WO2005080319A1

WO2005080319A1 - Hydrazino alcohol derivatives useful as inhibitors of copper-containing amine oxidases

Info

Publication number: WO2005080319A1
Application number: PCT/FI2005/050038
Authority: WO
Inventors: Ferenc Fulop; Laszlo Lazar; Marjo Pihlavisto; Marta Palko; Auni Juhakoski; Anne MARJAMÄKI; David John Smith
Original assignee: Biotie Therapies Corporation
Priority date: 2004-02-20
Filing date: 2005-02-18
Publication date: 2005-09-01
Also published as: FI20040270A0

Abstract

The invention relates to hydrazino alcohol derivatives of Formula (I) wherein ; R1 is hydrogen, lower alkyl or an optionally substituted phenyl or heteroaryl group; R2 is hydrogen or lower alkyl, or R1 and R2 may form together with the nitrogen atom to which they are attached a saturated heterocyclic ring; R3 - R5 represent each independently hydrogen, lower alkyl, aralkyl, optionally substituted phenyl or a heteroaryl group, or R2 and R3 may form together with the atoms to which they are attached a saturated heterocyclic ring, or R3 and R5 may form together with the carbon atoms to which they are attached a saturated carbocyclic ring; R6 is naphtyl, phenyl, substituted phenyl or a heteroaryl group; R7 is hydrogen, lower alkyl or aralkyl; n is 1, 2 or 3; and X = O, S, SO, SO2 or NR2. The compounds of Formula (I) are useful as inhibitors of copper-containing amine oxidases.

Description

Hydrazino alcohol derivatives useful as inhibitors of copper-containing amine oxidases

Field of the Invention The present invention relates to novel hydrazino alcohol derivatives and their use as inhibitors of copper-containing amine oxidases (E.G. 1.4.3.6) and enzymes of significant identity thereto. The compounds of the present invention have therapeutic utility as drugs for treating diseases including, but not limited to, inflammatory diseases. In particular, acute and chronic inflammatory conditions or diseases such as chronic arthritis, inflammatory bowel diseases and skin dermatoses as well as diseases related to carbohydrate metabolism and to aberrations in adipocyte differentiation or function and smooth muscle cell function may be treated with the compounds.

Background of the invention VAP-1 is a human endothelial cell adhesion molecule that has sev- eral unique properties that distinguish it from the other inflammation-related adhesion molecules. It has a unique and restricted expression pattern and mediates lymphocyte binding to vascular endothelium (Salmi, M., and Jalkanen, S., Science 257:1407-1409 (1992)). Inflammation induces the upregulation of VAP-1 to the surface of vascular endothelial cells mediating leukocyte entry to skin, gut and inflamed synovium (Salmi, M., and Jalkanen, S., Science 257: 1407-1409 (1992); Salmi, M., et al., J. Exp. Med 178:2255-2260 (1993); Arvil- lomi, A., et al., Eur. J. Immunol. 26:825-833 (1996); Salmi, M., et al., J. Clin. Invest. 99:2165-2172 (1997); (Salmi, M., and Jalkanen, S., J. Exp. Med. 183: 569-579 (1996); J. Exp. Med. 186:589-600 (1997)). One of the most interest- ing features of VAP-1 is a catalytic extracellular domain which contains a monoamine oxidase activity (Smith, D.J., et al., J. Exp. Med. 188:17-27 (1998). The cloning and sequencing of the human VAP-1 cDNA revealed that it encodes a transmembrane protein with homology to a class of enzymes called the copper-containing amine oxidases (E.C. 1.4.3.6). Enzyme assays have shown that VAP-1 possesses a monoamine oxidase (MAO) activity which is present in the extracellular domain of the protein (Smith, D.J., et al., J. Exp. Med. 188:17-27 (1998)). Thus, VAP-1 is an ecto-enzyme. Analysis of the VAP- 1 MAO activity showed that VAP-1 belongs to the class of membrane-bound MAO's termed semicarbazide-sensitive amine oxidases (SSAO). These are distinguished from the widely distributed mitochondrial MAO-A and B flavopro- teins by amino acid sequence, cofactor, substrate specificity and sensitivity to certain inhibitors. However, certain substrates and inhibitors are common to both SSAO and MAO activities. The mammalian SSAO's can metabolize various monoamines produced endogenously or absorbed as dietary or xenobiotic substances. They act principally on primary aliphatic or aromatic monoamines such as methylamine or benzylamine (Lyles, G.A., Int. J. Biochem. Cell Biol. 28:259-274 (1996)). Thus, VAP-1 located on the vascular endothelial cell surface can act on circulating primary monoamines with the following reaction pathway. RNH₂ + 0₂ + H₂0 ► RCHO + H₂0₂ + NH₃

