U.S. patent application number 09/779925 was filed with the patent office on 2002-10-10 for process for the treatment of erectile dysfunction and product therefor.
Invention is credited to Egerland, Ute, Hofgen, Norbert, Kronbach, Thomas, Marx, Degenhard, Szelenyi, Stefan.
Application Number | 20020147199 09/779925 |
Document ID | / |
Family ID | 22696438 |
Filed Date | 2002-10-10 |
United States Patent
Application |
20020147199 |
Kind Code |
A1 |
Marx, Degenhard ; et
al. |
October 10, 2002 |
PROCESS FOR THE TREATMENT OF ERECTILE DYSFUNCTION AND PRODUCT
THEREFOR
Abstract
The invention relates to the use of pyrido[3,2-e]-pyrazinones of
formula 1 as inhibitors of phosphodiesterase 5 for the treatment of
erectile dysfunction (impotence).
Inventors: |
Marx, Degenhard; (Radebeul,
DE) ; Hofgen, Norbert; (Medingen, DE) ;
Egerland, Ute; (Radebeul, DE) ; Szelenyi, Stefan;
(Schwaig, DE) ; Kronbach, Thomas; (Radebeul,
DE) |
Correspondence
Address: |
Gabriel P. Katona
Gabriel P. Katona L.L.P.
14th Floor
708 Third Avenue
New York
NY
10017
US
|
Family ID: |
22696438 |
Appl. No.: |
09/779925 |
Filed: |
February 8, 2001 |
Current U.S.
Class: |
514/250 |
Current CPC
Class: |
A61P 15/12 20180101;
A61K 31/4985 20130101 |
Class at
Publication: |
514/250 |
International
Class: |
A61K 031/4985 |
Claims
We claim:
1. A process for the treatment of erectile dysfunction, which
comprises administering to a patient in need therefor a
pyrido[2,3-e] pyrazinone of Formula (1) 8wherein R.sup.1, R.sup.2,
and R.sup.4 can be the same or different, and being a linear or
branched C.sub.1-4 alkyl residue, and R.sup.3 is --CH.sub.2--A
wherein A is a cyclohexyl residue; or a C.sub.6-10 monocyclic or
bicyclic hydrocarbon which can be substituted once or more times by
--F, --Cl, --Br, --NO.sub.2, --OH, --OCH.sub.3, --CH.sub.3, or
--CN; or a monocyclic or bicyclic C.sub.3-10 heteroaromatic
hydrocarbon containing 1-4 heteroatoms, and can be substituted one
or more times by --F, --Cl, --Br, --OH, --OCH.sub.3, --CH.sub.3, or
--CN, and pharmaceutically acceptable salts thereof.
2. The process of claim 1, wherein said heteroatom is N, S, or
O.
3. The process of claim 1, wherein said pyrido[2,3-e] pyrazinone is
a pharmaceutically acceptable salt of a compound of Formula (1),
obtained by neutralization of a base with an organic or inorganic
acid, or by neutralization of an acid with an organic or inorganic
base, or by quaternization of a tertiary amine to a quaternary
ammonium salt.
