U.S. patent application number 12/938163 was filed with the patent office on 2011-09-29 for thieno[2,3-c] isoquinolines for use as inhibitors of parp.
Invention is credited to FLAVIO MORONI, ROBERTO PELLICCIARI.
Application Number | 20110237617 12/938163 |
Document ID | / |
Family ID | 11446071 |
Filed Date | 2011-09-29 |
United States Patent
Application |
20110237617 |
Kind Code |
A1 |
PELLICCIARI; ROBERTO ; et
al. |
September 29, 2011 |
THIENO[2,3-C] ISOQUINOLINES FOR USE AS INHIBITORS OF PARP
Abstract
The present invention relates to compounds for the inhibition of
poly(ADP-ribose) polymerase (PARP). In some embodiments, the
compounds have the Formula: ##STR00001## wherein the constituent
variables are as defined herein. The invention further provides
methods for the use of the compounds disclosed herein.
Inventors: |
PELLICCIARI; ROBERTO;
(PERUGIA, IT) ; MORONI; FLAVIO; (FIRENZE,
IT) |
Family ID: |
11446071 |
Appl. No.: |
12/938163 |
Filed: |
November 2, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11264468 |
Nov 1, 2005 |
7825129 |
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12938163 |
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10415601 |
Dec 18, 2003 |
6989388 |
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PCT/IB01/02030 |
Oct 30, 2001 |
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11264468 |
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Current U.S.
Class: |
514/291 ;
546/80 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 35/00 20180101; A61P 25/14 20180101; C07D 495/04 20130101;
A61P 27/02 20180101; A61P 25/16 20180101; A61P 17/00 20180101; A61P
25/28 20180101; A61P 35/02 20180101; A61P 9/10 20180101; A61P 19/10
20180101; A61P 29/00 20180101; A61P 21/04 20180101 |
Class at
Publication: |
514/291 ;
546/80 |
International
Class: |
A61K 31/4743 20060101
A61K031/4743; C07D 495/04 20060101 C07D495/04; A61P 35/00 20060101
A61P035/00; A61P 29/00 20060101 A61P029/00; A61P 9/10 20060101
A61P009/10; A61P 27/02 20060101 A61P027/02 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 31, 2000 |
IT |
M100A002358 |
Claims
1.-59. (canceled)
60. A compound of formula I, ##STR00007## wherein Y is a hetero
atom selected from sulphur and nitrogen, each of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5 and R.sub.6, independently, is hydrogen,
hydroxy, OR.sub.7, carboxy, COOR.sub.7, amino, NHR.sub.7 or
halogen, or R.sub.5 and R.sub.6 taken together form a fused
aromatic 5-membered ring or a fused aromatic 6-membered
heterocyclic ring, and R.sub.7 is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl or C.sub.3-C.sub.7 cycloalkyl optionally
substituted with one or more groups selected from hydroxy,
C.sub.1-C.sub.4 alkoxy, carboxy, C.sub.1-C.sub.6 alkoxycarbonyl,
amino, C.sub.1-C.sub.6 mono-alkylamino, C.sub.1-C.sub.6
di-alkylamino and halogen; or a salt, solvate, or physiologically
equivalent derivative thereof.
61. The compound of claim 60, wherein Y is sulphur.
62. The compound of claim 60, wherein Y is nitrogen.
63. The compound of claim 60, wherein R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6 are each hydrogen.
64. The compound of claim 60, wherein one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5 and R.sub.6 is hydroxyl, OR.sub.7,
COOR.sub.7, amino, NHR.sub.7, or halogen, and the others are each
hydrogen.
65. The compound of claim 60, wherein R.sub.4 is hydrogen, hydroxy,
OR.sub.7, or amino, and R.sub.1, R.sub.2, R.sub.3, R.sub.5 and
R.sub.6 are each hydrogen.
66. The compound of claim 60, wherein R.sub.5 and R.sub.6 taken
together form a fused aromatic 5-membered ring.
67. The compound of claim 60, wherein R.sub.5 and R.sub.6 taken
together form a fused aromatic 6-membered heterocyclic ring.
68. A pharmaceutical composition comprising a compound of claim 60,
and a pharmaceutically acceptable vehicle.
69. A method for treating an animal or human who has a condition
selected from the group consisting of tumor disease, leukemia and
sarcoma, who has a tissue damage due to ischemia or reperfusion, or
who has an inflammatory disease, retinal ischemia, or cerebral
trauma, the method comprising administering an effective quantity
of a compound of Formula I to an animal or human, ##STR00008##
wherein Y is a hetero atom selected from oxygen, sulphur, and
nitrogen, each of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5 and
R.sub.6, independently, is hydrogen, hydroxy, OR.sub.7, carboxy,
COOR.sub.7, amino, NHR.sub.7 or halogen, or R.sub.5 and R.sub.6
taken together form a fused aromatic or non-aromatic 5- or
6-membered ring, and R.sub.7 is C.sub.1-C.sub.6 alkyl,
C.sub.2-C.sub.6 alkenyl or C.sub.3-C.sub.7 cycloalkyl optionally
substituted with one or more groups selected from hydroxy,
C.sub.1-C.sub.4 alkoxy, carboxy, C.sub.1-C.sub.6 alkoxycarbonyl,
amino, C.sub.1-C.sub.6 mono-alkylamino, C.sub.1-C.sub.6
di-alkylamino and halogen; or a salt, solvate, or physiologically
equivalent derivative thereof.