Although the physiological substrates of VAP-1 SSAO in man have not been clearly identified. Methylamine is a good substrate for VAP-1 SSAO. Methylamine is a product of various human biochemical pathways for the deg- radation of creatinine, sarcosine and adrenaline, and is found in various mammalian tissues and in blood. It can also be derived from the diet by gut bacterial degradation of dietary precursors. The concentration of methylamine in the blood can be increased in certain physiological and pathological situations such as diabetes. Another potential physiological substrates is aminoacetone. VAP-1 SSAO activity has been proposed to be directly involved in the pathway of leukocyte adhesion to endothelial cells by a novel mechanism involving direct interaction with an amine substrate presented on a VAP-1 ligand expressed on the surface of a leukocyte (Salmi et al. Immunity, (2001 )). This publication describes the direct involvement of VAP-1 SSAO activity in the process of adhesion of leukocytes to endothelium. Thus inhibitors of VAP-1 SSAO activity could be expected to reduce leukocyte adhesion in areas of inflammation and thereby reduce leukocyte trafficking into the inflamed region and therefore the inflammatory process itself. In human clinical tissue samples expression of VAP-1 is induced at sites of inflammation. This increased level of VAP-1 can lead to increased production of H₂0₂ generated from the action of the VAP-1 SSAO extracellular domain on monoamines present in the blood. This generation of H₂0₂ in the localised environment of the endothelial cell could initiate other cellular events. H₂0₂ is a known signalling molecule that can upregulate other adhesion mole- cules and this increased adhesion molecule expression may lead to enhanced leukocyte trafficking into areas in which VAP-1 is expressed. It also may be that other products of the VAP-1 SSAO reaction could have biological effects also contributing to the inflammatory process. Thus the products of the VAP-1 SSAO activity may be involved in an amplification of the inflammatory process which could be blocked by specific SSAO inhibitors. VAP-1 SSAO may be involved in a number of other pathological conditions associated with an increased level of circulating amine substrates of VAP-1 SSAO. The oxidative deamination of these substrates would lead to an increase in the level of toxic aldehydes and oxygen radicals in the local environment of the endothelial cell which could damage the cells leading to vascular damage. Increased levels of methylamine and aminoacetone have been reported in patients with Type I and Type II diabetes and it has been proposed that the vasculopathies such as retinopathy, neuropathy and nephropathy seen in late stage diabetes could be treated with specific inhibitors of SSAO activity. The development of specific VAP-1 SSAO inhibitors that modulate VAP-1 activity would be useful for the treatment of acute and chronic inflammatory conditions or diseases such as chronic arthritis, inflammatory bowel diseases, and skin dermatoses, as well as diseases related to carbohydrate metabolism (including diabetes and complications resulting from diabetes). In addition, aberrations in adipocyte differentiation or function and smooth muscle cell function (in particular, atherosclerosis), and various vascular diseases may be suitable for treatment with VAP-1 SSAO inhibitors. Compounds structurally similar to those of the present invention have been described in the following documents: Journal of Medicinal Chemistry (2002), 45 (22), Bondavalli, F. et al., s. 4875-4887; II Farmaco (1990), 45 (5), Bruno, O. et al., s. 527-543; Khimiko-Farmatsevticheskii Zhurnal (1989), 23 (5), Vostrova, L. N. et aL. s. 584-587; Archives Internationales de Pharmacodynamie et de Therapie (1960), 128, Schuler, W. & Wyss, E., s. 431-468; US 2002/0002152 A1 ; and European Journal of Medicinal Chemistry (2002), 37 (10), Sivaku- mar, R. et al., s. 793-801. However, the compounds disclosed in said documents are intended for use in fields totally different from that of the present invention. Document WO 02/02090 A3 discloses hydrazine derivatives, which are described to be useful as VAP-1 SSAO inhibitors. However, said deriva- fives have been found to decompose producing thereby significant amount of benzaldehyde, which is not the case in connection with the now claimed compounds having the structural element -CH₂-X-R^δ.

Summary of the invention The present invention relates to hydrazino alcohol derivatives of formula (I) and pharmaceutically acceptable salts thereof,

X R^c (I) wherein R¹ is hydrogen, lower alkyl or an optionally substituted phenyl or heteroaryl group; R² is hydrogen or lower alkyl, or R¹ and R² may form together with the nitrogen atom to which they are attached a saturated heterocyclic ring; R³ - R⁵ represent each independently hydrogen, lower alkyl, aralkyl, optionally substituted phenyl or a heteroaryl group, or R² and R³ may form together with the atoms to which they are attached a saturated heterocyclic ring, or R³ and R⁵ may form together with the carbon atoms to which they are attached a saturated carbocyclic ring; R⁶ is naphtyl, phenyl, substituted phenyl or a heteroaryl group; R⁷ is hydrogen, lower alkyl or aralkyl; n is 1 , 2 or 3; and X = 0, S, SO, S0₂ or NR²; with the proviso that (i) when X is O, R¹ to R⁵ and R⁷ are hydrogen and n is 1 , then R⁶ is not phenyl or 2-methoxy-phenyl, (ii) when X is O, R¹ is hydrogen, R² is methyl and R³ to R⁵ and R⁷ are hydrogen and n is 1 , then R⁶ is not 3-trifluoromethyl-phenyl; and (iii) when X is O, R¹ is phenyl, R² to R⁵ and R⁷ are hydrogen and n is 1 , then R^δ is not 4-hydroxy-phtalazinyl. The invention also relates to the use of a derivative of formula (I) for inhibiting copper-containing amine oxidases. Further the invention relates to a derivativ of formula (I) for use as a medicament. The invention also relates to a pharmaceutical composition comprising an effective amount of a derivative of formula (I) and a suitable carrier. Finally the invention relates to a process for preparing derivatives of formula (I).