4. The process of claim 1, wherein said pyrido[2,3-e] pyrazinone of
Formula (1) is
5-cyclohexyl-1-ethyl-8-methoxy-3-methyl-imidazo[1,5-a]-pyr-
ido[3,2-e]pyrazinone;
5-benzyl-1-ethyl-8-methoxy-3-methyl-imidazo[1,5-a]-p-
yrido[3,2-e]pyrazinone;
5-benzyl-8-ethoxy-1-ethyl-3-methyl-imidazo[1,5-a]--
pyrido[3,2e-]pyrazinone;
5-benzyl-8-ethoxy-1,3-diethyl-imidazo[1,5-a]-pyri-
do[3,2-e]pyrazinone;
1-ethyl-5-(2-fluorobenzyl)-8-methoxy-3-methyl-imidazo-
[1,5-a]-pyrido[3,2-e]pyrazinone;
5-(2-chlorobenzyl)-1-ethyl-8-methoxy-3-me-
thyl-imidazo[1,5-a]-pyrido[3,2-e]pyrazinone;
1-ethyl-8-methoxy-5-(4-methox-
ybenzyl)-8-methoxy-3-imidazo[1,5-a]-pyrido[3,2-e]pyrazinone;
1-ethyl-5-(4-fluorobenzyl)-3-methyl-imidazo[1,5-a]-pyrido[3,2-e]pyrazinon-
e;
5-(4-chlorobenzyl)-1-ethyl-8-methoxy-3-methyl-imidazo[1,5-a]-pyrido[3,2-
-e]pyrazinone;
5-(2,6-dichlrobenzyl)-1-ethyl-8-methoxy-3methyl-imidazo[1,5-
-a]-pyrido[3,2-e]pyrazinone;
5-(2-chloro-6-fluorobenzyl)-1-ethyl-8-methoxy-
-3-methyl-imidazo[1,5-a]-pyrido[3,2-e]pyrazinone;
5-(2,6-difluorobenzyl)-1-
-ethyl-8-methoxy-3-methyl-imidazo[1,5-a]-pyrido[3,2-e]pyrazinone;
1-ethyl-8-methoxy-3-methyl-5-(2,3,6-trifluorobenzyl)-imidazo[1,5-a]-pyrid-
o[3,2-e]pyrazinone;
5-(2-chloro-6-fluorobenzyl)-8-methoxy-3-methyl-1-propy-
l-imidazo[1,5-a]-pyrido[3,2-e]pyrazinone;
5-(2,6-difluorobenzyl)-8-methoxy-
-3-methyl-1-propyl-imidazo[1,5-a]-pyrido[3,2-e]pyrazinone;
8-methoxy-3-methyl-1-propyl-5-(2,3,6-trifluorobenzyl)-imidazo[1,5-a]-pyri-
do[3,2-e]pyrazinone;
5[(3,5-dimethylisoxazol-4-yl)-methyl-1-ethyl-8-methox-
y-3-methyl-imidazo[1,5-a]-pyrido[3,2-e]pyrazinone;
1-ethyl-8-methoxy-3-met-
hyl-5-(4-pyridylmethyl)-imidazo[1,5-a]-pyrido[3,2-e]pyrazinone;
1-ethyl-8-methoxy-2-methyl-5-(4-pyridylmethyl-imidazo[1,5-a]-pyrido[3,2-e-
]pyrazinone hydrochloride;
5-[(2,6-dichloropyrid-4-yl)-methyl]-1-ethyl-8-m-
ethoxy-3-methyl-imidazo[1,5-a]-pyrido[3,2-e]pyrazinone.
5. The pyrido[2,3-e] pyrazinone compound of
5-cyclohexyl-1-ethyl-8-methoxy-
-3-methyl-imidazo[1,5-a]-pyrido[3,2-e]pyrazinone.
Description
FIELD OF THE INVENTION
[0001] This invention relates to a therapeutic process for the use
of pyrido[3,2-e]-pyrazinones of Formula 1 as active ingredients for
treatment of erectile dysfunction (impotence), new
pyrido[3,2-e]pyrazinones, as well as to pharmaceutical compositions
containing these compounds. 1
BACKGROUND
[0002] Impotence in a man can be defined as the inability to engage
in sexual intercourse because of the absence of an erection and/or
because of the failure to ejaculate. One speaks of erectile
dysfunction, if the erection, with respect to strength or duration,
is insufficient for sexual intercourse.
[0003] Erectile disorders affect about 10% of the male population.
About 52% of men between the ages of 40 and 70 are affected.
Several million men worldwide suffer from this disease (about 7.5
million in Germany alone), which in most cases is due to organic
causes and less frequently due to mental causes. Erectile
dysfunction is a widespread problem among older men, particularly
if other chronic diseases are present, such as a high blood
pressure, arteriosclerosis and diabetes.
[0004] Although different active ingredients can induce an
erection, these act only after an injection directly into the penis
(intracavernous, i.c.) or instillation into the urethra
(intraurethral). This form of pharmacological therapy has been
available for more than 10 years and involves the i.c. injection of
vasoactive substances, such as papaverin, phenoxybenzamine,
phenotolamine, moxisylyte and prostaglandin E.sub.1 (PGE.sub.1).
