U.S. patent application number 10/441177 was filed with the patent office on 2004-03-04 for phthalazine derivatives as phosphodiesterase 4-inhibitors.
This patent application is currently assigned to Zambon Group S.p.A. Invention is credited to Alvarez-Builla, Julio G., Botta, Daniela, Garcia Navaio, Jose Luis, Grancini, Giancarlo, Morazzoni, Gabriele, Napoletano, Mauro, Norcini, Gabriele, Santangelo, Francesco, Siro Herrero, Jorge G..
Application Number | 20040043999 10/441177 |
Document ID | / |
Family ID | 11378396 |
Filed Date | 2004-03-04 |
United States Patent
Application |
20040043999 |
Kind Code |
A1 |
Napoletano, Mauro ; et
al. |
March 4, 2004 |
Phthalazine derivatives as phosphodiesterase 4-inhibitors
Abstract
A process for preparing a compound of formula I: 1 wherein G is
methylene, ethylene, CONH, amino or a bond; R is H, phenyl or a
(C.sub.1-4)alkyl group optionally substituted by phenyl; Cy is
phenyl or a 5- or 6-membered heterocycle of the group consisting of
pyrrole, imidazole, pyrazole, pyrrolidine, pyrroline, imidazoline,
imidazolidine, pyrazolidine, pyrazoline, pyridine, pyrazine,
pyrimidine, pyridazine, piperazine, piperidine, and triazine, the
Cy residue being optionally substituted by one or more
substituent(s) selected from the group consisting of keto, nitro,
carboxy, fluorine, chlorine, bromine, or iodine; R.sub.1 is a
(C.sub.1-6)alkyl or polyfluoro(C.sub.1-6)alkyl group; and R.sub.2
is aryl, aryl-(C.sub.1-10)-alkyl, (C.sub.4-7)cycloalkyl or
(C.sub.4-7)heterocycle wherein the heteroatom is an oxygen
atom.
Inventors: |
Napoletano, Mauro; (Milano,
IT) ; Norcini, Gabriele; (Vizzola Ticino, IT)
; Botta, Daniela; (Como, IT) ; Grancini,
Giancarlo; (Nova Milanese, IT) ; Morazzoni,
Gabriele; (Lainate, IT) ; Santangelo, Francesco;
(Milano, IT) ; Siro Herrero, Jorge G.; (Cala
d'Henares, ES) ; Garcia Navaio, Jose Luis; (Madrid,
ES) ; Alvarez-Builla, Julio G.; (Madrid, ES) |
Correspondence
Address: |
ARENT FOX KINTNER PLOTKIN & KAHN
1050 CONNECTICUT AVENUE, N.W.
SUITE 400
WASHINGTON
DC
20036
US
|
Assignee: |
Zambon Group S.p.A
|
Family ID: |
11378396 |
Appl. No.: |
10/441177 |
Filed: |
May 20, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10441177 |
May 20, 2003 |
|
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|
09581506 |
Aug 10, 2000 |
|
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6589951 |
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09581506 |
Aug 10, 2000 |
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PCT/EP98/08291 |
Dec 17, 1998 |
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Current U.S.
Class: |
514/248 ;
544/237 |
Current CPC
Class: |
A61P 29/00 20180101;
C07D 405/12 20130101; A61P 11/06 20180101; C07D 237/34 20130101;
A61P 37/08 20180101; A61P 11/00 20180101; C07D 237/30 20130101;
A61P 43/00 20180101; C07D 401/06 20130101; A61P 27/16 20180101;
C07D 401/12 20130101 |
Class at
Publication: |
514/248 ;
544/237 |
International
Class: |
C07D 237/30; A61K
031/502 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 1997 |
IT |
MI97A002806 |
Claims
1. A compound of formula I 33wherein B is methylene, ethylene,
amino, CONH or a bond; Cy is phenyl or a 5- or 6-membered
heterocycle containing from 1 to 3 nitrogen atom(s), being both the
residues optionally substituted by one or more substituent(s); R is
H, phenyl or a (C.sub.1-4)alkyl group optionally substituted by an
aromatic or hydrogenated ring containing from 5 to 7 members:
R.sub.1 is a (C.sub.1-6)alkyl or polyfluoro(C.sub.1-6)alkyl group;
R.sub.2 is aryl, aryl-(C.sub.1-10)-alkyl or a (C.sub.4-7)cycloalkyl
group optionally containing an oxygen atom and optionally
substituted by a polar substituent; and the N.fwdarw.O derivatives
and pharmaceutically acceptable salts thereof, with the proviso
that when R is H, R.sub.2 is not aryl-methyl.
2. A compound according to claim 1 wherein B is methylene or amino;
Cy is phenyl or a 5- or 6-membered heterocycle containing from 1 to
3 nitrogen atom(s), being both the residues substituted by one or
two halogen(s); R is H, phenyl or a (C.sub.1-4)alkyl group
optionally substituted by an aromatic or hydrogenated ring
containing from 5 to 7 members; R.sub.1 is a (C.sub.1-6)alkyl or
polyfluoro(C.sub.1-6)alkyl group; R.sub.2 is a
(C.sub.4-7)cycloalkyl group optionally containing an oxygen atom
and optionally substituted by a polar substituent; and the
N.fwdarw.O derivatives and pharmaceutically acceptable salts
thereof.
3. A compound according to claim 1 wherein B is methylene; Cy is
phenyl or a 6-membered heterocycle containing 1 nitrogen atom,
being both the residues substituted by one or two halogen(s); R is
phenyl or a (C.sub.1-4)alkyl group optionally substituted by an
aromatic or hydrogenated ring containing from 5 to 7 members;
R.sub.1 is a (C.sub.1-6)alkyl or polyfluoro(C.sub.1-6)alkyl group;
R.sub.2 is a (C.sub.4-7)cycloalkyl group optionally containing an
oxygen atom and optionally substituted by a polar substituent; and
the N.fwdarw.O derivatives and pharmaceutically acceptable salts
thereof.
4. A process for preparing a compound according to claim 1 wherein
B is methylene, ethylene, ammo or a bond, characterized in that a
benzaldehyde of formula II 34wherein R.sub.1 and R.sub.2 are as
defined in claim 1 is oxidised to give an acid of formula III
35wherein R.sub.1 and R.sub.2 are as defined above, which is
transformed into the corresponding acyl halide of formula IV
36wherein R.sub.1 and R.sub.2 are as defined above and X is
chlorine or bromine; this compound is reacted with diethylamine to
give a benzamide of formula V 37wherein R.sub.1 and R.sub.2 are as
defined above, which reacted with dimethylformamide in the presence
of a strong base yields a compound of formula VIa 38wherein R.sub.1
and R.sub.2 are as defined above, and R.sup.1 is hydrogen which,
when a compound of formula I wherein R is hydrogen is desired, is
reacted with tert.butylcarbazole to give the compound of formula
VIIa 39wherein R.sup.1, R.sub.1 and R.sub.2 are as defined above,
and R' is a protecting group of the carboxy moiety; instead when a
compound of formula I wherein R is over then hydrogen is desired,
the compound of formula VIa is treated with a R.sup.II-magnesium
halide or R.sup.II-lithium, wherein R.sup.II is phenyl or a
(C.sub.1-4)alkyl group optionally substituted by an aromatic or
hydrogenated ring having from 5 to 7 members, to give a compound of
formula XIII 40wherein R.sup.II, R.sub.1 and R.sub.2 are as defined
above, which treated with a suitable oxidising agent, yields a
compound of formula VIb 41wherein R.sub.1, R.sub.2 and R.sup.II are
as defined above, which is treated with tert.butylcarbazole to give
the compound of formula VIIb, differing from the compound VIIa in
that R has the meanings of formula I hydrogen excluded; the
compound of formula VIIa or VIIb is reacted with trifluoroacetic
acid to give the phthalazinone of formula VIII 42wherein R, R.sub.1
and R.sub.2 are as defined above; this is reacted with a
halogenating agent to give the phthalazine of formula IX 43wherein
R, R.sub.1 and R.sub.2 are as defined above, and X.sup.1 is a
halogen atom, which by treatment with a compound of formula
XIVCy-B'-Y (XIV)wherein Cy is as defined in claim 1, B' is
methylene, ethylene, amino or a bond and Y is hydrogen or halogen,
provides the desired compound.
5. A process for preparing compounds according to claim 1 wherein B
is methylene, ethylene, amino or a bond characterized in that a
compound of formula VIa or VIb 44wherein R.sub.1 and R.sub.2 are as
defined in claim 1, R.sup.I is hydrogen and R.sup.II is phenyl or a
(C.sub.1-4)-alkyl group optionally substituted by an aromatic or
hydrogenated ring of from 5 to 7 members, is reacted with acetic
acid in acidic medium to give a compound of formula X 45wherein R
and R.sub.1 are as defined above and R'.sub.2 is hydrogen, aryl,
aryl-(C.sub.1-10)-alkyl or a (C.sub.4-7)cycloalkyl group optionally
containing an oxygen atom and optionally substituted by a polar
substituent, which in turn is reacted with hydrazine to give a
phthalazinone of formula XI 46wherein R, R.sub.1 and R'.sub.2 are
as defined above, which only when R'.sub.2 is hydrogen, is treated
with the suitable compound of formula XIa or
XIIbR.sub.2OSO.sub.2CH.sub.3 (XIIa)R.sub.2X (XIIb)wherein R.sub.2
and X are as defined above, and yields a compound of formula VIII
47wherein R, R.sub.1 and R.sub.2 are as defined above; this is
reacted with a chlorinating agent to give the phthalazine of
formula IX 48wherein R, R.sub.1 and R.sub.2 are as defined above,
and X' is a halogen atom, which by treatment with a compound of
formula XIVCy-B'-Y (XIV)wherein Cy is as defined above, B' is
methylene, ethylene, amino or a bond and Y is hydrogen or halogen,
provides the desired compound.
6. A process for preparing a compound according to claim 1 wherein
B is CONH, characterized in that a compound IX is reacted with
carbon monoxide and methanol in the presence of a metal catalyst,
to give a compound of formula XV 49wherein R, R.sub.1 and R.sub.2
are as defined in claim 1, which is converted in the desired
compound of formula I by amidation.
7. A process for preparing a compound according to claim 1 wherein
B is other than amino, characterized in that the acid of formula
III 50wherein R.sub.1 and R.sub.2 are as defined in claim 1, is
reacted with formaldehyde/HCl to give a compound of formula XVI
51wherein R.sub.1 and R.sub.2 are as defined above, which is
oxidized, then hydrolyzed to give a compound of formula XVII
52wherein R.sub.1 and R.sub.2 are as defined above, which with an
hydrohalide acid and triphenylphosphine gives a compound of formula
XVIII 53wherein R.sub.1 and R.sub.2 are as defined above, which
treated with an aldehyde of formula XIXCy-B"-CHO (XIX)wherein Cy is
as defined in claim 1 and B" is methylene or is absent, in the
presence of an organic base, gives a compound of formula XX
54wherein R.sub.1, R.sub.2, B" and Cy are as defined above; this is
reacted with hydrazine to give a compound of formula XXI 55wherein
R.sub.1, R.sub.2, and Cy are as defined above and B is other than
amino, which is treated with a halogenating agent to give a
compound of formula XXII 56wherein R.sub.1, R.sub.2, X and Cy are
as defined above and B is other than amino, which is subdued to a
coupling reaction with the suitable metal-organic derivative in the
presence of a catalyst, or to a nucleophilic substitution.
8. A pharmaceutical composition containing a therapeutically
effective amount of a compound according to claim 1 in admixture
with a suitable carrier.
9. A pharmaceutical composition according to claim 8 for the
treatment of allergic and inflammatory pathologies.
10. A pharmaceutical composition according to claim 8 for the
treatment of respiratory diseases.
Description
[0001] The present invention relates to phthalazine derivatives, to
pharmaceutical compositions comprising them and to their use as
phosphodiesterase 4 inhibitors. Phosphodiesterases are a family of
isoenzymes which constitutes 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 the cell surface, the adenylated cyclase activates and turns
Mg.sup.+2-ATP into cAMP. cAMP modulates the activity of the
majority, if not of all, of the cells contributing to the
pathophysiology of various respiratory diseases both of allergic
origin and not. It follows that an increase of 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 capable 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.
[0002] In the phosphodiesterase family there is a distinct group of
isoenzymes, phosphodiesterases 4 (hereinafter PDE 4), specific for
the hydrolysis of the airway smooth muscle and inflammatory cells
cAMP (Torphy, "Phosphodiesterase Isoenzymes: Potential Targets for
Novel Anti-asthmatic Agents" in New Drugs for Asthma, Barnes, ed.
IBC Technical Services Ltd, 1989). Studies carried out on this
enzyme show that its inhibition yields not only airway smooth
muscle relaxation, but also mastocyte suppression, basophil and
neutrophil degranulation, thus inhibiting monocyte activation and
neutrophil activation. Furthermore, the PDE 4 inhibitors activity
is markedly improved when the adenylated cyclase activity of the
target cells is enhanced by endogenous hormones, as the case in
vivo. Thus, PDE 4 inhibitors should be effective in the therapy of
asthma. Such compounds would 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.
