PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`wo 97103675
`
`WORLD INTELLECfUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 6 :
`A61K 3V495
`
`Al
`
`(11) International Publication Number:
`
`(43) International Publication Date:
`
`6 February 1997 (06.02.97)
`
`(21) International Application Number:
`
`PCTIEP96/03024
`
`(22) International Filing Date:
`
`11 July 1996 (11.07.96)
`
`(30) Priority Data:
`9514464.8
`
`14 July 1995 (14.07.95)
`
`GB
`
`(71) Applicant (for all designated States except US): LABORA(cid:173)
`TOIRE GLAXO WELLCOME S.A. [FR/FR]; 43, rue
`Vineuse, F-75116 Paris (FR).
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): DAUGAN, Alain, Claude(cid:173)
`Marie [FRIFR]; Laboratoire Glaxo Wellcome S.A., Centre
`de Recherches, Z.A. de Courtaboeuf, 25, avenue de Quebec,
`F-91940 Les Ulis (FR).
`
`(74) Agents: FILLER, Wendy, Anne et al.; Glaxo Wellcome House,
`Berkeley Avenue, Greenford, Middlesex UB6 ONN (GB).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BB, BG, BR, BY,
`CA,CH,CN,CZ,DE,DK,EE,ES,H,GB,GE,HU,IL,
`IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV,
`MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU,
`SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, US, UZ,
`VN, ARIPO patent (KE, LS, MW, SD, SZ, UG), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, DE, DK, ES, H, FR, GB, GR, IE, IT,
`LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI,
`CM, GA, GN, ML, MR, NE, SN, TD, TG).
`
`Published
`With international search report.
`
`(54) Title: USE OF CGMP-PHOSPHODIESTERASE INHIBITORS TO TREAT IMPOTENCE
`
`(57) Abstract
`
`(I)
`fonnula
`The use of compounds of
`12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-
`(6R,
`6-(3,4-methylenedioxyphenyl)-pyrazino[2', 1 ':
`6,l]pyrido[3,4-b]indole-1,4-dione,
`(3S,
`12aR)-2,3,6,7, 12, 12a-hexahydro-2,3-dimethyl-
`6-(3,4-methylenedioxyphenyl)-pyrazino[2', 1 ':
`6, l]pyrido[3,4-b ]indole-I ,4-dione, and physiologically
`acceptable salts and solvates thereof, in the treatment
`of impotence.
`
`6R,
`
`0
`
`(I)
`
`~~
`
`DRL - EXHIBIT 1007
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AM
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cs
`cz
`DE
`DK
`EE
`ES
`Fl
`FR
`GA
`
`Annenia
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`COte d'Ivoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Gennany
`Denmark
`Estonia
`Spain
`Finland
`France
`Gabon
`
`GB
`GE
`GN
`GR
`HU
`IE
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LI
`LK
`LR
`LT
`LU
`LV
`MC
`MD
`MG
`ML
`MN
`MR
`
`United Kingdom
`Georgia
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Kenya
`Kyrgystan
`Democratic People's Republic
`of Korea
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Liberia
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`Mali
`Mongolia
`Mauritania
`
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TI'
`UA
`UG
`us
`uz
`VN
`
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Narn
`
`~
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`USE OF CGMP-PHOSPHODIESTERASE INHIBITORS TO TREAT IMPOTENCE
`
`This invention relates to the use of tetracyclic derivatives which are potent
`and selective inhibitors of cyclic guanosine 3' ,5'-monophosphate specific
`phosphodiesterase (cGMP specific POE) in the treatment of impotence.
`
`Impotence can be defined as a lack of power, in the male, to copulate and
`may involve an inability to achieve penile erection or ejaculation, or both. More
`specifically, erectile impotence or dysfunction may be defined as an inability to
`obtain or sustain an erection adequate for intercourse. Its prevalence is claimed
`to be between 2 and 7% of the human male population, increasing with age, up
`to 50 years, and between 18 and 75% between 55 and 80 years of age.
`
`Reports of well-controlled clinical trials in man are few and the efficacy of
`orally administered drugs is low. Although many different drugs have been
`shown to induce penile erection, they are only effective after direct injection into
`the penis, e.g. intraurethrally or intracavemosally (i.e.), and are not approved for
`erectile dysfunction. Current medical treatment is based on the i.e. injection of
`vasoactive substances and good
`results have been claimed with
`phenoxybenzamine, phentolamine, papaverine and prostaglandin E1, either
`alone or in combination; however, pain, priapism and fibrosis of the penis are
`associated with the i.e. administration of some of these agents. Potassium
`channel openers (KCO) and vasoactive intestinal polypeptide (VIP) have also
`been shown to be active i.e., but cost and stability issues could
`limit
`development ~f the latter. An alternative to the i.e. route is the use of glyceryl