However, the i.c. use of these substances frequently is accompanied
by serious side effects such as priapismus, pain or penile
fibrosis. PGE.sub.1 can be used intraurethrally and nitroglycerin
and minoxidil transdermally (on the penis). However, this can cause
side effect in the man as well as in the partner.
[0005] Surgical intervention by implanting a prosthesis is an
alternative to pharmacological therapy. However, because of the
anticipated late complications (infections, blood circulation
disorders), this form of therapy is hardly accepted by
patients.
[0006] The introduction of sildenafil (Viagra.RTM.) by Pfizer in
the USA and in Europe was a breakthrough in the treatment of
erectile dysfunction. Sildenafil is an orally effective
phosphodiesterase 5-inhibitor (PDE5 inhibitor), which does not
cause an erection directly, but intensifies the action of nitric
oxide (NO), which is released in the penis by sexual stimulation.
NO, like its second messenger cGMP, brings about a vascular
expansion in the corpus cavernosum (cavernous body), so that more
blood, which brings about the erection, can flow in.
[0007] Phosphodiesterases (PDE) are an isoenzyme family, to which
10 different isoenzymes can so far be assigned. By hydrolysis, PDE
enzymes split cyclic guanosine-3',5'-monophosphate (cGMP) or cyclic
adenosine-3',5'-monophosphate (cAMP), which occur as `second
messengers` in a plurality of cells. The phosphodiesterase 5 (PDE
5) is cGMP-specific and dominates in the tissue of human corpus
cavernosum.
[0008] The inhibition of the PDE 5 in human corpus cavernosum leads
to an increase in the intracellular cGMP level, induced by NO. A
relaxation of the smooth musculature of the corpus cavernosum and,
consequently, an erection is associated with this.
[0009] Inhibitors of PDE 5 accordingly are therapeutic agents,
which are suitable in the case of indication of erectile
dysfunction.
[0010] European patent 0 400 583 relates to imidazoquinoxalines of
the formula 2
[0011] wherein A is nitrogen or CH in positions 7 or 8, B and D are
nitrogen or CH or a substituted carbon atom and R, R.sup.1, R.sup.2
are hydrogen or different organic substituents. These compounds are
said to have a vasodilating effect.
[0012] In addition to various imidazo[1,2-a]-quinoxalinones, D. D.
Davey et al. (J. Med. Chem. 34 (1991), 2671-2677) also described
two imidazo[1,5-a]-pyrido[3,2-e]pyraxinones of formula 3
[0013] in which, on the one hand R.sup.1 is H, and R.sup.2 is
C.sub.2H.sub.5, and, on the other, R.sup.1 is 2-methylimidazolo-,
and R.sup.2 is CH.sub.3.
[0014] Both compounds are PDE 3 inhibitors with a positive
inotropic effect.
[0015] Patent application No. WO 93/20 077 relates to
imidazoquinoxalinones of the formula 4
[0016] wherein A is a 5-membered heterocyclic ring with 2 or 3
nitrogen atoms in the ring, R.sup.1 is NO.sub.2 or CF.sub.3, and X
is a variety of chains, with up to 4 chain elements, some of which
contain nitrogen. These compounds are described as glutamate
receptor antagonists with psychotropic and anti-ischemic
activity.
[0017] In German patent application No. 199 02 082,
imidazo[1,5-a]-pyrido[3,2-e]-pyrazinones of the formula 5
[0018] are described, which are inhibitors of PDE 5. For these
compounds, which in each case are unsubstituted in the 5 position,
the use for the treatment of erectile dysfunction is claimed.
According to German patent application No. 199 61 302,, some of
these compounds are dual inhibitors of PDE 3and PDE 5. For these
compounds, the use for different heart and circulatory diseases is
also claimed.
[0019] In German patent No. 195 10 965, describes
pyrido[3,2-e]-pyrazinone- s of the formula 6
[0020] These also include imidazo[1,5-a]-pyrido[3,2-e]-pyrazinones.
Antiasthmatic and antiallergic properties were described for the
described group of materials.