[0003] The excessive or irregular production of the tumor necrosis
factor (hereinafter TNF.sub..alpha.), a cytokine with
pro-inflammatory activity produced by various kind of cells,
affects the mediation or the exacerbation of many pathologies such
as, for example, the adult respiratory disease syndrome (ARDS) and
the chronic pulmonary inflammatory disease. Therefore, compounds
able to control the negative effects of TNF.sub..alpha., i.e. the
inhibitors of this cytokine, are to be considered useful against
many pathologies.
[0004] The patent application EP-0 722 936 (in the name of Eisai)
claims, inter alia, compounds of formula
[0005] The patent application EP-0 722 936 (in the name of Eisai)
claims, inter alia, compounds of formula 2
[0006] wherein n=0-4; R.sub.1, is an optionally substituted lower
alkoxy or cycloalkyl, or a --OR.sub.9 group
[0007] wherein R.sub.9 is an optionally substituted arylalkyl
group; Y is --CB.dbd. wherein B is an optionally substituted
heteroarylalkyl group or --NR.sub.7R.sub.8 wherein one of R.sub.7,
and R.sub.8 may be H and the other an optionally substituted
heteroarylalkyl group; A is hydrogen or a halogen atom, optionally
mono- or bi-substituted amino group, optionally substituted aryl,
heteroaryl or heteroarylalkyl group. Among the groups optionally
substituting the above mentioned residues halogen atoms are listed.
Such compounds are said to be active as cGMP-PDE inhibitors, i.e.
PDE 5, a phosphodiesterase acting by a cGMP-dependent mechanism and
whose field of action is markedly cardiovascular (Schudt C. et al.,
Phosphodiesterase Inhibitors, Academic Press).
[0008] wherein n=0-4; R.sub.1, is an optionally substituted lower
alkoxy or cycloalkyl, or a --OR.sub.9 group
[0009] wherein R.sub.9 is an optionally substituted arylalkyl
group; Y is --CB.dbd. wherein B is an optionally substituted
heteroarylalkyl group or --NR.sub.7R.sub.8 wherein one of R.sub.7,
and R.sub.8 may be H and the other an optionally substituted
heteroarylalkyl group; A is hydrogen or a halogen atom, optionally
mono- or bi-substituted amino group, optionally substituted aryl,
heteroaryl or heteroarylalkyl group. Among the groups optionally
substituting the above mentioned residues halogen atoms are listed.
Such compounds are said to be active as cGMP-PDE inhibitors, i.e.
PDE 5, a phosphodiesterase acting by a cGMP-dependent mechanism and
whose field of action is markedly cardiovascular (Schudt C. et al.,
Phosphodiesterase Inhibitors, Academic Press).
[0010] The patent application EP-0 498 723 (in the name of Roussel
Uclaf) discloses, inter alia, compounds of formula 3
[0011] wherein R.sub.2b and R.sub.3b are hydrogen, hydroxy, alkyl,
cycloalkyl, acyloxy; at least one but no more then two of A.sub.1b,
A.sub.2b, A.sub.3b and A.sub.4b are a nitrogen atom and at least
one of them is a methyne radical substituted by the --R.sub.5--YB
group wherein R.sub.5 is a divalent alkylene radical, and YB
represents the radical --Y.sub.1B--B--Y.sub.2B wherein Y.sub.1B is
a monocyclic aryl optionally containing nitrogen, B is a single
bond and Y.sub.2B is hydrogen or halogen. These compounds are said
to be effective for the treatment of arterial hypertension, heart
and renal failure and for the prevention of restenosis after
angioplasty.
[0012] The patent application EP-0 017 411 (in the name of Pfizer)
claims phthalazines of formula 4
[0013] wherein R.sub.1 is lower alkyl; Y is --(CH.sub.2).sub.m-Z
wherein m is 1 or 2, and Z is carbamoyloxy, carbonylamino,
sulfamoyl, ureido, amino-sulfamoyl, carboxamino substituted on the
terminal portion by a (C.sub.3-7)cycloalkyl. These compounds are
said to be phosphodiesterase inhibitors and to have a cardiac
muscle stimulating activity, thus their action does not relate to
PDE 4.
[0014] The patent U.S. Pat. No. 3,274,185 (in the name of
Messengill) describes, inter alia, phthalazines of formula 5
[0015] wherein Y and Y.sub.1 are lower alkoxy; Z is phenyl
optionally substituted by halogen or benzyl; and R is hydrogen.
These phthalazines are endowed with sedative and hypertensive
activity, without an explicit mechanism of action.
[0016] The patent U.S. Pat. No. 3,813,384 (in the name of
Asta-Werke) illustrates, inter alia, benzylphthalazinones of
formula 6
[0017] wherein R.sub.1 and R.sub.2 are lower alkoxy or halogen; X
is an optionally branched alkylene chain; m and n are 1-3; p is 0
or 1; and the 7
[0018] group is a C.sub.3-8 mono-, bi- or tricyclic residue
containing one or two nitrogen atom(s). Such compounds have a
hystaminolytic action and are useful, for example, in the treatment
of asthma.
[0019] The patent application NL 8005411 (in the name of Mitsubishi
Yuka) describes phthalazines of formula 8
[0020] wherein X is O or NH; R.sub.1, R.sub.2 and R.sub.3 are,
inter alia, (C.sub.1-5)alkyl, (C.sub.1-5)alkoxy, halogen or
CF.sub.3; n, m and p are 0-3. The use of these compounds is as
platelet aggregation inhibitors.
[0021] The patent application JP-56061365 (in the name of Showa
Denko) describes phthalazinones of formula 9
[0022] wherein, inter alia, R is halogen and n is 1-3, as
vasodilators and anti-ulcer agents. The patent application WO
97/40020 (in the name of Schering AG) illustrates, inter alia,
compounds of formula 10
[0023] wherein R.sub.1 and R.sub.2 are H, nitro, halogen, amino,
lower alkoxy or --CF.sub.3; R.sub.4 is H or lower alkyl; R.sub.5 is
lower alkyl. These compounds are uncompetitive antagonists of
excitatory aminoacids.
[0024] The patent application WO97/48697 (in the name of Rhone
Poulenc Rorer), published on Dec. 24, 1997, disloses, inter alia,
compounds of formula 11
[0025] wherein A is an azaheterocycle and B an azaheteroaryl ring
or an optionally halo-substituted benzene ring; Z.sup.1 is a bond
or an oxygen atom; R.sup.1 is H or lower alkyl optionally
substituted by halogen atom(s); A.sup.1 is a bond or a C.sub.1-6
alkylene optionally substituted by aryl, cycloalkyl or heteroaryl;
R.sup.2 may be H, aryl, heteroaryl; R.sup.3 may be aryl,
heteroaryl, aryl-methoxy, heteroaryl-methoxy; n and m are
alternatively 0 or 1. The aryl and heteroaryl moieties may be
substituted by halogen atoms. These compounds are PDE 4 and TNF
inhibitors.
[0026] Therefore the present invention relates to compounds of
formula I 12
[0027] wherein
[0028] G is methylene, ethylene, amino, CONH or a bond;
[0029] Cy is phenyl or a 5- or 6-membered heterocycle containing
from 1 to 3 nitrogen atom(s), being both the residues optionally
substituted by one or more substituent(s); R is H, phenyl or a
(C.sub.1-4)alkyl group optionally substituted by an aromatic or
hydrogenated ring containing from 5 to 7 members;
[0030] R.sub.1 is a (C.sub.1-6)alkyl or polyfluoro(C.sub.1-6)alkyl
group;
[0031] R.sub.2 is aryl, aryl-(C.sub.1-10)alkyl or a
(C.sub.4-7)cycloalkyl group optionally containing an oxygen atom
and optionally substituted by a polar substituent;
[0032] and the N.fwdarw.O derivatives and pharmaceutically
acceptable salts thereof;
[0033] with the proviso that when R is H, R.sub.2 is not
aryl-methyl.
[0034] The proviso is due to avoid the overlap with the patent
application WO97/48697 (in the name of Rhone Poulenc Rorer) said
above.
[0035] The compounds of formula I may have an asymmetric centre and
thus may be in the form of stereoisomers. The objects of the
present invention are the compounds of formula I in the form of
stereoisomeric mixtures as well as single stereoisomers.
[0036] The compounds of formula I are active as PDE 4 and
TNF.sub..alpha. inhibitors, and are thus used as therapeutic agents
in allergic and inflammatory pathologies such as, for example,
emphysema, chronic bronchitis, asthma and allergic rhinitis.
[0037] As for 5- or 6-membered heterocycle it is meant pyrrole,
imidazole, pyrazole, pyrrolidine, pyrroline, imidazoline,
imidazolidine, pymzolidine, pyrazoline, pyridine, pyrazine,
pyrimidine, pyridazine, piperazine, piperidine, triazine, and the
like, preferably pyridine and piperidine. The substituents
optionally present on Cy may be keto, nitro, carboxy, halogen, this
term embracing a fluorine, chlorine, bromine or iodine atom,
chlorine being the preferred substituent.
[0038] Specific examples of alkyl groups are methyl, ethyl,
n-propyl, i-propyl, n-butyl, s-butyl, tert-butyl, n-pentyl,
1-methyl-butyl, 2-ethyl-propyl, 3-ethyl-butyl, 3-ethyl-tyl, n-exyl
and the like. As for (C.sub.5-7) cycloalkyl group it is meant
cyclopentyl, cyclohexyl and cycloheptyl, and when it contains an
oxygen atom it is meant, for example, tetrahydrofuran or
tetrahydropyran, while aryl and aryl-(C.sub.1-10) mean a ring or a
C.sub.6-10 aromatic system such as, for example, phenyl, benzyl,
phenethyl, phenyl-pentyl, naphthyl, indanyl, indanyl-pentyl and the
like. For "polar substituent" it is meant those groups constituted
by atoms with different electronegativity thereby a dipole is
created, such as, for example, hydroxy or keto groups, are
meant.
[0039] The N.fwdarw.O groups optionally present in number of one or
more may regard both the nitrogen atoms of the phthalazine ring,
and the ones on the substituent Cy.
[0040] Pharmaceutically acceptable salts of the compounds of
formula I are those with organic and inorganic acids, such as, for
example, hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,
phosphoric, acetic, benzoic, maleic, fumaric, succinic, tartaric,
citric, aspartic, methansulfonic,
3,7-di-tert.butylnaphthalen-1,5-disulfonic (dibudinic acid).
Preferred compounds of formula I are those wherein G is methylene
or amino; Cy is phenyl or a 5- or 6-membered heterocycle containing
from 1 to 3 nitrogen atom(s), both the residues being optionally
substituted by 1 or 2 halogen(s); R is H, phenyl or a
(C.sub.1-4)alkyl group optionally substituted by an aromatic or
hydrogenated ring containing 5-7 members; R.sub.1 is a
(C.sub.1-6)alkyl or polyfluoro(C.sub.1-6)alkyl group; R.sub.2 is a
(C.sub.4-7)cycloalkyl group optionally containing an oxygen atom
and optionally substituted by a polar substituent; and the
N.fwdarw.O derivatives and pharmaceutically acceptable salts
thereof.
[0041] More preferred compounds of formula I are those wherein G is
methylene; Cy is phenyl or a 6-membered heterocycle containing 1
nitrogen atom, being both the residue substituted by 1 or 2
chlorine atom(s); R is phenyl or a (C.sub.1-4)alkyl group
optionally substituted by a 5-7 members aromatic or hydrogenated
ring; R.sub.1 is a (C.sub.1-6)alkyl, polyfluoro(C.sub.1-6)-alkyl
group; R.sub.2 is a (C.sub.4-7)-cycloalkyl group optionally
containing an oxygen atom and optionally substituted by a polar
substituent; and the N.fwdarw.O derivatives and the
pharmaceutically acceptable salts thereof.
[0042] The synthesis of the compounds of formula I proceeds
according to methods known to the skilled in the art. For example,
a benzaldehyde of formula II 13
[0043] wherein R.sub.1 and R.sub.2 are as defined above, is
oxidized, for example with potassium permanganate and
tetrabutylammonitun bromide, to give an acid of formula III 14
[0044] wherein R.sub.1 and R.sub.2 are as defined above, which, for
example by treatment with thionyl chloride, is turned into the
corresponding acyl halide of formula IV 15
[0045] wherein R.sub.1 and R.sub.2 are as defined above and X is
chlorine or bromine. This compound is reacted with diethylamine in
an at least equimolar amount to give a benzamide of formula V
16
[0046] wherein R.sub.1 and R.sub.2 are as defined, which reacted
with dimethylformamide in the presence of a strong organic base
such as, for example, n-butyl-lithium, tert butyllithium,
sec-butyl-lithium, optionally in the presence of a binding agent
such as, for example, tetramethylethylendiamine, yields a compound
of formula VIa 17
[0047] wherein R.sub.1 and R.sub.2 are as defined above, and
R.sub.1 is hydrogen.
[0048] When a compound of formula I wherein R.dbd.H is desired, the
compound of formula VIa is reacted with an equimolar amount of
tert-butylcarbazole to give a compound of formula VIIa 18
[0049] wherein R.sup.1, R.sub.1 and R.sub.2 are as defined above,
and R' is a protecting group of the carboxy moiety such as, for
example, tert-butyl.
