Lipoprotein(a) [Lp(a)j is a LDL-like lipoprotein where its major lipoprotein, apoB-100 is covalently linked to an unusual glycoprotein, apoprotein(a). Due to its structural similarity to plasminogen, apo(a) interfers with the normal physiological thrombosis- hemostasis process. The structural feature of Lp(a), where the LDL lipoprotein is linked to apo(a), is thought to be responsible for its atherogenic and thrombolytic activities.

Elevated levels of Lp(a) have been associated with the development of atherosclerosis, coronary heart disease, myocardial infarction, cerebral infarction, restenosis following balloon angioplasty and stroke. A recent epidemiologic study has provided the clinical proof of a positive correlation between plasma Lp(a) concentrations and the incidence of heart disease (see for instance: "Elevated Plasma Lipoprotein(a) and Coronary Heart Disease in Men Aged 55 Years and Younger"; A.G. Bostom, L. A. Cupples, J.L. Tenner, J.M. Ordovas, L.J. Seman, P.W.F. Wilson, E.J. Schaefer and W.P. Castelli; Journal of American Medical Association 1996, 276, p. 544-548).

Patients that have Lp(a) levels in excess of 20-30 mg/dl run a significantly increased risk of heart attacks and stroke. An effective therapy for lowering Lp(a) does not exist at present as potent hypocholesterolemic agents such as the HMGCoA reductase inhibitors do not affect Lp(a). Until recently, the only compound shown to lower Lp(a) was niacin.

The high doses necessary for activity however entail unacceptable side-effects. There is therefore an unmet therapeutic need for agents that effectively reduce elevated levels of Lp(a).

International application W097/02037 (Symphar SA; SmithKline Beecham plc, published 23 January 1997), published after the priority date of the present application, describes a group of aminophosphonates alpha substitued by phenol groups of the formula (A):

in which Xa is H, C(18)alkyl, hydroxy or C(1 g)alkoxy; Xbis C(1-8)alkyl or C(1-8)alkoxy; xC is H, C(1-4)alkyl, or X30 and one of the two other substituents xa or xb may form an alkylidene dioxy ring having from 1 to 4 carbon atoms; Ra and Rb which may be identical or different, are H or C(1 6)alkyl; B is CH2CH2, CH=CH, or CH2; n is zero or 1; Z is H or a C(1-8)alkyl group; mis 0 or an integer from 1 to 5; Xd is H, or C(1 g)alkyl, C(18)alkoxy or halo; and the pyridyl ring is attached by the ring carbon a- or - to the nitrogen (2- or 3-pyridyl). These have Lp(a) lowering activity.

Compounds of formula (A) fall within scope of the generic disclosure of EP-A-0 559 079.

This is directed towards aminophosphonates alpha substitued by phenol groups which are said to be of use in decreasing plasma cholesterol and blood peroxides. It has now been found that further modification of the substituents on the phenyl ring provides compounds with an improved biological profile.

Accordingly, the present invention provides compounds of formula (I): in which: X1, X2, which may be the same or different, are H, a straight or branched C1 -8 alkyl or C1-8 alkoxy group, OH; X3 is H, a C14 alkyl group, X30 and one ofthe two other substituents X1 and X2 may form a C1-4 alkylidene dioxy ring; X4 is a straight or branched C18 alkyl or alkoxy group, OH, F, C1, Br, I; or X4 and X2 may form a 5 or 6-membered alkylidene ring optionally substituted with C1 -4 alkyl groups or a 5 or 6-membered alkylidenedioxy ring optionally substituted with C1-4 alkyl groups,

X5 is H, a straight or branched C 1 -8 alkyl or alkoxy group, OH, F, C1, Br, I; R1, R2, which may be the same or different, are H, a straight or branched C1-6 alkyl group; B is CH2, CH2-CH2 or CH=CH, n is zero or 1; Z is H, a straight or branched C18 alkyl group; m is 0 or an integer from 1 to 5; yl y2, y3 and Y4, which may be the same or different, are H, a straight or branched C1- 8 alkyl or C18 alkoxy group, a cyano, trifluoromethyl, nitro, hydroxy, hydroxymethyl, C 1 -C4 alkoxymethyl, amino, C 1-4 alkylamino, C1-4 dialkylamino group, a halogen atom (F, C1, Br, I); or any two adjacent yl, Y2, Y3 and Y4 may form an optionally substituted C 1 -6 alkylidene or C 1 -6 alkylidenedioxy ring; or a pharmaceutically acceptable salt thereof.