DESCRIPTION OF THE INVENTION
[0021] The present invention relates to a therapeutic process for
the treatment of erectile dysfunction, which comprises
administering to a patient in need therefor, a
pyrido[3,2-e]-pyrazinone of Formula (1) 7
[0022] wherein
[0023] R.sup.1, R.sup.2, R.sup.4 can be the same or different,
being a branched or linear C.sub.1-4 alkyl residue
[0024] R.sup.3 is --CH.sub.2--A wherein
[0025] A is a cyclohexyl residue; or a C.sub.6-10 monocyclic or
bicyclic hydrocarbon which can be substituted once or more times by
--F, --Cl, --Br, --NO.sub.2, --OH, --OCH.sub.3, --CH.sub.3 or --CN;
or a monocyclic or bicyclic C.sub.3-10 heteroaromatic hydrocarbon
containing 1-4 heteroatoms such as N, S or O, and can be
substituted once or more times by --F, --Cl, --Br, --NO.sub.2,
--OH, --OCH.sub.3, --CH.sub.3 or --CN;
[0026] and pharmaceutically acceptable salts thereof.
[0027] Typical compounds known for other uses of the foregoing
formula include:
[0028]
5-benzyl-1-ethyl-8-methoxy-3-methyl-imidazo[1,5-a]-pyrido[3,2-e]pyr-
azinone;
[0029]
5-benzyl-8-ethoxy-1-ethyl-3-methyl-imidazo[1,5-a]-pyrido[3,2-e]pyra-
zinone;
[0030]
5-benzyl-8-ethoxy-1,3-diethyl-imidazo[1,5-a]-pyrido[3,2-e]pyrazinon-
e;
[0031]
1-ethyl-5-(2-fluorobenzyl)-8-methoxy-3-imidazo[1,5-a]-pyrido[3,2-e]-
pyrazinone;
[0032]
5-(2-chlorobenzyl)-1-ethyl-8-methoxy-3-methyl-imidazo[1,5-a]-pyrido-
[3,2-e]pyrazinone;
[0033]
1-ethyl-8-methoxy-5-(4-methoxybenzyl)8-methoxy-3-methyl-imidazo[1,5-
-a]pyrido[3,2-e]pyrazinone;
[0034]
1-ethyl-5-(4-fluorobenzyl)3-methyl-imidazo[1,5-a]-pyrido[3,2-e]pyra-
zinone;
[0035]
5-(4-chlorobenzyl)-1-ethyl-8-methoxy-3-methyl-imidazo[1,5-a]-pyrido-
[3,2-e]pyrazinone;
[0036]
5-(2,6-dichlrobenzyl)-1-ethyl-8-methoxy-3-methyl-imidazo[1,5-a]-pyr-
ido[3,2-e]pyrazinone;
[0037]
5-(2-chloro-6-fluorobenzyl)-1-ethyl-8-methoxy-3-methyl-imidazo[1,5--
a]-pyrido[3,2-e]pyrazinone;
[0038]
5-(2,6-difluorobenzyl)-1-ethyl-8-methoxy-3-methyl-imidazo[1,5-a]-py-
rido[3,2-e]pyrazinone;
[0039]
1-ethyl-8-methoxy-3-methyl-5-(2,3,6-trifluorobenzyl)-imidazo[1,5-a]-
-pyrido[3,2-e]pyrazinone;
[0040]
5-(2-chloro-6-fluorobenzyl)-8-methoxy-3-methyl-1-propyl-imidazo[1,5-
-a]-pyrido[3,2-e]pyrazinone;
[0041]
5-(2,6-difluorobenzyl)-8-methoxy-3-methyl-1-propyl-imidazo[1,5-a]-p-
yrido[3,2-e]pyrazinone;
[0042]
8-methoxy-3-methyl-1-propyl-5-(2,3,6-trifluorobenzyl)-imidazo[1,5-a-
]-pyrido[3,2-e]pyrazinone;
[0043]
5-[(3,5-dimethylisoxazol-4-yl)-methyl-1-ethyl-8-methoxy-3-methyl-im-
idazo[1,5-a]-pyrido[3,2-e]pyrazinone;
[0044]
1-ethyl-8-methoxy-3-methyl-5-(4-pyridylmethyl)-imidazo[1,5-a]-pyrid-
o[3,2-e]pyrazinone;
[0045]
1-ethyl-8-methoxy-2-methyl-5-(4-pyridylmethyl)-imidazo[1,5-a]-pyrid-
o[3,2-e]pyrazinone hydrochloride;
[0046]
5-[(2,6-dichloropyrid-4-yl)-methyl]-1-ethyl-8-methoxy-3-methyl-imid-
azo[1,5-a]-pyrido[3,2-e]pyrazinone.