`trinitrate (GTN) patches applied to the penis, which has been shown to be
`effective but produces side-effects in both patient and partner.
`
`As a general alternative to pharmacological intervention, a variety of penile
`prostheses has been used to assist achievement of an erection. The short term
`success rate is good, but problems with infection and ischaemia, especially in
`diabetic men, make this type of treatment a final option rather than first-line
`therapy.
`
`The compounds of the invention are potent inhibitors of cyclic guanosine 3',5'(cid:173)
`monophosphate phosphodiesterases (cGMP PDEs). GB 9514464.8, which is
`the priority document for the present application describes the syntheses of the
`compounds of the invention and their utility in impotence. W095/19978, which
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`PCT/EP96/03024
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`was unpublished at the priority date of the present application, also describes
`the syntheses of the compounds of the invention and their utility in other
`diseases associated with inhibition of cGMP PDEs. The compounds may be
`represented by the following general formula (I):
`
`0
`
`(I)
`
`and salts and.solvates (e.g. hydrates) thereof, in which:
`. RO represents hydrogen, halogen or c1-6 alkyl;
`R1 represents hydrogen, C1-6alkyl, C2-ealkenyl, C2_6 alkynyl, haloC1-6alkyl,
`C3-scycloalkyl, C3-scycloalkyiC1-3alkyl, aryiC1-3alkyl or heteroaryiC1-3alkyl;
`R2 represents an optionally substituted monocyclic aromatic ring selected
`from benzene, thiophene, furan and pyridine or an optionally substituted bicyclic
`
`m>
`
`attached to the rest of the molecule via one of the benzene
`ring
`ring carbon atoms and wherein the fused ring A is a 5- or 6-membered ring
`which may be saturated or partially or fully unsaturated and comprises carbon
`atoms and optionally one or two heteroatoms selected from oxygen, sulphur and
`nitrogen; and
`R3 represents hydrogen or C1_3 alkyl, or R 1 and R3 together represent a 3- or
`4- membered alkyl or alkenyl chain.
`
`Suitable individual compounds of the invention for use in the treatment of
`erectile dysfunction include:
`Cis-2,3,6, 7, 12, 12a-hexahydro-2-( 4-pyridylmethyl)-6-(3,4-methylenedioxyphenyl)(cid:173)
`pyrazino[2', 1': 6, 1]pyrido[3,4-b]indole-1 ,4-dione;
`Cis-2,3,6,7, 12, 12a-hexahydro-6-(2,3-dihydrobenzo[b ]furan-5-yl)-2-methyl(cid:173)
`pyrazino[2',1':6,1 ]pyrido[3,4-b ]indole -1 ,4-dione;
`Cis-2,3,6, 7 ,12,12a-hexahydro-6-(5-bromo-2-thienyl)-2-methyl(cid:173)
`pyrazino[2',1':6,1 ]pyrido[3,4-b ]indole -1 ,4-dione;
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`Cis-2,3,6, 7, 12, 12a-hexahydro-2-butyl-6-(4-methylphenyl)(cid:173)
`pyrazino[2', 1 ':6, 1 ]pyrido[3,4-b ]indole -1 ,4-dione;
`(6R, 12aR}-2,3,6, 7, 12, 12a-Hexahydro-2-isopropyl-6-(3,4-methylenedioxyphenyl}(cid:173)
`pyrazino[2', 1':6, 1]pyrido[3,4-b]indole -1 ,4-dione;
`(6R, 12aR}-2,3,6,7, 12, 12a-Hexahydro-2-cyclopentyl-6-(3,4-
`methylenedioxyphenyl)-pyrazino[2', 1 ':6, 1 ]pyrido[3,4-b ]indole -1 ,4-dione;
`(6R, 12aR}-2,3,6, 7, 12, 12a-Hexahydro-2-cyclopropylmethyl-6-( 4-methoxyphenyl)(cid:173)
`pyrazino[2', 1':6, 1]pyrido[3,4-b]indole -1 ,4-dione;
`(6R, 12aR)-2,3,6, 7, 12, 12a-Hexahydro-6-(3-chloro-4-methoxyphenyl)-2-methyl-
`pyrazino[2', 1 ':6, 1 ]pyrido[3,4-b]indole -1 ,4-dione;
`(6R, 12aR)-2,3,6, 7, 12, 12a-Hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)(cid:173)
`pyrazino[2', 1 ':6, 1 ]pyrido[3,4-b ]indole-1 ,4-dione;
`(6R, 12aR)-2,3,6,7, 12, 12a-Hexahydro-6-(3,4-methylenedioxyphenyl)(cid:173)
`pyrazino[2', 1': 6,1] pyrido [3,4-b] indole-1,4-dione;
`(5aR, 12R, 14a8)-1 ,2,3,5,6,11, 12, 14a-Octahydro-12-(3,4-
`methylenedioxyphenyl}-pyrrolo[1 ",2" : 4' 15']pyrazino[2' I 1' : 61 1 ]pyrido[3,4-
`b]indole-5-1 ,4-dione;
`Cis-213161 7 I 121 12a-hexahydro-2-cyclopropyl-6-(3~4-methylenedioxyphenyl)­
`pyrazino[2'1 1':61 1]pyrido[3~4-b]indole -1,4-dione;
`(38, 6R, 12aR)-21316,71 121 12a-hexahydro-3-methyl-6-(3~4-
`methylenedioxyphenyl)-pyrazino[2', 1 ':61 1 ]pyrido[3~4-b]indole -1 ,4-dione;
`and physiologically acceptable salts and solvates (e.g. hydrates) thereof.
`
`The specific compounds of the invention are:
`
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`
`(6RI 12aR}-213161 71121 12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-
`pyrazino[2', 1':61 1]pyrido[3,4-b]indole -1 ,4-dione (Compound A); and
`
`(38, 6R1 12aR)-2131617, 12, 12a-hexahydro-213-dimethyl-6-(3,4-
`methylenedioxyphenyl)-pyrazino[2' I 1' : 6, 1]pyrido[3,4-b]indole-1 ,4-dione
`(Compound B);
`
`and physiologically acceptable salts and solvates (e.g. hydrates) thereof.
`
`30
`
