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Phthalazine derivatives phosphodiesterase 4 inhibitors

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WO2004056798A1
WO2004056798A1 PCT/EP2003/014731 EP0314731W WO2004056798A1 WO 2004056798 A1 WO2004056798 A1 WO 2004056798A1 EP 0314731 W EP0314731 W EP 0314731W WO 2004056798 A1 WO2004056798 A1 WO 2004056798A1
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acid
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mg
mmol
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PCT/EP2003/014731
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French (fr)
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Mauro Napoletano
Franco Pellacini
Gabriele Morazzoni
Silvia Macecchini
Lara Pippo
Thomas Haak
Leopoldo Allievi
Ermanno Moriggi
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Zambon Group S.P.A.
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D401/00Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Abstract

Compounds of formula (I) wherein R is methyl or difluoromethyl; X is a methylene group, ethylene, -CH=CH-, -O-CH2-, -O-(CH2)2, -O-(CH2)3-, -NH-SO2-(CH2)3-, -NH-CO-(CH2)2-, -NH-CO-(CH2)3-, N-(ethane-sulfonylamino) pyperazinyl, the N-oxidised derivatives of -CH2-CH-NH2 | the compounds of formula I and the pharmaceutically acceptable salts thereof are described.The compounds of formula I are PDE 4 inhibitors.

Description

"PHTHALAZINE DERIVATIVES PHOSPHODIESTERASE 4 INHIBITORS"

The present invention relates to phthalazine derivatives, pharmaceutical compositions containing them and their use as phosphodiesterase 4 inhibitors.

Phosphodiesterases are a family of isoenzymes which constitute the basis of the main mechanism of cAMP (cyclic adenosine-3',5'-monophosphate) hydrolytic inactivation. cAMP has been shown to be the second messenger mediating the biologic response to many hormones, neurotransmitters and drugs [Krebs Endocrinology Proceedings of the 4th International Congress Excerpta Medica, 17-29, 1973]. When the suitable agonist binds to the cell surface, the adenylate cyclase activates and turns Mg2+-ATP into cAMP. cAMP modulates the activity of the majority, if not of all the cells contributing to the physiopathology of various respiratory diseases, both of allergic origin and not. It follows that an increase of the cAMP concentration yields beneficial effects such as airway smooth muscle relaxation, inhibition of the mast cell mediator release (basophil granulose cells), suppression of the neutrophil and basophil degranulation, inhibition of the monocyte and macrophage activation. Thus, compounds able of activating adenylate cyclase or of inhibiting phosphodiesterases could suppress the undesired activation of the airway smooth muscle and of a great number of inflammatory cells. In the phosphodiesterase family there is a distinct group of isoenzymes, phosphodiesterases 4 (hereinafter PDE 4), specific for the hydrolysis of cAMP in the airway smooth muscle and inflammatory cells (Torphy, "Phosphodiesterase Isoenzymes: Potential Targets for Novel Anti-asthmatic Agents" in New Drugs for Asthma, Barnes, ed. EBC Technical Services Ltd, 1989). Studies carried out on this enzyme show that its inhibition yields not only the airway smooth m uscle r elaxation, b ut a lso the s uppression of m astocyte, b asophil a nd n eutrophil degranulation, so as the inhibition of the monocyte and neutrophil activation. Thus PDE 4 inhibitors are effective in the therapy of asthma.

Selective inhibition of PDE 4 attenuates the functionality of inflammatory cells such as, for example, neutrophils, alveolar macrophages and T cells which possess, as it is known, a key role in COPD (chronic obstructive pulmonary disease) and such activity suggests how this class of compounds could provide an effective therapy in this kind of pathology (Duglas WP Hay, Curr. Opin. Chem. Biol., 2000, vol. 4, pages 412-419).

Such compounds offer a unique approach to the therapy of various respiratory diseases both of allergic origin and not, and possess significant therapeutic advantages over the current therapy.

The excessive or irregular production of tumour necrosis factor (hereinafter TNFα), a cytokine with pro-inflammatory activity produced by various types of cells, affects the mediation or the exacerbation of many pathologies such as, for example, the adult respiratory distress syndrome (A DS) and the chronic pulmonary inflammatory disease. Therefore, compounds able to control the negative effects of TNFα i.e. the inhibitors of this cytokine, are to be considered as useful against many pathologies.

The patent application EP 0722936 (in the name of Eisai) claims, inter alia, compounds of formula

wherein n = 0-4; Ri is optionally substituted lower alkoxy, optionally substituted cycloalkyl, or a - OR9 group wherein R9 represents an optionally substituted arylalkyl group; X is -N= or - NRg- wherein Re is hydrogen, a lower alkyl group, or optionally substituted arylalkyl or heteroarylalkyl groups; Y is -CO or -CB= wherein B is -NR7R8 wherein R7 and R8 represent each independently a hydrogen atom, an optionally substituted lower alkyl group, an optionally substituted heteroarylalkyl group or R7 and R8 together with the nitrogen atom to which they are bonded may form a ring which may be substituted; or B is a hydrogen atom or an optionally substituted aryl, heteroaryl, arylalkyl or heteroarylalkyl group; A is a hydrogen or halogen atom, or an optionally mono- or disubstituted amino group, an optionally substituted aryl, heteroaryl, arylalkyl or heteroarylalkyl group. Among the groups that optionally substitute the above mentioned residues, halogen atoms and the optionally protected carboxy group are cited. I?

- 3 - The exemplified compounds, included in a very wide general formula, show a predominant interest in the phthalazine nucleus substituted with (3-chloro-4-methoxy)-benzylamino or 3,4-methylenedioxy-benzylamino groups as meanings of A; halogen, nitro groups or cyano with n = 1 as meanings of Rf, a -NR7R8 group wherein R7 and R8 represent each independently a hydrogen atom, an optionally substituted lower alkyl group, an optionally substituted heteroarylalkyl group or R7 and R8 together with the nitrogen atom to which they are bonded may form a ring which may be substituted, as meanings of B. Therefore, no aspect of the description and no example lead to specific compounds wherein

10 Ri is methoxy or difluoromethoxy with n = 1, A is phenyl or heterocycle substituted with a carboxy group and optionally with another functional group and B represents a (3,5- dichloro)-pyridin-4-yl-methyl group .

Moreover, these compounds are said to be active as inhibitors of cGMP-PDE, i.e. PDE 5, a phosphodiesterase just acting through a cGMP-dependent mechanism and whose field of

15 application is markedly cardiovascular (Schudt C. et al., Phosphodiesterase Inhibitors, Academic Press).

The international patent application WO 00/05218 (in the name of Zambon Group S.p.A.) claims, inter alia, the compounds of formula

wherein

25 the bond between the carbon atom to which the substituent R2 is bonded and the adjacent nitrogen atom is single or double; R is a (Cι-C6)-alkyl or polyfluoro-(Cι-C6)-alkyl group; R] is absent when the bond between the carbon atom to which the substituent R2 is bonded and the adjacent nitrogen atom is double or, when the same bond is single, Ri is a hydrogen atom, an optionally substituted (Cι-C6)-alkyl group or a (Cι-C )-alkylsulfonyl group; R2

30 when the bond between the carbon atom to which the substituent R2 is bonded and the adjacent nitrogen atom is double is a hydrogen atom, a cyano group, amido, (Cι-C8)-alkyl,

(C2-C8)-alkenyl or (C2-C8)-alkynyl, alkoxy or optionally substituted aryl or heterocycle; R3 is hydrogen or a (C C8)-alkyl, (C2-C8)-alkenyl or (C2-C8)-alkynyl group optionally substituted; Z is NΗ, methylene or a (C2-C6)-alkylene chain optionally branched and/or unsaturated and/or interrupted by a (C5-C7)-cycloalkyl residue; A is phenyl or heterocycle optionally substituted by one or more substituent(s).

These compounds are very active as PDE 4 inhibitors and TNFα release inhibitors and, moreover, they do not show activity on PDE 3 and 5 enzymes. It is evident how this specificity and selectivity of action make these compounds suitable therapeutic agents for the treatment of pathologies involving PDE 4 and TNFα.

Nevertheless, the exemplified compounds of the above patent application, show some physico-chemical characteristics such as, for example, the water-solubility which are suitable for the preparation of a reduced number of compositions. In fact, the limited solubility of the active principle is the main cause of the difficulties that appear in the preparation of compositions which are able to warrant a sufficiently high bioavailability.

Moreover, in order to obviate the poor solubility in the in vitro and in vivo preclinical models, special compositions that make use of non physiological vehicles and, consequently, that make difficult the evaluation of both the compounds activity and the bioavailability, are commonly used.

It would be desirable, for the use these compounds are intended for, to have a wide range of formulative opportunities for which, in the most classic uses, it is necessary that the active ingredients are sufficiently water-soluble. The oral route, for example, is the most convenient and widely used method for the administration of drugs and, thus, it results of relevant importance to be able to improve the solubility of potential active ingredients and to permit the development of an oral composition without resorting to special formulative skillness.

We have now surprisingly found that by inserting a carboxy group bonded by a spacer chain (-X-), whose nature is hereinafter defined, to the phenyl substituent encompassed in the meanings of R2 (see WO 00/05218), compounds endowed of a potent inhibitory activity on the PDE 4 and on the TNFα release, inactive on PDE 1, 2, 3 and 5 and, therefore, selective and endowed with optimum water-solubility properties are obtained. Such compounds constitute a not exemplified sub-class in the ambit of the general formula of the above patent application in the name of Zambon Group and, therefore, they are per se new.