Detailed description of the invention In the definition of the compound group of formula (I) the term "lower alkyl" refers to branched or straight chain alkyl groups having suitably 1 - 6, preferably 1 - 4, carbon atoms, such as methyl, ethyl, n-propyl, isopro- pyl, n-butyl, sec-butyl, fetf-butyl and isobutyl, especially methyl. Illustrative examples of "aralkyl" are benzyl, p-methyl benzyl, p- chlorobenzyl, 2-phenylethyl and 3-phenylpropyl. Typical examples of "substituted phenyl" are o-tolyl, m-tolyl, p-tolyl, p-fluorophenyl, p-chlorophenyl, m- and o-metoxyphenyl. Illustrative examples of the meaning "heteroaryl group" are 2- pyridyl, 3-pyπdyl, 4-pyridyl, 2-furyl, 3-furyl, 1-thienyl, 2-thienyl. By the term "saturated carbocyclic ring" is meant e.g. cyclopentane, cyclohexane, 4-methyl-cyclohexane, cycloheptane, adamantane, indan and tetralin. Typical examples of the meaning "saturated heterocyclic ring" are pyrrolidine, piperidine, 1 ,2,3,4-tetrahydroisoquinoline, 6,7-dimethoxy-1 ,2,3,4- tetrahydro-isoquinoline, pyrazolidine and tetrahydropyridazine. The compound group of formula (I) encompasses racemic mixtures as well as optical isomers of the compounds in question. Preferred compounds if formula (I) are those where X is O or S or

NR², n is 1 , R¹ is hydrogen or phenyl, R² is hydrogen or lower alkyl, R³, R⁴ and R⁵ are hydrogen and R^δ is naphtyl or phenyl optionally substituted with one or more substituents selected from the group consisting of lower alkyl and lower alkoxi. Examples of preferred specific compounds of formula (I) are: 1 -(1 -methylhydrazino)-3-phenoxy-2-propanol, 1-(1-methylhydrazino)-3-(p-tolyloxy)-2-propanol, 3-(p-methoxyphenoxy)-1 -(1 -methylhydrazino)-2-propanol, 3-(p-methoxyphenoxy)-1 -(2-phenylhydrazino)-2-propanol, 1 -(1 -methylhydrazino)-3-phenyltio-2-propanol, 3-(3,4-dimethoxyphenyltio)-1 -(1 -methylhydrazino)-2-propanol, 3-(3-methoxyphenoxy)-1 -(1 -methylhydrazino)-2-propanol, (S)-3-(3-methoxyphenoxy)-1-(1-methylhydrazino)-2-propanol, and 1 -(1 -methylhydrazino)-3-methylphenylamino-2-propanol. According to the invention the hydrazino alcohol derivatives of for- mula (I) can be used for inhibiting copper-containing amine oxidases both in vitro and in vivo, for instance for diagnostic purposes.The derivatives of formula (I) are also useful as medicaments for treating diseases related to the inhibition of copper-containing amine oxidase, e.g. diseases such as inflammatory bowel diseases, skin dermatoses, as well as diseases related to carbohy- drate metabolism (including diabetes and complications resulting from diabetes). In addition, aberrations in adipocyte differention or function and smooth muscle cell function (in particular atherosclerosis), and various vascular diseases may be suitable for the treatment with the now described VAP-1 SSAO inhibitors. The pharmaceutical compositions according to the present invention contains an effective amount of a derivative of formula (I) together with a suitable carrier and optionally with suitable appropriate additives conventionally used in the field. The hydrazino alcohols of formula (I) can be prepared by a) reducing a nitroso compound of formula

R^c (III) wherein R² - R⁷, n and X are as defined above, or b) hydrolyzing an oxadiazine compound of formula

wherein R - R , n and X are as defined above, and R and R represent each independently lower alkyl or aralkyl. The compounds of formula (I) were synthesized starting from amino alcohols of formula