70. The method of claim 69, wherein Y is sulphur.
71. The method of claim 69, wherein Y is nitrogen.
72. The method of claim 69, wherein R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5 and R.sub.6 are each hydrogen.
73. The method of claim 69, wherein one of R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5 and R.sub.6 is hydroxyl, OR.sub.7,
COOR.sub.7, amino, NHR.sub.7, or halogen, and the others are each
hydrogen.
74. The method of claim 69, wherein R.sub.4 is hydrogen, hydroxy,
OR.sub.7, or amino, and R.sub.1, R.sub.2, R.sub.3, R.sub.5 and
R.sub.6 are each hydrogen.
75. The method of claim 69, wherein R.sub.5 and R.sub.6 taken
together form a fused aromatic 5-membered ring.
76. The method of claim 69, wherein R.sub.5 and R.sub.6 taken
together form a fused aromatic 6-membered heterocyclic ring.
77. The method of claim 69, wherein the compound of Formula I is
administered in combination with ionizing agents or
chemotherapeutic agents.
78. The method of claim 77, wherein the animal or human has a
condition selected from the group consisting of tumor disease,
leukemia and sarcoma.
79. The method of claim 69, wherein the animal or human has a
tissue damage due to ischemia or reperfusion, or has an
inflammatory disease, retinal ischemia, or cerebral trauma.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/415,601 filed Dec. 18, 2003, now pending,
which is a U.S. national phase of PCT/IB01/02030, filed Oct. 30,
2001, which claims priority to Italian Application No. M100A002358
filed Oct. 31, 2000. The entire disclosure of each of the foregoing
is hereby incorporated by reference. The contents of each of the
foregoing references are hereby incorporated in their entirety.
[0002] The subject of the present invention is certain heterocyclic
derivatives, including derivatives of
thieno[2,3-c]isoquinolin-3-one and their use in therapy as
inhibitors of poly(ADP-ribose) polymerase (PARP).
BACKGROUND OF INVENTION
[0003] Poly(ADP-ribose) polymerase or poly(ADP-ribose) synthase
(PARS) is one of the enzymes (EC 2.4.2.30) which catalyse the
transfer of ADP-ribose from NAD to the carboxyl groups of proteins.
It is mainly found in the cell nucleus and for many years it has
been considered that its activity is directed towards the
maintenance of the integrity of the DNA sequence by in some way
participating in the repair of damage undergone by the gene
sequence after toxic or infective insults. Some histones,
topoisomerase I and II, DNA ligase and other nuclear proteins are
substrates of this enzyme. Moreover, PARP itself can undergo
self-poly-ADP ribosylation.
[0004] The cDNA which codes for PARP has been cloned and the
protein has been purified from tissues of many animal species,
including man. The structure of PARP is available in various data
banks, and consists of: [0005] 1. an amino terminal domain capable
of binding DNA (42 kDa), [0006] 2. a central zone also called
self-modification domain (which thus can be ADP-ribosylated and
which on average weighs 16 kDa), and [0007] 3. a carboxy terminal
domain which contains the catalytic site (55 kDa) and which has an
amino acid composition and a tertiary structure highly conserved
from drosophila to man.
[0008] The necessary and sufficient stimulus for activating the
enzyme is damage to the DNA (formation of "nicks" or other lesions
of the double helix) (de Murcia et al., 1992; Menisser-de Murcia et
al., 1997). This commonly occurs by the action of strong oxidants
and/or of highly reactive molecules such as the free radicals which
form abundantly in the tissues in the course of various
pathological situations. Nitric oxide can also directly or
indirectly damage the DNA double helix, activate PARP and lead the
cell to massive utilization of NAD and to ATP depletion (Szabo et
al., 1996). Considering then that NAD is essential for oxidative
phosphorylation and the resynthesis of ATP, it can be understood
how cells which are in that situation are not capable of
maintaining their ionic equilibria and can undergo degeneration
both of necrotic and of apoptotic type. Excessive activation of
PARP under the action of newly formed nitric oxide can occur in the
central nervous system following activation of the glutamate
receptors of the NMDA type associated with neuronal nitric oxide
synthase (nNOS). It can also occur in other organs or tissues,
since nitric oxide can be synthesized by the NOS of the endothelium
or by the inducible NOS of the microglia and macrophages, and
diffuse into the surrounding cells. Much experimental data seems to
be in agreement with this hypothesis and it has been clearly
demonstrated that by inhibiting PARP it is possible to reduce
excitotoxic neuronal death or that due to free radicals in various
types of neuronal culture (Schraufstatter et al., 1986; Cosi et
al., 1994; Zhang et al., 1994). It has also been shown that baby
mice with this enzyme knocked out are particularly resistant to
cerebral tissue loss after occlusion of the medial cerebral artery
or endocrine cell loss after administration of toxins. It has also
been shown that cells taken from such mice are very resistant to
oxidative stress (Eliasson et al., 1997; Endres et al., 1997;
Pieper et al., 1999).