The additional polarity inserted in the molecules by the spaced carboxy group allowed to obtain compounds both endowed with physico-chemical properties more easily manageable from the formulation point of view and to evaluate the bioavailability of the compounds of formula I in vehicles suitable for pharmaceutical compositions; bioavailability that was proved, in an unexpected manner, comparable or in many cases higher than what found for the compounds of the above cited patent application which was carried out by dissolving the same compounds in non physiological vehicles. The insertion of the spaced carboxy group, endowed with a specific polar characteristic, on the phenyl in position 1, allowed the compounds of formula I to be endowed with the correct hydro-lipophilic molecular balance that is necessary to dissolve in fluids and to permeate biological membranes during the absorbption and distribution processes keeping a high inhibitory activity on the PDE 4 enzyme. Therefore, by the introduction of the spaced carboxy substituent, compounds endowed with optimun solubility properties, easy to formulate and endowed with inhibitory activity, selectivity and bioavailability which are comparable to or superior to the compounds of which they constitute a selection, were obtained. Therefore, object of the present invention are compounds of formula wherein

R is methyl or difluoromethyl;

X is a methylene group, ethylene, -CH=CH-, -O-CH2-, -O-(CH2)2, -O-(CH2)3-,

-NH-SO2-(CH2)3-, -NH-CO-(CH2)2-, -NH-CO-(CH2)3-, N-(ethane-sulfonylamino) pyperazinyl, _CH2-CH-NH2 the N- oxidised derivatives of the compounds of formula I and the pharmaceutically acceptable salts thereof.

The compounds of formula I may have one or more asymmetric centre(s) and thus be in form of stereoisomers. Object of the present invention are compounds of formula I in form of stereoisomeric mixtures so as of single stereoisomers.

The compounds of formula I are active as PDE 4 and TNFα inhibitors and thus they find a use as therapeutic agents in allergic and inflammatory pathologies such as, for example,

ARDS, COPD, asthma and allergic rhinitis.

The N-oxidised form, if it is present, may involve both the nitrogen atoms present on the phthalazine ring and that present on the pyridyl ring.

Pharmaceutically acceptable salts of the compounds of formula I are salts with alkaline or alkaline-earth metals, zinc salts and salts with pharmaceutically acceptable organic bases such as, for example, trometamol (2-amino-2-hydroxymethyl-propane-l,3-diol), N-methyl- glucamine. Specific examples of compounds object of the invention are:

[3 -[4-(3 ,5 -Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin- 1 -yl] -phenoxy] -acetic acid;

4-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenoxy]-butyric acid;

4-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenylsulfa-moyl]- butyric acid; l-[[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenylsulfa-moyl]- ethyl] -piperidine-4-carboxylic acid; N-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-succi-namic acid;

4-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenylcarba-moyl]- butyric acid;

2-Arnino-3-[4-[4-(3,5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]- propionic acid hydrochloride;

3-[4-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acrylic acid;

3-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acrylic acid;

3-[2-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acrylic acid;

3 -[4-[4-(3 ,5 -Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin- 1 -yl] -phenyl] -propionic acid;

3-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-propionic acid;

[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acetic acid;

3 -[2-[4-(3 ,5 -Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin- 1 -yl] -phenyl] -propionic acid;

[4-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acetic acid;

N-methyl-glucamine salt of 4-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy- phthalazin- 1 -yl] -phenoxy] -butyric acid;

N-methyl-glucamine salt of 3-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy- phthalazin-1-yl] -phenyl] -acrylic acid; (2-amino-2-hydroxymethyl-propan-l,3-diol) salt of 3-[3-[4-(3,5-Dichloro-pyridin-4- ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acrylic acid;

3 -[3 -[4-(3 ,5 -Dichloro- 1 -oxy-pyridin-4-ylmethyl)-7-methoxy-phthalazin- 1 -yl] -phenyl] - acrylic acid.

The compounds of formula I, object of the present invention, are prepared by an aromatic nucleophilic substitution reaction or a coupling reaction, in presence of a catalyst such as for example palladium, between a compound of formula

wherein

R has the meanings reported for the compounds of formula I; and a reactant such as a tin or boronic acid derivative, suitable for the substitution of the halogen atom directly bonded to the phthalazine nucleus with a phenyl substituted with a carboxy group bonded to the phenyl moiety by a spacer X defined in formula I.

The p hthalazine d erivatives o f formula 11 a re p repared a ccording t o t he s ynthetic s cheme described in the international patent application WO 00/05218 (in the name of Zambon

Group S.p.A.) example 45, page 35, and example 99, page 57.

In case tin derivatives in the coupling reaction are used, it would be better that they contain precursors of the free carboxy group such as, for example, a cyano group or an ester group.

Preferably, a Suzuki coupling reaction between the compounds of formula II and the proper boronic acid is carried out in presence of palladium, triphenylphosphine and an aqueous solution of potassium carbonate.

The used boronic acids are, for example, (carboxy-ethyl)-phenylboronic, (carboxy-vinyl)- phenylboronic acids or (hydroxy)-phenylboronic and (nitro)-phenylboronic acids which are then functionalised, according to common techniques, to give the corresponding compounds of formula I.

The preparation of the N-oxydised compounds of formula I is carried out by treatment with peracids such as, for example, m-chloroperbenzoic acid.

In case the oxidation reaction is directed to the nitrogen atom present on the pyridine ring, in order to warrant selectivity to the process, this is carried out on the isobenzofuranol nucleus, precursor of the phthalazine nucleus, according to what described in the international patent application WO 00/05218. The preparation of the salts of the compounds of formula I is carried out according to known methods.

The compounds of formula I are PDE 4 inhibitors as shown by the enzymatic inhibition tests (Example 36), and they are also able to inhibit the TNFα release (Example 37). Furthermore, the compounds of the present invention do not show any inhibitory activity on PDE 1, 2, 3 and 5 enzymes as shown by the enzymatic inhibition tests carried out.

It is evident how these enzimatic selectivity and specificity characteristics combined with the lack of activity on the cardiovascular system make the compounds of formula I specifically suitable for treating pathologies involving PDE 4 and TNFα such as asthma, COPD, ARDS, allergic rhinoconjunctivitis, psoriasis, atopic dermatitis, rheumatoid arthritis, septic shock, ulcerative cholitis, even if in the present contest the interest is particularly focused on the respiratory pathologies. In particular, the compounds of the invention are useful in the treatment o f allergic and inflammatory diseases and particularly in the therapy o f ARDS, COPD, asthma and allergic rhinitis. The therapeutic doses are generally comprised from 0.1 to 1.000 mg/day and from 1 to 200 mg by oral route for single administration.

The therapeutically effective amounts will depend on the age and on the general physiological conditions of patient, on the administration route and on the used pharmaceutical composition. The compounds of the present invention for their therapeutic or preventive use in the above mentioned pathologies will be preferably used in a pharmaceutical composition suitable for the oral, rectal, sublingual, parenteral, topical, transdermal and inhalatory administration. Therefore, a further object of the present invention are pharmaceutical compositions containing a therapeutically effective amount of a compound of formula I or a salt thereof in admixture with a pharmaceutically acceptable carrier.

The pharmaceutical compositions object of the present invention may be liquid, suitable for the oral and/or parenteral administration such as, for example, drops, sirups, solutions, injectable solutions ready to use or prepared by the dilution of a lyophilized preparation, and solid or semisolid such as tablets, capsules, granulates, powders, pellets, vaginal suppositories, suppositories, creams, ointments, gels, unguents; or still solutions, suspensions, emulsions, and other forms suitable for the inhalatory or transdermal administrations.

Depending on the type of composition, besides a therapeutically effective amount of one (or more) compounds of formula I, they will contain some solid or liquid excipients or diluents for pharmaceutical use and optionally further additives, commonly used in the preparation of pharmaceutical compositions, such as thickeners, binders, lubricants, disintegrators, flavouring and coloring agents.

The preparation of the pharmaceutical compositions object of the invention can be carried out according to common techniques. For better illustrating the invention the following examples are now given.

Example 1 Synthesis of 3- 4-(3.5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenol (intermediate 2) To a mixture of 4-chloro-l-(3,5-dichloro-pyridin-4-yl-methyl)-6-methoxy-phthalazine (intermediate 1) (450 mg, 1.26 mmol, 1 eq.), prepared according to what described in the international patent application WO 00/05218 example 45, page 35, 3-hydroxyphenylboronic acid (193 mg, 1.4 mmol, 1.1 eq.) and palladium tetrakis-(triphenylphosphine) (73 mg, 5 mol %), a mixture of DME (10 ml), ethanol (1 ml) and a 2 N aqueous solution of potassium carbonate (1.9 ml) previously flushed with nitrogen was added at room temperature under an inert argon atmosphere. The reaction mixture was centrifugated in a Bϋchi Syncore System (350 rpm) at 90°C for 16 hours. The reaction was quenched with a 5% aqueous solution of citric acid until pH 6 and then poured on a Varian Chem Elut CE1005 which was washed with dichloromethane (10 ml x 3 times). The solvent was evaporated in vacuo and the crude was purified on Varian Mega Bond Elut (SI 10 g / 60 ml cartridge) eluting with dichloromethane and methanol (from 0% to 3% gradient) to give the intermediate 2 as yellow solid (268 mg, 0.65 mmol, 54% yield). [M+H]+ 412. NMR DMSO_d6: 8.70 (s, 2H, Py); 8.54 (d, IH, JHH=9.5 Hz, *CH=CH-COMe); 7.76 (dd, IH, JHH=2.3 Hz, CH=*CH-COMe); 7.37-6.92 ( m, 5H, Ar); 5.01 (s, 2H, *CH2-Py); 3.88 (s, 3H, OMe).

