-methoxy-N-acetyl benzylamine

Pale yellow solid, yield 65%.

¹H NMR (DMSO-de) δ 1.81 (s, 3H), 3.42 (d, 3H), 3.79 (s, 3H), 3.90 (m, 4H), 4.08 (d, 2H), 6.79 (s, 1 H), 6.98 (s, 1 H), 7.45 (dd, 4H), 8.20 (bt, 1 H).

Example 5 5.1. Synthesis of 2-bromo-4-(2,3-dihydroxypropoxy)-5-methoxy-N- acetyl benzylamine

To a solution of 2-bromo-4-hydroxy-5-methoxy-Λ/-acetyl benzylamine 3 (0.3 g, 1.1 mmol) in dry DMF (10 ml) dry K2CO3 (2 mol eq) and 3-chloro-1 ,2- dihydroxypropane (2 mol eq) were added. The mixture was refluxed for 6 hours, then the solvent was evaporated off under reduced pressure. After addition of water the aqueous layer was extracted with EtOAc (3X 25 ml) and the organic phases were anhydrified over Na2SO4 and evaporated off under reduced pressure to furnish the title compound as pale yellow solid (0.35 g, 84% yield). 1H NMR (DMSO-de) δ 1.88 (s, 3H), 3.44 (t, 2H), 3.74 (s, 3H), 3.88-3.96

(m, 3H), 4.22 (d, 2H, J=6), 4.66 (t, 1 H), 4.96 (d, 1 H, J=6), 6.93 (s, 1 H), 7.14 (s, 1 H), 8.25 (t, 1 H).

5.2. Synthesis of 2-bromo-4-(2,3-dihydroxypropoxy)-5-methoxy- benzylamine hydrochloride 37% Hydrochloric acid (0.3 ml) was added to a solution of 2-bromo-4-

(2,3-dihydroxypropoxy)-5-methoxy-Λ/-acetyl benzylamine 4 (0.3 g, 0.86 mmol) in abs. ethanol (12 ml) and the mixture was refluxed for 12 hours. After cooling, the solvent was evaporated off under reduced pressure and the residue was recrystallized from a methanol/ethyl ether mixture to afford the title compound as pale orange solid in quantitative yield.

¹H NMR (DMSO-de) δ 3.42 (t, 2H), 3.74 (s, 3H), 3.74-3.95 (m, 4H), 4.21 (d, 2H, J=6), 4.98 (m, 4H), 7.13 (s, 1 H), 7.38 (s, 1 H). Example 6

General Procedure for the synthesis of 2-(phenyl/pyridine-3-yl/4- (chloro)phenyl)-4-(2,3-dihydroxypropoxy)-5-methoxy-benzylamine hydrochloride

37% Hydrochloric acid (5 ml) was added to a solution of 2-(phenyl/pyridine-3-yl/4-(chloro)phenyl)-4-(2,3-dihydroxypropoxy)-5- methoxy-N-acetyl benzylamine (8 mmol) in abs. ethanol (25 ml) and the mixture was refluxed for 12 hours. After cooling, the solvent was evaporated off under reduced pressure and the residue was recrystallized from a methanol/ethyl ether mixture to afford the title compound solids in a quantitative yield.

6.1. Synthesis of 2-chloro-4-acetoxy-5-methoxy-Λ/-acetyl benzylamine

Λ/-chlorosuccinimide (3.15 mmol, 0.42 g) was added to a solution of 4-acetoxy-3-methoxy-Λ/-acetyl-benzylamine of Example 1.1 (0.5 g, 2.1 mmol) in dry DMF (6 ml) and the mixture was stirred for 30' at 0⁰C and then for 16 hours at room temperature.

When water was added to the reaction (40 ml) the formation of a white precipitate was observed.

The solid was filtered off and washed twice with cold water (2 x 20 ml), then dried over P2O5 to afford the title compound as white solid (0.45 g, 83% yield).

¹H NMR (DMSO-de) δ 1.85 (s, 3H), 2.21 (s, 3H), 3.74 (s, 3H, OCH₃),4.21 (d, 2H, CH₂, J=8), 7.01 (s, 1 H, aromatic), 7.22 (s, 1 H, aromatic), 8.32 (t, 1 H, NH). Bidimensional NOESY (DMSO-dβ): coupling between the singlet at 2.21 ppm and the singlet at 7.22 ppm confirms that chlorine is at the 2-position of the aromatic ring.