`Unexpectedly, it has now been found that compounds of formula (1), and in
`particular compounds A and B, are useful
`in
`the treatment of erectile
`dysfunction. Furthermore the compounds may be administered orally, thereby
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`obviating the disadvantages associated with i.e. administration. Thus the
`present invention concerns the use of compounds of formula (I), and in
`particular compounds A and 8, or a pharmaceutically acceptable salt thereof, or
`a pharmaceutical composition containing either entity, for the manufacture of a
`medicament for the curative or prophylactic treatment of erectile dysfunction in a
`male animal, including man.
`
`The pharmaceutically acceptable salts of the compounds of formula (1), and in
`particular compounds A and 8 which contain a basic centre are acid addition
`salts formed with pharmaceutically acceptable acids. Examples include the
`hydrochloride, hydrobromide, sulphate or bisulphate, phosphate or hydrogen
`phosphate, acetate, benzoate, succinate, fumarate, maleate, lactate, citrate,
`tartrate,
`gluconate,
`methanesulphonate,
`benzenesulphonate
`and
`p-toluenesulphonate salts. Compounds of formula
`in particular
`(1), and
`compounds A and 8 can also provide pharmaceutically acceptable metal salts,
`in particular alkali metal salts, with bases. Examples include the sodium and
`potassium salts.
`
`It has been shown that compounds of the present invention are potent and
`It has now been surprisingly found
`selective inhibitors of cGMP specific POE.
`that human corpus cavernosum contains three distinct POE enzymes. The
`predominant POE has further surprisingly been found to be cGMP POE. As a
`consequence of the selective POE V inhibition exhibited by compounds of the
`present invention, the subject compounds can elevate cGMP levels, which in
`turn can mediate relaxation of the corpus cavernosum tissue and consequent
`penile erection.
`
`Although the compounds of the invention are envisaged primarily for the
`treatment of erectile dysfunction or male sexual dysfunction, they may also be
`useful for the treatment of female sexual dysfunction including orgasmic
`dysfunction related to clitoral disturbances.
`
`Generally, in man, oral administration of the compounds of the invention is
`the preferred route, being the most convenient and avoiding the disadvantages
`associated with i.e. administration. In circumstances where the recipient suffers
`from a swallowing disorder or from impairment of drug absorption after oral
`administration, the drug may be administered parenterally, e.g. sublingually or
`buccally.
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`For administration to man in the curative or prophylactic treatment of the
`disorders identified above, oral dosages of a compound of formula (1), and in
`particular compounds A and B will generally be in the range of from 0.5-BOOmg
`daily for an average adult patient (70kg). Thus for a typical adult patient,
`individual tablets or capsules contain from 0.2-400mg of active compound, in a
`suitable pharmaceutically acceptable vehicle or carrier, for administration in
`single or multiple doses, once or several times per day. Dosages for buccal or
`sublingual administration will typically be within the range of from 0.1-400 mg
`per single dose as required.
`In practice the physician will determine the actual
`dosing regimen which will be most suitable for an individual patient and it will
`vary with the age, weight and response of the particular patient. The above
`dosages are exemplary of the average case but there can be individual
`instances in which higher or lower dosage ranges may be merited, and such are
`within the scope of this invention.
`
`For human use, compounds of formula (I), and in particular compounds A and
`B can be administered alone, but will generally be administered in admixture
`with a pharmaceutical carrier selected with regard to the intended route of
`administration and standard pharmaceutical
`practice.
`For example, the
`compound may be administered orally, buccally or sublingually, in the form of
`tablets containing excipients such as starch or lactose, or in capsules or ovules
`either alone or in admixture with excipients, or in the form of elixirs or
`suspensions containing flavouring or colouring agents. Such liquid preparations
`may be prepared with pharmaceutically acceptable additives such as
`suspending agents (e.g. methylcellulose, a semi-synthetic glyceride such as
`witepsol or mixtures of glycerides such as a mixture of apricot kernel oil and
`PEG-6 esters or mixtures of PEG-8 and caprylic/capric glycerides).
`
`For veterinary use, a compound of formula (I), and in particular compound A