01) where R² - R⁷, n and X are as defined above, either via /V-nitroso derivatives of formula (III) or via oxadiazines of formula (IV). Nitroso compounds of formula (III) were obtained from amino alcohols of formula (II) in slightly acidic aqueous solution by using sodium nitrite (A. A. Potekhin, A. O. Safronov, Zhur. Org. Khim., 1981 , 17, 379-386; H. Ta- kahashi, T. Senda, K. Higashiyama, Chem. Pharm. Bull., 1991 , 39, 836-842; J- K. Shen, H. Katayama, N. Takatsu, I. Shiro, J. Chem. Soc. Perkin Trans. 1, 1993, 2087-2097) or by using other well known methods of A/-nitrosation (M. A. Zolfigol, M. H. Zebarjadian, G. Chehardoli, H. Keypour, S. Salehzadeh, M. Shamsipur, J. Org. Chem., 2000, 66, 3619-3620). Reduction of nitroso com- pounds of formula (III) were carried out either in tetrahydrofurane by using lithium aluminium hydride (H. Takahashi, T. Senda, K. Higashiyama, Chem. Pharm. Bull., 1991 , 39, 836-842) or in aqueous acetic acid by using zinc dust (D. L. Trepanier, V. Sprancmanis, K. G. Wiggs, J. Org. Chem., 1964, 29, 668- 672). Some hydrazino alcohols of formula (I) were prepared by acidic hydrolysis of oxadiazines of formula (IV) obtained from amino alcohols of formula (II) and oxaziridines of formula

where R⁸ and R⁹ are as defined above, (E. Schmitz, S. Schramm, Cs. Szantay, Zs. Kardos, Liebigs Ann. Chem., 1983, 1043-1046). Compounds of formula (I), where R² = H, may be prepared by hydrazinolysis of the corresponding oxirane derivatives of formula.

In the case of R³ ≠ R⁴, the amino alcohols of formula (II) were used as single diastereomers. The synthesis of the enantiomers of the compounds of formula (I) started from enantiomerically pure amino alcohols of formula (II) or epoxides of formula (VI). Transformations occurred without racemization. The compounds of formula (I) are useful in the form of acid addition salts. The expression "pharmaceutically acceptable acid addition salt" is intended to apply to any non-toxic organic and inorganic acid addition salts of the base compounds of formula (I). Illustrative inorganic acids, which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids. Illustrative organic acids, which form suitable salts include acetic, lactic, malo- nic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic, methanesulfonic and salicylic acids. The following examples illustrates the present invention.

Example 1

1 -(1 -Methylhydrazino)-3-phenoxy-2-propanol hydrogenmaleate (BTT- 2066) A solution of NaN0₂ (1.38 g, 20 mmol) in H₂0 (10 ml) was added dropwise to a suspension of 1-methylamino-3-phenoxy-2-propanol (1.81 g, 10 mmol) in H₂0 (50 ml) with vigorous stirring on an ice-cold bath, and then

AcOH (0.90 g, 15 mmol) was added dropwise. The mixture was stirred at room temperature for 8 h, then was extracted with EtOAc (4 x 50 ml). The combined organic phases were dried (Na₂S0 ) and evaporated under reduced pressure to give 2.04 g A/-nitroso derivative as an oily product which was used in the next step without further purification. A solution of 1-methylamino-/V-nitroso-3-phenoxy-2-propanol (2.04 g, 9.7 mmol) in THF (20 ml) was added dropwise to a stirred suspension of Li- AIH₄ (0.74 g, 19.5 mmol) in THF (50 ml), and the mixture was stirred and re- fluxed for 3 h. The excess of LiAIH was decomposed with a mixture of H₂0 (1.5 ml) and THF (20 ml), the resulting precipitate was filtered off and washed with EtOAc (2 x 75 ml). The combined filtrates were dried (Na S0 ) and evaporated under reduced pressure. The oily residue was treated with an equivalent amount of maleic acid in a mixture of EtOH and Et₂0 to give crystalline hydrogenmaleate salt which was filtered off and recrystallized. ¹H-NMR (400 MHz, D₂0) δ (ppm): 3.06 (3H, s, NCH₃), 3.34-3.48 (2H, m, NCH₂), 4.08 (1 H, dd, J = 10.4, 5.2 Hz, OCH₂), 4.15 (1 H, dd, J = 10.4, 4.2 Hz, OCH₂), 4.40-4.50 (1 H, m, OCH), 6.28 (2H, s, CHCOOH) 6.97-7.11 (3H, m, C₆H₅), 7.32-7.42 (2H, m, C₆H₅).