[0050] Instead, when a compound of formula I wherein R is other
than hydrogen is desired, the compound of formula VIa is treated
with R.sup.II-magnesium halide, for example, chloride, or
R.sup.II-lithium, wherein R.sup.II phenyl or a (C.sub.1-4)alkyl
group optionally substituted by an aromatic or hydrogenated ring
having from 5 to 7 members, to give a compound of formula XIII
19
[0051] wherein R.sup.II, R.sub.1 and R.sub.2 are as defined above.
The compound of formula XIII is treated with a suitable oxidising
agent such as, for example, pyridinium-chloro chromate, and yields
a compound of formula Vib 20
[0052] wherein R.sub.1, R.sub.2 and R.sup.II are as defined above,
which is treated with an equimolar amount of tert.butylcarbazole to
give a compound of formula VIIb, which differs from the compound
VIIIa in that R has the meanings of formula I but hydrogen.
[0053] The compound of formula VIIa or VIIb is reacted with
trifluoroacetic acid to give the phthalazinone of formula VIII
21
[0054] wherein R, R.sub.1 and R.sub.2 are as defined above. This
phthalazinone is reacted with a halogenating agent such as, for
example, phosphoryl chloride, to give the phthalazine of formula IX
22
[0055] wherein R, R.sub.1 and R.sub.2 are as defined above, and X'
is a halogen atom.
[0056] Alternatively, the compound of formula VIII may be directly
obtained from the compound of formula VIa or VIb by treatment with
hydrazine in acetic acid.
[0057] The compound of formula IX yields a compound of formula I by
treatment with a compound of formula XIV
Cy-G'-Y (XIV)
[0058] wherein Cy is as defined above, G' is methylene, ethylene,
amino or a bond and Y is hydrogen, halogen.
[0059] When a compound of formula I wherein G is CONH is desired,
the compound IX is reacted with carbon monoxide and methanol in the
presence of a catalyst such as metal palladium or nickel, to give a
compound of formula XV 23
[0060] wherein R, R.sub.1 and R.sub.2 are as defined above, which
is then turned into the desired compound of formula I through
methods known to those skilled in the art, for example by reaction
with an aryl-amine in the presence of bases.
[0061] Alternatively, the compounds of formula I may be synthesized
by treatment of a compound of formula VIa or VIb with acetic acid
in acidic medium to give a compound of formula X 24
[0062] wherein R and R.sub.1 are as defined above, and R'.sub.2 has
the meanings of R.sub.2 listed above plus hydrogen, which is
reacted with hydrazine to give a phthalazinone of formula XI 25
[0063] wherein R, R.sub.1 and R'.sub.2 are as defined above. When
R'.sub.2 is hydrogen this compound is treated with the due compound
of formula XIIa or XIIb
R.sub.2OSO.sub.2CH.sub.3 (XIIa)
R.sub.2X (XIIb)
[0064] wherein R.sub.2 and X are as defined above to give a
compound of formula VIII as described above.
[0065] Another alternative is for the compounds of formula I
wherein G is other than amino, which may be yielded starting from
the acid of formula III which reacting with formaldehyde/HCl which
forms a compound of formula XVI 26
[0066] wherein R.sub.1 and R.sub.2 are as defined above. This
compound is oxidized, for example with benzoyl
peroxide/N-bromo-succinimide, then hydrolyzed to give a compound of
formula XVII 27
[0067] wherein R.sub.1 and R.sub.2 are as defined above, which with
a halogenidric acid and triphenylphosphine gives a compound of
formula XVIII 28
[0068] wherein R.sub.1 and R.sub.2 are as defined above, which
treated with an aldehyde of formula XIX
Cy-G"-CHO (XIX)
[0069] wherein Cy is as defined above and G" is methylene or is
absent, in the presence of an organic base such as, for example,
triethylamine, gives a compound of formula XX 29
[0070] wherein R.sub.1, R.sub.2, G and Cy are as defined above.
This is reacted with hydrazine to give a compound of formula XXI
30
[0071] wherein R.sub.1, R.sub.2, and Cy are as defined above and G
is other than amino, which is treated with a halogenating agent,
such as phosphoryl chloride or bromide, to give a compound of
formula XXII 31
[0072] wherein R.sub.1, R.sub.2, X and Cy are as defined above and
G is other than amino. This compound is subjected to a coupling
reaction with the suitable metallorganic derivative in the presence
of a catalyst, for example, a palladium-based catalyst, or to a
nucleophilic substitution which gives a compound of formula I
wherein G is other than amino.
[0073] A choice for having a compound of formula I wherein R.sub.1
is a polyfluoro(C.sub.1-6)alkyl group consists in treating a
compound of formula XXIII 32
[0074] wherein R'.sub.1 is hydrogen, with bases, for example a
carbonate or bicarbonate, and a
polyfluoro(C.sub.1-6)alkyl-chloride, -bromide or -iodide at
70-75.degree. C. The compound of formula XXIII may be obtained from
a compound of formula I wherein R.sub.1 is (C.sub.1-6)alkyl for
example by treatment with sodium p-thiochresolate in the presence
of a base such as dimethylformamide.
[0075] The synthesis of the N-oxides of the compounds of formula I
is effected by treating the compounds of formula I with peracids
such as, for example m-chloroperbenzoic acid.
[0076] The preparation of the salts of the compounds of formula I
is effected by conventional methods.
[0077] The compounds of formula I are PDE 4 inhibitors as resultant
from the in vitro enzymatic inhibition activity tests (example 67),
without any effect on PDE 3 and PDE 5 (example 69). Moreover they
are able to inhibit the TNF.sub..alpha. release (example 68).
Comparisons with the following compounds have been carried out:
6,7-dimethoxy-4-(pyridin-4-yl-- methyl)-2H-phthalazin-1-one
(reference 1) and 6,7-dimethoxy-4-(piperidin-4-
-yl-methyl)-2H-phthalazin-1-one (reference 2) embraced by the
general formula of the patent application EP-0 722 936 (in the name
of Eisai) just cited above, chosen in view of the structural
affinity with the compounds of the invention. The reference
compounds, though chemically alike, did not show to be active on
PDE 4.
[0078] It is apparent how these receptorial selectivity and
specificity features joined with the lack of activity on the
cardiovascular system make the compounds of formula I particularly
suitable for the treatment of the pathologies involving PDE 4 and
TNF.sub..alpha. even if in the present context the interest is
specifically focused on the respiratory pathologies. Especially the
compounds of the invention are useful in the treatment of allergic
and inflammatory diseases and above all in the therapy of
emphysema, of chronic obstructive pulmonary disease and chronic
bronchitis in particular, of asthma and allergic rhinitis.
[0079] The therapeutical dosage shall generally be comprised
between 0.1 and 1,000 mg a day and between 1 and 100 mg by oral
route for a single administration.
[0080] A further object of the present invention are the
pharmaceutical compositions comprising a therapeutically effective
amount of the compounds of formula I or of the pharmaceutically
acceptable salts thereof in admixture with a suitable carrier.
[0081] The pharmaceutical compositions object of the invention may
be liquid, suitable for enteral or parenteral administration, and,
preferably, solid such as tablets, capsules, granulates, suitable
for oral administration, or in a form suitable for the transdermic
or inhalatory administration.
[0082] The preparation of the pharmaceutical compositions object of
the invention may be effected according to common techniques.
[0083] Provided below are examples in order to better illustrate
the present invention.
EXAMPLE 1
Synthesis of 3-cyclopentyloxy-4-methoxy benzoic acid
[0084] A solution of potassium permanganate (53.1 g, 0.336 moles)
in water (1 l) was added under stirring with a solution of bromide
tetrabutylammonium (111.68 g, 0.336 moles) in water (0.4 l). The
formed solid was isolated by filtration, washed with water and
dissolved in pyridine (0.5 l). The solution was dropped into a
solution of crude 3-cyclopentyloxy-4-methoxy benzaldehyde (obtained
as described in J.Med.Chem., 1995, 38, page 4851) (74 g, 0.336
moles) in pyridine (0.2 l) under stirring in water/ice. At the end
ice (3 l) was added and the whole was acidified by dropping 12N HCl
(0.85 l). The stirring was kept on for 1.5 hours more, then the
solid was isolated by filtration and extracted under stirring for
30 minutes in ethyl acetate (2 l). The solid residue was removed
and the mother liquor was further extracted in ethyl acetate
(2.times.0.7 l). The organic phases were washed with water,
anhydrified over sodium sulfate and brought to a small volume to
give 53.2 g of the title product (yield: 67%).
[0085] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 7.75-6.87
(m, 3H); 4.87-4.79 (m, 1H); 3.90 (s, 3H); 2.08-1.52 (m, 8H).
EXAMPLE 2
Synthesis of 3-cyclopentyloxy-4-methoxy benzoic acid chloride
[0086] A solution of 3-cyclopentyloxy-4-methoxy benzoic acid (53 g,
0.224 moles) obtained as described in example 1, in thionyl
chloride (200 ml), was refluxed for 2 hours under nitrogen, then
evaporated to dryness and taken up twice in toluene (100 ml) to
give 57 g of the title product which was used as such in the next
step.
[0087] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 7.81-7.72
(dd, 1H); 7.52-7.50 (d, 1H); 6.91-6.85 (d, 1H); 4.87-4.75 (m, 1H);
3.91 (s, 3H); 2.08-1.50 (m, 8H).
EXAMPLE 3
Synthesis of 3-cyclopentyloxy-N,N-diethyl-4-methoxy-benzamide
[0088] A solution of 3-cyclopentyloxy-4-methoxy benzoic acid
chloride (57 g, 0.224 moles) obtained as described in example 2, in
methylene chloride (250 ml) was dropwise added at 5-10.degree. C.
with diethylamine (69.2 g, 0.672 moles). The mixture was evaporated
to dryness, dissolved in ethyl acetate, washed with water, 2%
potassium bisulfate, water again and sodium bicarbonate,
anhydrified over sodium sulfate and dried. The residue was taken up
in petrolatum (250 ml) to give 61.2 g of the title product (yield:
94%).
[0089] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 6.94-6.78
(m, 3H); 4.80-4.70 (m, 1H); 3.82 (s, 3H); 3.50-3.22 (m, 4H);
1.98-1.50 (m, 8H); 1.17 (t broad, 6H).
EXAMPLE 4
Synthesis of
3-cyclopentyloxy-N,N-diethyl-2-formyl-4-methoxy-benzamide and
5-cyclopentyloxy N,N-diethyl 2-formyl-4-methoxy-benzamide
[0090] A solution of 3-cyclopentyloxy
N,N-diethyl-4-methoxy-benzamide (61.2 g, 0.21 moles), obtained as
described in example 3, and tetramethylethylendiamine (34.86 ml,
0.231 moles) in dry tetrahydrofuran (480 ml) was dropwise added
under stirring at -75.degree. C. with sec-butyl-lithium (195.76 ml,
0.231 moles) and, after 1 hour at the same temperature, with
dimethylformamide (53.66 ml, 0.693 moles). After a further half
hour at -75.degree. C., the reaction mixture was poured into a
phosphate buffer pH=7, ethyl acetate and concentrated HCl. The
extraction was repeated twice with fresh ethyl acetate, then the
extract was washed with 5% potassium bisulfate, then with water,
then anhydrified over sodium sulfate and dried, the residue was
chromatographed over silica gel (eluent petrolatum/ethyl acetate
1:1) to give 23.3 g of the first title product (product A; yield:
35%) and 6.7 g of the second title product (product B: yield:
10%).
[0091] Product A: .sup.1-NMR (200 MHz, CDCI.sub.3) .delta. (PPM):
10.44 (s, 1H); 7.09-6.85 (m. 2H); 5.05-4.98 (m, 1H); 3.84 (s, 3H);
3.59-3.47 and 3.05-2.92 (2q, 4H); 1.95-1.52 (m, 8H); 1.28 and 0.95
(2t, 6H).
[0092] Product B: .sup.1-NMR (200 MHz, CDCI.sub.3) .delta. (PPM):
9.86 (s, 1H); 7.3 (s, 1H); 6.73 (s, 1H); 4.87-4.76 (m, 1H); 3.88
(s, 3H); 3.58 and 3.12 (2q, 4H); 2.05-1.52 (m, 8H); 1.26 and 1.02
(2t, 6H).
EXAMPLE 5
Synthesis of
N'-(2-cyclopentyloxy-6-diethylcarbamoyl-3-methoxy-benzylidene-
)hydrazincarboxy acid tert-butyl ester
[0093] A solution of
3-cyclopentyloxy-N,N-diethyl-2-formyl-4-methoxy-benza- mide (21.7
g, 0.068 moles) obtained as described in example 4, product A, and
tert-butylcarbazole (12.84 g, 0.1 moles) in absolute ethanol (217
ml) was refluxed for 3 hours, then dried, taken up in petrolatum
and dried again. The residue was taken up in ethyl ether (100 ml)
and petrolatum (0.2 I), then filtered and the mother liquor was
dried to give 27.45 g of the title product (yield: 93%). .sup.1-NMR
(200 MHz, CDCI.sub.3) .delta. (ppm): 8.60(m, 1H); 8.05(s broad,
1H); 6.87(s, 2H); 4.87-4.72 (m, 1H); 3.82(s, 3H); 3.58-3.25(m, 4H);
1.90-1.50(m, 8H); 1.40(s, 9H); 1.22-1.06 (2t, 6H).