[0009] Thus the importance of having available pharmaceutically
acceptable PARP inhibitors is clear.
DESCRIPTION OF INVENTION
[0010] The subject of the invention is compounds of formula I
##STR00002##
where Y is a hetero atom selected from sulphur (S), nitrogen (N) or
oxygen (O), R.sub.1-R.sub.6 are the same or different, and
represent hydrogen, hydroxide, an OR.sub.7 group, carboxy,
COOR.sub.7 group, amino group, NHR.sub.7 group or halogen, or
R.sub.5 and R.sub.6 taken together form a fused aromatic or
non-aromatic 5- or 6-membered ring, R.sub.7 is a C.sub.1-C.sub.6
alkyl group, C.sub.2-C.sub.6 alkenyl group or C.sub.3-C.sub.7
cycloalkyl group possibly substituted with one or more hydroxy,
C.sub.1-C.sub.4 alkoxy, carboxy, C.sub.1-C.sub.4 alkoxycarbonyl,
amino, C.sub.1-C.sub.6 mono- or dialkylamino groups or halogen. The
invention further includes the non-toxic salts, solvates and
physiologically equivalent derivatives of the compounds of formula
I.
[0011] The compounds of formula I containing ionizable groups will
in fact be able to form salts with pharmaceutically acceptable
acids and bases such as hydrochloric, sulphuric, tartaric, maleic,
citric, succinic, acetic, phosphoric, benzoic, methanesulphonic
acid and the like or with bases such as alkali or alkaline earth
metals, ammonium, alkylammonium and the like.
[0012] Examples of suitable solvates include the hydrated forms of
the compounds I.
[0013] "Physiologically equivalent derivative" is understood to
mean a compound capable of liberating a compound of formula I or an
active metabolite thereof in vivo.
[0014] Examples of C.sub.1-C.sub.6 alkyl groups include methyl,
ethyl, isopropyl, n-propyl and butyl.
[0015] Examples of C.sub.2-C.sub.6 alkenyl groups include vinyl and
allyl.
[0016] Examples of C.sub.3-C.sub.7 cycloalkyl groups include
cyclopropyl, cyclopentyl and cyclohexyl.
[0017] Examples of C.sub.1-C.sub.4 alkoxy groups include methoxy
and ethoxy.
[0018] Examples of C.sub.1-C.sub.4 alkoxycarbonyl groups include
methoxycarbonyl and ethoxycarbonyl.
[0019] Examples of C.sub.1-C.sub.6 alkylamino groups include
methylamino, dimethylamino, methylethylamino and
isopropylamino.
[0020] The term halogen refers to chlorine, bromine, iodine or
fluorine, preferably chlorine or fluorine.
[0021] Preferably, in compounds of formula I, Y is an atom of
sulphur.
[0022] Compounds more particularly preferred are those wherein
R.sub.1-R.sub.6 are hydrogen or one of R.sub.1-R.sub.6 is different
from hydrogen, such as ioniz, alkoxy, carboxy, alkoxycarbonyl,
amino, alkylamino or halogen as defined above and the others are
hydrogen.
[0023] In a particularly preferred group of compounds, R.sub.4 is
hydrogen or a group different from hydrogen, preferably ioniz or
alkoxy or amino, Y is nitrogen, oxygen or sulphur, preferably
sulphur, and R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.6 are
hydrogen.
[0024] The invention also provides a method for the preparation of
the compounds of formula I, which comprises the reaction of a
compound of formula
##STR00003##
wherein R'.sub.1-R'.sub.6 are R.sub.1-R.sub.6 as defined above or
are groups convertible into R.sub.1-R.sub.6, with thionyl chloride
followed by reaction with sodium azide and subsequent Curtius
rearrangement (J. Heterocyclic Chem., 1978, 15, 301-305) by
thermolysis.
[0025] An example of a convertible group is nitro, which can be
reduced to amino.
[0026] The compounds II are known or can be obtained by known
methods. In the case of the compounds wherein Y is sulphur, for
example, the compounds of formula II can be obtained in accordance
with the following scheme:
##STR00004##
3-bromothiophen-2-aldehyde (2) was obtained from commercially
available 2,3-dibromothiophen (1) via 3-bromothienyllithium and
reaction with N,N-dimethylformamide. The aldehyde 2 was protected
by reaction with ethylene glycol to give
2-(3-bromo-2-thienyl)-1,3-dioxolane (3). The latter derivative was
converted into 2-formyl-3-thiopheneboronic acid (4) using
halogen-metal interconversion between the substrate (3) and
butyllithium at -70.degree. C., followed by treatment with tributyl
borate and subsequent hydrolysis. The thienylboronic derivative 4
is a very useful intermediate, in fact it can be condensed with
various bromo-aromatic compounds. Thus, palladium-catalysed
condensation reaction of the derivative 4 with bromobenzene in
aqueous sodium carbonate-ethanol mixture as solvent gave
3-phenyl-thiophen-2-carboxaldehyde (5). Oxidation of 5 with Jones'
reagent yielded 3-phenyl-thiophen-2-carboxylic acid (6).