MS: m/z 272.1 (M⁺ Ci₂Hi₄CINO₄). 6.2. Synthesis of 2-chloro-4-hydroxy-5-methoxy-Λ/-acetyl benzylamine

10% Aq. hydrochloric acid (2.5 ml) was added to a solution of 2-chloro- 4-acetoxy-5-methoxy-Λ/-acetyl-benzylamine 2 (0.45 g, 1.66 mmol) in dioxane (15 ml) and the mixture was refluxed for 2 hours. After cooling, the solvent was reduced under vacuum and the residue was basified with 10% aq. NaOH. The resulting solid was collected by filtration, washed with cold water and dried to furnish the title compound as white solid in quantitative yield.

¹H NMR (DMSO-de) δ 2.15 (s, 3H, CH₃), 3.82 (s, 3H, OCH₃), 3.99 (d, 2H, CH₂), 6.86 (s, 1 H, aromatic), 7.30 (s, 1 H, aromatic), 8.41 (t, 3H, NH₂), 9.77 (bs, 1 H₁ OH). M. p.: >300°C.

6.3. Synthesis of 2-chloro-4-(2,3-dihydroxypropoxy)-5-methoxy-/V- acetyl benzylamine

Dry K₂CO₃ (2 mol eq) and 2-iodoethanol (2 mol eq) were added to a solution of 2-chloro-4-hydroxy-5-methoxy-Λ/-acetyl benzylamine 3 (0.4 g, 1.46 mmol) in dry DMF (15 ml). The mixture was refluxed for 6 hours, then the solvent was evaporated off under reduced pressure. After addition of water the aqueous layer was extracted with EtOAc (3X 25 ml) and the organic phases were anhydrified over Na₂SO₄ and evaporated under reduced pressure to furnish the title compound as pale yellow solid (0.38 g, 81 % yield).

¹H NMR (CDCI₃) δ 2.00 (s, 3H), 3.84 (s, 3H), 3.92 (t, 2H, J=2), 4.09 (t, 2H₁ J=2.1), 4.44 (d, 2H, J=4), 5.21 (t, 1 H), 5.90 (bs, 1 H), 6.96 (s, 1 H), 7.07 (S, 1 H). 6.4. Synthesis of 2-chloro-4-(2,3-dihydroxypropoxy)-5-methoxy- benzylamine hydrochloride

37% Hydrochloric acid (0.2 ml) was added to a solution of 2-bromo-4- (2-hydroxyethoxy)-5-methoxy-Λ/-acetyl benzylamine 4 (0.1 g, 0.31 mmol) in abs. ethanol (5 ml) and the mixture was refluxed for 12 hours. After cooling, the solvent was evaporated off under reduced pressure and the residue was recrystallized from a mixture of methanol/ethyl ether to afford the title compound as pale yellow solid in quantitative yield. Example 7 General procedure for the synthesis of compounds 6a-6m Triphosgene

(0.37 mol eq) was dissolved in CH2CI2 (3 ml). A mixture of 4-terf- butyl/trifluoromethyl benzyl amine (0.33 mmol) and DIEA (2.2 mol eq) in CH2CI2 (2 ml) was slowly added to the stirred solution of triphosgene over a period of 30 min. using a syringe pump. After 5 min a solution of a suitable amine hydrochloride 5 (0.33 mmol) was added in one portion. The reaction mixture was stirred at room temperature for 2-4 h, evaporated under reduced pressure, diluted with EtOAc (20 ml), washed with 10% aq. KHSO₄, 5% aq. NaHCO3 and brine, dried over Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography (100% EtOAc) to furnish the title compound as solid.

7.1. 1 -[4-(2-Hydroxyethoxy)-2-bromo-5-methoxybenzyl]-3-[4-(terf- butyl)-benzyl] urea 6b

¹H NMR (DMSO-de) δ 1.26 (s, 9H), 3.37 (s, 3H), 3.70 (m, 4H), 3.98 (t, 2H, J=2), 4.61 (bs, 4H), 4.87 (t, 1 H, J=2.1), 6.98 (bs, 1 H), 7.21 (s, 1 H), 7.23 (d, 2H, J=7.8), 7.34 (d, 2H, J=8), 7.80 (bs, 1 H), 8.00 (bs, 1 H). Mp: 138°C. MS: m/z 481.4 (M⁺ 022H₂₉BrN₂O₃S).