EXAMPLE 6
Synthesis of 5-cyclopentyloxy-6-methoxy-2H-phthalazin-1-one
[0094] Trifluoroacetic acid (150 ml) was added with
N'-(2-cyclopentyloxy-6-diethylcarbamoyl-3
methoxy-benzylidene)-hydrazinca- rboxy acid tert-butyl ester (27.35
g, 0.063 moles) obtained as described in example 5, under stirring
at 5-10.degree. C., then the mixture was stirred for 15 minutes and
dried. The residue was dissolved in methylene chloride (750 ml) and
left at room temperature for 6 hours, then washed with 5% sodium
bicarbonate to alkalinity, then with water, anhydrified over sodium
sulfate and dried. The residue was taken up in ethyl ether (0.1 l)
and filtered to give 15.58 g of the title product (yield: 95%).
[0095] .sup.1-NMR (200 MHz, CDCI.sub.3) .delta. (ppm): 10.12 (m,
1H); 8.84 (s, 2H); 8.11 and 7.36 (2d. 2H); 5.11-5.01 (m, 1H); 3.98
(s, 3H); 2.00-1.60 (m, 8H).
EXAMPLE 7
Synthesis of 5-cyclopentyloxy-1-chloro-6-methoxy-2H-phthalazine
[0096] A slurry under nitrogen of
5-cyclopentyloxy-6-methoxy-2H-phthalazin- -1-one (7.5 g, 28.81
mmoles), obtained as described in example 6, in phosphoryl chloride
(30 ml) was heated under stirring to 80.degree. C., then
concentrated to dryness, and the residue was dissolved in ethyl
acetate and washed with potassium bicarbonate to alkalinity, then
with water, anhydrified over sodium sulfate and evaporated to
dryness to give 7.95 g of the title product (yield: 99%).
[0097] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 8.40 (s,
2H); 8.11 and 7.36 (2d, 2H); 5.11-5.01 (m, 1H); 3.98 (s, 3H);
2.00-1.60 (m, 8H).
EXAMPLE 8
Synthesis of
5-cyclopentyloxy-1-(3,5-dichloropyridin-4-ylmethyl)-6-methoxy-
-phthalazine (Compound 1)
[0098] A solution of 3,5-dichloro-4-methyl-pyridine (13.86 g, 85.56
mmoles) in dry dimethylformamide (100 ml) was added, under nitrogen
and stirring, with 55% NaH (3.73 g, 85.56 mmoles). The mixture was
stirred for 1 hour then added with a solution of
5-cyclopentyloxy-1-chloro-6-meth- oxy-2H-phthalazine (7.95 g, 28.52
mmoles), obtained as described in example 7, in dry
dimethylformamide (70 ml). The mixture was left to stand for one
night, quenched with water, diluted with water and extracted in
ethyl acetate. The extract was washed with water and anhydrified
over Na.sub.2SO.sub.4, then evaporated to dryness. The residue was
flash chromatographed (eluent: petrolatum/ethyl acetate 1:1) to
give 6.58 g of the title product (yield: 57%).
[0099] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 8.92(s,1H);
8.49(s,2H); 7.89(d,1H, JHH=9.1Hz); 7.63 (d,1H); 5.21-5.14(m,1H);
4.86(s,2H); 4.03(s,3H); 1.98-1.57(m,8H).
EXAMPLE 9
Synthesis of
5-cyclopentyloxy-1-(3,5-dichloropyridin-4-ylmethyl)-6-methoxy-
-phthalazin-3-oxide (Compound 2)
[0100] A solution under stirring of
5-cyclopentyloxy-1-(3,5-dichloropyridi-
n-4-ylmethyl)-6-methoxy-phthalazine (0.1 g, 0.741 mmoles), obtained
as described in example 8, in methylene chloride (2 ml), was added
with 55% m-chloroperbenzoic acid (255.75 mg, 0.816 mmoles). The
mixture was diluted with methylene chloride, washed with a solution
of NaHCO.sub.3, anhydrified and dried. The residue was flash
chromatographed over silica gel (eluent: methylene chloride/ethyl
acetate 1:1) and the eluate taken up in ether to give 0.2 g of the
title product (yield: 64%).
[0101] .sup.1H-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 8.64(s,1H);
8.50(s,2H); 7.82(d,1H,JHH=9Hz); 7.40(d, 1H); 5.16-5.09(m,1H);
4.79(s,2H); 4.02(s,3H); 1.92-1.55(m,8H).
EXAMPLE 10
Synthesis of
5-cyclopentyloxy-1-(3,5-dichloropyridin-4-yl-methyl)-6-hydrox-
y-phthalazine
[0102] A solution under nitrogen of
5-cyclopentyloxy-1-(3,5-dichloropyridi-
n-4-ylmethyl)-6-methoxy-phthalazine (1.2 g, 2.97 mmoles), obtained
as described in example 8, and sodium p-thiochresolate (866 mg,
5.94 mmoles) in dimethylformamide (20 ml), was heated to
90-95.degree. C. for 2 hours. The reaction mixture was left to
stand for 1 night, poured into water/ice, filtered, and the
precipitate was suspended in 95.degree. ethanol (30 ml) and stirred
for 0.5 hour. The resulting solid was separated by filtration,
dissolved by refluxing in methanol/chloroform 1:1 (100 ml) and
concentrated to small volume to give 410 mg of the title product
which were used as such in the next step.
[0103] .sup.1-NMR (200 MHz, DMSO) .delta. (ppm): 10.89 (m, 1H);
9.42 (s, 1H); 8.66 (s, 2H); 8.09 and 7.69 (dd, 2H, JHH=8.9Hz);
5.23-5.17 (m, 1H); 4.89 (s, 2H); 1.87-1.56 (m, 8H).
EXAMPLE 11
Synthesis of
5-cyclopentyloxy-1-(3,5-dichloropyridin-4-yl-methyl)-6-difluo-
romethoxy-phthalazine (Compound 3)
[0104] In a solution of
5-cyclopentyloxy-1-(3,5-dichloropyridin-4-ylmethyl-
)-6-hydroxyphthalazine (400 mg, 1.025 mmoles), obtained as
described in example 10, potassium carbonate (188.4 mg, 1.366
mmoles) and potassium iodide (68.3 mg) in dimethylformamide (20
ml), a stream of chloro difluoro methane was gurgled by heating at
70-75.degree. C. under stirring. After 4.5 hours, the mixture was
poured into water, extracted in ethyl acetate, the extract was
washed with water, anhydrified over sodium sulfate and dried. The
residue was chromatographed on silica gel (eluent: petrolatum/ethyl
acetate 7:3) to give an oil which was crystallized from ethyl
ether/40-60.degree. petrolatum 1:3 to give 96 mg of the title
product (yield: 21%).
[0105] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.68 (s,1H);
8.52 (s,2H); 7.90 and 7.82(dd, 2H, JHH=9Hz); 6.66(t, H,JHF=73.5Hz);
5.17-5.10(m,1H); 4.90(s,2H); 2.02-1.62 (m,8H).
EXAMPLE 12
Synthesis of
5-cyclopentyloxy-1-(3,5-dichloropyridin-4-ylamino)-6-methoxy--
phthalazine (Compound 4)
[0106] A solution of 2,5-dichloro-4-amino-pyridine (5.1 g, 31.4
mmoles) in dry dimethylformamide (100 ml) under nitrogen was added
under stirring with 55% sodium hydride (1.37 g, 31.4 mmoles). The
mixture was stirred for 1 hour, then dropwise added with a solution
of 5-cyclopentyloxy-1-chloro-6-methoxy-2H-phthalazine (1.75 g, 6.28
mmoles) in dry dimethylformamide (20 ml) and the mixture was heated
to 100.degree. C. for 2 hours. The whole was poured into water and
extracted in ethyl acetate. The extract was washed with water and
anhydrified over sodium sulfate, then dried. The residue was taken
up in few of methylene chloride, the insoluble was filtered off and
the solution dried, the residue was purified by silica gel
chromatography (eluent: methylene chloride+1% of methanol) and the
crude was chromatographed a second time (eluent: petrolatum/ethyl
acetate 7:3) to give 720 mg of the title product (yield: 22%)
[0107] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.59 (m,
1H); 8.32 (s, 2H); 8.28 (s, 1H); 8.23 and 7.33 (2d, 2H, JHH=8.9Hz);
5.11-5.03 (m, 1H); 3.98 (s, 3H); 1.96-1.57 (m, 8H).
EXAMPLE 13
Synthesis of 3-benzyloxy-4-methoxy benzoic acid
[0108] A solution of potassium permanganate (24.81 g, 0.157 moles)
in water (0.1 l) was added under stirring with a solution of
tetrabutylammonium bromide (50.61 g, 0.157 moles) in water (0.2 l).
The formed solid was separated by filtration, washed with water,
squeezed, then dissolved in pyridine (0.3 l). The solution was
dropped into a solution of 3-benzyloxy-4-methoxy benzaldehyde (38.2
g, 0.157 moles) in pyridine (0.15 l) in water bath. After 3 hours
the reaction mixture was brought to acidic pH by 1N HCl, the solid
was filtered off and the mother liquor extracted more times in
methylene chloride. The organic phases were anhydrified and
concentrated to dryness and the residue taken up with 1N NaOH and
washed with ethyl ether. The aqueous solution was acidified and
extracted twice with methylene chloride, brought to dryness,
discoloured with TONSIL.RTM. and concentrated to small volume. The
resulting precipitate was filtered to give 35.869 g of the title
product (yield: 88%)
[0109] .sup.1H-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 7.78-6.69
(m, 8H); 5.18 (s, 2H); 3.39 (s, 3H).
EXAMPLE 14
Synthesis of 3-benzyloxy-4-methoxy benzoic acid chloride
[0110] Operating analogously to example 2 starting from
3-benzyloxy-4-methoxy benzoic acid (35.86 g, 0.139 moles) obtained
as described in example 1, in thionyl chloride (150 ml), 35.01 g of
the title product were obtained (yield: 98%).
[0111] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 7.84-6.90
(m, 8H); 5.16 (s, 2H); 3.95 (s, 3H).
EXAMPLE 15
Synthesis of 3-benzyloxy-N,N-diethyl-4-methoxy-benzamide
[0112] Operating analogously to example 3 starting from
3-benzyloxy-4-methoxy benzoic acid chloride (35.01 g, 0.127 moles)
obtained as described in example 14, in methylene chloride (200
ml), and diethylamine (131 ml, 92.54 g, 0.127 moles) in methylene
chloride (130 ml), 37.65 g of the title product were obtained
(yield: 95%)
[0113] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 7.43-6.84
(m, 8H); 5.14 (s, 2H); 3.88 (s, 3H); 3.31 (m, 4H); 1.07 (m,
6H).
EXAMPLE 16
Synthesis of
3-benzyloxy-N,N-diethyl-2-formyl-4-methoxy-benzamide
[0114] A solution of 3-benzyloxy-N,N-diethyl-4-methoxy-benzamide
(39.54 g, 126.2 mmoles) obtained as described in example 15, and
tetramethylethylendiamine (16.13 g, 138.3 mmoles) in
tetrahydrofuran (4.25 l), cooled to -78.degree. C., was dropwise
added with 1.21M s-butyl-lithium (115.64 ml, 138.8 mmoles). After 2
hours N,N-dimethylformamide was added (43 ml, 40.59 g, 555 mmoles)
and the mixture was left at the same temperature for 4 hours, then
1 night while the temperature arose. The mixture was washed with
0.47M phosphate buffer pH=7 and the organic phases were separated,
the aqueous one was extracted in ethyl ether. The organic phases
were anhydrified and concentrated to give a solid which was
filtered off. The mother liquor was dried and the crude flash
chromatographed (eluent: ethyl acetate/petrolatum 1:1) to give
13.76 g of the title product (yield: 32%).
[0115] 1H-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 10.22(s,1H);
7.36-7.29(m,5H); 7.13 and 6.94 (2s, 2H, JHH=8.3Hz); 5.17(s,2H);
3.94(s,3H); 3.52 and 2.93(2q,4H); 1.26 and 0.93 (2t, 6H).
EXAMPLE 17
Synthesis of 3,4-dihydroxy-5-methoxy-3H-isobenzofuran-1-one
[0116] 3-Benzyloxy-N,N-diethyl-2-formyl-4-methoxy-benzamide (11.6
g, 34 mmoles) obtained as described in example 16 was dissolved in
10% HCl and acetic acid (80+80 ml) and the mixture was refluxed for
18 hours. The solvents were evaporated and the crude was taken up
in toluene. The solid was washed with ethyl ether and dried to give
6.66 g of the title product (quantitative yield).
[0117] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.64 and
7.82 (2s broad,2H); 7.28-7.16 (m, 2H); 3.89 (s, 3H); 6.53 (s broad,
1H).