Suitably, X1 is H, hydroxy, C(1 4)alkyl for instance methyl or t-butyl, or C(1-3)alkoxy, for instance methoxy.

Suitably, X2 is H, hydroxy, C(1-4)alkyl, for instance methyl or t-butyl, or C(1-3)alkoxy, for instance methoxy and X4 is hydroxy, C(1 4)alkyl for instance methyl or t-butyl, or C(1-3)alkoxy, for instance methoxy, or X2 and X4 together form a C4 alkylidene ring optionally substituted, for instance by one or two alkyl groups.

Suitably, X5 is hydrogen, C(13)alkyl, for instance methyl or C(1 3)alkoxy for instance methoxy.

Preferably, X3 is hydrogen.

Preferably, (B)n is a direct bond.

Preferably, R1 and R2 is each a C(1 3)alkyl group, more preferably, a C2 or C3 alkyl group, in particular R1 and R2 is ethyl or isopropyl.

Preferably, Z is hydrogen.

Representative values for yl to Y4 include alkyl, for instance methyl or t-butyl, methoxy or chloro.

Preferably, the pyridyl ring is attached to the ring carbon - to the nitrogen (3- or 5- pyridyl).

Pharmaceutically acceptable salts are well known in the art and include inorganic and organic salts, for instance salts with HC1, H2S04, oxalic acid, maleic acid, sulfonic acid, etc..

Compounds of formula (I) are found to be effective in decreasing Lp(a) production by primary cultures of Cynomolgus monkey hepatocytes. The Lp(a) of these primates is similar in immunologic properties to human Lp(a) and occurs in an almost identical frequency distribution of plasma concentrations (see "Plasma Lipoprotein(a) Concentration is Controlled by Apolipoprotein(a) Protein Size and the Abundance of Hepatic Apo(a) mRNA in a Cynomolgus Monkey Model", N. Azrolan et al, J. Biol.

Chem., 266, 13866-13872, 1991). The compounds of formula (I) are thus potentially useful for decreasing Lp(a) in man and thereby providing a therapeutic benefit.

Accordingly, in a further aspect, the present invention provides a compound of formula (I ) or a pharmaceutically acceptable salt thereof for use in therapy, in particular as an Lp(a) lowering agent. Elevated plasma and tissue levels of lipoprotein(a) is associated accelerated atherosclerosis, abnormal proliferation smooth muscle cells and increased thrombogenesis and expressed in disease states such as, for instance: coronary heart disease, peripheral artery disease : intermittent claudication, thrombosis, restenosis after angioplasty, extracranial carotid atherosclerosis, stroke and atherosclerosis occuring after heart transplant. Compounds of formula (I) may also be useful in treating inflammatory diseases and excessive wound healing.

For such therapeutic use, the compounds of the present invention will generally be administered in a standard pharmaceutical composition. Accordingly, in a further aspect, the present invention provides for a pharmaceutical composition comprising a compound of formaula (I) and a pharmaceutically acceptable excipient or carrier. Suitable excipients and carriers are well known in the art and will be selected with regard to the intended route of administration and standard pharmaceutical practice. For example, the compositions may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsule, ovules or lozenges either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents. They may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously. For parenteral administration, they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or

glucose to make the solution isotonic with blood. The choice of form for administration as well as effective dosages will vary depending, inter alia, on the condition being treated. The choice of mode of administration and dosage is within the skill of the art.