Example 2 Synthesis of (3-[4-(3 ,5 -dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin- 1 -yl]-phenoxyl - acetic acid tert-butyl ester (intermediate 3) A suspension of intermediate 2 (50 mg, 0.12 mmol, 1 eq.) and potassium carbonate (33 mg, 0.24 mmol, 2 eq.) in DMF (3 ml) was centrifugated at room temperature in a Bϋchi Syncore System (350 rpm) under an inert argon atmosphere for 30 minutes. Then, t-butyl bromoacetate (28 mg, 0.14 mmol, 1.2 eq) was added and the reaction mixture was centrifugated at room temperature for further 18 hours. The reaction was quenched with 10% HCl aqueous solution (1 ml) and the volume was reduced in vacuo. The crude was diluted with dichloromethane (10 ml) and washed with water (7 ml). After evaporation of the organic 1 ayer i n v acuo, t he p roduct w as p urified b y p reparative H PLC-MS ( method 1 ) t o give the intermediate 3 as a light orange solid (25 mg, 0.048 mmol, 40% yield). [M+H]+ 526 HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A/B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A/B = 95:5, t = 8 min., A/B = 5:95; Rt 6.03 min. Example 3 Synthesis of (3-r4-(3.5-dichloro-pyridin-4-yl-methyl)-7-methoxy-phthalazin-l-yl]- phenoxy) -acetic acid (compound 1) A solution of intermediate 3 (75 mg, 0.14 mmol) and 10% trifluoroacetic acid in dichloromethane (3 ml) was stirred at room temperature for 30 minutes. The solvent was evaporated in vacuo and the crude was purified on Varian Mega Bond Elut (SI 5 g cartridge) eluting with dichloromethane, methanol and formic acid (99: 1: 0.5) to give the compound 1 as a brown oil (58 mg, 0.12 mmol, 88% yield). [M+H]+ 470

NMR DMSO_d6: 8.70 (s, 2H, Py); 8.56 (d, IH, JHH=9.1 Hz, *CH=CH-COMe); 7.78 (dd, IH, JHH=2.6 Hz, CH=*CH-CO-Me); 7.51 (t, IH, JHH=7.8 Hz, CH=*CH-CH); 7.33-7.11 (m, 4H, AT); 5.03 (s, 2H, *CH2-Py); 4.77 (s, 2H, *CH2-COOH); 3.89 (s, 3H, OMe).

Example 4 Synthesis of 4-(3-|"4-(3.5-dichloro-pyridin-4-yl-methyl)-7-methoxy-phthalazin-l-yl]- phenoxy I -butyric acid tert-butyl ester (intermediate 4)

A suspension of intermediate 2 (70 mg, 0.17 mmol, 1 eq.) and potassium carbonate (47 mg, 0.34 mmol, 2 eq.) in DMF (3 ml) was centrifugated at room temperature in a Bϋchi Syncore System (350 rpm) under an inert argon atmosphere for 30 minutes. Then, 4-bromo-butyric acid t-butyl ester (189 mg, 0.85 mmol, 5 eq) was added and the reaction mixture was centrifugated at 40°C for further 5 days. The reaction was quenched with 10% HCl aqueous solution (1 ml) and the volume was reduced in vacuo. The crude was diluted with dichloromethane (15 ml) and washed with water (10 ml). After evaporation of the organic layer under reduced pressure, the product was purified on Varian Mega Bond Elut (SI 5 g cartridge) eluting with cyclohexane and ethyl acetate (from 9:1 to 6:4 gradient) to give intermediate 4 as a yellow oil (34 mg, 0.06 mmol, 36% yield). [M+H]+ 554

HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A/B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A B = 95:5, t = 8 min., A B

= 5:95; Rt 6.67 min. Example 5

Synthesis of 4-(3-r4-(3,5-dichloro-pyridin-4-yl-methyl)-7-methoxy-phthalazin-l-yl]- phenoxyl -butyric acid (compound 2)

A solution of intermediate 4 (34 mg, 0.06 mmol) and 10% trifluoroacetic acid in dichloromethane (2 ml) was stirred at room temperature overnight. The solvent was evaporated in vacuo and the crude was purified on Varian Mega Bond Elut (SI 5 g cartridge) by eluting with dichloromethane and methanol (from 99:1 to 90:10 gradient) to give the compound 2 as a yellow solid (29 mg, 0.058 mmol, 98% yield).

[M+H]+ 498

NMR DMSO_d6: 8.71 (s, 2H, Py); 8.55 (d, IH, JHH=9.1 Hz, *CH=CH-C-OMe); 7.77 (dd, IH, JHH=2.6 Hz, CH=*CH-C-OMe); 7.53-7.45 ( , IH, CH=*CH-CH); 7.34-7.11 (m, 4H,

Ar); 5.02 (s, 2H, *CH2-Py); 4.06 (t, 2H, JHH=6.6, O-*CH2-CH2-CH2); 3.88 (s, 3H, OMe);

2.42-2.35 (m, 2H, O-CH2-CH2-*CH2); 2.01-1.91 (m, 2H, O-CH2-*CH2-CH2).

Example 6

Synthesis of l-(3,5-dichloro-pyridin-4-ylmethyl -6-methoxy-4-(3-nitro-phenyl)-phthalazine (intermediate 5)

To a mixture of intermediate 1 (5 g, 14.4 mmol, 1 eq.), 3-nitrophenylboronic acid (2.65 g,

15.8 mmol, 1.1 eq.) and palladium tetrakis(triphenylphosphine) (832 mg, 5 mol %), a 2 N aqueous solution of potassium carbonate (21.6 ml), DME (70 ml) and ethanol (10 ml) previously flushed with nitrogen was added at room temperature under an inert argon atmosphere. The reaction mixture was stirred at 90°C for 2 hours. The reaction was quenched with a 5% aqueous solution of citric acid until pH=6 and then extracted with ethyl acetate (80 ml). The formation of a precipitate was observed and filtration gave intermediate

5 as a yellow solid (4.69 g, 10.6 mmol, 74% yield).

[M+H]+ 441 NMR CDC13: 8.70 (m, IH, C=*CH-CNO2); 8.56 (s, 2H, Py); 8.44-8.39 (m, IH, NO2- C=*CH-CH=CH); 8.29 (d, IH, JHH=9.0 Hz, *CH=CH-C-OMe); 8.21-8.17 (m, IH, NO2-

C=CH-CH=*CH); 7.77 (t, IH, JHH=7.8 Hz, NO2-C=CH-*CH=CH); 7.67 (dd, IH, JHH=2.5

Hz, 9.1 Hz, CH=*CH-C-OMe); 7.34 (d, IH, JHH=2.5 Hz,C=*CH-C); 5.01 (s, 2H, *CH2- Py); 3.94 (s, 3H, OMe).

Example 7 Synthesis of 3- 4-(3 ,5-dichloro-pyridin-4-yl-methyl)-7-methoxy-phthalazin-l -yl]- phenylamine (intermediate 6) A suspension of intermediate 5 (4.5 g, 10.9 mmol) and platinum oxide (200 mg, 0.88 mmol) in dioxane (250 ml) and acetic acid (10 ml) was stirred under hydrogen atmosphere (20 psi) for 8 hours. Then the catalyst was filtered off through celite and the solvent was evaporated in vacuo. The product was purified by Biotage flash chromatography (40 S cartridge) eluting with cyclohexane, ethyl acetate and 2 M methanolic ammonia (250:250:2) to afford intermediate 6 as a yellow solid (2.24 g, 5.4 mmol, 50% yield). [M+H]+ 411

HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC 18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A/B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A/B = 95:5, t = 8 min., A/B = 5:95; Rt 3.77 min.

Example 8 Synthesis of 4-{3-r4-(3,5-dichloro-pyridin-4-ylmethyl -7-methoxy-phthalazin-l-yl]- phenylsulfamovU -butyric acid methyl ester (intermediate 7) To a solution of intermediate 6 (200 mg, 0.48 mmol, 1 eq.) in DMF (6 ml) at 0°C, triethylamine (97 mg, 0.96 mmol, 2 eq.) was added dropwise. After 5 minutes, 4- chlorosulfonyl-butyric acid methyl ester (126 mg, 0.63 mmol, 1.3 eq.) was added and the temperature was allowed to rise to room temperature. The reaction mixture was stirred under nitrogen atmosphere for 48 hours. Water (10 ml) was added and the formation of a yellow precipitate was observed. The solid was collected by filtration and purified on Varian Mega Bond Elut (SI 5 g cartridge) eluting with ethyl acetate and cyclohexane (60:40) to give intermediate 7 as a yellow solid (42 mg, 0.07 mmol, 15% yield). [M+H]+ 575

HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A/B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A/B = 95:5, t = 8 min., A B = 5:95; Rt 5.00 min.

Example 9 Synthesis of 4-(3-[4-(3,5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl1- phenylsulfamoyl) -butyric acid (compound 3) An aqueous solution (1 ml) of lithium hydroxide (1.6 mg, 0.07 mmol, 1.1 eq.) was added to a solution of intermediate 7 (35 mg, 0.06 mmol, 1 eq.) in tetrahydrofuran (2 ml). The reaction mixture was stirred at room temperature for 2 hours, then it was quenched with a 5% aq. solution of citric acid (3 ml) and extracted with dichloromethane (7 ml x 2 times). The organic layers were combined and the solvent was evaporated in vacuo. The crude was washed with diethyl ether to give compound 3 as a yellow solid (28 mg, 0.05 mmol, 83% yield). [M+H]+ 561

HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A/B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A/B = 95:5, t = 8 min., A/B = 5:95; Rt 4.49 min.

Example 10 Synthesis of |3- 4-(3.5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyll- amide ethanesulfonic acid (intermediate 8)

2-Chloro-l-ethanesulfonyl chloride (535 mg, 3.28 mmol, 1.5 eq) was added dropwise to a solution of intermediate 6 (900 mg, 2.19 mmol, 1 eq.) and triethylamine (774 mg, 7.66 mmol, 3 .5 e q.) in NMP ( 12 ml) at 0 °C under nitrogen atmosphere. The temperature was allowed to rise to room temperature and the reaction mixture was stirred for 24 hours. The reaction was quenched with water (15 ml) and the formation of a precipitate was observed. It was filtered and washed with methanol/water (1:2) to give intermediate 8 as an orange solid (930 mg, 1.8 mmol, 85% yield). [M+H]+ 501

HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC 18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A B = 95:5, t = 8 min., A B = 5:95; Rt 4.88 min.