EXAMPLE 18
Synthesis of 5-hydroxy-6-methoxy-2H-phthalazin-1-one
[0118] 3,4-Dihydroxy-5-methoxy-3H-isobenzofuran-1-one (6.63 g, 34
mmoles) obtained as described in example 17 was dissolved in
ethanol (60 ml) and added with 98% hydrazine (8.5 ml). The mixture
was heated until limpidity and after 5 minutes the formed
precipitate was filtered, treated with 1N HCl. The mother liquor
was concentrated more times and the fractions of solid obtained
were joined to give 4.65 g of the title product (yield: 72%).
[0119] .sup.1-NMR (200 MHz, CDCI.sub.3) .delta. (ppm): 12.33 (m,
1H); 10.06 (s, 1H); 8.36 (s, 1H); 7.68 and 7.49 (2d, 2H,
JHH=8.6Hz); 3.94 (s, 3H).
EXAMPLE 19
Synthesis of methansulfonic acid 2,3-dihydro-1H-inden-2-yl
ester
[0120] 2-Indanol (5.14 g, 38.3 mmoles) in methylene chloride (20
ml) at 0.degree. C. was added with triethylamine (6.13 ml, 4.46 g,
44.1 mmoles) and a solution of mesyl chloride (3.26 ml, 4.83 g,
42.1 mmoles) in methylene chloride (4 ml). The temperature was
raised to room value, then the mixture was poured into water and
the organic phase separated, washed with water, anhydrified and
dried to give 8.12 g of the title product (yield: 99%).
[0121] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 7.27-7.16
(m, 4H); 5.55-5.46 (m, 1H); 3.42-3.18 (m, 4H); 3.00 (s, 3H).
EXAMPLE 20
Synthesis of 5-(indan-2-yloxy)-6-methoxy-2H-phthalazin-1-one
[0122] Methansulfonic acid 2,3-dihydro-1H-inden-2-yl ester (3.31 g,
15.61 mmoles) obtained as described in example 19, in
dimethylformamide (12 ml) was treated with Na.sub.2CO.sub.3 (2.21
g, 20.32 mmoles) and KI in catalytic amount at 90.degree. C. After
1 night at this temperature 5-hydroxy-6-methoxy-2H-phthalazin-1-one
(2 g, 10.41 mmoles) obtained as described in example 18 was added
and the mixture was left at 90.degree. C. for another night, then
poured into 1N HCl, and the precipitate was filtered, tritured in
petrolatum and dried to give 2.55 g of the title product (yield:
80%).
[0123] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 12.43(s,1H);
7.97 and 7.64 (2d, 2H, JHH=8.8Hz); 7.72 (s,1H); 7.30-7.16(m,4H);
5.42-5.34(m,1H); 3.99(s,3H); 3.22-2.99 (m,4H)
Example 21
Synthesis of
1-chloro-5-(indan-2-yloxy)-6-methoxy-2H-phthalazine
[0124] 5-(Indan-2-yloxy)-6-methoxy-2H-phthalazin-1-one (2.55 g, 8.3
mmoles) obtained as described in example 20, was suspended, under
nitrogen, in phosphoryl chloride (20 ml). The reaction mixture was
refluxed for 1 hour then poured into diluted NaOH (pH=8), extracted
in methylene chloride, anhydrified and dried. The crude was
tritured in petrolatum to give 2.57 g of the title product (yield:
95%).
[0125] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.20 (s,
1H); 8.02 and 7.65 (2d, 2H, JHH=8.8Hz); 7.27-7.17 (m, 4H);
5.58-5.50 (m, 1H); 4.07 (s, 3H); 3.18 (m, 4H).
EXAMPLE 22
Synthesis of
1-(3,5-dichloro-pyridin-4-methyl)-5-(indan-2-yloxy)-6-methoxy-
-2H-phthalazine (Compound 5)
[0126] A solution of 3,5-dichloro-4-methyl-pyridine (3.63 g, 22.4
mmoles) in dimethylformamide (50 ml) was added under stirring with
55% sodium hydride (0.977 g, 67.2 mmoles), then with a solution of
1-chloro-5-(indan-2-yloxy)-6-methoxy-2H-phthalazine (2.44 g, 7.5
mmoles) obtained as described in example 21, in dimethylformamide.
After stirring for 1.5 hours the mixture was poured into a buffer
at pH=7 and extracted in methylene chloride. The extract was washed
with water, anhydrified and evaporated to dryness. The residue was
flash chromatographed (eluent petrolatum/ethyl acetate 6:4) to give
1.7 g of the title product (yield:54%).
[0127] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.31(s,1H);
8.49(s,2H); 7.94 and 7.68(2d,2H, JHH=9.1Hz); 7.27-7.16(m, 4H);
5.57-5.47(m,1H); 4.87(s,2H); 4.07(s,3H); 3.23-3.20 (m, 4H).
EXAMPLE 23
Synthesis of methansulfonic acid 5-phenyl-pentyl ester
[0128] Operating analogously to example 19 using 5-phenylpentanol
(7.18 ml, 7 g, 42.6 mmoles) in methylene chloride (8 ml) and
methyl-sulfonyl chloride (3.63 ml, 5.37 g, 46.9 mmoles) in
methylene chloride (4 ml), and also using triethylamine (6.82 ml,
4.96 g, 49 mmoles) to adjust the pH after the addition of
methyl-sulfonyl chloride, 10.57 g of the title product were
obtained after half an hour of reaction (stoichiometric yield).
[0129] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 7.31-7.13
(m, 5H); 4.20 (t, 2H, JHH=6.5Hz); 2.96 (s, 3H); 2.65-2.57 (m, 2H);
1.83-1.34 (m, 6H).
EXAMPLE 24
Synthesis of
5-(5-phenyl-pentyl-1-oxy)-6-methoxy-2H-phthalazin-1-one
[0130] Operating analogously to example 20 using
5-hydroxy-6-methoxy-2H-ph- thalazin-1-one (2 g, 10.41 mmoles)
obtained as described in example 18, sodium carbonate (2.21 g,
20.82 mmoles), potassium iodide in catalytic amount, methansulfonic
acid 5-phenyl-pentyl ester (3.78 g, 15.61 mmoles) obtained as
described in example 23, and dimethylformamide (50 ml) under
nitrogen, 3.11 g of the title product were obtained (yield:
88%).
[0131] 1H-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 10.19(s
broad,1H); 8.41(s,1H); 8.13 and 7.35(2d,2H, JHH=8.8Hz);
7.30-7.11(m,5H); 4.11(t,2H,JHH=6.6Hz); 3.96(s,3H); 2.68-2.60(m,2H);
1.91-1.45 (m, 6H).
EXAMPLE 25
Synthesis of
1-chloro-5-(5-phenyl-pentyl-1-oxy)-6-methoxy-phthalazine
[0132] Operating analogously to example 21, using
5-(5-phenyl-pentyl-1-oxy- )-6-methoxy-2H-phthalazin-1-one (3.05 g,
9.01 mmoles), obtained as described in example 24, and phosphoryl
chloride (5 ml) the title product was obtained as a crude which was
used as such in the next step.
[0133] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.66(s,1H);
8.01 and 7.63 (2d, 2H, JHH=9Hz); 7.30-7.12 (m, 5H); 4.20
(t,2H,JHH=6.6Hz); 4.02 (s,3H); 2.68-2.60 (m,2H); 1.94-1.47 (m,
6H).
EXAMPLE 26
Synthesis of
1-(3,5-dichloro-pyridin-4-methyl)-5-(5-phenyl-pentyl-1-oxy)-6-
-methoxy-phthalazine (Compound 6)
[0134] Operating analogously to example 22 and using
1-chloro-5-(5-phenyl-pentyl-1-oxy)-6-methoxy-phthalazine (3.2 g,
9.01 mmoles) obtained as described in example 25,
3,5-dichloro-4-methyl-pyridi- ne (4.38 g, 27.03 mmoles), 55% sodium
hydride (1.18 g) and dimethylformamide (50 ml), 0.6 g of the title
product was obtained.
[0135] .sup.1H-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.68 (s,
1H); 8.51 (s, 2H); 8.93 and 7.65 (2d, 2H, JHH=9.2Hz); 7.30-7.12 (m,
5H); 4.88 (s, 2H); 4.21 (t, 2H, JHH=6.6Hz); 4.03 (s, 3H); 2.68-2.61
(m, 2H); 1.96-1.47 (m, 6H).
EXAMPLE 27
Synthesis of 7-cyclopentyloxy-6-methoxy-2H-phthalazin-1-one
[0136] Operating analogously to example 6 starting from
7-cyclopentyloxy-N,N-diethyl-2-formyl-4-methoxy-benzamide (obtained
similarly to the compound of example 4) the title product was
obtained (yield: 80%).
[0137] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 10.90 (s,
1H); 8.03 (s, 1H); 7.72 (s, 1H); 7.00 (s, 1H); 4.96 (m, 1H); 3.98
(s, 3H); 2-18-1.60 (m, 8H).
EXAMPLE 28
Synthesis of 7-cyclopentyloxy-1-chloro-6-methoxy-2H-phthalazine
[0138] Operating analogously to example 7 starting from
7-cyclopentyloxy-6-methoxy-2H-phthalazin-1-one, obtained as
described in example 27, the title product was obtained (yield:
98%).
[0139] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.21 (s,
1H); 7.42 (s, 1H); 7.13 (s, 1H); 5.00 (m, 1H); 4.02 (s, 3H);
2.20-1.60 (m, 8H).
EXAMPLE 29
Synthesis of
7-cyclopentyloxy-1-(3,5-dichloropyridin-4-ylmethyl)-6-methoxy-
-phthalazine (Compound 7)
[0140] Operating analogously to example 8 using
7-cyclopentyloxy-1-chloro-- 6-methoxy-2H-phthalazine, obtained as
described in example 28, the title product was obtained (yield:
59%).
[0141] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.20 (s,
1H); 8.49 (s, 2H); 7.28 (s, 1H); 7.14 (s, 1H); 4.97 (m, 1H); 4.83
(s, 2H); 4.02 (s, 3H); 2.18-1.60 (m, 8H).
EXAMPLE 30
Synthesis of
7-cyclopentyloxy-1-(3,5-dichloropyridin-4-ylamino)-6-methoxy--
phthalazine (Compound 8)
[0142] Operating analogously to example 12 using
7-cyclopentyloxy-1-chloro- -6-methoxy-2H-phthalazine, obtained as
described in example 28, the title product was obtained (yield:
28%).
[0143] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.40 (s
broad, 1H); 8.36 (s, 2H); 7.90 (s, 1H); 7.83 (s, 1H); 6.95 (s, 1H);
3.00 (m, 1H); 3.98 (s, 3H); 2.20-1.50 (m, 8H).
EXAMPLE 31
Synthesis of
N,N-diethyl-5-cyclopentyloxy-2-(1-hydroxyethyl)-4-methoxybenz-
amide
[0144] A solution of
5-cyclopentyloxy-N,N-diethyl-2-formyl-4-methoxy-benza- mide (3.2 g,
10 mmoles), obtained as described in example 4, product B, in dry
tetrahydrofuran (50 ml) at 0.degree. C., was dropwise added with a
3M solution of methyl-magnesium chloride (4 ml, 12 moles) in
tetrahydrofuran. The mixture was stirred at room temperature for 1
hour, then carefully poured into a mixture of ethyl acetate (0.1 l)
and water (0.2 l) and cooled to 3.degree. C. The organic phase was
washed with water, anhydrified over Na.sub.2SO.sub.4 and dried at
reduced pressure to give a residue which was chromatographed on
silica gel (eluent: ethyl acetate/hexane 1:1) to give 2.7 g of the
title product (yield: 80%).
[0145] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 7.40(s,1H);
6.82(s,1H); 5.41(q,1H); 4.86(m,1H); 4.01 (q, 2H); 3.93(s,3H);
3.55(q,2H); 2.18-1.70(m,8H); 1.58(d,3H); 1.40-1.10(m, 6H).
EXAMPLE 32
Synthesis of
N,N-diethyl-5-cyclopentyloxy-2-(1-hydroxypropyl)-4-methoxy-be-
nzamide
[0146] Operating analogously to example 31 using
5-cyclopentyloxy-N,N-diet- hyl-2-formyl-4-methoxy-benzamide,
obtained as described in example 4, product B, and ethylmagnesium
bromide the title product was obtained (yield 76%).
[0147] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 7.53(s,1H);
6.81(s,1H); 5.43(t,1H); 4.85(m,1H); 4.08 (q,2H); 3.95(s,3H);
3.75(q,2H); 2.33(m,2H); 2.20-1.60(m,8H); 1.51-1.10(m,9H).
EXAMPLE 33
Synthesis of
N,N-diethyl-5-cyclopentyloxy-4-methoxy-2-(1-oxoethyl)-benzami-
de
[0148] A solution of
N,N-diethyl-5-cyclopentyloxy-2-(1-hydroxyethyl)4-meth- oxybenzamide
(2 g, 6 mmoles) obtained as described in example 31, in
CH.sub.2Cl.sub.2 (25 ml) at 10.degree. C. was added with
pyridinium-chloro-chromate (1.9 g, 9 mmoles), and the mixture was
stirred at room temperature for 1 night, then added with ethyl
ether (50 ml) and the residue was washed with ethyl ether (30 ml).