The compounds of formula (I) and their pharmaceutically acceptable salts which are active when given orally can be formulated as liquids, for example syrups, suspensions or emulsions or as solids for example, tablets, capsules and lozenges. A liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in suitable liquid carrier(s) for example, ethanol, glycerine, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavoring or coloring agents. A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose. A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule. Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil.

Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration. A typical suppository formulation comprises a compound of formula (I) or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.

Preferably the composition is in unit dose form such as a tablet or capsule. Each dosage unit for oral administration contains preferably from 1 to 250 mg (and for parenteral administration contains preferably from 0.1 to 25 mg) of a compound of the structure (I) or a pharmaceutically acceptable salt thereof calculated as the free base.

The compounds of the invention will normally be administered to a subject in a daily dosage regimen. For an adult patient this may be, for example, an oral dose of between 1 mg and 500 mg, preferably between 1 mg and 250 mg, or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 25

mg, of the compound of the formula (I) or a pharmaceutically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per dav.

Compounds of formula (I) may be prepared by processes well known in the art, for instance those previously described in WO 97/02037.

Thus, for instance, compounds of formula (I) in which Z is hydrogen may be prepared by a process which comprises treating an imine of formula (it) : in which X1, X2, X3. X4, X5, B, n, m. yl, Y2, Y3, Y4 are as previously defined: with a dialkyl phosphite of structure (III) H-PO(OR1)(OR2) (III) in which R1, R2 are as previously defined; or a trialkyl silyl derivative thereof, preferably the trimethyl silyl phosphite or a metal thereof, for instance the sodium salt, formed in situ by treatment of the compound of structure (III) with a suitable base, for instance sodium hydride, ethoxide or methoxide.

The reaction may be carried out in presence or absence of a catalyst. Suitable catalysts incude an amine such as diethylamine or triethylamine. The reaction may be carried out in presence or in absence of a solvent.

Suitable solvents include petroleum ether, benzene. toluene, diethyl ether, tetrahydrofuran, 1 ,2-dimethoxyethane. Suitable reaction temperatures are in the range of 30 to 140"C.

The imine compound of structure (II) may be obtained by condensing an aldehyde compound of structure (IV): in which xl, X2, X3, X4, X5, B and n are as previously defined ; with a primary amine of structure (V)

in which Z, m, y1, Y2, Y3 and Y4 are as previously described; under imine forming conditions.

Suitably, the condensation may be effected with or without a catalyst in a solvent such as ether, tetrahydrofuran, benzene, toluene or ethanol. Suitable catalysts include molecular sieve, an acid such as glacial acetic acid, p-toluenesulfonic acid, thionyl chloride, titanium tetrachloride, boron trifluoride etherate, or a base such as potassium carbonate. The reaction is suitably carried out in the range of 0°C to the boiling point of the solvent being used. For less reactive amines or aldehydes, the reaction may be usefully carried out in a Dean-Stark apparatus.

Novel compounds of structure (I) may also be prepared by a process which comprises treating equimolar amounts of an aldehyde of structure (IV) , an amine of structure (V); and a dialkyl phosphite of structure (III), in which X1, X2, X3, X4, X5, B ,n, Z, m, yl.

y2, y3 ,y4, R1 and R2 are as previously described, suitably in the presence of p- toluenesulfonic acid as a catalyst, in a hydrocarbon solvent such as petroleum ether, benzene, toluene or xylene, at a temperature between ambient room temperature and the boiling point of the solvent being used, and with concomitant elimination of water, for instance, by using a Dean-Stark apparatus.

Compounds of formula (I) in which m is not zero may also be prepared by a process which comprises treating a compound of formula (VI): in which X1, X2, X3, X4, X5, B and n are as previously defined; with an aldehyde of formula (VII)

in which m is an integer from 1 to 5 and yl, Y2, Y3 and Y4 are as previously described; under reductive amination condiions.