Example 11 Synthesis of 1 -( ( 3 ~r4-(3.5 -dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin- 1 -yl] - phenylsulfamovU-ethylVpiperidine-4-carboxylic acid (compound 4) Piperidine-4-carboxylic acid (39 mg, 0.3 mmol, 1.5 eq.) was added to a suspension of intermediate 8 (100 mg, 0.2 mmol, 1 eq.) in ethanol (2 ml). The reaction mixture was centrifugated in a Bϋchi Syncore System (350 rpm), at 80°C for 6 hours then the temperature was cooled to 40°C and centrifugation was continued overnight. The solvent was evaporated in vacuo and the product was purified by preparative HPLC-MS to give compound 4 as a white solid (7 mg, 0.011 mmol, 6% yield). [M+H]+ 630 - NMR DMSO_d6 at 100°C: 8.64 (s, 2H, Py); 8.51 (d, IH, JHH=9.2 Hz, *CH=CH-C-OMe); 7.73 (dd, IH, THH=2.4 Hz, CH=*CH-C-OMe); 7.60-7.35 (m, 5H, Ar); 5.02 (s, 2H, *CH2- Py); 3.93 (s, 3H, OMe); 3.31 (m, 2H, NH-S02-*CH2); 2.77-1.97 (m, 5H, *CH-COOH, S- CH2-*CH-N-*(CH2)2); 1.73-1.45 (m, 4H, CH-*(CH2)2). Example 12 Synthesis of N- (3 - [4-f 3.5 -dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin- 1 -yl] - phenyl) -succinamic acid (compound 5) Succinic anhydride (48 mg, 0.48 mmol, 2 eq.) was added to a solution of intermediate 6 (100 mg, 0.24 mmol, 1 eq.) in dichloromethane (2 ml). The reaction mixture was stirred at 40°C for 5 hours, then at room temperature overnight. The formation of a precipitate was observed and filtration gave compound 5 as a white solid (55 mg, 0.1 mmol, 45%yield). [M+H]+ 511 NMR DMSO_d6: 12.19 (s-broad, IH, OH); 10.20 (s, IH, NH); 8.70 (s, 2H, Py); 8.56 (d, IH, IHH=9.3 HZ, *CH=CH-C-OMe); 8.07 (m, IH, C-*CH=C-NH); 7.77 (dd, IH, JHH=2.3 Hz, CH=*CH-C-OMe); 7.68-7.64 (m, IH, NH-C=*CH-CH=CH); 7.52 (t, IH, JHH=7.5 Hz, NH- C=CH-*CH=CH); 7.44-7.40 (m, 2H, NH-C=CH-CH=*CH, C=*CH-C-OMe); 5.03 (s, 2H, *CH2-Py); 3.91 (s, 3H, OMe); 2.62-2.53 (m, 4H, CO-*CH2*CH2-CO). Example 13

Synthesis of 4-|3-[4-(3.5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]- phenylcarbamoyll -butyric acid (compound 6)

Glutaric anhydride (55 mg, 0.48 mmol, 2 eq.) was added to a solution of intermediate 6 (100 mg, 0.24 mmol, 1 eq.) in dichloromethane (2 ml). The reaction mixture was stirred at 40°C for 5 hours, then at room temperature overnight. The formation of a precipitate was observed and filtration gave compound 6 as a white solid (80 mg, 0.15 mmol, 64% yield). [M+H]+ 525

NMR DMSO_d6: 12.19 (s-broad, IH, OH); 10.20 (s, IH, NH); 8.70 (s, 2H, Py); 8.56 (d, IH, JHH=9.3 Hz, *CH=CH-C-OMe); 8.07 (m, IH, C-*CH=C-NH); 7.77 (dd, IH, JHH=2.3 Hz, CH=*CH-C-OMe); 7.68-7.64 (m, IH, NH-C=*CH-CH=CH); 7.52 (t, IH, JΗH=7.5 Hz, NH- C=CH-*CH=CH); 7.44-7.40 (m, 2H, NH-C=CH-CH=*CH, C=*CH-C-OMe); 5.03 (s, 2H, *CH2-Py); 3.91 (s, 3H, OMe); 2.62-2.53 (m, 4H, CO-*CH2*CH2-CO).

Example 14 Synthesis of 4-(2-Benzyloxycarbonylamino-2-carboxy-ethyl -boronic acid (intermediate 9) A solution of di-tert-butyl dicarbonate (345 mg, 1.58 mmol, 1.1 eq.) in methanol (1 ml) was added dropwise to amixture of water (4 ml), methanol (1.2 ml) and aOH (0.28 ml of a 10.8 N aq. sol.) at 10°C. The temperature was allowed to rise to room temperature and the reaction mixture was stirred for 3 hours. Water (10 ml) was added and the aqueous phase was washed with diethyl ether (15 ml) and acidified with citric acid (5% aq. sol.). The product was extracted with ethyl acetate, dried over Na2SO4 and the solvent evaporated in vacuo to give intermediate 9 (370 mg, 1.19 mmol, 83% yield).

NMR DMSO_d6: 7.69 (d, 2H, JHH=8.0 Hz, B-C-*(CH)2); 7.20 (d, 2H, B-C-(CH-*CH)2); 7.10 (d, IH, JHH=8.0 Hz, *NH); 4.14-4.02 (m, IH, *CH-CH2); 3.06-2.75 (m, 2H, CH2 ); 1.32 (s, 9H, 3Me)

Example 15 Synthesis of 2-tert-Butoxycarbonylamino-3 - 14-[4-(3 ,5-dichloro-pyridin-4-ylmethyl)-7- methoxy-phthalazin-l-yl -phenv -propionic acid (intermediate 10) To a mixture of intermediate 1 (300 mg, 0.84 mmol, 1 eq.), intermediate 9 (267 mg, 0.86 mmol, 1.02 eq.) and palladium tetrakis(triphenylphosphine) (49 mg, 5% mol), a mixture of 2 N aqueous solution of potassium carbonate (1.27 ml), DME (10 ml) and ethanol (0.5 ml) previously flushed with nitrogen was added at room temperature under an inert argon atmosphere. The reaction mixture was stirred at 90°C for 6 hours and then water (15 ml) was added. The aqueous phase was washed with ethyl acetate (15 ml x 3) and acidified with a 5% aqueous solution of citric acid until pH=6. The product was extracted with dichloromethane (15 ml x 2) and the combined organic layers were dried over Na2SO4. The solvent was evaporated in vacuo to give intermediate 10 (450 mg, 0.77 mmol, 92% yield). [M+H]+ 583 HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A/B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A/B = 95:5, t = 8 min., A/B = 5:95; Rt 5.09 min. Example 16 Svnthesis of 2-Amino-3 - (4-14-(3 ,5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin- 1 - yl]-phenyl}-propionic acid hydrochloride (compound 7)

A I M solution of HCl in diethyl ether (5 ml) was added at room temperature to a solution of intermediate 10 (420 mg, 0.72 mmol) in ethyl acetate (7 ml) and methanol (2 ml). The reaction mixture was stirred for 3 hours, then the solvent was evaporated in vacuo. The product was purified by flash chromatography eluting with dichloromethane, methanol and 50% aqueous solution of formic acid (90:10:1) to give compound 7 (225 mg, 0.43 mmol, 60% yield). [M+H]+ 483

NMR DMSO_d6: 8.72 (s, 2H, Py); 8.66 (d, IH, JHH=9.3 Hz, *CH=CH-C-OMe); 7.90 (dd, IH, JHH=2.6 Hz, CH=*CH-C-OMe); 7.75 (d, 2H, JHH=8.0 Hz, N=C-*(CH)2); 7.54 (d, 2H, JHH=8.0 Hz,N=C-(CH)2-*(CH)2); 7.39 (d, IH, JHH=2.4 Hz,C=*CH-C); 5.04 (s, 2H, *CH2-Py); 3.89 (s, 3H, OMe); 4.28 (m-broad, IH, CH-N); 3.29 (d, 2H, JHH=6.4 Hz, CH2). Example 17

Synthesis of 3 - (4-r4-(3 ,5 -dichloro-pyridin-4-ylmethyl -7-methoxy-phthalazin- 1 -yl] -phenyl } - acrylic acid (compound 8)

To a mixture of intermediate 1 (300 mg, 0.84 mmol, 1 eq.), 4-(2-carboxy-vinyl)- phenylboronic acid (178 mg, 0.92 mmol, 1.1 eq.) and palladium tetrakis(triphenylphosphine) (49 mg, 5% mol), a mixture of 2 N aqueous solution of potassium carbonate (1.27 ml), DME (10 ml) and ethanol (0.5 ml) previously flushed with nitrogen was added at room temperature under an inert argon atmosphere. The reaction mixture was stirred at 90°C for 6 hours, then water (15 ml) was added. The aqueous phase was washed with ethyl acetate (15 ml x 3) and acidified with a 5% aqueous solution of citric acid until pH=6. The product was extracted with dichloromethane (15 ml x 2) and the combined organic layers were dried over Na2SO4. The solvent was evaporated in vacuo to give compound 8 (195 mg, 0.41 mmol, 50% yield). [M+H]+ 466 NMR DMSO_d6: 8.70 (s, 2H, Py); 8.57 (d, IH, JHH=9.4 Hz, *CH=CH-C-OMe); 7.92-7.77 (m, 4H, C-*(CH=CH)2); 7.81-7.76 (m, IH, CH=*CH-C-Ome); 7.71 (d, IH, JHH=16.3,Ar- *CH); 7.32 (d, IH, JHH=2.5 Hz, C=*CH-C); 6.66 (d, IH, CH=*CH-COOH); 5.03 (s, 2H,

*CH2-Py); 3.90 (s, 3H, OMe).

Example 18 Synthesis of 3-{3-r4-r3.5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyll- acrylic acid (compound 9)

Compound 9 was synthesized by Suzuki coupling of intermediate 1 (300 mg, 0.84 mmol, 1 eq.) and 3-(2-carboxy-vinyl)-phenylboronic acid (178 mg, 0.92 mmol, 1.1 eq.) according to what described in example 17. After evaporation of the solvent the solid was washed with a solution of methanol and chloroform (2:1) to afford compound 9 (273 mg, 0.58 mmol, 70% yield).

