AU628400B2 - Prostaglandin-derivatives having antithrombotic activity - Google Patents

Description

I ANNOI II~FcgAFzITt- 7t--- *TI-4F I A T~0 011 I/ A~ 1IP~A 4,5 1 5r/ F OF INTERNATIONAL SEARCH REPORTS WORLD INTELLECTUAL PROPERTY ORGANIZATION International Bureau

PC

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 90/03170 A61K 31/557, C07C 229/46 A3 (43) International Publication Date: 5 April 1990 (05.04.90) (21) International Application Number: PCT/1T89/00061 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), (22) International Filing Date: 12 September 1989 (12.09.89) DK, FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), SE (European patent), SU, US.

Priority data: 22012 A/88 20 September 1988 (20.09.88) IT 20927 A/89 20 June 1989 (20.06.89) IT Published (71) Applicant (for all designated States except US): IBI ISTITU- With international search reporL TO BIOCHIMICO ITALIANO GIOVANNI LOREN- Before the expiration of the time limit for amending the ZINI S.P.A. [IT/IT]; Via G. Lorenzini, 2-4, I-Milan claims and to be republished in the event of the receipt of amendments.

(72) Inventor; and Inventor/Applicant (for US only) VALCAVI, Umberto [IT/ (88) Date of publication of the international search report: IT]; V. le Bianca Maria, 21, I-Milan 17 May 1990 (17.05.90) (74) Agents: KLAUSNER, Erich et al.; Ufficio Internazionale Brevetti Ing. C. Gregorj Via Dogana, 1, 1-20123 Milan (IT).AGS H G (54)Title: PROSTAGLANDIN-DERIVATIVES HAVING ANTITHROMBOTIC ACTIVITY (57) Abstract The compounds of formula wherein: n 2, 3; m 4, 5, 6, 7; X NHR, CH 2 NHR, NHNH 2 R H, a linear or branched (C 1

-C

4 )alkyl, a linear or branched (Ci-C 4 )acyl; Y CH 2

NH

2 COOH, CONH 2 (with the exclusion of the compounds wherein n 2, m 5, X NHR with R H, a linear or branched (Ci-C 4 )alkyl, a linear or branched (Ci-C 4 )acyl; Y COOH, CONH 2 including all optical and/or geometrical isomers, alone or in admixture, and their salts, which inhibit the platelet aggregation in the blood and which can be prepared for use as antiplatelet, antithrombotic agents.

r 'WO 90/03170 PCT/IT89/00061 1 PROSTAGLANDIN-DERIVATIVES HAVING ANTITHROMBOTIC ACTIVITY The instant invention refers to the activity and use of compounds having a prostaglandin-like structure as well as pharmaceutical compositions thereof, having platelet antiaggregating, antithrombotic activity.

It is known that substances such as ADP, collagen, thrombin arachidonic acid, favour platelet aggregating phenomena causing the formation of thrombi which are mainly responsible for ischemic,cardio- and cerebro-vascular disorders,atherosclerotic peripheral arterial disorders and venous thrombosis. Thus, substances which are capable of antagonizing their effects are of considerable therapeutic interest.

In the course of studies carried out by the Applicants on prostaglandin-like compounds, possessing antiulcer activity, described in IT 1,190,400 filed on October 4,1985 and in IT 1,205,183 filed on June 25,1987 (corresponding to the PCT patent application No. IT88/00010 of February 10,1988), both owned by the Applicants, it has been discovered that some of the compounds, disclosed therein and corresponding to formula(I) of the instant application, possess antiplatelet, antithrombotic activity.

The compounds of formula (I)

CH

2 m-CH 3 WO 90/03170 PCT/IT89/00061 -2 wherein: n 2,3; m 4,5,6,7; X NHR, CH 2NHR, NHNH2, wherein R H, a linear or branched (C -C )alkyl, a linear or branched (CI-C acyl; Y CH2NH2, COOH, CONH 2 (with the exclusion of the compounds wherein n 2, m 5, X NHR with R H, a linear or branched (C -C )alkyl, a linear or branched (C -C )acyl; Y COOH, CONH2) possess 3 asymmetric carbon atoms identified by an asterisc, wherefore 8 stereoisomers are possible.

Therefore, when not otherwise specified, the above formula includes all possible stereoisomers, all possible combinations of two or more such stereoisomers, of enanthiomers or mixtures of enanthiomers in any proportion.