7.2. 1-[4-(2,3-Dihydroxypropoxy)-2-chloro-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea 6c

White solid, yield 80%.

¹H NMR (DMSO-de) δ 3.44 (t, 2H, J=6), 3.68 (s, 1 H), 3.74-4.00 (m, 5H), 4.22 (d, 2H, J=6), 4.32 (d, 2H, J=6), 4.67 (t, 1 H), 4.94 (d, 1 H), 6.45 (bt, 1 H), 6.65 (bt, 1 H), 6.90 (s, 1 H), 7.00 (s, 1 H), 7.48 (d, 2H, J=7.8), 7.69 (d, 2H, J=8).

MS: m/z 462 (M⁺ C20H22CIF3N2O5).

Mp: 154-5°C.

(R)-(-)6c: [α]D²⁰ = -8.33 (95% EtOH). 7.3. 1 -[4-(2,3-Dihydroxypropoxy)-2-bromo-5-methoxybenzyl]-3-[4-

(trifluoromethyl)-benzyl] urea 6d

White solid, yield 84%.

¹H NMR (DMSO-de) δ 3.41 (t, 2H, J=6), 3.74 (s, 1 H), 4.00-3.85 (m, 5H), 4.19 (d, 2H, J=6), 4.32 (d, 2H, J=6), 4.62 (t, 1 H), 4.95 (d, 1 H), 6.49 (bt, 1 H), 6.68 (bt, 1 H), 6.90 (s, 1 H), 7.13 (s, 1 H), 7.45 (d, 2H, J=7.8), 7.65 (d, 2H, J=8).

MS: m/z 507 (M⁺ C₂OH₂₂BrF₃N₂O₅).

Mp: 164°C.

(R)-(-)6d: [α]_D ²⁰ = -8.5 (95% EtOH). 7.4. 1-[4-(2-Hydroxyethoxy)-2-bromo-5-methoxybenzyl]-3-[4-

(trifluoromethyl)-benzyl] urea 6h

White solid, yield 73%.

¹H NMR (DMSO-de) δ 3.67 (s, 3H), 3.47 (m, 2H), 3.94 (t, 2H, J=4), 4.16 (d, 2H, J=6), 4.32 (d, 2H, J=6), 4.85 (t, 1 H, J=2), 6.52 (bt, 1 H), 6.68 (bt, 1 H), 6.90 (s, 1 H), 7.14 (s, 1 H), 7.49 (d, 2H, J=8), 7.65 (d, 2H, J=8).

MS: m/z 477 (M⁺ Ci₉H₂₀BrF₃N₂O₄).

Mp: 162°C.

7.5. 1 -[4-(2,3-dihydroxypropoxy)-2-phenyl-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea 6i

White solid, yield 35%.

¹H NMR (DMSO-de) δ 3.41 (t, 2H), 3.78 (s, 3H), 4.06 (m, 3H), 4.23 (d, 2H), 4.26 (d, 2H), 4.61 (t, 1 H), 4.95 (d, 1 H), 6.40 (t, 1 H), 6.51 (t, 1 H), 6.80 (s, 1 H), 6.99 (s, 1 H), 7.36 (m, 7H), 7.68 (d, 2H).

MS: m/z 504 (M⁺ C26H27F3N2O5).

Mp: 168°C.

7.6. 1-[4-(2,3-dihydroxypropoxy)-2-(pyridin-3-yl)-5-methoxybenzyl]-3- [4-(trifluoromethyl)-benzyl] urea 6I Pale yellow solid, yield 30%.

¹H NMR (DMSO-de) δ 3.44 (t, 2H), 3.75 (s, 3H), 3.91 (m, 4H), 4.05 (d, 2H, J=4.3), 4.29 (d, 2H, J04.2), 4.61 (t, 1 H), 4.92 (d, 1 H), 6.51 (m, 2H), 6.84 (s, 1 H), 7.03 (s, 1 H), 4.45 (m, 3H), 7.68 (d, 2H), 7.77 (m, 1 H), 8.56 (m, 1 H).

MS: m/z 505 (M⁺ C25H26F3N3O5). Mp: 201⁰C.

7.7. 1-[4-(2,3-dihydroxypropoxy)-2-(4-chloro)phenyl-5-methoxybenzyl]- 3-[4-(trifluoromethyl)-benzyl] urea 6m

White solid, yield 45%.