[0047] Compounds of Formula (1) in which A is a cyclohexyl residue,
are new, such as for example
5-cyclohexyl-1-ethyl-8-methoxy-3-methyl-imidazo[-
1,5-a]-pyrido[3,2-e]pyrazinone.
[0048] It is an important feature of the present invention that the
compounds of Formula 1 have a nitrogen atom in position 9 as an
essential structural prerequisite for the present inventive use for
the treatment of erectile dysfunction.
[0049] The pharmaceutically acceptable salts of the compounds of
Formula 1, can be suitably obtained by neutralizing the bases with
inorganic or organic acids, or by neutralizing the acids with
inorganic or organic bases, or by quaternizing tertiary amines to
quaternary ammonium salts. Some of the compounds of Formula 1 of
the present invention are known from German patent No. 195 10 965,
to which reference was already made hereinabove in describing the
state of the art. Pyrido[3,2-e]-pyrazinones were referred thereto
as dual inhibitors of PDE 4 and PDE 5, as anti-asthmatic or
anti-allergic agents.
[0050] The compounds of Formula 1 are distinguished in that their
inhibitory effect on PDE 5 is particularly pronounced. It is the
essence of the present invention that the compounds of Formula 1,
due to their principle of action, are particularly suitable for use
as therapeutic agents for the treatment of erectile
dysfunction.
[0051] It is a particular advantage of the compounds of the present
invention that they highly selectively affect the cGMP level in
human tissue, but not the cAMP level. This was shown for human
tissue of the heart as well as of the penis. With this selectivity,
the risk of cardiovascular side effects is minimized. In regard to
the cGMP selectivity, the compounds of the present invention are
superior to the standard therapeutic agent sildenafil.
[0052] The compounds of Formula 1 can be administered systemically,
for example, intravenously, intramuscularly or subcutaneously, as
well as orally, bucally or sublingually. A topical application, for
example, by inhalation or intranasally, is also possible. The oral
administration of 5 to 200 mg of the compound before sexual
intercourse represents a suitable treatment regimen.
[0053] Drugs which contain one or more of the compounds of Formula
1 contain in addition to the conventional, physiologically
tolerated carriers and/or diluents or inactive ingredients. The
present invention also relates to a method for producing these
drugs.
[0054] The compounds of Formula 1 and the drugs, which contain the
compounds of Formula 1, can be used individually or in combination
with one another.
[0055] As a further feature of the present invention, the compounds
of Formula 1 can also be used as veterinary therapeutic agents for
the prophylaxis and treatment of erectile dysfunction in male
mammals. In determining the dosage, the administration schedule and
the pharmaceutical formulation of the compound, species differences
and the requirements of veterinary practice are taken into
consideration.
[0056] The compounds of Formula 1 are strong inhibitors of
phosphodiesterase 5. Their therapeutic potential is confirmed in
vitro, for example, by the reinforcement of the action of NO on the
intracellular cGMP level in fibroblasts of the rat, the selectivity
of the effect on the cAMP and cGMP levels in human tissue and by
the relaxation of the human corpus cavernosum.