The four pairs of possible enanthiomers indicated by the code Nos.IBI-P-(I)A, IBI-P-(I)B, IBI-P-(I)C, IBI-P-(I)D, are represented as follows:

I

IBI-P-(I)A

(CH2

)MCH

2 3 ~(CH 2

)CH

3

(CH

2 )n (CH H 'H x 2

X

(cis-cis) (cis-cis) (CH CH3 IBI-P-(I)B (CH n

X

(trans-trans) CH CH 3

(CH

2 n (CH 2 mCH3 (trans-trans) i *-liam^.Btw WO 90/03170

IBI-P-(I)C

PCT/1T89/00061 3 (CH)mCH3 (CH,)n 2sE

I

:CH 2 )mCH 3 (trans-cis) (trans-cis)

IBI-P-(I)D

C H 2 mC

H

3

(CH

2 n

Y

X

(CH2

CH

(CH (cis-trans) (cis-trens) wherein, considering the planar or almost planar ring, lying in the horizontal plane of the sheet, as is customary in chemical literature, the broken lines indicate that a particular substituent lies below the molecular plane, while the full lines in the form of a wedge indicate that a particular substituent lies above the molecular plane. The prefices cis and trans are used to indicate the relative position of the substituents with respect to one another.

More particularly,the instant invention relates to prostanoic compounds of the above cited formula useful for preparing pharmaceutical compositions having antiplatelet, antithromboti 1- WO 90/03170 PL-r/M/00061 -4ic activity.

It is reiterated that the compounds of formula.(I) are the subject matter of IT 1,190,400 filed on October 4,1985 and of IT 1,205,183 filed on June 25,1987, of which Applicants are the proprietors, wherein their activity as antiulcer agents and their use for preparing pharmaceutical compositions are described, and, therefore, they are not being claimed, per se, herein, whereas Applicants wish to protect their use as inhibitors of platelet aggregation and antithrombotic agents.

The compounds of formula wherein n 2, m 5, X NHR with R H, a linear or branched (C -C4)alkyl, a linear or branched (C1-C4)acyl, Y COOH, CONH 2 fall within the scope of IT 1,060,366 filed on August 7,1974, owned by Applicants, which discloses their activity as inhibitors of platelet aggregation and hence such compounds are excluded from the present invention.

The present invention includes also the pharmaceutically acceptable cationic salts of the compounds of formula when Y COOH and, in general, the pharmaceutically acceptable anionic salts.

As used herein, the expression "pharmaceutically acceptable cationic salts" refers to the alkali and alkaline-earth metal salts such as, e.g. sodium; potassium, calcium, magnesium, or salts of aluminum, ammonium, zinc and of organic amines, including the amino acids such as, e.g. lysine,arginine, phenylalaline and proline, triethanol amine, inner salts and salts of basic resins.

As used herein, the expression "pharmaceutically acceptable anionic salts" refers to salts obtained by the addition of hydrochloric, hydrobromic, nitric, phosphoric, sulfuric, a

IYI

WO 90/03170 5 pCT/IT89/00061 benzenesulfonic, benzoic, citric, laurylsulfonic, fumaric,oxalic, maleic, methanesulfonic, tartaric, ascorbic, p-toluenesulfonic, salicylic and succinic acid.

With polybasic acids, the salt may include more than one mole of base per mole of acid. However, the salts obtained from one mole of acid per mole of the inventive compound are preferred.

The present invention, lastly, refers to pharmaceutical compositions containing a compound of formula or a pharmaceutically acceptable anionic or cationic salt thereof.

The preparation of some of the compounds of formula has already been described in IT 1,190,400 of October 4,1985,and IT 1,205,183 filed on June 25,1987.

For example, in said patents is described the preparation of the compounds of formula wherein: n 2, m 5, X CH2NH2, Y COOH (IBI-P-01058) n 2, m 5, X NHNH 2 Y COOH (IBI-P-01062) n 2, m 5, X NH 2 Y CH2NH 2 (IBI-P-01068) n 3, m 5, X NH 2 Y COOH (IBI-P-05006) n 3, m 5, X CH2NH 2 Y COOH (IBI-P-05011) n 3, m 5, X NH 2 Y CH2NH 2 (IBI-P-05012) n 2, m 6, X NH2, Y COOH (IBI-P-12004) Here below is given, for illustrative purposes only, the preparation of the compounds of formula wherein: n 2, m 4, X NH2, Y COOH (IBI-P-12003) n 2, m 7, X NH2, Y COOH (IBI-P-12005) n 3, m 5, X NH2, Y COOH (IBI-P-05006) which may be prepared according to the reaction scheme I SCHEME I -)Cn YCH __R.Cn 2 mH3 1o y, ec C y, 1

H

2

IV

WO 90/03170 pCTr/1T9/00061 wheeinY' CH2 NH2' CONH 2 COORI and RI a linear or branched H(C C4 )alkcyl.

The cycloalkenone of formula when n 2, may be prepared as described in IT patent application 19043 A/84 of January 5,1984 (continuation-in-part U.S.S.N. 117669 of November 1987 of U.S. application S.N. 744406 of June 13,1985) owned by Applicants.

It has now been found that when n 3, Y' COOCH 3 the cycloalkenone of formula (II) may be prepared according to the following reaction scheme II: SCHEME II 2 CH -N,CH OH DO H 0 C H 2 Hal (VI) Hal VI I ,.CH"O"CH p~ C N C 1 2 1 2 2p Hal Mg.Had.