Suitable such conditions include carrying out the reaction in the presence of sodium cyanoborohydride in an alcoholic solvent, preferably methanol. at a pH between 3 to 6 and at a temperature between 0°C and 25"C.

A compound of formula (VI) may be obtained according to the process previously described for a compound of structure (I) from an aldehyde of formula (IV), an amine of structure (VIII) A-NH2 (VIII) in which A is a protecting group which can be removed by hydrogenolysis, for instance an a substituted benzyl or benzyloxycarbonyl and a phosphite of structure (III). This forms an intermediate which is then subjected to hydrogenolysis according to standard conditions, to give a compound of formula (VI).

It will be appreciated that the aminophosphonate ester of formula (I) have a basic centre and can form salts, for instance with inorganic acids such as HC1, H2SO4 and with organic acids such as oxalic acid, maleic acid, sulfonic acids, etc... All these salts are integral part of this invention.

Compounds of structure (I) are racemates as they have at least one chiral center which is the carbon atom in position alpha to the phosphonate group. The compounds of formula ( I) therefore exist in the two enantiomeric forms. The racemic mixtures (50% of each enantiomer), the pure enantiomers and other mixtures thereof all form part of the present invention. Mixtures of enantiomers, including racemates, may be resolved into its constituent enantiomers according to procedures well known in the art, including for instance, chiral chromatography. Unless otherwise indicated, the physical constants and biological data given for compounds of structure (I) refer to racemates.

The structure of compounds of formula (I) described in the following Examples was established by their infrared (IR), mass (MS) and nuclear magnetic resonance (NMR) spectra. The purity of the compounds was checked by thin layer, gas liquid or high performance liquid chromatography.

The invention is further described in the following examples which are intended to illustrate the invention without limiting its scope.

The abbreviations used in this application are the following: In the tables, n is normal, i is iso, s is secondary and t is tertiary. In the description of the NMR spectra, respectively 's' is singlet, 'd' is doublet, 'dd' is double doublet, 't' is triplet and 'm' is multiplet. TsOH is p-toluenesulfonic acid monohydrate. The temperatures were recorded in degrees Celsius and the melting points are not corrected.

Examples Example 1: Diethvl a-(3 4ert-butyl-4-hydroxy-5,6,8-tetrahydronaphthyl)-N-(3- pyridyl-aminomethylphosphonate A mixture of 35g (0.15 mol) of 3-tert-butyl-4-hydroxy-5,6,7,8-tetrahydronaphthaldehyde and 14.9 g (0.158mol) of 3-aminopyridine dissolved in 120 ml toluene and a catalytic amount of p-toluenesulfonic acid (ca. 50 mg) contained in a flask connected to a Dean Stark apparatus was refluxed for 4 h. The solution was evaporated to dryness to give a solid which was purified by recrystallisation from a mixture of toluene and ligroin : 35g, mp = 185-186", IR (KBr) 1590 cm-1 : CH=N.

Diethyl phosphite (40.3 g, 0.29 mol) was added to 30 g (0.097mol) of the previously described imine dissolved in 100 ml THF and the mixture was refluxed for 16 h. The solvent was evaporated and the residue was purified by column chromatography (SiO2, 9/1 CH2C12/MeOH). Recrystallisation from a mixture of CH2C12/AcOEt gave a white solid, mp = 175-1760C.

MS (m/e) = 447 : M+ + 1, 309 : M+ - PO3Et2, 57 : t-Bu (100%) NMR (CDC13): 6 = 8.0, 7.94, 7.02 and 6.81 (4m, 1H each): aromatic H, 3-pyridyl 7.29 (d, J = 2Hz, 1H): aromatic H, substituted tetrahydronaphthyl 5.1 (broad, 1H) : OH 4.93 (d x d, 1H, J=7.5 and 23Hz): Ci:f-PO3Et2 4.74 (m, 1H): N-H