`or 8 or a non-toxic salt thereof is administered as a suitably acceptable
`formulation in accordance with normal veterinary practice and the veterinary
`surgeon will determine the dosing regimen and route of administration which will
`be most appropriate for a particular male animal.
`
`Thus the invention includes a pharmaceutical composition for the curative or
`prophylactic treatment of erectile dysfunction in a male animal, including man,
`comprising a compound of formula (1), and in particular compound A or 8, or a
`pharmaceutically acceptable salt thereof, together with a pharmaceutically
`acceptable diluent or carrier.
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`There is further provided a process for the preparation of a pharmaceutical
`composition for the curative or prophylactic treatment of erectile dysfunction in a
`male animal, including man, comprising formulating a compound of formula (1),
`and in particular compound A or 8, or a pharmaceutically acceptable salt
`thereof, with a pharmaceutically acceptable diluent or carrier.
`
`The invention also provides a method of treating a male animal, including
`man, to cure or prevent erectile dysfunction which comprises treating said male
`animal with an effective amount of a compound of formula (1), and in particular
`compound A or 8, or a pharmaceutically acceptable salt thereof, or a
`pharmaceutical composition containing either entity.
`
`Moreover, the invention includes the use of a compound of formula (1), and in
`particular compound A or 8, or a pharmaceutically acceptable salt thereof, or a
`pharmaceutical composition containing either entity, for the manufacture of a
`medicament for the curative or prophylactic treatment of erectile dysfunction in a
`male animal, including man.
`
`A compound of formula (1), and in particular compound A or 8, may also be
`used in combination with other therapeutic agents which may be useful in the
`treatment of erectile dysfunction substantially as hereinbefore described. The
`invention thus provides, in another aspect, a combination of a compound of
`formula
`(1), and
`in particular compound A or 8
`together with another
`therapeutically active agent.
`
`The combination referred to above may conveniently be presented for use in
`the form of a pharmaceutical formulation and thus pharmaceutical compositions
`comprising a combination as defined above together with a pharmaceutically
`acceptable diluent or carrier comprise a further aspect of the invention.
`
`The individual components of such a combination may also be administered
`either sequentially or simultaneously in separate pharmaceutical formulations.
`
`Appropriate doses of known therapeutic agents for use in combination with a
`compound of the invention will be readily appreciated by those skilled in the art.
`
`The compounds of the invention may be prepared by any suitable method
`known in the art or by the following process which forms part of the present
`invention. The process has been previously substantially described in the
`priority document of the present invention 889514464.8, and in W095/19978.
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`Thus, a process for preparing a compound of formula (I) comprises treating a
`compound of formula (II)
`
`0
`
`(II)
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`(in which Alk represents C1-salkyl, e.g. methyl or ethyl and Hal is a halogen
`atom, e.g. chlorine) with a primary amine R1NH2 in a suitable solvent such as
`an alcohol (e.g. methanol or ethanol) or a mixture of solvents, conveniently at a
`temperature of from 2ooc to reflux (e.g. at about sooc).
`
`A compound of formula (II) may conveniently be prepared by treating a
`compound of formula (Ill) with a compound of formula (IV)
`
`0
`
`OAik
`
`0
`
`HaiYyHal
`3
`(IV) R
`
`in a suitable solvent such as a halogenated hydrocarbon (e.g. trichloromethane
`or dichloromethane), or an ether (e.g. tetrahydrofuran), preferably in the
`presence of a base such as an organic amine (e.g. a trialkylamine such as
`triethylamine) or an alkali metal carbonate or bicarbonate (e.g. NaHC03). The
`reaction may conveniently be effected at a temperature of from -20°C to +200C
`(e.g. at about OOC).
`
`A compound of formula (I) may also be prepared from a compound of formula
`(Ill) in a two-step procedure via a compound of formula (II) isolated without
`purification.
`
`Compounds of formula (I) may be prepared as individual enantiomers in two
`steps from the appropriate enantiomer of formula (Ill) or as mixtures (e.g.
`racemates) of either pairs of cis or trans isomers from the correspondong