(VIII)

(IX)

(Ox Y. -Coc. CH G 0

N(CH

2

(X)

xi 11 with n 3;Y COOCH 3; Hal Cl,Br,I.

7 r-c /ifon /nnag1 S WO 90/03170 r Lo1107 v.u The monochloride of 8-carbomethoxy-octanoic acid (X)(prepared as disclosed in IT patent application 19043 A/84) is condensed as a Grignard compound of formula (IX) Hal-Mg-(CH 2 3 CH 0 >(CH 2

(IX)

wherein Hal Cl,Br,I; p 2, 3 in inert polar solvents such as, e.g. tetrahydrofuran, ethyl ether, butyl ether, dioxane, dimethylformamide.

The acetal of the 1,5-dicarbonyl compound(XI),prepared by Grignard condensation, can be cyclized by acid catalyzed,intramolecular crotonic condensation, in a suitable solvent, yielding the desired compound of formula (II).

The compounds of formula (IX) may be prepared according to methods known from the literature, for example by monohalogenation of the 1,4-butanediol(V) performed as described by Suk-Ku-Kang in Synthesis,1161 (1985),oxydation of the aldehyde (VII) of the (6-halobutanol (VI) with a catalytic process employing sodium hypochlorite as the oxydizing agent and the free radical 2,2,6,6-tetramethyl-piperidinyl-1-oxyl as the catalyst, as described by P.L.Anelli,C.Biffi, F.Montanari in J.Org.Chem.52,2559 (1987) and the subsequent protection of the carbonyl group of the ccmpounds of formula (VII), with ethylene glycol or with 1,3-propanediol, and the subsequent transformation into a Grignard reagent, viz.

the compound of formula (IX).

As described in IT 1,205,183 filed on June 25,1987, the cycloalkenone of formula (II) may be transformed into the cycloalkanone of formula (III) wherein m 4,5,6 and 7, using a Grignard reagent such as, e.g. CH 3

(CH

2 m MgHal (Hal= C1,B,I) in the presence of copper salts such as,e.g. CuC1, Cu(OAc) 2 ,CuCN, CuI,CuCl 2 Polar 1 WO 90/03170 PCr/T89/00061 8 and inert solvents are generally used such as, e.g. tetrahydrofuran, dimethyl-formamide, ethyl ether, butyl ether.

The compounds of formula (IV) can be prepared from the corresponding cycloalkanones (III) by using per se known reactions such as e.g. reductive aminations with ammonia, hydrogen and metal catalysts, in alcoholic solvents, at a temperature of from 300 to 100°C and at a pressure of from 1 to 20 atms according to reaction scheme I, or according to methods known from the technical literature, according to scheme III: SCHEME III

(CH

2 mCH 3

(CH

2

(CH

2 )mCH 3 Y' (CH )n 1 C3

OH

(XII)

(CH

2 mCH 3 ((CH2XI 0= 0

(XIII)

(III)

_,(CH

2

CH

3 (CH )n

N

3

(XIV)

(CH mCH3 (CH)n

NH

2

(IV)

through reduction of the cycloalkanones of formula (III) to the alcohols (XII) with hydrides selected among lithium aluminum hydride, sodium boronhydride, lithium tri-sec-butyl boronhydride, lithium dicyclohexyl-tert-butyl boronhydride, lithium di-sec-

L

WO 90/03170 PCT/IT89/00061 9 butyl hexyl boronhydride, lithium diisobutyl-tert-butyl boronhydr:de; preparation of the mesilate (XIII),substitution of the mesilate with sodium azide to yield the compounds of formula(XIV) and subsequent reduction with metal catalysts and hydrogen. As disclosed in IT 1,205,183 filed on June 25,1987, the compounds of formula (IV) can subsequently be converted into the compounds of formula e.g. wherein Y COOH and X NH 2 in aqueous, or aqueous alcoholic, or alcoholic solutions, of bases such as,e.g.

NaOH,KOH, K2CO 3 which are capable of hydrolizing the carbalcoxy 2 3' group.

The compounds of formula prepared according to the processes of the instant invention, when not otherwise specified, generally consist of mixtures of the stereoisomers in ratios depending on the route of synthesis, on the reagents used and the experimental conditions.

The desired enanthiomer pairs can be prepared by separating the stereoisomeric mixtures by methods known to the man skilled in the art.

It is, for example, possible to isolate chromatographically a pair of enanthiomers from a mixture containing all the stereoisomers of the compounds of formula or of formula as stationary phase one can use e.g. acid alumina, neutral alumina, basic alumina, cellulose, charcoal, silica, Amberlite XAD 2 (Fluka), Amberlite IRC 50 (Fluka); and as eluent it is possible to use a solvent or a solvent mixture selected among e.g.ethanol, methanol, ethyl acetate, acetone, ethyl ether,methylene chloride, chloroform, hexane, petrol ether, per se or in admixture with bases such as e.g. diethylamine, isopropylamine, triethylamine and ammonium hydroxide. The thus separated enanthiomer pairs of the compounds of formula (IV) yield the corresponding enanthiomer i i~il:_LYiiiC I 1 WO 90/03170 10 PCT/IT89/00061 pairs of formula by saponification with suitable bases, in alcoholic or hydroalcoholic solvents.