The joined solutions were evaporated at reduced pressure and the
residue chromatographed on silica gel (eluent: hexane/ethyl acetate
6:4) to give 1.5 g of the title product (yield: 77%).
[0149] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 7.6 (s, 1H);
6.87 (s, 1H); 4.9 (m, 1H); 4.1 (q, 2H); 4 (s, 3H); 3.76 (q, 2H);
2.5 (s, 3H); 2.2-1.6 (m, 8H); 1.4-1.1 (m, 6H).
EXAMPLE 34
Synthesis of
N,N-diethyl-5-cyclopentyloxy-4-methoxy-2-(1-oxopropyl)-benzam-
ide
[0150] Operating analogously to example 33 starting from
N,N-diethyl-5-cyclopentyloxy-2-(1-hydroxypropyl)-4-methoxy-benzamide,
obtained as described in example 32, the title product was obtained
(yield: 82%).
[0151] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 7.58(s,1H);
6.85(s,1H); 4.85(m,1H); 4.15(q,2H); 3.96 (s,3H); 3.74(q,2H);
3.42(q,2H); 2.20-1.60(m,8H); 1.53(t,3H); 1.40-1.10(m,6H).
EXAMPLE 35
Synthesis of
7-cyclopentyloxy-4-methyl-6-methoxy-2H-phthalazin-1-one
[0152] Operating analogously to example 6 using
N,N-diethyl-5-cyclopentylo- xy-4-methoxy-2-(1-oxoethyl)-benzamide
obtained as described in example 33, the title product was
obtained.
EXAMPLE 36
Synthesis of
7-cyclopentyloxy-4-ethyl-6-methoxy-2H-phthalazin-1-one
[0153] Operating analogously to example 6 using from
N,N-diethyl-5-cyclopentyloxy-4-methoxy-2-(1-oxopropyl)-benzamide,
obtained as described in example 34, the title product was
obtained.
EXAMPLE 37
Synthesis of
1-chloro-7-cyclopentyloxy-4-methyl-6-methoxy-phthalazine
[0154] Operating analogously to example 7 starting from
7-cyclopentyloxy-4-methyl-6-methoxy-2H-phthalazin-1-one, obtained
as described in example 35, the title product was obtained.
EXAMPLE 38
Synthesis of
1-chloro-7-cyclopentyloxy-4-ethyl-6-methoxy-phthalazine
[0155] Operating analogously to example 7 starting from
7-cyclopentyloxy-4-ethyl-6-methoxy-2H-phthalazin-1-one, obtained as
described in example 36, the title product was obtained.
EXAMPLE 39
Synthesis of
7-cyclopentyloxy-1-(3,5-dichloro-pyridin-4-ylmethyl)-4-methyl-
-6-methoxy-phthalazine (Compound 9)
[0156] Operating analogously to example 8 starting from
1-chloro-7-cyclopentyloxy-4-methyl-6-methoxy-phthalazine, obtained
as described in example 37, the title product was obtained.
[0157] .sup.1-NMR (300 MHz, CDCl.sub.3) .delta. (ppm): 8.50(s,2H);
7.30(s,1H); 7.23(s,1H); 5.04-5.00(m,1H); 4.82(s,2H); 4.05(s,3H);
2.88(s,3H); 2.10-1.81(m,6H); 1.80-1.60(m,2H).
EXAMPLE 40
Synthesis of
7-cyclopentyloxy-1-(3,5-dichloro-pyridin-4-ylmethyl)-4-ethyl--
6-methoxy-phthalazine (Compound 10)
[0158] Operating analogously to example 8 using
1-chloro-7-cyclopentyloxy-- 4-ethyl-6-methoxy-phthalazine, obtained
as described in example 38, the title product was obtained.
[0159] .sup.1-NMR (300 MHz, CDCl.sub.3) .delta. (ppm): 8.50(s,2H);
7.32(s,1H); 7.29(s,1H); 5.01-5.05 (m, 1H); 4.81(s,2H); 4.05(s,3H);
3.26(q,2H,J=7.5Hz); 2.13-1.67(m,8H); 1.48(t,3H,J=7.5Hz).
EXAMPLE 41
Synthesis of methansulfonic acid tetrahydro-pyran-2-ylmethyl
ester
[0160] A solution of (tetrahydropyran-2-yl)methanol (6 g, 51.65
moles) in methylene chloride (80 ml) at 0.degree. C. was added with
triethylamine (8.27 ml, 59.4 moles) then with mesyl chloride (4.4
ml, 56.8 moles) in methylene chloride (5 ml). The temperature was
left to rise to room value and after 30 minutes the mixture was
poured into water and extracted in methylene chloride. The organic
phases were anhydrified and dried to give 10 g of the title product
(quantitative yield).
EXAMPLE 42
Synthesis of
6-methoxy-5-(tetrahydro-pyran-2-yl-methoxy)-2H-phthalazin-1-o-
ne
[0161] A solution of 5-hydroxy-6-methoxy-2H-phthalazin-1-one (1.5
g, 7.81 mmoles), obtained as described in example 18, in
dimethylformamide (40 ml) was added with Na.sub.2CO.sub.3 (1.65 g,
15.61 mmoles) and KI in catalytic amount and the mixture was heated
to 90.degree. C. in nitrogen atmosphere. A solution of
methansulfonic acid tetrahydropyran-2-ylmethyl ester (6.1 g, 31.46
mmoles), obtained as described in example 41, in dimethylformamide
(14 ml) was dropped therein. After 24 hours the mixture was poured
into water and thrice extracted in ethyl acetate. The organic
phases were washed with water till neutrality then anhydrified and
concentrated under vacuum to give a solid which tritured in ethyl
ether yielded 1.66 g of the title product (yield: 66%)
EXAMPLE 43
Synthesis of
1-chloro-6-methoxy-5-(tetrahydro-pyran-2-yl-methoxy)-phthalaz-
ine
[0162] Operating analogously to example 7 starting from
6-methoxy-5-(tetrahydropyran-2-yl-methoxy)-2H-phthalazin-1-one
(1.48 g, 5.12 mmoles), obtained as described in example 42, and
phosphoryl chloride (6 ml) 1.65 g of the title product as a crude
were obtained and used as such in the next step.
EXAMPLE 44
Synthesis of
1-(3,5-dichloropyridin-4-yl-methyl)-6-methoxy-5-(tetrahydro-p-
yran-2-yl-methoxy)-phthalazine (Compound 11)
[0163] Operating analogously to example 8 using
1-chloro-6-methoxy-5-(tetr- ahydropyran-2-yl-methoxy)-phthalazine,
obtained as described in example 43, in dimethylformamide, 55% NaH
and 3,5-dichloro-4-methyl-pyridine in dimethylformamide, the title
product was obtained (yield: 35%).
[0164] .sup.1-NMR (300 MHz, CDCl.sub.3) .delta. (ppm): 9.77(s,1H);
8.50(s,2H); 7.91 (d, 1H, JHH=9.0Hz); 7.62 (d, 1 H); 4.86(s,2H);
4.18-4.05(m,2H); 4.06-3.42(m,3H); 4.04(s,3H); 1.94-1.38(m,6H).
EXAMPLE 45
Synthesis of
1-(3,5-dichloropyridin-4-yl-methyl)-6-methoxy-5-(tetrahydro-p-
yran-2-yl-methoxy)-phthalazin-3-oxide (Compound 12)
[0165] Operating analogously to example 9 starting from
1-(3,5-dichloropyridin-4-ylmethyl)-6-methoxy-5-(tetrahydropyran-2-yl-meth-
oxy)-phthalazine (0.2 g, 0.48 mmoles) obtained as described in
example 44, in methylene chloride (2 ml) and m-chloroperbenzoic
acid (0.143 g, 0.58 mmoles) 0.104 g of the title product was
obtained (yield: 48%).
[0166] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 8.82(s,1H);
8.50(s,2H); 7.84(d,1H,JHH=9.0Hz); 7.39 (d,1H); 4.78(s,2H);
4.13-3.98(m,3H); 4.02(s,3H); 3.74-3.41(m,2H); 1.91-1.35(m,6H).
EXAMPLE 46
Synthesis of methansulfonic acid tetrahydro-furan-3-yl ester
[0167] A solution of tetrahydrofuran-3-ol (4.6 g, 52.2 moles) in
methylene chloride (70 ml) at 0.degree. C. was added with
triethylamine (8.36 ml, 60 moles) and mesyl chloride (4.44. ml,
57.4 moles) in methylene chloride (85 ml). The temperature was left
to rise to room value and after 1 hour the mixture was poured into
water and extracted in methylene chloride. The organic phases were
anhydrified and dried to give 8.12 g of the title product (yield:
93%).
EXAMPLE 47
Synthesis of
6-methoxy-5-(tetrahydro-furan-3-yloxy)-2H-phthalazin-1-one
[0168] Operating analogously to example 42 using
5-hydroxy-6-methoxy-2H-ph- thalazin-1-one (1.55 g, 8.07 mmoles)
obtained as described in example 18, in dimethylformamide (35 ml),
sodium carbonate (1.71 g, 16.13 mmoles), potassium iodide in
catalytic amount and methansulfonic acid tetrahydrofuran-3-yl ester
(2.01 g, 12.1 mmoles) obtained as described in example 46, in
dimethylformamide (10 ml), 0.73 g of the title product was obtained
(yield: 86%).
EXAMPLE 48
Synthesis of
1-chloro-6-methoxy-5-(tetrahydro-furan-3-yloxy)-phthalazine
[0169] Operating analogously to example 7 starting from
6-methoxy-5-(tetrahydro-furan-3-yloxy)-2H-phthalazin-1-one (1.73 g,
6.6 mmoles) and phosphoryl chloride (6.7 ml, 73 mmoles), 1.63 g of
the title product were obtained (yield: 88%).
EXAMPLE 49
Synthesis of
1-(3,5-dichloropyridin-4-ylmethyl)-6-methoxy-5-(tetrahydrofur-
an-3-yl-oxy)-phthalazine (Compound 13)
[0170] Operating analogously to example 8 starting from
3,5-dichloro-4-methyl-pyridine (2.47 g, 16.89 mmoles), sodium
hydride (0.74 g, 16.89 mmoles),
1-chloro-6-methoxy-5-(tetrahydro-furan-3-yloxy)-p- hthalazine (1.58
g, 5.63 mmoles) obtained as described in example 48, in
dimethylformamide (35 ml) 0.74 g of the title product (yield: 63%)
were obtained.
[0171] .sup.1-NMR (300 MHz, CDCl.sub.3) .delta. (ppm): 9.65(s,1H);
8.49(s,2H); 7.93 and 7.65 (2d,2H,JHH=9.0Hz); 5.36-5.28(m,1H);
4.86(s,2H); 4.22-3.80(m,4H); 4.05(s,3H); 2.32-2.00(m,4H).
EXAMPLE 50
Synthesis of
1-(3,5-dichloropyridin-4-ylmethyl)-6-methoxy-5-(tetrahydrofur-
an-3-yl-oxy)-phthalazin-3-oxide and
1-(3,5-dichloro-1-oxy-pyridin-4-ylmeth-
yl)-6-methoxy-5-(tetrahydro-furan-2-yloxy)-phthalazine 3-oxide
(Compound 14 and 19)
[0172] Operating analogously to example 9 starting from
1-(3,5-dichloropyridin-4-ylmethyl)-6-methoxy-5-(tetrahydrofuran-3-yloxy)--
phthalazine (0.74 g, 1.82 mmoles) obtained as described in example
49, in methylene chloride (4 ml) and m-chloroperbenzoic acid (0.72
g, 2.93 mmoles), 0.331 g of the first title product was obtained.
From the chromatographic column 0.207 g of the second title product
was eluted (eluent: methylene chloride/methanol 9:1)
[0173] First product: .sup.1-NMR (200 MHz, CDCl.sub.3) .delta.
(ppm): 8.67(s,1H); 8.50(s,2H); 7.86 and 7.41 (2d,2H,JHH=8.9Hz);
5.30-5.24(m,1H); 4.80(s,2H); 4.15-3.76(m,4H); 4.04 (s,3H);
2.26-2.00(m,4H).
[0174] Second product: .sup.1-NMR (200 MHz, CDCl.sub.3) .delta.
(ppm): 8.68(s,1H); 8.19(s,2H); 7.85 (d,1H, JHH=9.1Hz); 7.42(d,1H);
5.32-5.25(m,1H); 4.73(s,2H); 4.05(s,3H); 4.16-3.76(m,4H);
2.20-2.00(m,2H).
EXAMPLE 51
Synthesis of
6-hydroxy-5-(tetrahydrofuran-2-yloxy)-2H-phthalazin-1-one
[0175] A solution of
6-methoxy-5-(tetrahydrofuran-2-yloxy)-2H-phthalazin-1- -one (680
mg, 2.6 mmoles), obtained as described in example 47, in DMF (15
ml) and sodium p-thiochresolate (378.6 mg, 2.6 mmoles) was heated
to 90.degree. C. for 10 hours, poured into water, acidified at pH
6-7 with 1M HCl, dried, taken up in water and dried again. The
crude was chromatographed (eluent: methylene chloride/methanol 9:1)
to give 330 mg of the title product (yield: 52%).