Chlorination of 6 with thionyl chloride followed by reaction with
sodium azide yielded the acyl azide intermediate which on
thermolysis in boiling o-dichlorobenzene gave the derivative
thieno[2,3-c]isoquinolin-5(4H)-one (7).
[0027] In the case of the synthesis of compounds in which R.sub.4
are amino groups or in the case where suitable bromo-aromatic
intermediates are not available, the following alternative route
can be utilized:
##STR00005##
[0028] The intermediate 3-bromothiophen-2-aldehyde (2) can be
condensed with various commercially available or synthetic
2-substituted phenylboronic acids, in particular with
2-nitrophenylboronic acid to obtain
3-(2-nitrophenyl)-thiophen-2-aldehyde (8). Oxidation of 8 with
Jones' reagent yielded 3-(2-nitrophenyl)-thiophen-2-carboxylic acid
(9). Chlorination of 9 with thionyl chloride followed by reaction
with sodium azide yielded the acyl azide intermediate which on
thermolysis in refluxing o-dichlorobenzene gave the intermediate
9-nitrothieno[2,3-c]isoquinolin-5(4H)-one (10) which on reduction
with hydrazine and Raney nickel as catalyst gave the derivative
9-aminothieno[2,3-c]isoquinolin-5(4H)-one (11).
[0029] In the case of compounds in which Y is nitrogen, for
example, the compounds of formula II can be obtained by following
schemes 1 and 2 analogously replacing the compound
3-bromo-thiophen-2-aldehyde (2) with the intermediate
3-iodopyrrole-2-aldehyde obtainable by the following scheme (Bull.
Soc. Chim. 1973, 1, 351-359):
##STR00006##
[0030] In the case of compounds in which Y is oxygen, the compounds
of formula II can be obtained in a manner similar to that reported
in the literature for furo[2,3-c]isoquinolinone derivatives (J.
Heterocyclic Chem., 1978, 15; 301-305).
[0031] The compounds of formula I can be used both for the
prevention and for the treatment of vast sectors of human and
animal pathology. In particular, they can be utilized in the
prevention and treatment of:
[0032] 1) Tissue Damage due to Ischaemia and Reperfusion. Such
damage, with consequent apoptopic or necrotic cell death, can give
rise to various neurological diseases such as: stroke, cerebral or
spinal trauma, epileptic events, cerebral damage due to cardiac
arrest and/or to situations of prolonged hypotension, respiratory
arrest, carbon monoxide or cyanide poisoning, drowning or
hydrocephalus. The cerebral insult can also be of a toxic nature
(excitotoxins and other chemical products), iatrogenic (including
surgical) and due to ionizing radiation. Tissue damage due to
ischaemia and reperfusion can also affect the myocardium and be
present in many cardiopathies such as post-infarction, during and
after coronary by-pass surgery, on the resumption of perfusion in
transplanted hearts and indeed any time when for surgical reasons
cardiac arrest is performed, and blood reperfusion is initiated.
The kidney, the liver, the intestine and skeletal musculature are
susceptible to damage due to ischaemia and reperfusion. This occurs
in septic, endotoxic, haemorrhagic and compression shock. It also
occurs in strangulated hernia, strangulation of intestinal loops,
and after prolonged compression of joints in multiply traumatized
patients.
[0033] 2) Degenerative Diseases. The inhibition of PARP can extend
the reproductive capacity of various cells and hence be utilized to
prevent diseases typically associated with aging. Particularly to
be mentioned are degenerative diseases of the central nervous
system such as Parkinson's disease, Alzheimer's dementia,
Huntington's chorea, amyotrophic lateral sclerosis, macular
degeneration and retinal ischaemia. Other degenerative diseases
include the aging of the skin, degenerative diseases of the muscles
(muscular dystrophy), bones (osteoporosis) and vascular system
(atherosclerosis), diabetes and diseases of the immune system
present during senescence.
[0034] 3) Inflammatory Diseases. Excessive activation of PARP can
be harmful in various diseases of predominantly inflammatory
nature, both of the central nervous system and of peripheral
organs. The compounds of the invention are thus useful in the
following pathological situations: multiple sclerosis and other
demyelinizing diseases, Guillain-Barre syndrome, neuralgias of the
trigeminus and/or other cranial nerves, peripheral neuropathies and
other chronic pain, osteoarthritis, inflammatory diseases of the
intestine (Crohn's disease, ulcerative colitis, and other forms of
colitis).