It is possible to prepare an enthiometer pair of formula(I) by fractional crystallization of salts of stereoisomer mixtures of the compounds of formula or of formula prepared by addition of inorganic or organic acids selected e.g. among the following acids: hyd:ochloric, hydrobromic, nitric, phosphoric, sulfuric, benzenesulfonic, p-toluenesulfonic, methanesulfonic, acetic, trifluoroacetic,oxalic, maleic, benzoic, in solvents or solvent mixtures selected among e.g. isopropanol, ethanol, methanol, ethyl ether, diisopropyl ether, dioxane, tetrahydrofuran, chloroform, methylene chloride, benzene, toluene, at a temperature of from -200 and +40 0 C and subsequent reaction with bases to yield the desired enanthiomer pairs in their free form.

It is furthermore possible to prepare each enanthiomer pair of the compounds of formula by chromatographically separating the mixture of the stereoisomer alcohols (XII) prepared by reduction with stereoselective reducing agents as described by C.H.Brown, S.Kirishnamurthy in J.Am.Chem.Soc. 7159 (1972), or by reduction with reducing agents having a low steric demand (Scheme III) of the compounds of formula (III) and subsequent conversion into the corresponding mesilate, substitution with sodium azide and reduction with hydrogen and metal catalysts.

The instant invention shall now be further described by the following examples illustrating the preparation of the compounds of formula of the intermediates of formula as well as of the enanthiomer pairs.

Im WO 90/03170 PCT/IT89/00061 11 Example 1 2-(6 -carbomethoxy-n-hexyl)-2-cyclohexene-l-one (a derivative of formula(II) wherein n 3 and Y' CO.OCH 3 a) A suspension of magnesium (19.6 g) in tetrahydrofuran (220 ml) is added with a solution of 2-(4-bromobutyl)-1,3-dioxolane (prepared according to reaction scheme II) in tetrahydrofuran (1600 ml) keeping the temperature at 20-25 C, at the end of which the reaction mixture is kept under stirring for further min. The reaction mixture is cooled to -45 0 C and added with the monochloride of 8-carbomethoxy-octanoic acid (prepared according to the method disclosed in IT patent application 19043 A/84 of January 5,1984), keeping the temperature between -400 and -45 0 C. The reaction mixture is stirred for 5 hrs at -150C, at the end of which a 15% solution of sodium chloride(120 ml) and ethyl ether (120 ml) is added thereto.

The organic phase is separated, washed to neutrality with a sodium chloride aqueous solution; it is then anhydrated on sodium sulfate and the solvent is evaporated under 'educed pressure yielding 125 g of crude 2-(ll-carbomethoxy)4-oxo-nundecyl)-1,3-dioxolane which is used without any further purification.

b) A solution of the compound (125 g) prepared according to the preceding step dissolved in tetrahydrofuran (1000 ml) is reacted 16 hrs at room temperature with 6N hydrochloric acid (300 ml).

At the end, the reaction mixture is extracted with methylene chloride (2 x 300 ml), and the organic phase is washed to neutrality with a 10% brine solution.

It is then anhydrated over Na2SO 4 and the solvent is distilled off under reduced pressure. The thus obtained crude material WO 90/03170 WO 9003170PCT/rr89/00061 12 contains, as the main bY-ProduCt, .carboxy-n.hexyl)-cyclo.

hexene-2-one which is converted into the title compound by reaction with methanol and p-toluenesulfonic acid. The thus prepared compound is distilled at 0.3 mmHg whereby the fraction having a b.p. of 140 0 -142*C is collected; 60 g of the title compound is obtained.

Analytical Data B.p. :140*-l42'C at, 0.3 mmHg I.R. N )max) 1740 cm ,1675 cm NMR (S 75 (rm,1H) 3. 7 3M); 2. 2 8H) 15 Calculated C =70.56% H 9.30% Found .C 70.47% H- 9.21% Example 2 2-(6'-carbomethoxy--n-hexyl)-3-n-hexyl-cyclohexanone (a derivative of formula(III) wherein n 3, Y' COOCH 3).

This derivative is prepared according to the process disclosed in IT Pat.Appln. 19043 A/84 filed on January 5,1984, by reacting 2-(6'-carbomethoxy-hexyl)-2-cyclohexene-1..one) (41 g) with bromohexane (52 g) and Cul (3.3 48.3 g of the title compound is obtained.