[0176] .sup.1-NMR (200 MHz, DMSO) .delta. (ppm): 12.41(s,1H);
11.07(s-broad,1H); 8.23 (s, 1H); 7.82 (d, 1H, JHH=8.8Hz);
7.44(d,1H); 5.25-5.20(m,1H); 4.03-3.59(m,4H); 2.11-2.00(m, 2H)
EXAMPLE 52
Synthesis of
6-difluoromethoxy-5-(tetrahydro-furan-2-yloxy)-2H-phthalazin--
1-one
[0177] 6-Hydroxy-5-(tetrahydrofuran-2-yloxy)-2H-phthalazin-1-one
(320 mg, 1.29 mmoles), obtained as described in example 51, in dry
DMF (80 ml) and K.sub.2CO.sub.3 (267 mg, 1.93 mmoles) were treated
with Freon-22 (5 g, 58 mmoles) at 0.5 bar. The mixture was brought
to 115.degree. C. for 1 night, then dried and chromatographed
(eluent: benzene/ethyl acetate 1:1, then ethyl acetate only) to
give 300 mg of the title product (yield: 78%).
[0178] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 10.62(s,1H);
8.44(s,1H); 8.16(d,1H, JHH=9.2Hz); 7.55 (d,1H); 6.63(t,1H,JHF=72
Hz); 5.24-5.18(m,1H); 4.18-3.75(m,4H); 2.29-2.09(m,2H).
EXAMPLE 53
Synthesis of
1-chloro-6-difluoromethoxy-5-(tetrahydro-furan-2-yloxy)-phtha-
lazine
[0179] A solution of
6-difluoromethoxy-5-(tetrahydrofuran-2-yloxy)-2H-phth- alazin-1-one
(280 mg, 0.94 mmoles), obtained as described in example 52, and
POCl.sub.3 (1.2 ml) was heated to 90.degree. C. for 1.5 hours, then
dried, taken up in methylene chloride, washed with a saturated
solution of NaHCO.sub.3, then twice with water, anhydrified and
dried to give 296 g of the title product (yield: 99.7%).
EXAMPLE 54
Synthesis of
1-(3,5-dichloro-pyridin-4-ylmethyl)-6-difluoromethoxy-5-(tetr-
ahydrofuran-3-yloxy)-phthalazine (Compound 15)
[0180] A solution under nitrogen of 3,5-dichloro-4-methyl-pyridine
(457 mg, 2.82 mmoles) in dry DMF (10 ml) was portionwise added with
60% NaH (113 mg, 2.82 mmoles) and, after 1 hour at room
temperature, was dropwise added, at a temperature <10.degree.
C., with 1-chloro-6-difluoromethox-
y-5-(tetrahydrofuran-2-yloxy)-phthalazine (296 mg, 0.937 mmoles),
obtained as described in example 53, in dry DMF (10 ml). After 2
hours at room temperature the mixture was poured into a 0.4M buffer
at pH=7 (50 ml), extracted in ethyl acetate (2.times.50 ml) and the
organic phase was washed with water, anhydrified and concentrated
under vacuum. The residue was chromatographed (eluent:
benzene/ethyl acetate 1:1, then ethyl acetate only), then
crystallized from ethyl ether/benzene to give 180 mg of the title
product (yield: 43.5%).
[0181] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.72(s,1H);
8.52(s,2H); 7.96(d,1H,JHH=9.2Hz); 7.80 (d,1H); 6.68(t,1H,JHF=74Hz);
5.34-5.29(m,1H); 4.91(s,2H); 4.22-3.81(m,4H); 2.33-2.08(m, 2H).
EXAMPLE 55
Synthesis of
1-(3,5-dichloro-pyridin-4-ylmethyl)-6-difluoromethoxy-5-(tetr-
ahydrofuran-3-yloxy)-phthalazine 3-oxide and
1-(3,5-dichloro-1-oxy-pyridin-
-4-ylmethyl)-6-difluoromethoxy-5-(tetrahydro-furan-3-yloxy)-phthalazine
3-oxide (Compound 16 and 17)
[0182] A solution of
1-(3,5-dichloro-pyridin-4-ylmethyl)-6-difluoromethoxy-
-5-(tetrahydrofuran-3-yloxy)-phthalazine (1.5 g, 3.39 mmoles) in
methylene chloride (20 ml) and 55% m-chloroperbenzoic acid (1.56 g,
4.99 mmoles) was kept under nitrogen for 2 hours at room
temperature.
[0183] The mixture was evaporated to dryness, taken up in water,
neutralized with NaHCO.sub.3, extracted in ethyl acetate,
anhydrified and dried. The crude oil was chromatographed (eluent:
ethyl acetate, then ethyl acetate/methanol 7:3), then tritured in
petrolatum/ethyl ether 8:2, filtered, washed with petrolatum and
dried under vacuum to give 0.5 g of the first title product (yield:
32.25%) and 0.6 g of the second title product (yield: 37.33%).
1 First product: .sup.1H-NMR(200MHz, CDCI.sub.3).delta.(ppm):
8.67(m, 1H); 8.47s, 2H); 7.86(d, 1H, JHH=9.2Hz); 7.53(d, 1H);
6.69(t, 1H, JHF-72Hz); 5.29-5.23(m, 1H); 4.80.sctn., 2H);
4.13-3.75(m, 4H); 2.23-2.11(m, 2H). Second product:
.sup.1H-NMR(200MHz, CDCI.sub.3).delta.(ppm): 8.68(m,1H);
8.19.sctn., 2H); 7.84(d, 1H, JHH=9.2Hz); 7.54(d, 1H); 6.68(t, 1H,
JHF=72Hz); 5.30-5.25(m, 1H); 4.75.sctn., 2H); 4.15-3.76(m, 4H);
2.24-2.16(m, 2H).
EXAMPLE 56
Synthesis of
[1,4-bis(diphenylphosphino(butane]-dichloropalladium(II)
[0184] A solution of PdCl.sub.2(CH.sub.3CN).sub.2 (405 mg, 1.561
mmoles) in benzene under nitrogen and magnetic stirring was added
with 1,4-(diphenylphosphanyl)butane (665 mg, 1.561 mmoles). After
1.5 hours a precipitate formed and was added with hexane (10 ml),
filtered, washed with hexane and dried under vacuum to give 620 mg
of the title product (66%).
EXAMPLE 57
Synthesis of
6-methoxy-1-phenyl-5-(tetrahydro-furan-2-yloxy)-phthalazine
(Compound 18)
[0185] A suspension of magnesium (0.078 g, 3.21 mmoles) in
tetrahydrofuran (1 ml) was dropwise added, under nitrogen and
stirring, with a solution of 1,2-dibromoethane (0.3 g, 1.6 mmoles),
3-bromo-pyridine (0.25 g, 1.6 mmoles) in tetrahydrofuran (1 ml). 30
Minutes after the end of the dropping a solution of ZnCl.sub.2
(0.22 g, 1.6 mmoles) in tetrahydrofuran (3 ml) was added. After 20
minutes [1,4-bis(diphenylphosphino(butane]-dic- hloropalladium(II)
(0.016 g, 0.026 mmole), obtained as described in example 57, and
1-chloro-6-methoxy-5-(tetrahydro-furan-3-yloxy)-phthalazi- ne (0.15
g, 0.53 mmole) obtained as described in example 48, were added and
the mixture was refluxed for 1.5 hours, then cooled, washed with
water and extracted in ethyl acetate. The organic phase was
anhydrified and dried. The aqueous phase was evaporated and taken
up in ethyl acetate. The joined organic phases were dried and the
residue flash chromatographed (eluent: methylene chloride/methanol
9:1) to give 0.071 g of the title product.
[0186] .sup.1-NMR (200 MHz, CDCl.sub.3) .delta. (ppm): 9.79(s,1H);
8.96-7.35(m,4H); 7.77-7.54(m,2H); 5.37-5.32 (m,1H);
4.24-3.80(m,6H); 4.02(s,3H); 2.34-2.01(m,2H).
EXAMPLE 58
Synthesis of 5,6-dimethoxy-3H-isobenzofuran-1-one
[0187] Under mechanical stirring a slurry of 3,4-dimethoxybenzoic
acid (353.5 g, 1.94 moles) in HCHO (1.7 l, 24.5 moles) was prepared
and cooled in ice, saturated with gaseous HCl (340 g, 9.32 moles),
then gradually brought to 60.degree. C. After 1 night the
temperature was brought to room value and further HCl (300 g) was
gurgled therein, then the temperature was brought to 60.degree. C.
again overnight. The mixture was brought to small volume, taken up
in water (1 l), neutralized with 28% NH.sub.4OH (1.5 l) and kept at
cold for 2 hours, then filtered. The filtrate was washed with water
till neutrality, then crystallized from methanol (2 l) and dried
under vacuum at 60.degree. C. to give 220 g of the title product
(yield: 58.65%).
[0188] .sup.1-NMR (200 MHz, CDCl3) .delta. (ppm): 7.28 and
6.28(2s,2H); 5.20(s,2H); 3.95 and 3.91(2s, 6H).
EXAMPLE 59
Synthesis of 2-formyl-4,5-dimethoxy-benzoic acid
[0189] A mixture under nitrogen of
5,6-dimethoxy-3H-isobenzofuran-1-one (10 g, 51.5 mmoles), obtained
as described in example 58, in carbon tetrachloride (250 ml),
N-bromo-succinimide (13.88 g, 77.25 mmoles) and benzoyl peroxide
(320 mg, 1.23 mmoles) was refluxed for 2 hours, then cooled,
filtered and washed with a solution of 10% Na.sub.2SO.sub.3 (200
ml), then with water, anhydrified and dried. The residue was taken
up in 5% HCl (100 ml) and refluxed for 4 hours, then the solution
was cooled, alkalinized with NaOH, washed with ethyl acetate and
slowly acidified to give a precipitate which was filtered, washed
with water and dried over P.sub.2O.sub.5 under vacuum to give 6.43
g of the title product (yield: 60%).
EXAMPLE 60
Synthesis of
5,6-dimethoxy-3-(triphenyl-.lambda..sup.6-phosphanyl)-3H-isob-
enzofuran-1-one
[0190] A slurry under nitrogen of 2-formyl-4,5-dimethoxy-benzoic
acid (6.43 g, 30.62 mmoles), obtained as described in example 59,
triphenyl-phosphine (8.3 g, 30.62 mmoles), HBr in 30% acetic acid
(8.26 ml, 30.62 mmoles) and glacial acetic acid (20 ml) was heated
to 90.degree. C. for 4.5 hours. The mixture was dried, dissolved in
acetonitrile (50 ml) and diluted with ethyl ether till slurry, then
cooled and filtered, and the filtrate was washed with ethyl ether
and dried under vacuum to give 13.6 g of the title product (yield:
83%).
[0191] .sup.1H-NMR (200 MHz, DMSO) .delta. (ppm): 8.35 and
7.31(2s,2H); 8.03-7.66(m,15H); 6.01 (s,1H); 3.84 and
3.45(2s,6H).
EXAMPLE 61
Synthesis of
5,6-dimethoxy-3-pyridin-4-ylmethylen-3H-isobenzofuran-1-one
[0192] In a slurry of
5,6-dimethoxy-3-(triphenyl-.lambda..sup.6-phosphanyl-
)-3H-isobenzofuran-1-one (78 g, 145 mmoles), obtained as described
in example 60, and 4-pyridincarboxaldehyde (13 ml, 145 mmoles) in
methylene chloride (1 l), triethylamine (20 ml, 145 mmoles) was
dropped at room temperature under stirring. After 1.5 hours the
mixture was filtered and dried and the residue treated with ethanol
at reflux, cooled and filtered. The mother liquor was
chromatographed (eluent: methylene chloride then with 1% of
methanol) and the residue dried and joined with the filtrate above
to give 25 g of the title product.
EXAMPLE 62
Synthesis of
6,7-dimethoxy-4-pyridin-4-ilmethyl-2H-phthalazin-1-one
[0193] 5,6-Dimethoxy-3-pyridin-4-ylmethylen-3H-isobenzofuran-1-one
(25 g, 88.34 mmoles), obtained as described in example 61, was
reacted with hydrate hydrazine (500 ml) for 2 hours at room
temperature under stirring, then for 1 hour at reflux. The mixture
was diluted with water (300 ml), cooled and filtered to give 23 g
of the title product (yield: 87%).
EXAMPLE 63
Synthesis of
1-chloro-6,7-dimethoxy-4-pyridin-4-ylmethyl-phthalazine
[0194] A slurry of
6,7-dimethoxy-4-pyridin-4-ylmethyl-2H-phthalazin-1-one (10 g, 33.6
mmoles), obtained as described in example 62, in POCl.sub.3 (70 ml)
was heated to 90.degree. C. for 4 hours. POCl.sub.3 was evaporated
and the residue dissolved in water, a saturated solution of
NaHCO.sub.3 and NaOH till the formation of a precipitate which was
filtered and suspended in methanol, dried, suspended in acetone and
filtered again. The residue was dried at 45.degree. C. under vacuum
to give 9.56 g of the title product.