¹H NMR (DMSO-de) δ 3.44 (t, 2H), 3.74 (s, 3H), 3.93 (m, 4H), 4.07 (d, 2H), 4.30 (d, 2H), 4.62 (t, 1 H), 4.92 (d, 1 H), 6.41 (m, 2H), 6.78 (s, 1 H), 6.98 (s, 1 H), 7.43 (m, 6H), 7.69 (d, 2H).

MS: m/z 538 (M⁺ C26H26CIF3N2O5).

Mp: 174°C.

Biological Assays Animals

In vivo experiments were conducted with PharmEste srl (Ferrara, Italy) and with the University of Ferrara, following protocols approved by the Animal Care and Use Committee of the University of Ferrara. Radioligand binding assay

Male Sprague-Dawley rats with body weight between 250 to 350 g were used. For binding assays the rats were decapitated under anesthesia and the spinal cord was removed and disrupted using a Polytron tissue homogenizer in ice cold buffer containing 5 mM KCI, 5.8 mM NaCI, 0.75 mM CaCb, 2 mM MgCb, 320 mM sucrose, 10 mM Hepes, pH 8.6 (Szallasi and Blunberg, 1992; 1993). In competition experiments, the membranes were incubated at 37°C for 60 min with [³H]RTX (0.4 nM) and with increasing concentrations of test compounds in the range from 0.1 nM to 3 μM. Non-specific binding was evaluated in the presence of 1 μM RTX. Saturation and competition studies were analyzed with the Ligand program (Bradford, 1976; Munson and Rodbard, 1980).

Ca²⁺ fluorescence measurements in cultured rat trigeminal ganglia

The calibration curve was determined using a buffer containing Fura-2- AM-ester and definite concentrations of free Ca²⁺. This curve was then used to convert the data obtained from F340/F380 ratio to [Ca²⁺Jj (nM) (Kudo, Y).

The effects of pretreatments with compounds 6a-6m on the increase in

[Ca²⁺]i produced by 30 nM capsaicin were studied.

Capsaicin-induced secondary allodynia in rat Capsaicin (5 nmols/50 μl/paw) was injected in the plantar surface of the glabrous skin of the right paw of rats anesthetized with diethyl ether (Chaplan et al., 1994). Compounds 6c and 6d were orally administrated 2 hours prior to capsaicin injection. Tactile allodynia was evaluated 90 min after capsaicin challenge. Reagents

The stock concentrations of capsaicin (10 mM) was prepared in absolute ethanol. Compounds 6a-6m were prepared in 50% DMSO and 50% Tween 80. Fura-2-AM-ester and ionomycin were dissolved in 100% DMSO. All the other drugs were dissolved in distilled water. The appropriate dilutions were then made in Krebs buffer solution.

Results

Radioligand binding assays

Compounds 6a-6m displaced [³H]RTX from its binding site in rat spinal cord membranes at low concentrations, as indicated by the Ki values reported in Table 1.

Ca²⁺ fluorescence Assay

Capsaicin (30 nM) increased [Ca²⁺]i in the vast majority (95%) of rat trigeminal neuron cells, which were therefore identified as TRPV1 -expressing neurons. IC50 values of inhibiting capsaicin-evoked [Ca²⁺]i mobilization are summarized in Table 1.

Table 1: Ki and IC50 values of some representative compounds of the invention.

The (R)-(-) and (S)-(+)-isomers of compounds 6c and 6d were also synthesized in order to appreciate the difference in activity with the respect to the racemic compounds. The most active isomer was the (R)-(-) form whereas as the (S)-(+)-form was at least 300 fold less active as shown in Table 2.

Due to the results obtained with the synthesis of the two separate isomers, compounds 6i-6m where directly synthesized in the active (R)-(-) forms. Table 2. Comparison between compounds 6c and 6d with their active isomers.