Analytical Data I.R. :1740 cm 1, 1710 cm- NMR 3.7 3H);3.6-1.1 (in, 30H); 0.95 3H) Example 3 2- (6 '-carbomethoxy-hexyl 3 -pentyl-cyclopentanone This derivative is prepared, as described in example 2, by react- WO90/03170 PC/IT89/00061 13 ing 2-(6'-carbomethoxy-hexyl)-2-cyclopentene-l-one (67 g) with Mg (14.4 bromopentane (96.6 g) and CuI (5.7 g) yielding 73 g of the title compound.

I.R. max) 1740 cm Example 4 2-(6'-carboxyhexyl)-3-pentyl-cyclopentanone The compound of example 3 is dissolved in methanol (300 ml) and treated with a solution of NaOH (24 g) in water (300 ml).

The reaction mixture is refluxed under stirring to completion of the reaction. Then it is cooled, acidified to pH 2, extracted with rethylene chloride (250 ml) and washed with water to neutrality. The reaction mixture is anhydrated and the solvent is distilled off; 68 g of the title compound is obtained.

Example 2-(6'-carboxyhexyl )-3-pentyl-cyclopentylamine (a derivative of formula wherein n 2, m 4, X NH Y COOH(IBI-P-12003) The compound of example 4(68 g) is reacted with methanol (1000 ml), ammonia (200 Pt02 (3 at a temperature of from 40 60 0

C,

in an autoclave, and left for 3 days under hydrogen pressure, yielding 42 g of a solid white compound.

Analytical data: M.p. 166°-168°C -1 -1 I.R. max) 3300 cm 1560 cm NMR S 4.6 3H); 2.8 1H); 1.2(m, 26H); 0.9(t,3H) Titer (as base) 96% Calculated: C 72.08% H 11.66% N 4.95% Found C 71.92% H 11.72% N 4.98% Example 6 2-(6'-carbomethoxy-hexyl)-3-octyl-cyclopentanone hecompounds of formula wherein: n 2, 3; m 4,5,6, 7; X NHR, CH 2 NHR, NHNH2; K a li,near or branched (C-C 4 )alkyl, a linear or branched (CI-C 4 )acyl; Y CH 2 NH COOH, CONH 2 (with the exclusion of the compounds wherein n 2 m 5, X NHR with R H, a linear or branched (Ci-C 4 )alkyl, a linear or branched

(CI-C

4 )acyl; Y COOH, CONH 2 including all optical and/or geometrical isomers, alone or in admixture, and their salts, which inhibit the platelet aggregation in the blood and which can be prepared for use as antiplatelet, antithrombotic agents.

WO 90/03170 PCT/IT89/00061 14 COOlMe This derivative is prepared in an analogous manner to that described in example 3, using as the starting material 2-(6'-carbomethoxy-hexyl)-2-cyclopentene-l-one (67 yield 78 g of the title compound.

-1 I.R. '0 max) 1740 cm Example 7 2-(6'-carboxyhexyl)-3-octyl-cyclopentylamine (a derivative of formula wherein n= 2,m 7, X NH., Y COOH (IBI-P-12005) This derivative is prepared in analogous manner to that described in example 5, using as the starting material 2-(6'-carboxyhexyl)- 3-octyl-cyclopentanone (75 Finally, 34 g of a solid white material is yielded.

Analytical Data m.p. 1520 154 0

C

-1 -1 I.R. max) 3300 cm 1560 cm NMR 4.6 2.8 1.2 (m,32H); 0.9 (t,3H) Titer (as base) 98% Example 8 2-(6'-carbomethoxy-hexyl)-3-n-hexyl-cyclohexylamine A suspension of 2-(6'-carboxyhexyl)-3-n-hexyl-cyclohexylamine (200 g) (IBI-P-05006, a compound disclosed in IT 1,205,183 filed on June 25,1987), in methanol (100 ml), is added with p-toluenesulfonic acid.

The reaction mixture is refluxed three hours, cooled to room temperature, then the solvent is concentrated under reduced pressure and the reaction mixture is added with a iN NaOH aqueous solution (1000 ml) and extractedwith ethyl ether (2x300 ml).

The organic phase is washed to neutrality with a 20% brine and anhydrated over Na2SO 4 the solvent is distilled off under WO 90/03170 PCT/M9/00061 15 reduced pressure, yielding 203 g of a colorless oil.

Analytical Data I.R. max) 1745 cm 1670 cm Calculated C 73.85% H 12.00% N 4.31% Found C 73.90% H 12.11% N 4.27% Example 9 R,S-cis-2-(6'-carbomethoxy-hexyl)-cis-3-n-hexyl-cyclohexylamine A solution of 2-(6'-carboxymethoxy-hexyl)-3-n-hexyl-cyclohexylamine(100 g)in methanol(500 g)is added with p-toluenesulfonic acid (61.4 g) and stirred for 15 min at room temperature, whereupon the solvent is distilled off under reduced pressure: a white solid material is obtained.