[0057] The following compounds represent suitable examples of the
compounds of Formula 1 which can be used in accordance with the
therapeutic process of the present invention:
1 Ex- Melting am- point ple --R.sup.1 --R.sup.2 --R.sup.3 --R.sup.4
[.degree. C.] 1 --C.sub.2H.sub.5 --CH.sub.3
--CH.sub.2C.sub.6H.sub.11 --CH.sub.3 136-138 (Ethanol) 2
--C.sub.2H.sub.5 --CH.sub.3 --CH.sub.2C.sub.6H.sub.5 --CH.sub.3
166-167 (Acetone) 3 --C.sub.2H.sub.5 --CH.sub.3
--CH.sub.2C.sub.6H.sub.5 --C.sub.2H.sub.5 162-163 (Acetone) 4
--C.sub.2H.sub.5 --C.sub.2H.sub.5 --CH.sub.2C.sub.6H.sub.5
--C.sub.2H.sub.5 159-161 (Acetone) 5 --C.sub.2H.sub.5 --CH.sub.3
--CH.sub.2C.sub.6H.sub.4(2-F) --CH.sub.3 186-187 (Ethanol) 6
--C.sub.2H.sub.5 --CH.sub.3 --CH.sub.2C.sub.6H.sub.4(2-Cl)
--CH.sub.3 245-246 (DMF) 7 --C.sub.2H.sub.5 --CH.sub.3
--CH.sub.2C.sub.6H.sub.4(4-OCH- .sub.3) --CH.sub.3 156-158
(Ethanol) 8 --C.sub.2H.sub.5 --CH.sub.3
--CH.sub.2C.sub.6H.sub.4(4-F) --CH.sub.3 189-191 (DMF) 9
--C.sub.2H.sub.5 --CH.sub.3 --CH.sub.2C.sub.6H.sub.4(4-Cl)
--CH.sub.3 201-202 (DMF) 10 --C.sub.2H.sub.5 --CH.sub.3
--CH.sub.2C.sub.6H.sub.4(2,6-di-Cl) --CH.sub.3 209-212 (Toluene) 11
--C.sub.2H.sub.5 --CH.sub.3 --CH.sub.2C.sub.6H.sub.4(2-Cl,6-F)
--CH.sub.3 197-200 (Acetone) 12 --C.sub.2H.sub.5 --CH.sub.3
--CH.sub.2C.sub.6H.sub.4(- 2,6-di-F) --CH.sub.3 177-180 (Acetone)
13 --C.sub.2H.sub.5 --CH.sub.3 --CH.sub.2C.sub.6H.sub.4 --CH.sub.3
183-185 (2,3,6-tri-F) (Acetone) 14 --C.sub.2H.sub.7 --CH.sub.3
--CH.sub.2C.sub.6H.sub.4(2-Cl,6-F) --CH.sub.3 159-162 (Acetone) 15
--C.sub.2H.sub.7 --CH.sub.3 --CH.sub.2C.sub.6H.sub.4(- 2,6-di-F)
--CH.sub.3 171-172 (Acetone) 16 --C.sub.2H.sub.7 --CH.sub.3
--CH.sub.2C.sub.6H.sub.4 --CH.sub.3 197-199 (2,3,6-tri-F) (Acetone)
17 --C.sub.2H.sub.5 --CH.sub.3 --CH.sub.3 188-190 (Ethanol) 18
--C.sub.2H.sub.5 --CH.sub.3 --CH.sub.3 198-200 (Ethanol) 19
--C.sub.2H.sub.5 --CH.sub.3 --CH.sub.3 237-240 zers. (DMF) 20
--C.sub.2H.sub.5 --CH.sub.3 --CH.sub.3 254-256 (Ethanol)
[0058] PDE 5 activity is determined in enzyme preparations from
human thrombocytes. Human blood was anti-coagulated with citrate.
By centrifuging at 700 g for 20 minutes at room temperature, the
thrombocyte-rich plasma in the supernatant is separated from the
erythrocytes and leukocytes. The thrombocytes are lysed by
ultrasound and used in the PDE 5 assay. With a few modifications,
the phosphodiesterase activity is determined by the method
described by Thompson et al. (Thompson, W. J., Appleman, M. M.,
Assay of cyclic nucleotide phosphodiesterase and resolution of
multiple molecular forms of the enzyme, Adv. Cycl. Nucl. Res. 1979,
10, 69-92). The reaction mixtures contain 50 mM of tris HCl (pH
7.4), 5.5 mM of MgCl.sub.2, the inhibitors in variable
concentrations, the enzyme preparation and the further components
necessary for determining the individual isoenzymes PDE 5 (see
below). The reaction is started by the addition of the substrate,
0.5 .mu.M of [.sup.3H] cGMP (approximately 6,000 CPM/test). The
final volume is 100 ml. Test substances are prepared as stock
solutions in DMSO. The DMSO concentration in the reaction mixture
is 1% v/v. The PDE 5 activity is not affected by this concentration
of DMSO. After the reaction is started by the addition of
substrate, the samples are incubated for 30 minutes at 37.degree.