[M+H 466

NMR DMSO_d6: 12.50 (s-broad, IH, OH); 8.71 (s, 2H, Py); 8.58 (d, IH, JHH=8.8 Hz,

*CH=CH-C-OMe); 8.05-7.59 (m, 6H, Ar, CH=*CH-C-Ome, *CH-CH-COOH); 7.29 (d, IH, JHH=2.8 Hz, MeO-C=*CH-C); 6.66 (d, IH, JHH=16.1, CH=*CH-COOH); 5.04 (s, 2H,

*CH2-Py); 3.89 (s, 3H, OMe).

Example 19

Synthesis of 3-{4-r4-(3.5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyll- propionic acid (compound 10) Compound 10 was synthesized by Suzuki coupling of intermediate 1 (300 mg, 0.84 mmol, 1 eq.) and 4-(2-carboxy-ethyl)-phenylboronic acid (178 mg, 0.92 mmol, 1.1 eq.) according to what described in example 17. Evaporation of the solvent afforded compound 10 (235 mg,

0.5 mmol, 60% yield).

[M+H]+ 468 NMR DMSO_d6: 8.70 (s, 2H, Py); 8.55 (d, IH, JHH=9.0 Hz, *CH=CH-C-OMe); 7.76 (dd,

IH, JHH=2.5 Hz, 9.3 Hz, CH=*CH-C-OMe); 7.67 (d, 2H, JHH=8.1 Hz, CH2-(CH)2-

*(CH)2); 7.44 (d, 2H, CH2-*(CH)2-(CH)2); 7.33 (d, IH, JHH=2.5 Hz, *CH-CH=C-OMe);

5.01 (s, 2H, *CH2-Py); 3.89 (s, 3H, OMe); 2.95 (t, 2H, JHH=7.4, *CH2-CH2-COOH); 2.64

(t, 2H, JHH=7.4, CH2-*CH2-COOH). Example 20 Svnthesis of 3-{2-r4-(3,5-dichloro-pyridin-4-ylmethyl)-7-methoxy-ρhthalazin-l-yl]-phenyl>- acrylic acid (compound 11)

Compound 11 was synthesized by Suzuki coupling of intermediate 1 (300 mg, 0.84 mmol, 1 eq.) and 2-(2-carboxy-vinyl)-phenylboronic acid (178 mg, 0.92 mmol, 1.1 eq.) according to what described in example 17. After evaporation of the solvent the solid was washed with methanol (12 ml) to afford compound 11 (210 mg, 0.45 mmol, 52% yield). [M+H]+ 466 NMR DMSO_d6: 12.31 (s-broad, IH, OH); 8.70 (s, 2H, Py); 8.59 (d, IH, JHH=9.4 Hz, *CH=CH-C-OMe); 7.78 (dd, IH, JHH=2.8 Hz, CH=*CH-C-OMe); 7.64-7.48 (m, 4H, Ar); 7.05 (d, IH, JHH=15.9 Hz, *CH=CH-COOH); 6.77 (d, IH, C=*CH-C); 6.46 (d, IH, CH=*CH-COOH); 5.08 (s, 2H, *CH2-Py); 3.77 (s, 3H, OMe).

Example 21 Synthesis of 3-(3-[4-(3,5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl|- propionic acid (compound 12)

A suspension of 3-(2-carboxy-vinyl)-phenylboronic acid (520 mg, 2.7 mmol) and 10% Pd on charcoal (30 mg) in methanol was stirred under hydrogen atmosphere (20 psi) at room temperature for 3 hours. The catalyst was filtered off through celite and the solvent was evaporated in vacuo to give the 3-(2-Carboxy-ethyl)-phenylboronic acid (360 mg, 1.85 mmol, 69% yield).

Compound 12 was synthesized by Suzuki coupling of intermediate 1 (300 mg, 0.84 mmol, 1 eq.) and previously prepared boronic acid (178 mg, 0.92 mmol, 1.1 eq.) according to what described in example 17. After evaporation of the solvent the solid was washed with a solution of methanol (14 ml) and chloroform (2 ml) to afford compound 12 (306 mg, 0.65 mmol, 78% yield). [M+H]+ 468

NMR DMSO_d6: 12.10 (s-broad, IH, OH); 8.71 (s, 2H, Py); 8.55 (d, IH, JHH=8.9 Hz, *CH=CH-C-OMe); 7.76 (dd, IH, JHH=2.9 Hz, CH=*CH-C-OMe); 7.60-7.42 (m, 4H, Ar); 7.31 (d, IH, JHH=2.5 Hz, MeO-C=*CH-C); 5.03 (s, 2H, *CH2-Py); 3.89 (s, 3H ,OMe); 2.94 (t, 2H, JHH=7.3,Ar-*CH2-CH2); 2.62 (t, 2H, Ar-CH2-*CH2). Example 22 Synthesis of |3-r4-(3.5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl|- acetonitrile (intermediate 11) Hexamethyldistannane (323 mg, 0.98 mmol, 1.2 eq.) was added to a suspension of (3-iodo- phenyl)-acetonitrile (200 mg, 0.82 mmol, 1 eq.) and palladium tetrakis(triphenylphosphine) (38 mg, 0.03 mmol, 0.04 eq) in dry toluene (12 ml). The reaction mixture was stirred at room temperature for 10 minutes, then it was taken to reflux temperature for 24 hours. Ethyl acetate (15 ml) was added and the organic phase was washed with KH2PO4 buffer solution pH 7 (10 ml). Evaporation of the solvent under reduced pressure gave (3-trimethylstannanyl- phenyl)-acetonitrile which was dissolved in toluene (2 ml) and to a suspension of intermediate 1 (290 mg, 0.82 mmol, 1 eq.) and palladium tetrakis(triphenylphosphine) (47 mg, 0.04 mmol, 0.05eq.) in toluene (10 ml) was added. The reaction mixture was stirred at 110°C for 18 hours under argon atmosphere. Ethyl acetate (15 ml) was added and the organic phase was washed with KH2PO buffer solution pH 7 (10 ml). After evaporation of the solvent in vacuo the product was purified by flash chromatography by eluting with ethyl acetate, petroleum ether and methanol (50:50:1) to give intermediate 11 (180 mg, 0.41 mmol, 50% yield). [M+H]+ 435 HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow

, rate: 1 mL/min; column temperature: 40°C; gradient elution A/B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A/B = 95:5, t = 8 min., A B = 5:95; Rt 4.99 min.

Example 23 Synthesis of (3-r4-(3,5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yll-phenyll- acetic acid (compound 13) Intermediate 1 1 w as a dded t o a s olution o f H 20 a nd H CI c oncentrated ( 1:1, 4 m 1) u nder argon atmosphere. The reaction mixture was stirred at 90°C for 10 hours then it was diluted with a KH2PO buffer solution pH 7 (5 ml) and the product was extracted with ethyl acetate

(10 ml x 2). The organic layers were combined and the solvent was evaporated in vacuo to give compound 13 (100 mg, 0.22 mmol, 90%). [M+H]+ 454

NMR DMSO_d6: 12.40 (s-broad, IH, OH); 8.70 (s, 2H, Py); 8.55 (d, IH, JHH=9.2 Hz,

*CH=CH-C-OMe); 7.76 (dd, IH, JHH=2.7 Hz, CH=*CH-C-OMe); 7.65-7.43 (m, 4H, Ar);

7.38 (d, IH, JHH=2.7 Hz, MeO-C=*CH-C); 5.03 (s, 2H, *CH2-Py); 3.89 (s, 3H, OMe); 2.62

(s, 2H, *CH2-COOH). Example 24

Synthesis of 3-{2-r4-(3.5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl|- propionic acid (compound 14)

A suspension of 2-(2-carboxy-vinyl)-phenylboronic acid (50 mg, 0.25 mmol) and 10% Pd on charcoal (5 mg) in methanol was stirred under hydrogen atmosphere (20 psi) at room temperature for 3 hours. The catalyst was filtered o ff through celite and the solvent was evaporated in vacuo to give the 2-(2-carboxy-ethyl)-phenylboronic acid (35 mg, 0.18 mmol,

70% yield).

Compound 14 was synthesized by Suzuki coupling of intermediate 1 (58 mg, 0.16 mmol, 1 eq.) and the previously prepared boronic acid (35 mg, 0.18 mmol, 1.1 eq.) according to what is d escribed in e xample 1 7. A fter e vaporation o f the s olvent the p roduct w as p urified b y flash chromatography eluting with dichloromethane, methanol and 50% aqueous solution of formic acid (95:5:0.5) to afford compound 14 (12 mg, 0.02 mmol, 16% yield).

[M+H]+ 468

HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A/B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A B = 95:5, t = 8 min., A B

= 5:95; Rt 4.47 min. Example 25 Svnthesis of {4-["4-(3.5-dichloro-pyridin-4-ylmethylV7-methoxy-phthalazin-l -yll-phenyl} - acetonitrile (intermediate 12)

Performing the reaction with (4-iodo-phenyl)-acetonitrile (500 mg, 2.06 mmol, 1 eq.), intermediate 12 (710 mg, 1.6 mmol, 82% yield) following the method described for intermediate 11 was synthesized.

[M+H 436

HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A B = 95:5, t = 8 min., A/B

= 5:95; Rt 4.91 min.

Example 26 Synthesis of {4- 4-(3,5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl1-phenyl)- acetic acid (compound 15)

Intermediate 12 (590 mg, 1.36 mmol) was added to a solution of H2O and HCl concentrated

(1:1, 24 ml) under argon atmosphere. The reaction mixture was stirred at 90°C for 10 hours then it was diluted with a 5% NaHCO3 aq. solution until alkaline pH. The aqueous phase was washed with ethyl acetate (2 x 20 ml), acidified with 10% HCl aq. solution and the product was e xtracted w ith e thyl a cetate (2 x 20 ml). T he o rganic 1 ayers w ere c ombined a nd t he solvent was evaporated in vacuo to give compound 15 (300 mg, 0.66 mmol, 49% yield).