The thus obtained solid material is suspended in ethyl ether (500 ml) and stirred for 60 min at room temperature.

The reaction mixture is filtered on a BUchner funnel and the solid material is dessicated, yielding 104 g R,S-cis-2-(6'carbomethoxy-hexyl)-cis-3-n-hexyl-cyclohexylamine p-toluenesulfonate.

The thus obtained solid is treated with water (250 ml) and with a 1N NaOH aqueous solution (250 ml),stirred 10 min and extracted with ethyl ether (500 ml): the organic phase is then washed to neutrality with a 20% brine,anhydrated over Na SO and the solvent is distilled off under reduced pressure.

Yield: 43.5 g of a colorless oil.

Elemental Data I.R. max) 1745 cm 1675 cm NMR (6 3.6 2.69 2.27 2H); 1.6-0.9(broad, 0.85 3H) Calculated: C 73.85% H 12.00% N 4.31% Found C 73.79% H 11.95% N 4.37% WO 90/03170 PCT/IT89/00061 16 Example R,S-cis-2-(6-carboxyhexyl)-cis-3-n-hexyl-cyclohexylamine (IBI-P-05006A) 00

NH

NH

2 (racemate) The compound (40 g) of example 9 is dissolved in methanol(400 ml) and added with a potassium carbonate (34.2g) water (60 ml) solution. The reaction mixture is refluxed three hours; it is then cooled and the solvent is distilled off under reduced pressure.

The reaction mixture is then added with water (250 ml),acidified to pH 5.6-5.8 with a 3N HC1 solution, filtered off, washed with water, acetone, and dessicated under reduced pressure yielding 36 g of a white solid compound.

Analytical data m.p. 2086-2100 Titer (as base) 98.67% Calculated C 73.31% H 11.90% N 4.50% Found C 73.41% H 12.00% N 4.61% Example 11 R,S-cis-2-(6'-carbomethoxy-hexyl)-cis-3-n-hexyl-cyclohexanol a) 2-(6'-carboxyhexyl)-3-n-hexyl-cyclcjhexano (125 g) is reacted with a 1N lithium-tri-sec-butylboronhydride solution in THF (800 ml) according to the method described by H.C.Brown et al in J.Am.Chem.Soc. 7159 (1972),yielding 130 g of an oily material essentially consisting of a.mixture of R,S-cis-2-(6'carboxyhexyl)-cis-3-n-hexyl-cyclohexanol and of R,S-cis-2- (6'-carboxyhexyl)-trans-3-n-hexyl-cyclohexanol.

Analytical data: -1 -1 I.R. max) 3400 cm 1710 cm I;I r WO 90/0370 PCr/M~T9/00061 17 b) The thus obtained compound is dissolved in methanol (750 ml) and added with p-toluenesulfonic acid (12.5 g).

The reaction mixture is refluxed 90 min, cooled and the methanol is concentrated under reduced pressure; the reaction mixture is then added with water (600 ml) and ethyl ether (400 ml).

The organic phase is washed with a 20% brine, anhydrated over Na SO and the solvent is distilled off under reduced pressure, yielding a yellow oil (124.3 g) consisting of a mixture of R,S-cis-2-(6'-carbomethoxyhexyl)-cis-3-n-hexyl-cyclohexanol and of R,S-cis-2-(6'-carbomethoxyhexyl)-trans-3-n-hexyl-cyclohexanol.

Chromatographing the stereoisomer mixture by silica T.L.C., using as the eluent a 7:3 hexane:ethyl acetate mixture, the cis-cis enanthiomer pair gives a R.f. 0.50 whereas the cistrans enanthiomer pair gives a R.f. 0.59.

The stereoisomer mixture is chromatographed on silica gel column using as the eluent a 7:3 hexane:ethyl acetate mixture, recovering the enanthiomer pair having a R.f. 0.50.

Yield: 70 g of the title compound.

Analytical Data -1 -1 I.R. max): 3450 cm- 1742 cm 1 NMR 3.70 1H); 3.68 2.3 1.8-1(broad, 29H); 0.87 (t,3H).

Calculated: C 73.61% H 11.66% Found C 73.55% H 11.71% Example 12 R,S-trans-2-(6'-carbomethoxyhexyl)-cis-3-n-hexyl-cyclohexylazide A solution of R,S-cis-2-(6'-carbomethoexhexyl)-cis-3-hexyl i I WO 90/03170 PCT/IT89/00061 18 cyclohexanol (70 g) in methylene chloride (350 ml) is added with triethylamine (51.7 ml) and methanesulfonyl chloride (34.3 g) is added dropwise,at a temperature of from 00 to 5 0

C;

the reaction mixture is reacted 30 min at the same temperature and at the end it is poured into water (600 ml). The various phases are separated; the organic phase is washed to neutrality with a 10% brine, anhydrated over Na SO4 and the solvent is distilled off under reduced pressure, whereby 93.9 g of a clear -1 -1 -1 oil is obtained. max): 1740 cm ,134'0 cm ,1170 cm That residue is taken up with water (850 ml), added with NaN 3 (37.4 hexadecyltributylphosphonium bromide (12.22 g) and stirred 4 hours at 65 0

C.