Example 2

1 -(1 -Methylhydrazino)-3-(p-tolyloxy)-2-propanol fumarate To a solution of 1-oxa-2-azaspiro[2.5]octane (0.60 g, 5.3 mmol) in ether (20 ml) a solution of 1-methylamino-3-(p-tolyloxy)-2-propanol (1.03 g, 5.3 mmol) in ether (10 ml) was added. The reaction mixture was stirred at room temperature for 30 minutes then evaporated to dryness. 5% Hydrochloric acid (30 ml) was added to the residue and the mixture was stirred at ambient temperature for 1 h. The mixture was washed with Et₂0 (2 x 30 ml), made alkaline with Na₂C0₃ under ice-cooling and extracted with EtOAc (3 x 50 ml). The combined EtOAc extracts were dried and evaporated to give a oily residue which was treated with 0.5 equivalent amount of fumaric acid in a mixture of EtOH and Et₂0 to give crystalline fumarate salt which was filtered off and re- crystallized. ¹H-NMR (400 MHz, D₂0) δ (ppm): 2.31 (3H, s, CCH₃), 3.07 (3H, s,

NCH₃), 3.34-3.48 (2H, m, NCH₂), 4.08 (1 H, dd, J = 10.4, 5.2 Hz, OCH₂), 4.16 (1 H, dd, J = 10.4, 4.0 Hz, OCH₂), 4.42-4.51 (1 H, m, OCH), 6.56 (1 H, s, CHCOOH), 6.98 (2H, d, J = 8.3 Hz, C₆H₄), 7.24 (2H, d, J = 8.3 Hz, C₆H₄). Example 3

3-(p-Methoxyphenoxy)-1 -(1 -methylhydrazino)-2-propanol hydrogenmale- ate To an ice-cooled and stirred suspension of zinc dust (2.62 g, 40 mmol) in H₂0 (10 ml) a solution of 3-(p-methoxyphenyl)-1-methylamino-/V- nitroso-2-propanol (2.30 g, 9.6 mmol, prepared according to Example 1 starting from 10 mmol 3-(p-methoxyphenyl)-1-methylamino-2-propanol) in AcOH (18 ml) was added dropwise over a period of 45 min. During the addition, the temperature of the reaction mixture was maintained at 20-25 °C by external cooling. After the addition was completed, the mixture was stirred at 50 °C for 1 h, then filtered by suction, and the zinc residue was washed with a mixture of H₂0 (15 ml) and AcOH (5 ml). The combined filtrate and washings were concentrated to ca. 10 ml in vacuo. The iced-cooled solution was made basic with 10% Na₂C0₃ solution and extracted with EtOAc (4 x 50 ml). The combined ethereal extracts were dried and under reduced pressure. The oily residue was treated with an equivalent amount of maleic acid in a mixture of EtOH and Et₂0 to give crystalline hydrogenmaleate salt which was filtered off and recrys- tallized. ¹H-NMR (400 MHz, D₂0): 3.06 (3H, s, NCH₃), 3.30-3.50 (2H, m, NCH₂), 3.80 (3H, s, OCH₃), 4.03 (1 H, dd, J = 10.4, 5.1 Hz, OCH₂), 4.10 (1 H, dd, J = 10.4, 4.1 Hz, OCH₂), 4.36-4.51 (1 H, m, OCH), 6.27 (2H, s, CHCOOH), 6.84-7.08 (4H, m, C₆H₄).

Example 4

3-(p-Methoxyphenoxy)-1 -(2-phenylhydrazino)-2-propanoI hydrochloride To a stirred solution of phenyl hydrazine (1.08 g, 10 mmol) in EtOH

(20 ml) 3-(p-methoxy-phenoxy)-1 ,2-epoxypropane (1.80 g, 10 mmol) was added dropwise and the mixture was heated under reflux for 1 h. The solvent was evaporated off and the oily residue was dissolved in methanol (5 ml) and converted to the crystalline hydrochloride salt by using 22% ethanolic hydro- gen chloride (2 ml) and diethyl ether. Crystals were filtered off and recrystal- lized. ¹H-NMR (400 MHz, D₂0) δ (ppm): 3.35-3.60 (2H, m, NCH₂), 3.80 (3H, s OCH₃), 4.02-4.13 (2H, m, OCH₂), 4.40-4.48 (1 H, m, OCH). Example 5