`mixtures of either pairs of cis or trans isomers of formula (Ill).
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`Individual enantiomers of the compounds of the invention may be prepared
`from racemates by resolution using methods known in the art for the separation
`of racemic mixtures into their constituent enantiomers, for example using HPLC
`(high perform~nce liquid chromatography) on a chiral column such as Hypersil
`naphthylurea.
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`A compound of formula (Ill) may conveniently be prepared from a tryptophan
`alkyl ester of formula M
`
`0
`
`OAik
`
`(V)
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`(where Alk is as previously defined) or a salt thereof (e.g. the hydrochloride salt)
`with an aldehyde R2CHO. The reaction may conveniently be effected in a
`suitable solvent such as a halogenated hydrocarbon (e.g. dichloromethane) or
`an aromatic hydrocarbon (e.g. toluene) in the presence of an acid such as
`trifluoroacetic acid. The reaction may conveniently be carried out at a
`temperature of from -2ooc to reflux to provide a compound of formula (Ill) in one
`step. The reaction may also be carried out in a solvent such as an aromatic
`hydrocarbon (e.g. benzene or toluene) under reflux, optionally using a Dean(cid:173)
`Stark apparatus to trap the water produced.
`
`The reaction provides a mixture of cis and trans isomers which may be either
`individual enantiomers or racemates of pairs of cis or trans isomers depending
`upon whether racemic or enantiomerically pure tryptophan alkyl ester was used
`as the starting material. Individual cis or trans enantiomers may conveniently be
`separated
`from mixtures
`thereof by
`fractional crystallisation or by
`chromatography (e.g. flash column chromatography) using appropriate solvents
`and eluents. Similarly, pairs of cis and· trans isomers may be separated by
`chromatography (e.g. flash column chromatography) using appropriate eluents.
`An optically pure trans isomer may also be converted to an optically pure cis
`isomer using suitable epimerisation procedures. One such procedure comprises
`treating the trans isomer or a mixture (e.g. 1 : 1 mixture) of cis and trans isomers
`with methanolic or aqueous hydrogen chloride at a temperature of from ooc to
`the refluxing temperature of the solution. The mixture may then be subjected to
`chromatography (e.g. flash column chromatography) to separate the resulting
`diastereoisomers, or in the procedure utilising aqueous hydrogen chloride the
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`desired cis isomer precipitates out as the hydrochloride salt which may then be
`isolated by filtration.
`
`The pharmaceutically acceptable acid addition salts of a compound of
`formula (1), and in particular compound A or 8 which contain a basic centre may
`be prepared in a conventional manner. For example, a solution of the free base
`may be treated with a suitable acid, either neat or in a suitable solution, and the
`resulting salt isolated either by filtration or by evaporation under vacuum of the
`reaction solvent. Pharmaceutically acceptable base addition salts may be
`obtained in an analogous manner by treating a solution of compound A or 8
`with a suitable base. Both types of salt may be formed or interconverted using
`ion-exchange resin techniques.
`
`Compounds of the invention may be isolated in association with solvent
`molecules by crystallisation from or evaporation of an appropriate solvent.
`
`The syntheses of compounds A and 8 and of the intermediates for use
`therein are illustrated by the following examples. The examples have been
`in
`the priority document of the
`instant
`invention
`previously described
`GB9514464.8, and the corresponding Intermediate or Example numbers therein
`are shown in parentheses ·next to the current Intermediate or Example number.
`
`In the Examples section hereinafter the following abbreviations are used:
`
`20
`
`MeOH (methanol) and EtOH (ethanol),
`
`Intermediate 1 (54)
`
`( 1 R. 3R)-Methyl 1.2. 3 .4-tetrahydro-1-(3 .4-methylenedjoxyphenyi)-9H-pyrido[3 .4-
`b]indole-3-carboxylate. cis isomer
`
`25
`
`30
`
`To a stirred solution of 0-tryptophan methyl ester (11 g) and piperonal (7.9 g) in
`anhydrous CH2CI2 (400 ml) cooled at ooc was added dropwise trifluoroacetic
`acid (7.7 ml) and the solution was allowed to react at ambient temperature.
`After 4 days, the yellow solution was diluted with CH2CI2 (200 ml) and washed
`with a saturated aqueous solution of NaHC03, then with water (3x200 ml) and
`dried over Na2S04. The organic layer was evaporated under reduced pressure
`and the residue containing the two geometric isomers was purified by flash
`
`