[0035] 4) Tumour Diseases. PARP inhibitors can facilitate the death
of tumour cells induced by ionizing agents or by chemotherapeutic
agents and will be used, both alone and in combination with other
treatments, in the prevention and in the therapy of various forms
of cancer, leukaemia and/or sarcoma, whether these are primary or
associated with AIDS.
[0036] For the intended pharmaceutical uses, the compounds of
formula I can be administered alone or in the form of a
conventional pharmaceutical preparation, the form of which
obviously depends on the choice of the administration route. For
example, for oral administration, they can be formulated in the
form of tablets, capsules, granules, powders, syrups or the like,
or, for parenteral administration, they can be formulated in the
form of injections, suppositories or the like. These pharmaceutical
preparations can be produced by conventional methods using
ingredients generally known in the technology, such as excipients,
binders, disintegration agents, lubricants, stabilizers, modifiers
and the like. Although the dosage can vary depending on the
symptoms and the age of the patient, the nature and severity of the
disease or disorder and on the route and mode of administration, in
the case of oral administration to an adult human patient, the
compounds of the present invention can commonly be administered at
a total daily dosage lying between 1 and 1000 mg, preferably
between 5 and 500 mg, either in a single dose or in subdivided
doses, for example from one to three times a day; in the case of an
intravenous injection, a dosage lying between 0.1 and 100 mg,
preferably lying between 0.5 and 50 mg, can be administered from
one to three times a day.
[0037] The following Examples illustrate the invention in more
detail.
Example 1
Thieno[2,3-c]isoquinolin-5(4H)-one
a. 3-Bromothiophen-2-aldehyde (2)
[0038] A 1.4 N solution of n-butyllithium in hexane (34 ml) was
added dropwise to 2,3-dibromo-thiophen (1) (10 g, 41.1 mmol) in
anhydrous ether (20 ml) at -70.degree. C. After 10 mins, the
solution was transferred into a solution of N,N-dimethylformamide
(4.8 ml) in anhydrous ether (15 ml) and the reaction mixture was
left for 30 mins with stirring. The reaction was stopped by
addition of a 10% solution of hydrochloric acid (20 ml). The ether
layer was washed with a solution of bicarbonate, dried
(Na.sub.2SO.sub.4), and evaporated under reduced pressure to give
the crude aldehyde compound 2 (7.54 g, 39.5 mmol, 96% yield) as an
oil.
[0039] .sup.1H-NMR (CDCl.sub.3) a 7.19 (d J=5.1 Hz, 1H, th-H), 7.45
(d J=5.2, 1H, th-H), 10.02 (s, 1H, CHO).
b. 2-(3-Bromo-2-thienyl)-1,3-dioxolane (3)
[0040] 3-Bromothiophen-2-aldehyde (2) (7.44 g, 39 mmol), ethylene
glycol (2.7 ml, 48 mmol), anhydrous benzene (130 ml) and a few
crystals of p-toluenesulphonic acid were refluxed in a Dean-Stark
apparatus until no more water separated. The benzene layer was
washed with a solution of bicarbonate, dried (Na.sub.2SO.sub.4),
and evaporated under reduced pressure to give the derivative 3 (6.8
g, 28.9 mmol, 74% yield) as an oil.
[0041] .sup.1H-NMR (CDCl.sub.3) a 3.80-4.06 (m, 4H, diox), 6.03 (s,
1H, diox), 6.85 (d J=5.2 Hz, 1H, th-H), 7.16 (d J=5.7 Hz, 1H,
th-H).
c. 2-Formylthiophen-3-boronic acid (4)
[0042] A 1.1 N solution of n-butyllithium in hexane (28 ml) was
added dropwise (10 mins) to a solution of
2-(3-bromo-2-thienyl)-1,3-dioxalane (3) (6.6 g, 28.1 mmol) in
anhydrous ether (30 ml) at -70.degree. C. After stirring for 10
mins, butyl borate (9.1 ml, 33.7 mmol) in anhydrous ether (20 ml)
was added in a single portion. The reaction mixture was stirred for
4 hrs at -70.degree. C. and then left to return to room
temperature. 1 N hydrochloric acid (20 ml) was added, and the
mixture was left for 1 hr with stirring. The aqueous phase was
extracted with ether and the combined ether phases were washed with
a solution of sodium bicarbonate, dried (Na.sub.2SO.sub.4), and
evaporated under reduced pressure to give the intermediate (4) (2.2
g, 14.1 mmol, 50% yield) as a brown solid.