1 -[2-Methoxy-3-(p-tolyloxy)propyI]-1 -methylhydrazine hydrogenfumarate 55% Sodium hydride suspension (1.33 g, 30.5 mmol) was washed with π-hexane and suspended in THF (35 ml). A solution of 2-methylamino-/V- nitroso-1-(m-metoxyphenyl)-1-ethanol (2.13 g, 9.5 mmol) in THF (60 ml) was degassed with N₂ flushing and added dropwise to the NaH suspension with stirring and continuous N₂ flushing at 0°C over a period of 1 h. Stirring was continued at 0 °C for 2 h, then a solution of Mel (2.30 g, 16.2 mmol) in THF (20 ml) was added dropwise to the stirred suspension at 0 °C. The mixture was allowed to warm to room temperature and the excess of NaH decomposed by addition of MeOH. The solution evaporated to dryness, the residue was dissolved in H₂0 (40 ml) and extracted with EtOAc (3 x 40 ml). The combined EtOAc extracts were washed with H₂0 (40 ml) then dried (Na₂S0 ) and evaporated under reduced pressure to give 1.86 g oily product which was used in the next step without further purification. A solution of A/-methyl-2-methoxy-3-(p-tolyloxy)-/V-nitrosopropyl- amine (1.86 g, 7.8 mmol) in THF (20 ml) was added dropwise to a stirred and ice-cooled suspension of LiAIH₄ (1.20 g, 31.6 mmol) in THF (60 ml). The mixture was stirred at 0 °C for 3 h, then allowed to warm to room temperature. The excess of LiAIH₄ was decomposed with a mixture of H₂0 (2.4 ml) and THF (20 ml), the resulting precipitate was filtered off and washed with EtOAc (2 x 75 ml). The combined filtrate were dried (Na₂S0 ) and evaporated under reduced pressure. The oily residue was treated with an equivalent amount of fumaric acid in a mixture of EtOH and Et₂0 to give crystalline hydrogenfumarate salt which was filtered off and recrystallized. ¹H-NMR (400 MHz, D₂0) δ (ppm): 2.31 (3H, s, CCH₃), 3.07 (3H, s, NCH₃), 3.38-3.63 (5H, om, NCH_2, OCH₃), 4.05-4.21 (2H, om, OCH₂₎ OCH), 4.39 (1 H, dd, J = 10.7, 3.2 Hz, OCH₂), 6.71 (2H, s, CHCOOH), 6.98 (2H, d, J = 8.3 Hz, C₆H₄), 7.24 (2H, d, J = 8.3 Hz, C₆H₄).

Example 6

1 -(1 -Methylhydrazino)-3-methyIphenylamino-2-propanol fumarate A solution of NaN0₂ (1.04 g, 15 mmol) in H₂0 (10 ml) was added dropwise to a solution of 1-methylamino-3-methylphenylamino-2-propanol

(1.94 g, 10 mmol) in H₂0 (10 ml) with vigorous stirring on an ice-cold bath, and then AcOH (1.20 g, 20 mmol) was added dropwise. The mixture was stirred at room temperature for 8 h, then was extracted with EtOAc (4 x 50 ml). The combined organic phases were dried (Na₂S0 ) and evaporated under reduced pressure to give a dark oil. The crude product was purified by means of column chromatography on silica gel 60 (Merck, 0.063-0.200 mm) using a 1 : 1 mixture of Pr₂0 and EtOAc as eluent. Evaporation of the appropriate fractions resulted in the Λ/-nitroso derivative as a green oil (0.92 g). A solution of 1-methylamino-/V-nitroso-3-methylphenylamino-2-pro- panol (0.92 g, 4.1 mmol) in THF (5 ml) was added dropwise to a stirred suspension of LiAIH₄ (0.46 g, 12 mmol) in THF (20 ml), and the mixture was stirred at room temperature for 1 h. The excess of LiAIH was decomposed with a mixture of H₂0 (1.0 ml) and THF (10 ml), the resulting precipitate was filtered off and washed with EtOAc (2 x 30 ml). The combined filtrates were dried (Na₂S0 ) and evaporated under reduced pressure. The oily residue was treated with 0.55 equivalent amount of fumaric acid in a mixture of MeOH and Et 0 to give crystalline fumarate salt which was filtered off and recrystallized. ¹H-NMR (400 MHz, D₂0) δ (ppm): 2.98 (3H, s, NCH₃), 2.99 (3H, s, NCH₃), 3.20-3.32 (2H, m, NCH₂), 3.39-3.54 (2H, m, NCH₂), 4.30-4.40 (1 H, m, OCH), 6.53 (1 H, s, CHCOOH), 6.87 (1 H, t, J = 7.3 Hz, C₆H₅), 6.89-6.97 (2H, m, C₆H₅), 7.28-7.40 (2H, m, C₆H₅). The compounds presented in Table 1 below were prepared using methods described above.

Table 1.