EXAMPLE 64
Synthesis of
1-benzyl-6,7-dimethoxy-4-pyridin-4-ylmethyl-phthalazine (Compound
20)
[0195] In anhydrous environment, ZnCl.sub.2 (863 mg, 6.33 mmoles)
was dissolved in tetrahydrofuran (20 ml), and a 2M solution of
benzyl magnesium chloride in tetrahydrofuran (3.2 ml, 2.5
equivalents) was dropped therein. After 30 minutes
1-chloro-6,7-dimethoxy-4-pyridin-4-ylme- thyl-phthalazine (800 mg,
2.53 mmoles), obtained as described in example 63, palladium
acetate (71 mg, 0.316 mmole) and triphenylphosphine (166 mg, 0.633
mmole) were added and the whole was refluxed. After 1 hour the
mixture was poured into a saturated solution of NH.sub.4Cl and by
adding ethyl acetate a solid precipitated. The whole was evaporated
to dryness and the residue taken up in methylene chloride and
extracted with concentrated ammonia. The organic phase was dried to
yield a residue which was chromatographed (eluent: methylene
chloride/2% methanol, then methylene chloride/5% methanol). The
oily eluate was dissolved in methylene chloride and by adding
HCl/ethyl ether a precipitate formed which was tritured in ethyl
acetate and filtered to give 788 mg of the title product (yield:
64.8%).
[0196] .sup.1-NMR (200 MHz, DMSO) .delta. (ppm): 8.89-8.06(m,4H);
7.96 and 7.86(2s,2H); 7.54-7.19(m, 5H); 5.23 and 5.04(2s,4H); 4.08
and 4.03(2s,6H).
EXAMPLE 65
Synthesis of
1-bromo-6,7-dimethoxy-4-pyridin-4-ylmethyl-phthalazine
[0197] 6,7-Dimethoxy-4-pyridin-4-ylmethyl-2H-phthalazin-1-one (5 g,
16.8 mmoles), obtained as described in example 62, was suspended in
acetonitrile (30 ml) and added with POBr.sub.3 (14.5 g, 50.4
mmoles). More acetonitrile was added (total 65 ml) and the mixture
was heated to 70.degree. C. for 16 hours. More POBr.sub.3 (8.1 g)
was added and the heating kept on for 7 hours. The solid was
filtered off and the mother liquor poured into 1N KOH and extracted
with ethyl acetate. The filtrate was slowly put in KOH and
extracted with CH.sub.2Cl.sub.2. The organic extracts were joined,
anhydrified over Na.sub.2SO.sub.4 and the solvent evaporated. The
residue was crystallized from CH.sub.2Cl.sub.2, and the mother
liquor was dissolved in methylene chloride/1% methanol and
chromatographed (eluent: methylene chloride/2% methanol, then
methylene chloride/3% methanol). Joining the crystallized and the
eluate 1.89 g of the title product were obtained (yield: 31%).
EXAMPLE 66
Synthesis of
6,7-dimethoxy-1-phenyl-4-pyridin-4-ylmethyl-phthalazine (Compound
21)
[0198] In dry environment, ZnCl.sub.2 (379 mg, 2.78 mmoles) was
dissolved in tetrahydrofuran (20 ml), then the temperature was
brought to 0.degree. C. and a 2M solution of phenyl lithium in
hexane (1.4 ml, 2.8 mmoles) was dropped therein. The temperature
was left to rise to room value and
1-bromo-6,7-dimethoxy-4-pyridin-4-ylmethyl-phthalazine (500 mg,
1.39 mmoles), obtained as described in example 65, palladium
acetate (15 mg, 0.0695 mmole) and triphenylphosphine (36 mg, 0.139
mmole) were added and the whole was refluxed for 1.5 hour. The
reaction was stopped with some drops of NaOH, then the mixture was
dried aid the residue taken up in water and CH.sub.2Cl.sub.2. The
organic phase was dried to yield a residue which was
chromatographed (eluent: methylene chloride, then methylene
chloride/2% methanol, then methylene chloride/5% methanol). The
eluate was dissolved in methylene chloride, treated with
TONSIL.RTM. and filtered over celite. The mother liquor was dried
yielding 172 mg of the title product.
[0199] .sup.1-NMR (200 MHz, DMSO) .delta. (ppm): 8.83-7.96(m,4H)
7.85-7.64(m,5H); 7.82(s,1H); 7.37(s, 1H); 5.00(s,2H); 4.07 and
3.89(2s,6H).
EXAMPLE 67
[0200] PDE 4 Enzyme Inhibition Test
[0201] a) Purification of Human Polymorphonucleate Leukocytes
[0202] The polymorphonucleate leukocytes (PMNs) were isolated from
peripheral blood of healthy volunteers according to what described
by Boyum A., Scand. J. Immunol., 1976, 5.degree. suppl., 9).
Shortly, the isolation of the PMNs was effected by Ficoll-Paque
gradient centrifugation followed by sedimentation on dextrane and
the erythrocyte contamination was eliminated by hypotonic
lysis.
[0203] b) PDE 4 Enzyme Purification
[0204] The human PMNs were resuspended in TRIS/HCl buffer (10 mM pH
7.8) containing MgCl.sub.2 (5mM), EGTA (4mM), mercaptoethanol
(5mM), TRITON-X100 (1%), pepstatin A (1 .mu.M), PMSF (100 .mu.M)
and leupeptin (1 .mu.M), and homogenised by Polytron. The
homogenate was centrifuged at 25,000.times.g for 30 minutes at
4.degree. C. and the surnatant was used for the PDE 4 enzyme
purification by ion exchange chromatography using the FPLC
technique according to what described by Schudt C. et al.,
Naunyn-Schmidberg's Arch. Pharmacol., 1991, 334, 682. The surnatant
was seeded on a UNO Q12 column (Bio-Rad) and the enzyme was eluted
by sodium acetate gradient from 50 mM to 1M. The fractions
containing enzymatic activity were joined, dialysed against water
and concentrated. The resulting PDE 4 enzyme was stored at
-20.degree. C. in the presence of ethylenglycole (30%) v/v) until
use.
[0205] c) PDE 4 Enzyme Inhibition
[0206] The enzyme activity was assessed with an Amersham kit based
on the SPA technique (Scintillation Proximity Assay). The enzymatic
reaction was effected in a total volume of 100 .mu.l of TRIS/HCl
buffer (50 mM, pH7.5), MgCl.sub.2 (8.3mM), EGTA (1.7 mM), CAMP (1
.mu.M) and [.sup.3H]cAMP (.about.100.000 dpm). The compounds of the
invention and the reference ones were added at the wanted
concentrations. As reference compounds
6,7-dimethoxy-4-(pyridin-4-ylmethyl)-2H-phthalazin-1-one (reference
1) and 6,7-dimethoxy-4-(piperidin-4-ylmethyl)-2H-phthalazin-1-- one
(reference 2) embraced by the general formula of the patent
application EP-0 722 036 (in the name of Eisai) were used. The
reaction was started by adding the enzyme (15 .mu.g protein/ml) in
40 minutes at 30.degree. C. and stopped with 50 .mu.l of SPA
particles suspension. The radioactivity of the particles was
measured in .beta.-emitting counter. The IC.sub.50 values were
computed over 9 equidistant concentrations in logarithmic scale
using a 4-parameters logistic function by a software. The results
are set forth in Table 1.
2 TABLE 1 PDE 4% inhibition(M) Compound 10.sup.-5 10.sup.-6
10.sup.-8 IC.sub.50 nM 1 84 19 53.1 .+-. 10 2 97 94 25 42.4 .+-. 5
3 100 34 38.9 .+-. 7 4 73 58.9 .+-. 5 5 80 33 29.5 .+-. 4 6 79 27
56.8 .+-. 9 7 186 .+-. 43 9 146 .+-. 38 10 93 80 33 74.9 .+-. 8 13
153 .+-. 85 14 298 .+-. 37 15 282 .+-. 36 19 206 .+-. 124 Reference
1 >100 .mu.M Reference 2 >100 .mu.M
EXAMPLE 68
[0207] TNF.sub..alpha. Inhibition Test
[0208] a) Human Monocytes Isolation
[0209] The monocytes were isolated from peripheral blood of healthy
volunteers according the procedure of Schreek L., J. Natl. Cancer
Inst., 1964, 32, 507. The monocytes and lymphocytes population was
isolated by Ficoll gradient centrifugation, and the cells diluted
at a density of 2.5.times.10.sup.6 cells/ml in RPMI1640 incubation
containing 1% inactivated bovine fetal serum, penicillin (100 U/ml)
and streptomycin (100 U/ml) were placed in 24-wells plates (1
ml/well) and left to adhere for 1 hour at 37.degree. C. with 5%
CO.sub.2. At the end of the incubation the lymphocytes not adhering
were removed by aspiration and the monocytes adhered to the plate
were used in the next step.
[0210] b) TNF.sub..alpha. Synthesis Inhibition
[0211] The TNF.sub..alpha. synthesis from human monocytes was
measured according to the method of Barnette M. et al., Biochemical
Pharmacology, 1996, 51, 949. The monocytes were incubated for 1
hour with 1 ml of RPMI1640 incubation medium (1% inactivated bovine
serum, 100 U/ml penicillin and streptomycin) containing different
concentrations of the products according to the present invention
or the carrier only for the controls. The TNF.sub..alpha. synthesis
from monocytes was induced by adding 1 ng/ml of LPS
(lipopolysaccharide of E. Coli) and after 16 hours of incubation at
37.degree. C., 5% CO.sub.2, the incubation medium was removed, and
the surnatant stored at -80.degree. C. until the measurement. The
TNF.sub..alpha. levels were determined by ELISA test with an
Amersham kit. The results are set forth in Table 2 as IC.sub.50
measured with the same calculation of example 67.
3 TABLE 2 Compound IC.sub.50 nM 1 254 .+-. 84 2 139 .+-. 55 3 167
.+-. 79 4 187 .+-. 35 5 252 .+-. 120 6 83.5 .+-. 24 10 259 .+-. 86
14 389 .+-. 47
EXAMPLE 69
[0212] PDE 3 and 5 Enzymes Inhibition Test
[0213] a) Human Platelet Preparation
[0214] Human platelets were prepared from platelet rich plasma
(PRP) obtained from the Hematological Dept. of the "L.Sacco"
Hospital of Milan. The PRP was centrifuged at 2,200 rpm for 15
minutes at 4.degree. C. and the pellet was suspended in lysis
solution (15 ml; 155 mM NH.sub.4Cl, 10 mM KHCO.sub.3 and 0.1 mM
Na.sub.2EDTA, pH=7.4) and incubated for 10 minutes on ice-bath to
remove the erythrocyte contamination. After a centrifuging at 1,400
rpm for 10 minutes at 4.degree. C., platelets were suspended in 10
ml of 145 mM NaCl, 5 mM KCl, 1 mM MgSO.sub.4, 10 mM glucose, 10 mM
HEPES and 0.05 U/ml of hirudin (pH=7.4), and stored at -20.degree.
C. until homogenization and chromatography.
[0215] b) Purification of PDE 3 and PDE 5 with Fast Protein Liquid
Chromatography (FPLC)
[0216] Before the chromatographic step, platelets were thawed and
50 ml of 20 mM TRIS (pH=6.5) containing 5 mM P-mercapto-ethanol, 2
mM EDTA, 50 mM sodium acetate and 50 .mu.M PMSF (homogenization
buffer) were added. The platelet suspension was then homogenized by
a Polytron homogenizer (Polytron PT 1200) for 20 seconds. The
homogenate was centrifuged at 14,500 rpm for 20 minutes at
4.degree. C., and the supernatant was applied to a UNO Q12 column
(Bio-Rad) pre-equilibrated with the PMSF. A flow rate of 4.5 ml/min
was used throughout the ion exchange chromatography procedure. The
column was washed with PMSF (180 ml) and PDE 3 and PDE 5 were
eluted by sodium acetate linear gradient from 0.05M to 1M. The
fractions containing enzymatic activity were joined, dialysed
against water and concentrated 10 times by ultrafiltration. The
resulting solution was stored at -20.degree. C. in the presence of
ethylenglycole (30%) v/v) until use.
[0217] c) Assay of Phosphodiesterase Activity
[0218] The enzyme activity was assessed with an Amersham kit based
on the SPA technique (Scintillation Proximity Assay). The enzymatic
reaction was effected in a total volume of 100 .mu.l of TRIS/HCl
buffer (50 mM, pH7.5), MgCl.sub.2 (8.3mM), EGTA (1.7 mM), cAMP (for
PDE 3 assay) or cGMP (for PDE 5 assay) (1 .mu.M), [.sup.3H]cAMP or
[3H]cGMP (10 .mu.l), and and 10 .mu.l of the compounds of the
invention. The reaction was started by adding the enzyme (10 .mu.l)
incubated for 40 minutes at 30.degree. C. and stopped by adding 50
.mu.l of SPA beads. The radioactivity due to the particles was
measured in .beta.-emitting counter. The results are expressed as
percent activity versus the control present in each test.
4TABLE 3 PDE 3 PDE 5 % inhibition % inhibition Compound at
10.sup.-6 M at 10 .sup.-6 M 1 0 20 2 20 4 3 5 14 4 8 19 6 25 20 10
20 20 13 4 2 15 11 0 19 -- 8
* * * * *