[0043] .sup.1H-NMR (CDCl.sub.3) a 7.0 (brs, 2H, 0H), 7.77 (d J=5.1
Hz, 1H, th-H), 7.85 (d J=5.2, 1H, th-H), 9.75 (s, 1H, CHO).
d. 3-Phenylthiophen-2-aldehyde (5)
[0044] 2-Formylthiophen-3-boronic acid (4) (0.89 g, 5.7 mmol)
dissolved in 96% ethanol (15 ml) was added dropwise to a mixture of
2-bromobenzene (0.66 ml, 6.2 mmol) in benzene (12 ml),
(Ph.sub.3P).sub.4Pd (197 mg, 0.15 mmol) and an aqueous solution of
2M Na.sub.2CO.sub.3 (6.3 ml). The reaction mixture was then
refluxed for 6 hrs. Then the mixture was cooled to room
temperature, the solvent was partially removed under reduced
pressure and the resulting mixture was extracted with ethyl ether
(4.times.100 ml). The combined organic layer was washed with a
saturated solution of NaCl, dried (Na.sub.2SO.sub.4), and
evaporated under reduced pressure. The mixture was chromatographed
using 95/5 petroleum ether/ethyl acetate, thus giving the
derivative (5) (0.575 g, [lacuna] mmol, 53% yield). .sup.1H-NMR
(CDCl.sub.3) a 7.19 (d J=5.0 Hz, 1H, th-H), 7.40-7.43 (m, 5H,
Ph-H), 7.70 (d J=5.2, 1H, th-H), 9.83 (s, 1H, CHO).
e. 3-Phenylthiophen-2-carboxylic acid (6)
[0045] 8N Jones' reagent (ca. 10 ml) was added dropwise to a
solution of 3-phenylthiophen-2-aldehyde (5) (0.58 g, 3.1 mmol) in
32 ml of acetone, until an orange colour persisted. The mixture was
stirred at room temperature for 18 hrs. Isopropanol (5 ml) was
added to remove the excess Jones' reagent and the mixture was then
filtered, the organic solvents were evaporated under vacuum and
extracted with ethyl acetate (4.times.100 ml). The combined organic
layer was washed with a saturated solution of NaCl, dried
(Na.sub.2SO.sub.4), and evaporated under reduced pressure. The
mixture was then chromatographed using 95/5 chloroform/methanol,
thus giving the compound (6) (0.44 g, 1.36 mmol, 46% yield) (M.Pt.
200-202.degree. C.) .sup.1H-NMR (CDCl.sub.3) a 6.87-7.37 (m, 5H,
Ph-H), 7.05 (d J=5.0 Hz, 1H, th-H4), 7.53 (d J=5.0 Hz, 1H,
th-H5).
f. Thieno[2,3-c]isoquinolin-5(4H)-one (7)
[0046] 0.08 ml of thionyl chloride were added to a solution of
3-phenylthiophen-2-carboxylic acid (6) (0.138 g, 0.68 mmol) in 2 ml
of anhydrous benzene and the mixture was refluxed for 2 hrs. The
solvent and the excess thionyl chloride were removed under vacuum
and the residue was taken up using 3 ml of THF, and NaN.sub.3
(0.066 g, 1.02 mmol) in 1 ml of water was added rapidly to this
stirred solution at 0.degree. C. The mixture was left for 1 hr at
room temperature with stirring, poured into 10 ml of crushed ice
and water and extracted with ethyl ether (4.times.10 ml) and the
separated organic layer was washed, dried and evaporated under
vacuum. The residue was dissolved in 1 ml of o-dichlorobenzene and
then added dropwise to 3 ml of boiling o-dichlorobenzene with
stirring. The mixture was refluxed for 5 hrs, then cooled and
evaporated. The mixture was subjected to flash chromatography,
elution with 90:10 petroleum ether-ethyl acetate giving the end
compound (7) (0.061 g, 0.3 mmol, 44% yield) as a pure solid (M.Pt.
266-268.degree. C.); IR: 2849, 1647 cm.sup.-1 (NHCO).
[0047] .sup.1H-NMR (DMSO) a 7.23 (d J=5.6 Hz, 1H, th-H1), 7.51 (m,
1H, th-H), 7.70 (d J=5.3 Hz, 1H, th-H1), 7.78 (m, 1H, Ph-H), 8.10
(dd J=7.4, 1.1 Hz, 1H, Ph-H), 8.25 (dd, J=8.0, 0.9 Hz, 1H, Ph-H),
12.3 (s, 1H, NH).
[0048] .sup.13C-NMR (CDCl.sub.3) a 116.4, 119.2, 120.6, 122.6,
123.3, 126.2, 128.4, 133.1, 134.1, 139.9, 163.2.
[0049] The following compounds were prepared by condensation of the
derivative (4) with 2-methoxybromobenzene, according to the
procedure of Example 1.
Example 2
g. 9-Hydroxythieno[2,3-c]isoquinolin-5(4H)-one
[0050] (M.Pt.: 298-301.degree. C.)
[0051] .sup.1H-NMR (CDCl.sub.3) a 7.01 (d, J=5.7 Hz, 1H, th-H1),
7.15 (dd, J=7.83 Hz, J=1.1 Hz, 1H, Ph-H6), 7.28 (t, J=7.9 Hz, 1H,
Ph-H7), 7.82 (dd J=7.8 Hz, J=1.1 Hz, 1H, Ph-H8), 8.05 (d J=5.7 Hz,
1H, th-H2).