[M+H]+ 454

HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A/B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A B = 95:5, t = 8 min., A/B

= 5:95; Rt 4.32 min. Example 27 Svnthesis of N-methyl-D-glucamine salt of compound 2 (compound 16) A solution of compound 2 (280 mg, 0.56 mmol, 1 eq.) and D(-)-N-mehylglucamine (112 mg, 0.56 mmol, 1 eq.) in ethanol 96 (5 ml) was heated to 70°C for 30 minutes under stirring. It was cooled down to room temperature and the obtained precipitate was filtered in a buckner washing with cool ethanol and diethyl ether. The obtained solid was dried in oven under vacuum at 45 °C to give compound 16 (353 mg, 0.51 mmol, resa 91%). NMR DMSO_d6: 8.30 (s, 2H, Py); 7.85 (d, IH, JHH=9.1 Hz, *CH=CH-C-OMe); 7.29-7.20 (m, IH, CH=*CH-CH); 7.17-7.08 (m, IH, Ar); 7.02-6.94 (m, IH, Ar); 6.87-6.74 (m, 3H, Ar); 4.58 (s, 2H, *CH2-Py); 4.00-3.92 (m, IH, *CHH-OH); 4.06 (t, 2H, JHH=6.6, O-*CH2- CH2-CH2); 3.73-3.47 (m, 5H, HO-*CHH-*CH(OH)-*CH(OH)-*CH(OH)-*CH(OH)), 3.44 (s, 3H, OMe); 3.09-3.02 (m, 2H, CH2-NH); 2.62 (s, 3H, NHMe); 2.11-2.20 (m, 2H, O-CH2- CH2-*CH2); 1.77-2.91 (m, 2H, O-CH2-*CH2-CH2).

Example 28 Synthesis of 2-amino-2-hvdroxymethyl-propane-l,3-diol salt of compound 9 (compound 17) To a suspension of compound 9 (200 mg, 0.43 mmol, 1 eq.) in methanol (5 ml), 2-amino-2- hydroxymethyl-propane-l,3-diol was added at room temperature under stirring. After 10 minutes was obtained a solution which was filtered and dried. The solid was treated with isopropylic ester and then filtered to give compound 17 as pale yellow solid (155 mg, 0.26 mmol, resa 61%).

NMR DMSO_d6: 8.69 (s, 2H, Py); 8.55 (d, IH, JHH=8.8 Hz, *CH=CH-C-OMe); 7.89-7.26 (m, 6H, Ar, CH=*CH-C-OMe, *CH-CH-COOH); 7.29 (d, IH, JHH=2.8 Hz, MeO-C=*CH- C); 6.54 (d, IH, JHH=16.1, CH=*CH-COOH); 5.02 (s, 2H, *CH2-Py); 3.86 (s, 3H, OMe); 3.35 (s, 6H, C(CH2)3). Example 29

Synthesis of N-methyl-D-glucamine salt of compound 9 (compound 18) To a suspension of compound 9 (200 mg, 0.43 mmol, 1 eq.) in methanol (5 ml), D(-)-N- methyl-glucamine was added at room temperature and under stirring. After 10 minutes was obtained a solution which was filtered and dried. The solid was treated with isopropylic ester and then filtered to give compound 18 as pale yellow solid (155 mg, 0.26 mmol, resa 61%). NMR DMSO_d6: 8.66 (s, 2H, Py); 8.53 (d, IH, JHH=8.8 Hz, *CH=CH-C-OMe); 7.82-7.54 (m, 5H, Ar, CH=*CH-C-OMe); 7.34-7.25 (m, 2H, *CH-CH-COOH, MeO-C=*CH-C); 6.48 (d, IH, JHH=16.1, CH=*CH-COOH); 5.00 (s, 2H, *CH2-Py); 3.93-3.75 (m, 4H, *CHH-OH, OMe); 3.68-3.31 (m, 5H, HO-*CHH-*CH(OH)-*CH(OH)-*CH(OH)-*CH(OH)), 2.90-2.83 ( , 2H, CH2-NH); 2.47 (s, 3H, NHMe).

Example 30 Synthesis of 3-(3.5 -dichloro-pyridin-4-yl-methylV6-methoxy- 1 -13 -([1 ,3]-dioxolan-2-vD- phenyl]-l,3-dihvdro-isobenzofuran-l-ol (intermediate 13) To a solution of 2-(3-Bromo-phenyl)-[l,3]dioxolane (3.6 ml, 23.9 mmol, 1.1 eq) in THF (50 ml) under nitrogen atmosphere and at -75°C, a 2.5 M solution of butyl-litio in exane (18.23 ml, 45.5 mmol, 2.1 eq ) was added dropwise. The reaction was kept under stirring at -75°C for about 40 minutes and then a solution of 3-(3,5-dichloro-pyridin-4-yl-methyl)-6-methoxy- 3H-isobenzofuran-l-one (7 g, 21.7 mmol, 1 eq) prepared as described in the international patent application WO 00/05218 example 37 page 32, in THF (80 ml) was added dropwise under stirring at that temperature for 4 hours.

The reaction mixture was treated with a saturated solution of NH4C1 (100 ml), it was poured in H2O (50 ml) and it was estracted with ethyl acetate (3 x 60 ml). The organic phase was dried over Na2SO4 and the solvent evaporated. The obtained crude was purified by chromatography on silice eluting with petroleum ether / ethyl acetate / methanol (50:50:1) to give the desired compound (2.7 g, 5.7 mmol, yield 26%).

NMR CDC13: 8.42 e 7.97 (2s, 2H, Py); 7.90 (d, IH, JHH=8.6 Hz, *CH=CH-C-OMe); 7.31- 7.23 (m, 2H, Ar); 7.19 (dd, IH, CH-*CH=C-OMe); 7.11-7.09 (m, 2H, Ar); 7.02 (d, IH, JHH=2.3 Hz, C=*CH-C=CH); 5.62 (s, IH, CH); 5.08 (s, IH, CH); 3.95 (s, 4H, OCH2CH2O); 3.91 (s, 3H, OCH3); 3.42 (bs, IH, OH).

Example 31 Synthesis of 3-(3,5-dichloro-l-oxy-pyridin-4-yl-methyl)-6-methoxy-l- 3-(r 31-dioxolan-2- yl)-phenyl]-L3-dihvdro-isobenzofuran-l-ol (intermediate 14) A solution of intermediate 13 (2.2 g, 4.6 mmol, 1 eq) and 3-chloro-perbenzoic acid (1.13 g at 77%, 5 mmol, 1.1 eq) in CHC13 (70 ml) was stirred under nitrogen atmosphere at room temperature for 72 hours.

It was dilued with CH2C12 (30 ml), washed with H2O (50 ml) and then with an aqueous solution of NaHCO3 (50 ml). The organic phase was dried over Na2SO4 and the solvent evaporated. The obtained crude was purified by chromatography on silice eluting with CH2Cl2/methanol (100:5) to give the desired compound (1.15 g, 2.3 mmol, resa 51%). HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A/B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A/B = 95:5, t = 8 min., A/B = 5:95; Rt 3.28 min.

Example 32 Synthesis of 1 -(3 ,5-dichloro- 1 -oxy-pyridin-4-yl-methyl)-6-methoxy-4-[3 -(\ 1 ,3]-dioxolan-2- yl)-phenyl]-phthalazine (intermediate 15)

To a solution of intermediate 14 (850 mg, 1.7 mmol, 1 eq) in methanol (15 ml) under nitrogen atmosphere and at 0°C, acetic acid (157 mg, 2.6 mmol, 1.5 eq) and then hydrazine monohydrated (130 mg, 2.6 mmol, 1.5 eq) were added. The reaction was stirred at reflux temperature for 5 hours and then the temperature was risen to room temperature. It was poured in H2O (10 ml) and it was estracted with ethyl acetate (2 x 10 ml). It was washed with an aqueous solution of NaHCO3 (10 ml), the organic phase was anhydrified over Na2SO4 and dried to give the desired compound (800 mg, purity 65%) which was used as it was in the next synthetic step. [M+l] = 485 HPLC/MS: Gilson instrument equipped with a C18 column Zorbax SBC18 (3.5 μm, 2.1 x 50 mm) coupled with a diode array UV detector (220 nm) and a Finnigan Aqa mass spectrometer (electron spray, positive ionization). The following settings were used: flow rate: 1 mL/min; column temperature: 40°C; gradient elution A B (eluent A: 0.5% formic acid in water; eluent B: 0.5% formic acid in acetonitrile); t = 0 min., A/B = 95:5, t = 8 min., A/B = 5:95; Rt 3.84 min. Example 33 Synthesis of 3-r4-(3,5-dichloro-l-oxy-pyridin-4-yl-methyl)-7-methoxy-phthalazin-l-yl] benzaldeyde (intermediate 16) A solution of intermediate 15 (800 mg of pure crude at 65%) and HCl concentrated (1 ml) in THF (50 ml) and H20 (15 ml) was stirred under nitrogen atmosphere at room temperature for 5 hours. It was poured in H2O (20 ml), extracted with CH C12 (2 x 30 ml), the organic phase was washed with an aqueous solution of NaHCO3 (30 ml), it was anhydrified with Na2SO and dried. The crude was purified by chromathography on silice eluting with CH2Cl /methanol (100:3) to give the desired compound (260 mg, 0.6 mmol, resa 57%).

NMR DMSO_d6: 10.13 (s, IH, CHO); 8.70 (s, 2H, Py); 8.57 (d, IH, JHH=9.0 Hz, *CH=CH-C-OMe); 8.29-7.80 (m, 4H, Ar-CHO); 7.82-7.76 (dd, IH, CH-*CH=C-Ome); 7.28 (d, IH, JHH=2.65 Hz, MeO-C-*CH=C); 4.96 (s, 2H, *CH2-Py); 3.88 (s, 3H, OMe).