Example 7

In Vitro Inhibition of VAP-1 SSAO Activity VAP-1 SSAO activity was measured using the coupled colorimetnc method essentially as described for monoamine oxidase and related enzymes (Holt, A , et al , Anal Biochem 244 384-392 (1997)) Recombmant human VAP-1 SSAO expressed in Chinese Hamster Ovary (CHO) cells was used as a source of VAP-1 SSAO for activity measurements Native CHO cells have negligible SSAO activity These cells and their culture have previously been described (Smith, D J , et al , J Exp Med. 188 17-27 (1998)) A cell lysate was prepared by suspending approximately 3 6 x 10⁸ cells in 25ml lysis buffer (150mM NaCI, 10 mM Tπs-Base pH 7 2, 1 5 mM MgCI₂, 1 % NP40) and incubating at 4°C overnight on a rotating table The lysate was clarified by centπfu- gation at 18000g for 5 mm at room temperature and the supernatant used directly in the assay The VAP-1 SSAO assay was performed in 96 well microti- tre plates as follows To each well was added a predetermined amount of inhibitor if required The amount of inhibitor varied in each assay but was generally at a final concentration of between 1 nM and 50μM Controls lacked inhibitor The inhibitor was in a total volume of 20 I in water The following reagents were then added 0 2M potassium phosphate buffer pH 7 6 to a total reaction volume of 200μl, 45 μl of freshly made chromogenic solution containing I mM 2,4-dιchlorophenol, 500 μM 4-amιnoantιpyπne and 4 U/ml horseradish peroxi- dase and an amount of CHO cell lysate containing VAP-1 SSAO that caused a change of 0 6 A₄₉₀ per h This was within the linear response range of the assay. The plates were incubated for 30 mm at 37°C and the background ab- sorbance measured at 490 nm using a Wallac Victor II multilabel counter To initiate the enzyme reaction 20 μl 10mM benzylamine (final concentration = 1 mM) was added and the plate incubated for 1 h at 37°C The increase in ab- sorbance, reflecting VAP-1 SSAO activity, was measured at 490nm Inhibition was presented as percent inhibition compared to control after correcting for background absorbance and IC₅₀ values calculated using GraphPad Prism

Example 8

Comparison of VAP-1 SSAO activity versus total rat MAO activity Rat MAO was prepared from rat liver by rinsing a 1 g liver sample several times in 14 ml KCI-EDTA-solution to remove all blood Then 1 g liver sample was homogenised in 4 ml ice-cold potassium phosphate buffer (0.1 M, pH 7.4) with an Ultra-Turrax homogenizer (setting 11 000 rpm, 4 x 10s). After centrifugation at 500 g for 10 min at 4°C the supernatant was carefully withdrawn and was centrifuged at 12 300 g for 15 min at 4°C. The supernatant was discharged and sedimented mitochondria were resuspended in 4 ml fresh phosphate buffer and centrifuged as previously. The mitochondria were suspended in 4 ml phosphate buffer and homogenized with an Ultra-Turrax ho- mogeniser (setting 11 000 rpm, 2 x 10s). Mitochondrial preparate was ali- quoted and stored at -70°C. Total MAO activity was measured in a similar way as for VAP-1 SSAO except that 2,4-dichlorophenol was replaced by 1 mM va- nillic acid. To each well was added a predetermined amount of inhibitor if required. The amount of inhibitor varied in each assay but was generally at a final concentration of between 10 nM and 800 μM. Controls lacked inhibitor. The inhibitor was in a total volume of 20:l in water. The following reagents were then added. 0.2 M potassium phosphate buffer pH 7.6 for a total reaction volume of 300 μl, 50 μl of freshly made chromogenic solution (as above) and 50 μl of MAO preparation. The plates were incubated for 30 min at 37°C and the background absorbance measured at 490 nm using a Wallac Victor II multilabel counter. To initiate the enzyme reaction 20 μl of 5 mM tyramine (final con- centration 0.5 mM) was added and the plate incubated for 1 h at 37°C. The increase in absorbance, reflecting MAO activity, was measured at 490nm. Inhibition was presented as percent inhibition compared to control after correcting for background absorbance and IC ₀ values calculated using GraphPad Prism. Clorgyline and pargyline (inhibitors of MAO-A and -B respectively) at 0.5 μM were added to some wells as positive controls for MAO inhibition. The ability of compounds 1 to 20 (Table 1 ) to inhibit VAP-1 SSAO activity with specificity for VAP-1 SSAO over rat MAO is shown in Table 2. The results indicate that the compounds of the invention are specific inhibitors of human VAP-1 SSAO activity. The compounds of the present invention are therefore expected to have therapeutic utility in the treatment of diseases and conditions in which the SSAO activity of the human adhesion molecule VAP-1 plays a role. Table 2 Potency and specificity of compounds 1 to 20

1 (BTT-2066) 0.37 13 35 4 0.43 10 23 7 0.44 6.0 14 10 0.55 6.0 1 1 13 0.64 3.3 5 16 0.35 7.4 21 2 0.52 8.7 17 5 0.52 4.0 8 8 0.65 10 15 11 (BTT-2067) 0.27 11 41 14 0.37 3.4 9 17 0.43 6.1 14 3 0.26 3.3 13 6 0.09 3.3 37 9 (BTT-2071 ) 0.21 41 195 12 (BTT-2072) 0.33 37 112 15 (BTT-2072), 0.34 32 94 S-enantiomer 18 (BTT-2072), 0.39 21 62 R-enantiomer 19 (BTT-2073) 0.20 8.8 45 20 0.34 9.6 28

Claims

Claims 1. A hydrazino alcohol derivative having formula,

'

(I)

or a pharmaceutically acceptable salt thereof, wherein

R¹ is hydrogen, lower alkyl or an optionally substituted phenyl or heteroaryl group;