Example 3
h. 9-Methoxythieno[2,3-c]isoquinolin-5(4H)-one
[0052] (M.Pt.: 198-200.degree. C.)
[0053] .sup.1H-NMR (CDCl.sub.3) a 4.00 (s, 3H, OCH.sub.3), 6.95 (d
J=5.7 Hz, 1H, th-H1), 7.20 (m, 1H, Ph-H6), 7.44 (t, J=8.0 Hz, 1H,
Ph-H7), 7.98 (d J=5.7, Hz, 1H, th-H2), 8.13 (dd J=8.0, 1.1 Hz, 1H,
Ph-H8).
[0054] The compound of Example 2 is obtained from the compound of
Example 3 by reaction with boron tribromide.
Example 4
i. 3-(2-Nitrophenyl)-thiophen-2-aldehyde (8)
[0055] The compound 3-bromothiophen-2-aldehyde (2) (5.20 g, 27
mmol), solubilized in ethylene glycol dimethyl ether (105 ml) was
treated with (Ph.sub.3P).sub.4Pd (936 mg, 0.81 mmol), with
2-nitrophenylboronic acid (5 g, 30 mmol) and an aqueous solution of
2M NaHCO.sub.3 (70 ml). The reaction mixture was then refluxed for
5 hrs. Then the mixture was cooled to room temperature, the solvent
was partially removed under reduced pressure and the resulting
mixture was extracted with ethyl acetate (4.times.100 ml). The
combined organic layer was washed with a saturated solution of
NaCl, dried (Na.sub.2SO.sub.4), and evaporated under reduced
pressure. The mixture was chromatographed using 80/20 petroleum
ether/ethyl acetate, thus giving the derivative (8) (4.5 g, 83%
yield).
[0056] .sup.1H-NMR (CDCl.sub.3) a 7.04 (d J=5.1 Hz, 1H, th-H), 7.45
(dd J=5.2, 0.9 Hz, 1H, th-H), 7.58-7.74 (m, 3H, Ph-H), 7.98 (dd
J=8.0, 0.9 Hz, 1H, Ph-H), 9.61 (s, 1H, CHO).
l. 3-Phenylthiophen-2-carboxylic acid (9) [sic]
[0057] 8N Jones' reagent (ca. 30 ml) was added dropwise to a
solution of 3-(2-nitrophenyl)-thiophen-2-aldehyde (8) (4.4 g, 3.1
mmol) in 32 ml of acetone, until an orange colour persisted. The
mixture was left at room temperature for 18 hrs with stirring.
Isopropanol (20 ml) was added to remove the excess Jones' reagent
and the mixture was then filtered, the organic solvents were
removed under vacuum and extracted with ethyl acetate (4.times.100
ml). The combined organic layer was washed with a saturated
solution of NaCl, dried (Na.sub.2SO.sub.4), and evaporated under
reduced pressure. The mixture was then chromatographed using 95/5
chloroform/methanol, thus giving the compound (9) (3.6 g, 66%
yield).
[0058] .sup.1H-NMR (CDCl.sub.3) a 6.80 (d J=5.1 Hz, 1H, th-H), 7.25
(dd J=5.2, 0.9 Hz, 1H, th-H), 7.30-7.60 (m, 3H, Ph-H), 7.95 (dd
J=8.0, 0.9 Hz, 1H, Ph-H).
m. 9-Nitrothieno[2,3-c]isoquinolin-5(4H)-one (10)
[0059] 2 ml of thionyl chloride were added to a solution of
3-(2-nitrophenyl)thiophen-2-carboxylic acid (9) (3.5 g, 14 mmol) in
20 ml of anhydrous benzene and the mixture was refluxed for 2 hrs.
The solvent and the excess thionyl chloride were removed under
vacuum and the residue was taken up using 50 ml of THF, and
NaN.sub.3 (1.37 g, 21 mmol) in 10 ml of water was added rapidly to
this stirred solution at 0.degree. C. The mixture was left for 1 hr
at room temperature with stirring, poured into 100 ml of ice and
water and extracted with ethyl ether (4.times.50 ml), the separated
organic layer was dried over anhydrous sodium sulphate and leaving
it for 2 hrs with stirring. It was evaporated under vacuum and
coevaporated with anhydrous benzene (3.times.50 ml). It is
evaporated and the residue was solubilized in 60 ml of
o-dichlorobenzene and refluxed using a Dean-Stark apparatus. The
mixture was left under reflux for 20 hrs with stirring, then cooled
and evaporated. The mixture was subjected to flash chromatography,
and elution with chloroform yielded the compound
9-nitrothieno-[2,3-c]-isoquinolin-5(4H)-one (0.075 g, 2.2% yield)
as a yellow solid (M.Pt. 255-258.degree. C.).