Example 34 Synthesis of 3 -[3 -|"4-(3 ,5 -dichloro- 1 -oxy-pyridin-4-yl-methyl)-7-methoxy-phthalazin- 1 -yll - phenyl] acrilic acid (compound 19)

A solution of intermediate 16 (250 mg, 0.57 mmol, 1 eq), malonic acid (118 mg, 1.13 mmol, 2 eq), pyridine (27 mg, 0.34 mmol, 0.6 eq) in DMF (2 ml) was stirred under nitrogen atmosphere at 100°C for 7 hours. It was cooled and then poured into a 5% aqueous solution of citric acid (5 ml). It was filtered washing with H2O, toluene and petroleum ether in this order and the obtained solid was purified by chromatography on silice eluting with CH2Cl2/methanol/HCOOH ( 100:5:0.5) to give the desired c ompound ( 80 mg, 0.16 mmol, resa 29%). [M+l]= 482, 484. NMR DMSO_d6: 8.70 (s, 2H, Py); 8.54 (d, IH, JHH=8.9 Hz, *CH=CH-C-OMe); 8.05-7.60 (m, 5H, Ar, *CH=CH-COOH); 7.80-7.75 (dd, IH, CH-*CH=C-Ome); 7.28 (d, IH, JHH=2.5 Hz, MeO-C=*CH-C); 6.65 (d, IH, JHH=15.7, CH=*CH-COOH); 4.95 (s, 2H, *CH2-Py); 3.87 (s, 3H, OMe).

Example 35 Synthesis of 3-["3-r4-(3,5-dichloro-pyridin-4-yl-methyl)-7-difluoromethoxy-phthalazin-l-yl]- phenyl] acrilic acid (compound 20)

To a mixture of 4-chloro-l-(3,5-dichloro-pyridin-4-yl-methyl)-6-difluoromethoxy- phthalazine (130 mg, 0.33 mM, 1 eq.), prepared according to what described in the international patent application WO 00/05218 example 99, page 57, 3-(2-carboxy-vinyl)- phenylboronic acid (64 mg, 0.33 mM, 1 eq.) and palladium tetrakis-(triphenylphosphine) (20 mg, 0.016 mM, 0.05 eq.), a mixture of DME (6 ml), ethanol (1 ml) and a 2 N aqueous solution of potassium carbonate (0.5 ml) previously flushed with nitrogen was added at room temperature under an inert nitrogen atmosphere. The reaction mixture was stirred at 80°C for 5 hours. It was then poured in water and extracted with EtOAc. The aqueous phase was quenched with a 5% aqueous solution of citric acid and extracted with EtOAc. It was dried over sodium sulphate and the solvent was evaporated. It was purified by chromatography eluting with dichloromethane, methanol and 50% formic acid (100/3/0.3) to give compound 20 (35 mg, 20% yield). [M+H]+ 503.

NMR DMSO_d6: 8.74 (d, 1 H, JHH=9.0 Hz); 8.69 (s, 2H, Py); 7.49 (t, 1 H, JHF=72 Hz, CHF2); 8.02-7.37 (m, 7H, Ar and *CH-COOH); 6.63 (d, IH, JHH=16.5 Hz, COOH- CH=*CH); 5.07 (s, 2H, CH2).

Example 36 PDE 4 enzyme inhibition a) PDE 4 enzyme purification

PDE 4 enzyme was isolated from U937 cell line according to the method of Nielson et al. (J. Allergy Clin. Immunol. 1990, vol. 86, pages 801-807) partially modified for the FPLC technique (Fast Protein Liquid Chromatography). U-937cell line (Istituto Zooprofilattico Sperimentale, Brescia, Italy) was maintained in RPMI 1640, with 10% foetal calf serum and 2 mM glutamine, at a cell density between lx 106 and 8xl06 cells per ml in an incubator at 37°C with 5% CO2.

U93 suspension was homogenised in a buffer containing 10 mM TRIS (tri- (hydroxymethyl)-aminomethane), 5 mM MgCl2, 4 mM EGTA (ethyleneglycol-bis-(β- aminoethylether)-N,N,N'N'-tetra-acetic acid), 5 mM β-mercaptoethanol, 1 μM leupeptin, 1 μM pepstatin, 1% TRITON x-100, and 100 μM phenylmethyl sulfonyl fluoride (PMSF) at pH 7.8.

The homogenate was centrifuged and the supernatant was used for the purification of the PDE 4 enzyme; it was seeded on a column connected to a biologic chromatography system

(FPLC, BIO RAD).

PDE 4 enzyme was eluted with a linear gradient, from 0.05 M to 1 M of sodium acetate, and elution fractions were collected for PDE 4 activity assay.

The fractions containing PDE4 activity were collected and, after an overnight dialysis against water to remove sodium acetate, were concentrated to 30% volume with an Amicon filtration system (using YM10 membrane filter).

Ethylene glycol (30% v/v) was added and the sample was stored at -20°C in a single aliquot until the use. b) PDE 4 activity assay PDE 4 activity assay was performed using Scintillation Proximity Assay (SPA) Amersham kit consisting of tracer 3H-cAMP, PDE assay buffer (10 x solution: 500 mM TRIS/HC1 pH

7.5, 83 mM MgCl2 and 17 mM EGTA) and Yttrium SPA PDE beads (containing 18 mM zinc sulphate).

The radioactivity of the beads was measured using a scintillation counter (Packard model MINAXI β TRI-CARB 4000 SERIES).

The IC50 values were calculated from concentration-inhibition curves by non linear regression analysis using the program ORIGIN 3.5 (MICROCAL SOFTWARE INC.) and each fitting was the mean ± SEM of 3 experiments using different PDE 4 preparations.

The compounds of formula I of the present invention showed to selectively inhibit PDE 4 enzyme.

The results are reported in the following table 1. Table 1

Example 37 TNFα release in whole human blood The blood samples obtained from healthy volunteers were collected in heparinised tubes and diluted 1 : 5 with RPMI 1640 without serum addition.

The test was carried out in 96-well plates and samples containing 150 μl of diluted blood and 150 μl of RPMI 1640 with control vehicle or with different concentration of the compounds of the present invention were incubated at 37°C in a 5% CO2 humidified atmosphere for 30 minutes. The whole blood in the assay samples was diluted 1:10 (v/v). The samples were stimulated with LPS (lipopolysaccharide from E. Coli: serotype B 0:55, Sigma) 0.25 μg/ml and incubated for 24 hours.

After centrifugation, the supernatants were collected for TNFα levels determination by a commercially available ELISA kit (Biosource).

The test objects were dissolved in DMSO at 10"2 M and further diluted in RPMI 1640. The final concentration of DMSO did not exceed 0.1% and did not affect TNFα release. Each compound was tested in duplicate at nine concentrations and the data obtained were further confirmed using blood samples from different donors.

The percent inhibition of TNFα production at each concentration was calculated and IC50 was determined using a 4-parameters logistic equation (Origin calculation program, Microcal Software Inc.)

The compounds of formula I of the present invention showed to inhibit the TNFα release. The results, expressed as IC 50 at two different concentration, are reported in the following table 2. Table 2

Example 38 PDE 3 and PDE 5 enzyme inhibition a) PDE 3 and PDE 5 enzyme purification

PDE 3 and PDE 5 enzymes were purified from platelet rich plasma (PRP) obtained from healthy volunteers. PDE 3 and PDE 5 enzymes were purified according to Simpson A.W.M. et al. (Biochem.

Pharmacol. 1988, 37, 2315-2320) partially modified for the FPLC technique (Fast Protein

Liquid Chromatography). The PRP was diluted 1:2 in saline solution and centrifuged at 2000 x g for 15 minutes at room temperature.

The pellet was suspended in 10 ml of lysis solution (155 mM NH4C1, 10 mM KHCO3 and

0.1 mM Na2EDTA pH 7.4) and incubated 10 for minutes on ice-bath to remove erythrocyte contamination. After centrifugation, platelets were suspended in 10 ml of 20 mM Bis-Tris, 2 mM EDTA, 5 mM β-mercaptoethanol, 50 mM sodium acetate, 2 mM benzamidine) and homogenised with a Polytron homogeniser on ice-bath.

The homogenate was centrifuged and the supernatant applied to a UNO Q12 column connected to a Biologic Chromatography system (FPLC, BIO RAD). PDE 3 and PDE 5 enzymes were eluted with a linear 0.05-lM sodium acetate gradient and the elution fractions were collected for PDE enzymes activity assay.

Fractions containing PDE activities were collected, dialysed overnight against distilled water and concentrated 10 times by an Amicon filtration system (using YM10 membrane filter).

Ethylene glycol was added to a final concentration of 30% v/v and the solution stored at -20°C.

Enzymatic activity was stable for several weeks under these conditions. b) PDE 3 and PDE 5 activity assay

Enzymes activity assay were performed using Scintillation Proximity Assay (SPA)

Amersham kit consisting of tracer 3H-cAMP for PDE3 or 3H-cGMP for PDE5, PDE assay buffer (lOx solution: 500 mM TRIS/HCl pH 7.5, 83 mM MgCl2 and 17 mM EGTA) and

Yttrium SPA PDE beads (containing 18 mM zinc sulphate).

The radioactivity of the beads was measured using a scintillation counter (Packard model

MINAXI β TRI-CARB 4000 SERIES).

The results, expressed as IC 50 of some compounds which are representative of the entire class of compounds, are reported in the following table 3. Table 3

Example 39

PDE 2 enzyme inhibition a) PDE 2 enzyme purification

PDE 2 was purified from murine PC 12 cells according to Whalin et al. (Molecular

Pharmacol. 1991, 39, 711-717). PC12 cells from Istituto Zooprofilattico Brescia (Italy) were maintained in 150 cm3 flasks at

37 °C and 5 % C O2 in D MEM c ontaining 5 % F CS, 15% horse s erum, 1 % p enicillin, 1 % streptomycin and 1% glutamine.