4.18-3.65 (3m, 4H total): P-O-CH2-CH3 2.9, 2.8, 2.6 and 1.8 (4 m): c-C4H8 1.34 (s, 9H): t-Bu 1.30 and 1.09: (2t, J=7Hz): P-O-CH2-Cf:i3 Example 2: Diethvl a-(3 -tert-butyl-5 .5-dimethvl-4-hvdroxy-5 .6.7 8-tetrahvdronaphthvl)- N-(3 -pvridyl-aminomethylphosphonate A mixture of 2.0g (7.7mmol) of 3 -tert-butyl-5,5-dimethyl-4-hydroxy-5,6,7,8- tetrahydronaphthaldehyde and 0.76 g (8. lmmol) of 3-aminopyridine dissolved in 20 ml toluene and 5 mg of TsOH contained in a flask connected to a Dean Stark apparatus was refluxed for 16 h. The solution was evaporated to dryness to give 2.85g of an oil which was directly used for the next step.

Diethyl phosphite (2.05 g, 14.9 mmol) was added to 2.5 g (7.4 mmol) of the crude imine dissolved in 20 ml THF and the mixture was refluxed for 7 h. A further amount of diethyl phosphite (4.8 g, 35 mmol) was added and the mixture was refluxed overnight (total reaction time 17 h). The solvent and the excess of diethyl phosphite were evaporated and the residue was purified by column chromatography (Si02, 9/1 CHC13/MeOH) to give 2.25g (64%) of a tan solid.

MS (m/e) = 475 : M+ - 1, 337 : M+ - PO3Et2, 57 : t-Bu (100%) NMR (CDC13): 6 = 8.14, 7.94, 7.24 and 7.09 (4m, 1H each): aromatic H, 3-pyridyl 7.30 (d, J = 2Hz, 1H): aromatic H, substituted tetrahydronaphthyl 5.5 (broad): OH 5.25 (broad, 1H): N-H 4.94 (d, 1H, J = 22Hz): CH-PO3Et2 4.18-3.5 (4m, 4H total): P-O-CH2-CH3 2.85, 2.76, 1.8 and 1.68 (4 m): c-C4H6 1.46 and 1.44 (2s, 6H total): c-C4H6-(CH3)2 1.34 (s, 9H): t-Bu

1.29 and 1.11: (2t, J=7Hz): P-O-CH2-CHa Example 3 : Diethvl a-(4-hvdroxv-3.56-trimethvlphenvl)-N-(3-pvridvl!-amino- methvlphosphonate A mixture of 15.3 g (0.09 mol) of 4-hydroxy-3,5,6-trimethylbenzaldehyde and 8.77 g (0.09 mol) of 3-aminopyridine dissolved in 100 ml toluene and 5 mg of TsOH contained in a flask connected to a Dean Stark apparatus was refluxed for 16 h. The solution was evaporated to dryness to give 20.3 g (83%) of a yellow solid which was directly used for the next step.

Diethyl phosphite (34.5 g, 25 mmol) was added to 20 g (8.3 mmol) of the crude imine dissolved in 150 ml THF and the mixture was refluxed for 22 h. At the end of the refluxing period a precipitate which has formed was collected . Recrystallization from a mixture of ligroine and ethanol gave 20.4 g (65%) of a white solid.

MS (m/e) = 378 : M+, 241: M+ - PO3Et2 NMR (CDC13): 6 = 7.96, 7.92, 7.0 and 6.76 (4m, 1H each): aromatic H. 3-pyridyl 7.06 (d, J = 2Hz, 1H): aromatic H, substituted phenyl 5.5 (broad, 1H): OH 4.99 and 4.90 (partially overlapped peaks): CH-PO3Et2 and N-H 4.18-3.50 (3m, 4H total): P-O-CH2-CH3 2.35, 2.18 and 2.12 (3s,9H total) : aromatic Me 1.31 and 1.09: (2t, 6H, J=7Hz): P-O-CH2-CH3 Further compounds of formula (I) were prepared by following procedures anologous to those described in the foregoing examples. The are included in the following Table 1, along with the preceding examples. The left hand column refers to a 'Compound' rather than the Example number, the same compound numbers being then used in the Biological Data section.