The results are expressed as Mean and 95% fiducial limits. Capsaicin-induced secondary allodynia in rat In a more extended study, compounds 6c and 6d were tested against capsaicin-induced secondary allodynia in rats. 90 Min after the capsaicin challenge, compounds 6c and 6d (both at 30 μmol/kg, p.o), significantly prevented the pro-allodinic effect of capsaicin (53.1 % and 47.9% of inhibition, respectively). ADME Studies

In order to select suitable drug candidates, ADME studies in vitro were performed on selected compounds 6c, 6d along with their active isomers, so as to assess the properties of these compounds according to the substituents. LogD Values at pH=7.0 were calculated in silico, while the in vitro tests analysed:

- metabolic stability in cryopreserved human hepatocytes;

- cytotoxicity on Hep G2 cells;

- cassette pharmacokinetics in rat. The data of the compounds of the invention were compared to those obtained on two structurally different compounds recently disclosed as TRPV1 antagonists, namely JYL 1421 (Jakab et al., 2005) and SB-705498 (Rami et al., 2006) and to those obtained with two widely used drugs, one with short half life (naloxone) and one with long half-life (tolbutamide). The most relevant ADME data allow rapid comparison of the influence of specific substituents, especially on metabolic stability. Hepatocytes preparation

The cells were rapidly and carefully thawed and diluted in ice-cold Krebs-Henseleit Buffer (KHB). After centrifugation (50 g, 5 min.) the supernatant was discharged and the cells were resuspended in a volume of ice-cold KHB to a greater density than 2X (with respect to the final concentration of incubation) of viable cells/ml based on nominal concentration in cryopreserved vials. The viable cells were counted by Trypan Blue exclusion with a haemocytometer and the concentration of viable hepatocytes was accurately corrected to 2X concentration with KHB. Hep G2 cells preparation

The cells were cultured for 3 days, trypsinized and re-suspended in 20 ml of culture medium. The cells were then counted and diluted to obtain a final concentration suitable for seeding 40.000 cells/well in 96-well cell culture plates (200 μl/well). The cells were seeded in columns 1 to 11 (column 12 contained medium without cells), then placed for 16-24 hours at 37°C with 5% of CO2. Compounds preparation

Test and reference compounds were prepared at 2X incubation concentrations (10 and 1 μM) diluting 10 μl of stock solution in 0.99 ml of KHB to a concentration of 10 μM and 5 μl of stock solution in 0.995 μl of KHB to a concentration 5 μM. 300 μl of 10 μM and 1 μM solutions were then dispensed respectively in 2 and 1 incubation test tubes (Sterilin T.C. tube 17 x 100 mm). Preliminary cassette pharmacokinetic study in catheterized conscious rats Compounds were administered together to rats. The compounds were stored at -20⁰C when not used. The formulation, route of administration, plasma samples identification and pharmacokinetic analysis were performed according to standard protocols (Raynaud, Fl et al, 2004; Manitpisitkul, P. et al., 2004; Singh S. et al., 2006).

Results

With respect to compounds Ia, Ib, Ic disclosed in WO 2005/123666 A1 , this new series of O-hydroxyalkyl urea derivatives showed a clear unexpected improvement in terms of metabolic stability and cytotoxicity comparable to reference compounds JYL 1421 and SB-705498 and a good half-life time, their clearance being relatively slow. Also cytotoxicity values expressed as micromolar IC50 were acceptable. Table 3 reports the ADME profile of two compounds of the invention with respect to compounds disclosed in WO 2005/123666.

Furthermore, the (fϊ)-(-)-isomers of compounds 6c and 6d showed a further improvement in terms of half-life, maintaining at the same time good values of metabolic stability and low cytotoxicity (Table 3).

Table 3. ADME profile of compounds 6c, 6d and their active isomers with the respect to selected reference compounds

References

1. PharmEste S.r.l. WO 2005/123666 A1.

2. Bradford MM. Anal Biochem. 1976, 72, 248-254. 3. Munson PJ. et al., Anal Biochem 1980, 107, 220-239.

4. Rigoni, M. et al., Br. J. Pharmacol. 2003, 138, 977-985.

5. Kudo, Y. et al., Jap. J. Pharmacol. 1986, 41, 345-351.

6. Chaplan N. et al., J Neurosci Methods. 1994, 53, 55-63

7. Jakab B. et al. Eur. J. Pharmacol. 2005, 517, 35-44. 8. Rami HK. et al. Bioorg. Med. Chem. Lett. 2006, 16, 3287-3291.

9. Raynaud Fl. et al. MoI. Cancer Ther. 2004, 3, 353-362.

10. Manitpisitkul, P. et al. Drug Discov. Today, 2004, 9, 652-658.

11. Singh S. Curr. Drug Metab. 2006, 7, 165-182.

Claims

1. Compounds of formula (I)

in which

R is selected from halogen, alkyl, alkoxy, aryl and heteroaryl;