`

`W097/03675
`
`PCTIEP96/03024
`
`10
`
`chromatography eluting with dichloromethane/ethyl acetate (97/3) to give as the
`firsr eluting product the title compound (6.5 g)
`
`m.p.: 154°C
`
`Intermediate 2 (83)
`
`5
`
`(1 R. 3R)-Methyl 1 .2.3.4-tetrahydro-2-(2-chloropropionyl)-1-(3.4-
`methylenedioxyphenyi)-9H-pyrjdo[3.4-b]indole-3-carboxylate
`
`1 0
`
`To a solution of (R)-(+)-2-chloropropionic acid (191 ~1. 2.2 mmol) in anhydrous
`dichloromethane (30 mL), was added dicyclohexylcarbodiimide (0.45 g,
`2.2. mol). Intermediate 1 (0,7 g, 2 mmol) was then added and the mixture was
`formed precipitate of
`stirred at room
`temperature
`for 20 hours. The
`dicyclohexylurea was removed by filtration, the filtrate was evaporated in vacuo
`and the crude product was purified by flash chromatography eluting with
`toluene/ethyl acetate : 95/5. The oily compound obtained was then crystallised
`from ether/hexane to give the title compound as pale yellow crystals (0.74 g)
`
`15
`
`m.p. : 126-128°C.
`
`Example 1 {78) {Compound A)
`
`(6R.12aR)-2,3,6. 7, 12, 12a-Hexahydro-2-methyl-6-(3.4-methylenedioxyphenyl)(cid:173)
`pyrazino[2' ,1':6.1 ]pyrido[3.4-b]indole -1.4-djone
`
`20
`
`25
`
`30
`
`a) To a stirred solution of intermediate 1 (0.5 g) and NaHC03 (0.14 g) in
`anhydrous CHCI3 (20 mL) was added dropwise chloroacetyl chloride (0.27 mL)
`at 0°C. The resulting mixture was stirred for 1 hour at the same temperature
`and diluted with CHCI3 (20 mL). Water (1 0 mL) was then added dropwise with
`stirring to the mixture, followed by a saturated solution of NaHC03. The organic
`layer was washed with water until neutrality and dried over Na2S04. After
`evaporation of the solvent under reduced pressure, {6R.12aR)-methyl 1 ,2,3.4-
`tetrahydro-2-chloroacetyl-1-(3.4-methylenedjoxyphenyi)-9H-pyrido[3.4-b)indole-
`3-carboxylate was obtained as an oil which was crystallised from ether to give a
`solid (0.38 g, m.p. : 233°C) which was used without further purification in the
`next step.
`
`b) To a stirred suspension of the chloroacetyl intermediate (0.37 g) in MeOH
`(20 ml) was added at room temperature a solution of methylamine (33% in
`
`