R² is hydrogen or lower alkyl, or

R¹ and R² may form together with the nitrogen atoms to which they are attached a saturated heterocyclic ring; R³ - R⁵ represent each independently hydrogen, lower alkyl, aralkyl, optionally substituted phenyl or a heteroaryl group, or

R² and R³ may form together with the atoms to which they are attached a saturated heterocyclic ring, or

R³ and R⁵ may form together with the carbon atoms to which they are attached a saturated carbocyclic ring; R^δ is naphtyl, phenyl, substituted phenyl or a heteroaryl group; R⁷ is hydrogen, lower alkyl or aralkyl; n is 1 , 2 or 3; and X is O, S, SO, S0₂ or NR²; with the proviso that (i) when X is O, R¹ to R⁵ and R⁷ are hydrogen and n is 1 , then R⁶ is not phenyl or 2-methoxy-phenyl,

(ii) when X is O, R¹ is hydrogen, R² is methyl and R³ to R⁵ and R⁷ are hydrogen and n is 1 , then R⁶ is not 3-trifluoromethyl-phenyl; and (iii) when X is O, R¹ is phenyl, R² to R⁵ and R⁷ are hydrogen and n is 1 , then R⁶ is not 4-hydroxy-phtalazinyl.

2. A derivative according to claim 1 , where

R¹ is hydrogen or phenyl; R² is hydrogen or lower alkyl; R³, R⁴ and R⁵ are hydrogen;

R^δ is phenyl optionally substituted with one or more substituents selected from the group consisting of lower alkyl and lower alkoxy.

3. A derivative according to claim 1 , which is 1-(1-methylhydrazino)- 3-phenoxy-2-propanol.

4. A derivative according to claim 1 , which is 1-(1-methylhydrazino)-

3-(p-tolyloxy)-2-propanol.

5. A derivative according to claim 1 , which is 3-(p-methoxyphen- oxy)-1-(1-methylhydrazino)-2-propanol.

6. A derivative according to claim 1 , which is 3-(p-methoxyphen- oxy)-1-(2-phenylhydrazino)-2-propanol.

7. A derivative according to claim 1 , which is 1-[2-methoxy-3-(p-tol- yloxy)propyl]-1-methylhydrazine.

8. A derivative according to claim 1 , which is 3-(3,4-dimethoxy- phenyltio)-1-(1-methylhydrazino)-2-propanol.

9. A derivative according to claim 1 , which is 3-(3-methoxyphen- oxy)-1 -(1-methylhydrazino)-2-propanol.

10. A derivative according to claim 1 , which is (S)-3-(3-methoxy- phenoxy)-1 -(1 -methylhydrazino)-2-propanol.

11. The use of a hydrazino alcohol derivative according to any of claims 1 to 10 for inhibiting copper-containing amine oxidases.

12. The use according to claim 1 1 where the use is carried out in vitro.

13. A hydrazino alcohol derivative according to any of claims 1 to 10 for use as a medicament.

14. A derivative according to claim 13 for use as a medicament for treating diseases related to the inhibition of copper containing amine oxidases.

15. A derivative according to claim 14 for use as a medicament for treating diseases selected from the group comprising acute and chronic inflammatory conditions or diseases such as chronic arthritis, inflammatory bowel diseases, skin dermatoses and multiple sclerosis; diseases related to carbohydrate metabolism, including diabetes and complications resulting from diabetes, e.g. vasculopathies, such as retinopathy, nepropathy and neuropa- thy; aberrations in adipocyte differentation or function, and smooth muscle cell function, in particular atherosclerosis; and various vascular diseases.

16. The use of a hydrazino alcohol derivative having formula

(I^')

or a pharmaceutically acceptable salt thereof, wherein R¹ is hydrogen, lower alkyl or an optionally substituted phenyl or heteroaryl group;

R² is hydrogen or lower alkyl, or

R³ and R⁵ may form together with the carbon atoms to which they are attached a saturated carbocyclic ring;

R⁶ is naphtyl, phenyl, substituted phenyl or a heteroaryl group;

R⁷ is hydrogen, lower alkyl or aralkyl; n is 1 , 2 or 3; and

X is O, S, SO, S0₂ or NR², for preparing a pharmaceutical composition for treating a disease related to the inhibition of copper-containing amine oxidases.

17. The use according to claim 16 where the disease is an inflammatory disease.

18. A pharmaceutical composition comprising an effective amount of a hydrazino alcohol derivative according to any of claims 1 to 10 and a suitable carrier.

19. A process for preparing a hydrazino alcohol deπvative according to claim 1 comprising a) reducing a nitroso compound of formula

(III)

wherein R - R , n and X are as defined in claim 1 , or b) hydrolizing an oxadiazine compound of formula

wherein R - R n and X are as defined in claiml , and R and R represent each independently lower alkyl or aralkyl.