Ri is selected from 2-hydroxyethyl, 2,3-dihydroxypropyl, 3-hydroxypropyl, 2,2-dihydroxyethyl, 3,3-dihydroxypropyl, 1 ,3-dioxolane- ethyl, 1 ,3-dioxane-methyl, 1 ,3-dioxolane-methyl, 1 ,3-dioxane-ethyl, 3-fluoro- 2-hydroxypropyl, 3-carboxy-2-hydroxy-propyl, 3-chloro-2-hydroxypropyl, 2-hydroxypropyl, 2-hydroxy-propen-2-yl, morpholinoethyl, piperazinoethyl, hydroxymethyl, benzyl, 4-(hydroxymethyl)benzyl, 4-chlorobenzyl, 4-fluorobenzyl, and 4-hydroxybenzyl R2 is terf-butyl or trifluoromethyl;

R3 is independently selected from hydrogen, carboxy, cyano, alkyl or hydroxyalkyl, including all possible optical isomers and diastereoisomers thereof, wherein "halogen" is a halogen atom selected from fluorine, chlorine, bromine and iodine;

"alkyl" is a straight or branched (Ci-C-i)alkyl group;

"alkoxy" is a straight or branched (Ci-C4)alkoxy group;

"aryl" is phenyl, optionally substituted with one or more halogen, alkyl, alkoxy groups as defined heirein before, cyano or amino groups, which can be the same or different from one another; and

"heteroaryl" is a 5- or 6-membered heterocycle containing one ore more nitrogen, oxygen or sulphur atoms, which can be the same or different from one another.

2. Compounds according to claim 1 wherein: R is chlorine or bromine;

Ri is 2-hydroxyethyl; R2 is tert-butyl or trifluoromethyl; R3 Js hydrogen.

3. Compounds according to claim 1 wherein: R is chlorine or bromine;

Ri is 2,3-dihydroxypropyl; R2 is trifluoromethyl; R3 Js hydrogen.

4. Compounds according to claim 1 wherein:

R is methyl, phenyl, pyridine or 4-(substituted)-phenyl; Ri is (R)-(-)-2,3-dihydroxypropyl; R2 is trifluoromethyl; R3 Js hydrogen.

5. Compounds according to claim 1 wherein: R is chlorine or bromine;

Ri is (f?)-(-)-2,3-dihydroxypropyl; R2 is trifluoromethyl; R3 is hydrogen.

6. A compound selected from: 1-[4-(2-hydroxyethoxy)-2-bromo-5-methoxybenzyl]-3-[4-

(trifluoromethyl)-benzyl] urea; 1-[4-(2-hydroxyethoxy)-2-chloro-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea;

1-[4-(2-hydroxyethoxy)-2-bromo-5-methoxybenzyl]-3-[4-(terf-butyl)- benzyl] urea; 1-[4-(2-hydroxyethoxy)-2-chloro-5-methoxybenzyl]-3-[4-(terf-butyl)- benzyl] urea;

1-[4-((R)-(-)-2,3-dihydroxypropoxy)-2-phenyl-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea; 1-[4-((R)-(-)-2,3-dihydroxypropoxy)-2-(pyridin-3-yl)-5-methoxybenzyl]-

3-[4-(trifluoromethyl)-benzyl] urea;

1-[4-((R)-(-)-2,3-dihydroxypropoxy)-2-bromo-5-methoxybenzyl]-3-[4- (trifluoromethyl)-benzyl] urea.

7. Compounds of formula (I) as defined in any one of claims 1 to 6 for use as medicaments.

8. Use of compounds of formula (I) as defined in any one of claims 1 to 6 as vanilloid TRPV1 receptor antagonists.

9. Use of compounds of formula (I) as defined in any one of claims 1 to 6 for the preparation of pharmaceutical compositions for the therapy of inflammatory states.

10. The use according to claim 9 wherein the inflammatory state is selected from chronic neuropathic pain, over-active bladder syndrome, tumour pain, haemorrhoids, inflammatory hyperalgesia, post-intervention pain, dental extraction, airway and gastro-intestinal diseases.

11. Pharmaceutical compositions containing compounds of formula (I) as defined in any one of claims 1 to 6 in admixture with suitable excipients and/or vehicles.