At the end, the reaction mixture is cooled and extracted with ethyl ether (500 ml). The organic phase is then washed with a 20% brine, anhydrated over Na2SO4, and the solvent is distilled off under reduced pressure, yielding 96 g of an orange oil.

The material is purified by silica gel chromatography, eluting with 95:5 hexane:ethyl acetate.

Yield: 42 g of the title compound.

Analytical Data -I -1 I.R. mx) 2100 cm 1745 cm Example 13 R, S-trans-2-(6'-carbomethoxyhexyl)-cis-3-n-hexyl-cyclohexvlamine A solution of R,S-trans-2(6'-carbomethoxyhexyl)-cis-3-n-hexylcyclohexylazide (42 g) in methanol (400 ml) ethyl ether (200 ml) is added with 5% Pd/C (4 g).

The reaction mixture is then shaken 5 hours under hydrogen atmosphere, then the catalyst is filtered off and the solvent is distilled off under reduced pressure; the residue is chromato-

I

WO 90/03170 19 PCT/IT89/00061 graphed on silica gel using an 80:20:0.5 hexane:acetone:ammonia mixture as the eluent.

Yield: 9.6 g of an oily material consisting essentially of the title compound.

Analytical Data -1 -1 I.R. max): 1745 cm 1675 cm NMR (6 3.65 2.65 2.25 1.7-1(broad, 0.82 3H) Example 14 R,S-trans-2-(6'-carboxyhexyl)-cis-3-n-hexyl-cyclohexylamine (IBI-P-05006C)

C

COOH

RH

2 (racemate) 9.6 g of R,S-trans-2-(6'-carbomethoxyhexyl)-cis-3-n-hexyl-cyclohexylamine is treated in the same way as described in example Yield: 5.2 g of the title compound as a white solid.

Analytical Data m.p. 154 0 -156 0

C.

-1 I.R. max): 1580 cm Calculated: C 73.31% H 11.90% N Found C 73.28% H 11.97% N 4.45% Titer (as base) 99.3% The activity of the inventive compounds as platelet aggregation inhibitors and as antithrombotic agents has been investigated in vitro determining the percent inhibition of the platelet aggregation induced by ADP, collagen, arachidonic acid and thrombin using platelet-rich plasma (hereinafter referred to as PRP) of mammals according to G.V.R.Born: Nature 194, 927-929 (1962) and G.V.R.Born and M.J.Cross:Physiol.168,178-195(1963).

L~ WO 90/03170 PCr/MT9/0061 20 1) Inhibition of platelet aggregation by added ADP with rat PRP 3 The tests are conducted with rat PRP (500,000 platelets/mm 3 adding a compound of formula or a pharmacologically acceptable salt thereof so that its plasma concentration be 10_ 4incubating 9 min at room temperature; whereupon as aggregating agent ADP (3/uM) is added and the percent inhibition of the platelet aggregation is measured using the turbidimetric method of Born and Cross (Table 2) Inhibition of platelet aggregation by added collagen with rat PRP The test is carried out as described sub 1) (inhibition of platelet aggregation by added ADP), using collagen (3 mcg/ml) as the'aggregating agent (Table 1).

3) Inhibition of platalet aggregation by added arachidonic acid with rat PRP The test is carried out as described sub 1) (inhibition of platelet aggregation by added ADP), using arachidonic acid (200 uM) as the aggregating agent (Table 1).

4) Inhibition of the platalet aggregation by added thrombin with rat PRP The test is carried out as described sub 1) (inhibition of platelet aggregation by added ADP), using thrombin (0.1 U/ml) as the aggregating agent. The transmittance measurements have been effected with a Chromo-Log or Elvi 840 aggregometer.