C. The reaction is stopped by heating the test tubes for 2 minutes
at 110.degree. C. The samples remain for a further 10 minutes in
ice. The addition of 30 .mu.L of 5'-nucleotidase (1 mg/mL, from a
Crotalus adamanteus snake poison suspension) is followed by an
incubation for 10 minutes at 37.degree. C. The samples are stopped
on ice. After the addition of 400 .mu.L of a (1:1:1) mixture of
Dowex, water and ethanol, the samples are mixed well and incubated
once again for 15 minutes on ice. The reaction vessels are
centrifuged for 20 minutes at 3,000 g. Subsequently, 200 .mu.L
aliquots of the supernatant are transferred directly to
scintillation vessels. After the addition of 3 mL of scintillator,
the samples are measured in the Betacounter. The in each case
nonspecific enzyme activities are determined in the presence of 100
.mu.M of IBMX for the determination of the PDE 5 and subtracted
from the test values.
[0059] For the inventive compounds, IC.sub.50 values ranging from
10.sup.-9 to 10.sup.-5 M were determined for the inhibition of the
phosphodiesterase 5. For example, the following values were
determined for selected values:
2 Example IC.sub.50 (.mu.moles/.lambda.) 1 0.025 2 0.203 5 0.059 7
0.104 8 0.126 9 0.112 10 0.132 11 0.019 12 0.038 13 0.016 14 0.008
15 0.002 16 0.005
[0060] Induction of NO Production in Fibroblasts (rat)
[0061] Rat fetal lung fibroblast cells (RFL-6) represent a suitable
medium for investigating influence of the action of NO on the
intracellular cGMP level (Ishii et al. 1991). The basic mechanism
can be transferred to the smooth vascular musculature of the corpus
cavernosum.
[0062] As a function of the concentration, the inventive compounds
reinforce the increase in the intracellular cGMP level induced by
the NO donor S-nitroso-N-acetyl-D,L-penicillamine.
[0063] For example, compound 13, at a concentration of 0.10
.mu.moles/l significantly induces an increase in the cGMP level.
The effectiveness of compound 13 in this connection is 1000 times
that achieved by the use of the nonspecific PDE inhibitor
3-isobutyl-1-ethylxanthine (IBMX).
[0064] Influencing the cCAMP and cGMP Levels in Human Tissue
[0065] The selectivity of the effect on the cGMP level in
comparison to the cCAMP level by the PDE 5 inhibitors gives
indications or possible side effects of the substances, above all
with respect to the cardiovascular system.
[0066] Strips of human atrium and human corpus cavernosum are
incubated for 10 minutes with the test compounds at a concentration
of 1.0 .mu.moles/L The preparations are frozen with liquid nitrogen
and the resulting levels of the cyclic nucleotides are
determined.
[0067] In both tissues, the inventive compounds selectively affect
the cGMP level. In human atrium tissue for example, the cGMP level
is increased by 247% by compound 11 and, on the other hand, the
cCAMP level only by 11%. In human corpus cavernosum tissue, the
cGMP level, on the other hand, is increased by 214% by compound 11
and the cCAMP level only by 80%.
[0068] With respect to this selectivity, the inventive compounds
are superior to the standard therapeutic agent, sildenafil. In
human atrium tissue, the cGMP level is increased by 147% by
sildenafil and the cCAMP level at the same time by 240%. In human
corpus cavernosum tissue, the cGMP level is increased only by 15%
by sildenafil and the cCAMP level, on the other hand, by 238%.
[0069] In Vitro Relaxation of Human Corpus Cavernosum
[0070] Strips of human corpus cavernosum are pre-contracted in an
organ bath with noradrenalin. For test compounds, the relaxation is
determined as a function of the concentration.
[0071] The inventive compounds have a relaxing effect, as a
function of the concentration, on the corpus cavernosum strips
pre-contracted with noradrenalin. For example, an EC.sub.50 value
of 0.15 .mu.moles/l was determined for compound 13.
* * * * *