The cells were expanded paying attention to split them only at reached confluency.

After washing the cells were detached with 0.05% trypsin/0.02%EDTA and collected in a clean test tube.

After centrifugation, the cells were suspended in buffer containing Bis-Tris 20 mM, EDTA

2 mM, 2-mercaptoethanol 5 mM, leupeptin 1 μM, pepstatin A 1 μM, PMSF 100 μM, pH 6.5, and homogenised using a Polytron homogeniser.

After centrifugation, the supernatant fraction was applied to a UNO Q-12 column connected to a Biologic Chromatography system (FPLC, BIO RAD).

PDE 2 enzyme was eluted with a linear 0.05-lM sodium acetate gradient and elution fractions were collected for PDE 2 enzyme activity assay.

Fractions containing PDE 2 enzyme activity were collected, dialysed overnight against distilled water and concentrated 10 times by an Amicon filtration system (using YM10 membrane filter). Ethylene glycol was added to a final concentration of 30% v/v and the solution stored at -20°C.

Enzymatic activity was stable for several weeks under these conditions. b) PDE 2 activity assay

PDE 2 activity assay was performed using Scintillation Proximity Assay (SPA) Amersham kit consisting of tracer 3H-cAMP, PDE assay buffer (10 x solution: 500 mM TRIS/HCl pH 7.5, 83 mM MgCl2 and 17 mM EGTA) and Yttrium SPA PDE beads (containing 18 mM zinc sulphate).

The radioactivity of the beads was measured using a scintillation counter (Packard model MDSfAXI β TRI-CARB 4000 SERIES).

The results, expressed as IC 50 of some compounds, which are representative of the entire class of compounds, are reported in the following table 4. Table 4

Example 40

PDE 1 enzyme inhibition a) PDE 1 enzyme purification

Lyophilised PDE 1 enzyme, partially purified from bovine heart, was purchased from Sigma

(P0520).

The enzyme consists of the PI fraction reported by Ho et al. (Biochim Biophys Acta, 1976, vol. 429(2), 461-473). The lyophilised enzyme was reconstituted with 30% v/v ethylene glycol at a concentration of 500g/ml and stored at -20°C. b) PDE 1 activity assay

PDE 1 activity was determined using the Scintillation Proximity Assay (SPA) Amersham kit consisting of tracer 3H-cAMP, PDE assay buffer (10 x solution: 500 mM TRIS/HCl pH 7.5, 83 mM MgCl2 and 17 mM EGTA) and Yttrium SPA PDE beads (containing 18 mM zinc sulphate).

The radioactivity of the beads was measured using a scintillation counter (Packard model MINAXI β TRI-CARB 4000 SERIES).

The results, expressed as IC 50 of some compounds, which are representative of the entire class of compounds, are reported in the following table 5. Table 5

Example 41 The data concerning the solubility of the compounds of formula I of the present invention in comparison with those exemplified in the international patent application WO 00/05218 are hereinafter reported. HPLC method used to test the solubility.

Solubility test was performed by HPLC analysis (column 4.6 X 15mm) water/0.1% TFA - CH3CN/0.06% TFA gradient) against standard. A 10 M solution of the substance dissolved in DMSO was prepared.

In order to prepare the sample 190 μl of PBS (Phosphate Buffer Saline, pH 7.4) were added to 10 μl of the above solution; instead (while) in order to prepare the standard 190 μl of DMSO to other 10 μl of solution were added. The results obtained for the compounds of formula I are reported in the following table 6. Table 6

The results obtained for the compounds of the international patent application WO 00/05218 in the following table 7 are reported.

R is a -CH3 group when not differently specified. Table 7

Follo Table 7

The compound identified in the international patent application WO 00/05218 with number 75 is endowed with a PDE 4 inhibitory activity lower than that reported for the compounds of the present invention.

Example 42

Pharmacokinetic study in rats

A mixture containing 3-5 compounds according to what described by Ward K.W. et al. (Drug Metab. Dispos. 2001, vol. 29, 82-88) was administered to rats at 10 mg/kg dosage by oral route (1 mg/ml in 77% N-methylglucosamine 25 mM and 20% PG containing 3%

Tween 80) or 3 mg/kg intravenously (3 mg/ml in 15% DMSO/85% PEG).

The animals received only one treatment per study.

The rats permanently implanted with jugular catheter (SilasticR, Dow Corning) were treated with the test mixture by an intravenous bolus (via implanted catheter) or by oral gavage.

Blood s amples w ere c ollected from t he jugular c atheter a t d ifferent times u p t o 6 h a fter administration.

Plasma s amples w ere a nalyzed a fter p rotein p recipitation b y t wo v olumes o f CH3CN a nd plasma concentrations were determined by liquid chromatography/mass spectrometry. The results obtained with some of the compounds of formula I are reported in table 8. Table 8

The increase of the solubility of the compounds of the present invention allowed to perform the bioavailability studies by means of physiologic vehicles normally used in the most common pharmaceutical compositions.

The performed tests by means of a physiologic vehicle on the compounds comprised in the general formula of the international patent application WO 00/05218 showed bioavailability values equal to zero.

Being poor the solubility of the compounds comprised in the general formula of the international patent application WO 00/05218, it was necessary to performe the pharmacoldnetic studies using a 15% DMSO / 85% PEG solution of the compounds both for intravenous and for oral route, i.e. by an absolutely non physiological vehicle. The following table 9 reports the bioavailability values obtained in such conditions. Table 9

Therefore, the compounds of formula I object of the present invention when administered in a physiological vehicle showed good bioavailability values comparable and in many cases higher than the c ompounds c omprised in the g eneral formula o f the patent a plication o f which they are a selection, underlying that the bioavailability of these last ones was measured in non physiological DMSO / PEG solution.

Claims

Claims
1) A compound of formula
wherein R is methyl or difluoromethyl; X is a methylene group, ethylene, -CH=CH-, -O-CH2-, -O-(CH2)2, -O-(CH2)3-, -NH-SO2-
(CH2)3-, -NH-CO-(CH2)2-, -NH-CO-(CH2)3-, N-(ethane-sulfonylamino) pyperazinyl,
-CH2-CH-NH2 the N- I oxidised derivatives of the compounds of formula I and the pharmaceutically acceptable salts thereof.
2) A compound according to claim 1 selected from:
[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenoxy]-acetic acid;
4-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenoxy]-butyric acid; 4-[3 -[4-(3 ,5 -Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin- 1 -yl] -phenylsulfa-moyl] - butyric acid; l-[[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenylsulfa-moyl]- ethyl] -piperidine-4-carboxylic acid;
N-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-succi-namic acid;
4-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenylcarba-moyl]- butyric acid; 2-Amino-3-[4-[4-(3,5-dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]- propionic acid hydrochloride;
3-[4-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acrylic acid; 3-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acrylic acid;
3-[2-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acrylic acid;
3-[4-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-propionic acid;
3-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-propionic acid;
[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acetic acid;
3-[2-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-propionic acid;
[4-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acetic acid; N-methyl-glucamine salt of 4-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy- phthalazin- 1 -yl] -phenoxy] -butyric acid;
N-methyl-glucamine salt of 3-[3-[4-(3,5-Dichloro-pyridin-4-ylmethyl)-7-methoxy- phthalazin- 1 -yl] -phenyl] -acrylic acid;
(2-amino-2-hydroxymethyl-propan-l,3-diol) salt of 3-[3-[4-(3,5-Dichloro-pyridin-4- ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]-acrylic acid;
3-[3-[4-(3,5-Dichloro-l-oxy-pyridin-4-ylmethyl)-7-methoxy-phthalazin-l-yl]-phenyl]- acrylic acid.
3) A process for the preparation of a compound according to claim 1 by an aromatic nucleophilic substitution reaction or a coupling reaction, in presence of a catalyst such as for example palladium, between a compound of formula
wherein
R has the meanings reported for the compounds of formula I; and a reactant such as a tin or boronic acid derivative, suitable for the substitution of the halogen atom directly bonded to the phthalazine nucleus with a phenyl substituted with a carboxy group bonded to the phenyl moiety by a spacer X defined in formula I. 4) A process according to claim 3 wherein the coupling reaction is carried out between the compounds of formula II and the proper boronic acid in presence of palladium, triphenylphosphine and an aqueous solution of potassium carbonate.
5) A pharmaceutical composition containing a therapeutically effective amount of a compound according to claim 1 in admixture with a pharmaceutically acceptable carrier. 6) A pharmaceutical composition according to claim 5 for the treatment of allergic and inflammatory diseases
7) A pharmaceutical composition according to claim 5 for the treatment of respiratory diseases.
PCT/EP2003/014731 2002-12-23 2003-12-22 Phthalazine derivatives phosphodiesterase 4 inhibitors WO2004056798A1 (en)

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WO2000005218A1 (en) * 1998-07-21 2000-02-03 Zambon Group S.P.A. Phthalazine derivatives phosphodiesterase 4 inhibitors

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NAPOLETANO M ET AL: "Phthalazine PDE4 inhibitors. Part 2: The synthesis and biological evaluation of 6-methoxy-1,4-disubstituted derivatives" BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, OXFORD, GB, vol. 11, no. 1, 8 January 2001 (2001-01-08), pages 33-37, XP004225316 ISSN: 0960-894X *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7723345B2 (en) 2005-12-29 2010-05-25 Lexicon Pharmaceuticals, Inc. Multicyclic amino acid derivatives and methods of their use
EP2386547A1 (en) 2005-12-29 2011-11-16 Lexicon Pharmaceuticals, Inc. Multicyclic amino acid derivatives and methods of their use
US8063057B2 (en) 2005-12-29 2011-11-22 Lexicon Pharmaceuticals, Inc. Multicyclic amino acid derivatives and methods of their use
US8629156B2 (en) 2005-12-29 2014-01-14 Lexicon Pharmaceuticals, Inc. Tryptophan hydroxylase inhibitors

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