`

`

`

`

`

`W097/03675
`
`PCT/EP96/03024
`
`13
`
`2.
`
`mg/tablet
`
`Active ingredient
`
`Colloidal Silicon Dioxide
`
`Crospovidone
`
`Sodium Lauryl Sulphate
`
`Magnesium Stearate Ph Eur
`
`Microcrystalline Cellulose USNF
`
`50.0
`
`0.5
`
`8.0
`
`1.0
`
`1.0
`
`139.5
`
`The active ingredient was sieved and blended with the excipients. The
`resultant mix was compressed into tablets.
`
`B.
`
`WET GRANULATION
`
`5
`
`1.
`
`Active ingredient
`
`Polyvinyl pyrollidone
`
`Polyethylene glycol
`
`Polysorbate 80
`
`Magnesium Stearate Ph Eur
`
`Croscarmellose Sodium
`
`Colloidal Silicon Dioxide
`
`Microcrystalline Cellulose USNF
`
`mg/tablet
`
`50.0
`
`150.0
`
`50.0
`
`10.0
`
`2.5
`
`25.0
`
`2.5
`
`210.0
`
`

`

`W097/03675
`
`PCT/EP96/03024
`
`14
`
`The polyvinyl pyrollidone, polyethylene glycol and polysorbate 80 were
`dissolved in water. The resultant solution was used to granulate the
`active ingredient. After drying the granules were screened, then extruded
`at elevated temperatures and pressures. The extrudate was milled
`and/or screened then was blended with the microcrystalline cellulose,
`croscarmellose sodium, colloidal silicon dioxide and magnesium stearate.
`The resultant mix was compressed into tablets.
`
`2.
`
`Active ingredient
`
`Polysorbate 80
`
`Lactose Ph Eur
`
`Starch BP
`
`Pregelatinised Maize Starch BP
`
`Magnesium Stearate BP
`
`mg/tablet
`
`50.0
`
`3.0
`
`178.0
`
`45.0
`
`22.5
`
`1.5
`
`The active ingredient was sieved and blended with the lactose, starch
`and pregelatinised maize starch. The polysorbate 80 was dissolved in
`purified water. Suitable volumes of the polysorbate 80 solution were
`added and the powders were granulated. After drying, the granules were
`screened and blended with the magnesium stearate. The granules were
`then compressed into tablets.
`
`Tablets of other strengths may be prepared by altering the ratio of active
`ingredient to the other excipients.
`
`5
`
`10
`
`15
`
`FILM COATED TABLETS
`
`The aforementioned tablet formulations were film coated.
`
`Coating Suspension
`
`%w/w
`
`

`

`wo 97/03675
`
`PCT/EP96/03024
`
`15
`
`•
`
`Opadry whitet
`
`Purified water Ph Eur
`
`13.2
`
`to 100.0*
`
`* The water did not appear in the final product. The maximum theoretical weight
`of solids applied during coating was 20mg/tablet.
`
`t Opadry white is a proprietary material obtainable from Colorcon Limited, UK
`which contains hydroxypropyl methylcellulose, titanium dioxide and triacetin.
`
`5
`
`The tablets were film coated using the coating suspension in conventional film
`coating equipment.
`
`CAPSULES
`
`10
`
`1.
`
`Active ingredient
`
`Lactose
`
`Polyvinyl pyrollidone
`
`Magnesium Stearate
`
`mg/capsule
`
`50.0
`
`148.5
`
`100.0
`
`1.5
`
`The active ingredient was sieved and blended with the excipients. The mix was
`filled into size No. 1 hard gelatin capsules using suitable equipment.
`
`2.
`
`Active ingredient
`
`Microcrystalline Cellulose
`
`Sodium Lauryl Sulphate
`
`mg/capsule
`
`50.0
`
`233.5
`
`3.0
`
`