[0060] .sup.1H-NMR (DMSO) a 6.83 (d J=5.8 Hz, 1H, Th--H), 7.22 (d
J=5.8 Hz, 1H, Th--H), 7.61 (t J=7.9 Hz, 1H, Ph-H), 8.16 (dd J=7.8,
0.8 Hz, 1H, Ph-H), 8.47 (dd, J=8.0, 0.8 Hz, 1H, Ph-H), 12.8 (bs,
1H, NH).
n. 9-Aminothieno[2,3-c]isoquinolin-5(4H)-one (11)
[0061] The compound 9-nitrothieno[2,3-c]isoquinolin-5(4H)-one
(0.028 g, 0.113 mmol) was solubilized in dimethoxyethanol (20 ml),
Raney nickel (ca. 10 mg) was added, and the mixture treated
dropwise with hydrazine hydrate (ca. 1 ml). The mixture was left
for 3 hrs with stirring, filtrated on Celite and evaporated under
vacuum. The residue was chromatographed eluting with 99/1
chloroform/methanol to give
9-aminothieno[2,3-c]isoquinolin-5(4H)-one (10) [sic] as the pure
solid (0.015 g, 61% yield) (M.Pt. 297-299.degree. C.).
[0062] .sup.1H-NMR (DMSO) a 5.31 (s, 2H, NH.sub.2), 7.11 (m, 2H,
Ph-H and Th--H), 7.17 (t J=7.8 Hz, 1H, Ph-H), 7.57 (dd J=7.8, 1.3
Hz, 1H, Ph-H), 7.83 (d, J=5.8 Hz, 1H, Th--H), 12.21 (bs, 1H,
NH)
Pharmacological Activity
[0063] The inhibitory activity of the compounds of Examples 1-3 on
the enzyme poly(ADP-ribose) polymerase (PARP) was evaluated in
hippocampus section culture models of organotypical cerebral
ischaemia, as was their activity in models of cerebral ischaemia in
vitro (primary neuronal cultures) and in vivo (occlusion of the
medial cerebral artery (MCAO) in rats).
The Methods Used were:
1) Purification and Measurement of Activity Against PARP
[0064] The activity on PARP was evaluated using purified PARP
derived from foetal calf thymus, as per Ito et al. (J. Biol. Chem.
274: 3647, 1979). A portion (ca. 1 g) of thymus was homogenized in
4 volumes of a buffer containing 50 mM Tris-HCl, 0.3M NaCl, 10%
glycerol, 10 mM mercaptoethanol and 50 mM sodium bisulphite. After
centrifugation (12,000 rpm/15 mins), the supernatant was treated
with 150 ml of 3.75% protamine sulphate and stirred in ice for 5
minutes. The enzymatic activity was further purified by affinity
chromatography on a DNA-agarose column (25.times.0.5 cm; 0.75 mg
DNA/ml of 4% agarose bed). The active fractions were used for the
activity tests carried out as per Banasik et al. (J. Biol. Chem.
267: 1569, 1992). The test mixture (100 ml) containing 100 mM
Tris-HCl pH 8, 50 mg of sonicated DNA, 60 ml of partially purified
enzyme preparation and 0.2 mC of 3[H]NAD (1-5 Ci/mmol, New England
Nuclear, Boston, Mass., USA) was incubated at 37.degree. C. for 30
mins. The reaction was terminated with 10% of trichloroacetic acid
and the radioactivity associated with the protein was evaluated in
a liquid scintillation spectrometer.
[0065] 2) The biological activity was evaluated in primary neuronal
cultures exposed to oxygen and glucose deprivation (see:
Neuropharmacology 38: 1007-1619) and in vivo in rats with MCA
[sic]. The volume of the infarct was measured 72 hrs after the
occlusion using a method described previously (Cozzi et al., J.
Cerebrali Blood Flow Metabolism 19: 771-777; 1999).
TABLE-US-00001 TABLE IC.sub.50 (.mu.m) of PARP inhibitors tested on
enzyme prepared from bovine thymus Compound IC.sub.50 (.mu.m)
Benzamide 90 Phenanthridinone 12 DPQ (UPF 707) 10 Example 1 0.3
Example 2 0.1 Example 3 0.3 Example 4 0.05
CONCLUSION
[0066] The compounds of the invention are potent PARP
inhibitors.
3) Biological Activity of PARP Inhibitors in Ischaemia Models
[0067] Some PARP inhibitors were tested on neuronal death induced
by ischaemia in primary neuronal cultures exposed to a period of
oxygen and glucose deprivation (see: Neuropharmacology 38:
1007-1619) and the results showed that the PARP inhibitors exert a
potent inhibitory activity on post-ischaemic neuronal death of the
necrotic type.
[0068] Among the compounds of the invention, the compound of
Example 1 was found to be extremely potent in reducing
post-ischaemic neuronal death, having a ca. 3 micromolar
IC.sub.50.
[0069] The neuroprotective activity of the compound of Example 1
was also tested in the MCA0 model in rats. In this stroke model,
the compound (3-10 mg/kg i.p.) reduced by 40% the volume of
necrotic brain induced by the permanent occlusion of the medial
cerebral artery.
* * * * *