Table 1: Aminophosphonates of formula (I) (where n is 0, X3 and X5 are H) Y 21 y mp(0C) Cpd X1 X2 X4 R1,R2 (CH2 tBu CH2CH2 CH2CH2 Et 3-pyridyl 175-176 2 tBu CH2CH2 CH2CH2 Me 3-pyridyl 183-185 3 tBu CH2CH2 CH2CH2 iPr 3-pyridyl 137-138 4 tBu CHOCK2 CH2CH2 Et 2-pyridyl 145-146 5 tBu CH2CH2 CH2CH2 Et 4-pyridyl 183-184 6 tBu C(Me)2CH2 CH2CH2 Et 3-pyridyl 131-132 7 Me Me | Me Et 3-pyridyl 183-184 Biological Data The compounds of formula (I) were assayed for lowering the production of Lp(a) in primary cultures of Cynomolgus hepatocytes.

Assay - Hepatocytes were isolated from livers of adult Cynomolgus monkeys by the two- step collagenase perfusion method according to C. Guguen-Guillouzo and A. Guillouzo "Methods for preparation of adult and fetal hepatocytes" p.1-12 in "Isolated and Cultured Hepatocytes", les editions Inserm Paris and John Libbey Eurotext London (1986).

The viability of cells was determined by Trypan blue staining. The cells were then seeded at a density from 0.7. 105 to 1.105 viable cells per cm2 in tissue culture plates in Williams E tissue culture medium containing 10% fetal calf serum. Cells were incubated for 4-6 hours and 24 hours at 370C in a CO2 incubator (5% CO2) in the presence of 20pM of the test compounds dissolved in ethanol. Four to six wells were used for each compound. Nicotinic acid and steroid hormones were used as references to validate the assay system since they are known to decrease Lp(a) in man. Control cells were incubated in the presence of ethanol only.

Results Lp(a) concentration - The amount of Lp(a) secreted in culture medium was directly assayed by ELISA using a commercially available kit. Cells were washed and lysed as described by A.L. White et al, Journal of Lipid Research vol 34, p. 509-517, (1993) and the cellular content of Lp(a) was assayed as described above.

Changes in Lp(a) concentration in culture medium are given as the percentage of value measured for the control plates at 24h.

The compounds No. 1, 2, 3, 4 and 7 tested at 20AM were found to decrease the Lp(a) secretion in the range from 19% to 22%.

In vivo Results Study Protocol - Male cynomolgus monkeys weighing between 3 and 7 kg were divided into groups of 3 to 4 animals each. Prior to treatment their plasma Lp(a) levels were followed over a two-month period to ascertain a constant baseline value. The Lp(a) values measured at Day -7 and Day -1 were comparable and served as predose values. Test compounds were given orally in gelatin capsules by gavage at the dose of 25 mg/kg/day for 4 weeks and Lp(a) was measured at weekly intervals (Day 7, 14, 21 and 28). At the end of the dosing period, animals were maintained for a treatment free period of 4 weeks, whereupon their plasma Lp(a) levels returned to pretreatment levels. This control provided proof that the decrease in Lp(a) measured was caused by the pharmacological activity of the test compounds.

Results - At Days -7-1, 7, 14, 21 and 28, after an overnight fast blood samples were collected on EDTA and Lp(a) was measured by the highly sensitive and specific ELISA test. Results (mean of 3-4 values of each group ) were expressed as % of predose values.

Selected compounds of formula (I) were tested under the experimental conditions to investigate their pharmacological activity in vivo.

Compounds No 1 and 7 were tested at 25mg/kg/day for 4 weeks and lowered plasma Lp(a) in the range of 35% to 40% (value measured at Day 28, % change from predose).

Compound No 2 was tested at 25mg/kg/day for 4 weeks and lowered plasma Lp(a) by 22 % (value measured at Day 14, % change from predose).