`

`W097/03675
`
`PCTIEP96/03024
`
`16
`
`Crospovidone
`
`Magnesium Stearate
`
`12.0
`
`1.5
`
`•
`
`The active ingredient was sieved and blended with the excipients. The mix was
`filled into size No. 1 hard gelatin capsules using suitable equipment.
`
`Other doses may be prepared by altering the ratio of active ingredient to
`excipient, the fill weight and if necessary changing the capsule size.
`
`5
`
`3.
`
`Active ingredient
`
`Labrafil M 1944CS
`
`mg/capsule
`
`50.0
`
`to 1.0 ml
`
`The active ingredient was sieved and blended with the Labrafil. The suspension
`was filled into soft gelatin capsules using appropriate equipment.
`
`10
`
`15
`
`20
`
`Inhibitory effect on cGMP-PDE
`cGMP-PDE activity of compounds of the present invention was measured using
`a one-step assay adapted from Wells at al. (Wells, J. N., Baird, C. E., Wu, Y. J.
`and Hardman, J. G., Biochim. Biophys. Acta 384, 430 (1975)). The reaction
`medium contained 50mM Tris-HCI,pH 7.5, 5mM Mg-acetate, 250J..Lglml 5'-
`Nucleotidase, 1mM EGTA and 0.15J..LM 8-[H3]-cGMP. The enzyme used was a
`human recombinant PDE V (ICOS, Seattle USA).
`
`Compounds of the invention were dissolved in DMSO finally present at 2% in
`the assay. The incubation time was 30 minutes during which the total substrate
`conversion did not exceed 30%.
`
`The
`IC50 values
`from
`the compounds examined were determined
`for
`concentration-response curves using typically concentrations ranging from 1 OnM
`to 1 OJ..LM. Tests against other PDE enzymes using standard methodology also
`
`

`

`W097/03675
`
`PCT/EP96/03024
`
`17
`
`showed that compounds of the invention are highly selective for the cGMP
`specific POE enzyme.
`
`-cGMP level measurements
`Rat aortic smooth muscle cells (RSMC) prepared according to Charnley et al. in
`Cell Tissue Res. 177, 503 - 522 (1977) were used between the 10th and 25th
`passage at confluence in 24-well culture dishes. Culture media was aspirated
`and replaced with PBS (0.5ml) containing
`the compound tested at the
`appropriate concentration. After 30 minutes at 37°C, particulates guanylate
`cyclase was stimulated by addition of ANF (1 OOnM) for 10 minutes. At the end
`of incubation, the medium was withdrawn and two extractions were performed
`by addition of 65% ethanol (0.25ml). The two ethanolic extracts were pooled
`and evaporated until dryness, using a Speed-vac system.
`c-GMP was
`measured
`after acetylation
`by
`scintillation
`proximity
`immunoassay
`(AMERSHAM).
`
`The compounds according to the present invention were typically found to
`exhibit an IC50 value of less than 500nM, and an EC50 value of less than 5.
`In
`vitro test data for representative compounds of the invention is given in following
`Table 1:
`
`5
`
`10
`
`15
`
`20
`
`Table 1
`
`Example No.
`1
`2
`
`IC50 nM
`2
`2
`
`EC50 J.LM
`0.2
`0.2
`
`25
`
`The above data demonstrates the ability of the subject compounds of the
`invention to inhibit cGMP POE, and hence their utility in the treatment of erectile
`dysfunction substantially as hereinbefore described.
`
`

`

`W097/03675
`
`PCT/EP96/03024
`
`18
`
`CLAIMS
`
`1. Use of a compound of formula (I):
`
`Ro
`
`0
`
`N-R1
`
`3
`.Nv-R
`0
`
`(I)
`
`and salts and solvates (e.g. hydrates) thereof, in which:
`RO represents hydrogen, halogen or c1-6 alkyl;
`R 1 represents hydrogen, C1-5alkyl, C2_6 alkenyl, C2_6 alkynyl, haloC1-6alkyl,
`C3-8cycloalkyl, C3-scycloalkyiC1-3alkyl, aryiC1-3alkyl or heteroaryiC1-3alkyl;
`R2 represents an optionally substituted monocyclic aromatic ring selected
`from benzene, thiophene, furan and pyridine or an optionally substituted bicyclic
`
`0~
`
`attached to the rest of the molecule via one of the benzene
`ring
`ring carbon atoms and wherein the fused ring A is a 5- or 6-membered ring
`which may be saturated or partially or fully unsaturated and comprises carbon
`atoms and optionally one or two heteroatoms selected from oxygen, sulphur and
`nitrogen; and
`R3 represents hydrogen or C1_3 alkyl, or R 1 and R3 together represent a 3- or
`4- membered alkyl or alkenyl chain;
`
`for the manufacture of a medicament for the curative or prophylactic treatment
`of er