WO090/03170 21pL-/MT9/00061 T A BLE 1 C 0 MP 0 N DPercentage of inhibition of platelet aggre- C 0 M P 0 U N DIgation with-rat PRP, with added: ADP Collagen Arachidonic IThrombin 2- (6 -cartoxyhexyl )-3-hexyvl-aminomethylcyclopentane (IBI-0-01058) 18 26 96 (6 -carboxyhexyl )7-3-hexyvl-cyclopentyl hydrazine chloride (IBI-P-01062) 35,6 82 62 47 2- -arinoheptyl )-2-hexyl-cyclopentyvlamine (IBI-P-01068) 100 100 100 100 2- (6 1 -carboxyh e xy 1) -3-hexyl--cyclohexyvlamine (IBI-P-05XJ6) 10 100 100 67,5 2- (6 '-carboxyhe xy1) -3-hexyvl-aminomnethyl-cyclohexane (IBI-P-05011) 11,6 69.7 100 100 2- (7'-arninoheptyvl )-3-hexyvl-cyclohexylamine (IBI-P-05012) 100 98*--98 2-(6 -cartoxyhexyl )-3-pentvl-cyclopentylamine (IBI-P-12003) 10 73 66 2- (6 '-cartoxyhexyl )-3-heptylcyclopentylamine (IBI-P-12004) 24 93 100 96,6 2-(6 1 -cartoxyhexvl )-3--octyvl-cyclopentyvlamine (IBI-P-12005) 13,5 70 100 100 R, S-cis-2- (6 1-carboxyhexyl) -c is-3-n-hexvl-cyclohexylamidne (IBI-P-05006 A) (010101 R,S-trans-2-(6' -cartoxyhexyl )-cis-3-nhexyl-cyclohexylamine (IBI-P-05006 C) (10 100 100 45,5 I, ii*i' WO 90/03170 22 PC/1T89/00061 The compounds of formula wherein n 2, m 5, X NHR with R H, a linear or branched (C -C )alkyl, a linear or branched (C -C4)acyl; Y =COOH ,CONH 2 disclosed in IT 1,060,366 filed on August 7,1974 of which Applicants are the owner, have shown a very low inhibition of platelet function, antithrombotic activity.

Table 2 illustrates the percent inhibition of platelet function with rat PRP, determined under the same conditions and at the -3 same compound concentrations (10 M)as well as at the same concentration of the aggregation favoring agent as used in the above described tests of platelet function, for example for the compounds according to the above referred formula (I) wherein n 2, m 5, X NH 2 Y COOH (IBI-P-01009) n 2, m 5, X NHCOCH 3 Y COOH (IBI-P-01012) n 2, m 5, X NHCH 3 Y COOH (IBI-P-01013) n 2, m 5, X NHCH(CH 3 2 Y COOH (IBI-P-01015 n 2, m 5, X NH2, Y CONH 2 (IBI-P-013036).

22 WO 90/03170 PT18/06 PCT/IT'89/00061 23 T A BLE 2

(COMPARISON)

COMPOUNDS OF IT 1,060,366 JPercentage of inhibition of the platelet ag- ExCUJDED FROM THE INSTANT gregat ion with rat PRP ,added with

*INVENTION

CollIagen Arachidonic Acid Thronb in' 2- (6 '-carboxyvethyl )-3-hexylcyclopentylamine N- -cartoxvhexxl) hexyl-cfopentyl/acetamide -carboxyhexyl hexyl-cyclopentyl I-N -mthylamine N-/2-(61-carboxvhexyl hexyl-cyclopentyl -isopropylamine 2- (6 1 -caramo:ylhexyl -3hexyl-cyclopentylamine 10 10 <10 (10 15,4 inactive 61 inactive 75,6 16,3 12 25 1 60 1 65 It therefore can be seen that the compounds subject of the instant invention show an antiplatelet, antithrombotic activity which is surprisingly higher(more than 10 times)over that of the compounds disclosed in IT 1,060,366 filed on August 7,1984.

WO 90/03170 PCr/IT89/00061 24 The compounds of the instant invention are preferably administered as pharmaceutical compositions in admixture with one or more pharmacologically acceptable diluents and/or carriers.

Within the scope of the instant invention the expression "pharmacologically acceptable diluents and/or carriers" refers to substances such as e.g. starch and derivatives thereof (e.

g. maize starch, STA RX 150U, which is a registered tradename of Colacon Ltd., Orpington, Kent, rice starch,carboxymethyl starch); cellulose and derivatives thereof Avice registered tradename of FMC Corporation, Philadelphia, U.S.A., and methyl cellulose, ethyl cellulose, hydroxypropylmethyl cellulose); silicates and silica oxides (e.g.talc,hydrated calcium silicate,Mg and Al silicates, Aerosil registered tradename of Wacker-Chemie GmbH, Munich, Fed.Repub.of Germany, Syloico, registered tradename of W.R.Grace Co., New York, ionic solid surfactants (e.g.sodium laurylsulfate) and non-ionic surfactants fatty acid esters of saccharose); acrylic derivatives and polymers thereof; alkali and alkaline-earth metal sulfates, carbonates and phosphates; polyvinyl pyrrolidone and derivatives thereof. Preferably, the inventive compounds are administered orally as tablets, capsules, granules, syrups) or parenterally or Although the dosages may vary according to the symptoms, sex, body weight and conditions of the patient as well as to the frequency of the route of administration, the inventive compounds can be administered orally to an adult at a daily dose of from 1 to 2000 mg, preferably of from 10 to 1000 mg, per single dose, or in doses subdivided over a period of 24 hours.

i

Claims (10)

1. A compound cf formula (I) (CH 2 )CH (CH2)n x (1) including all possible optical and/or geometrical isomers wherein: n 2,3; m 4,5,6,7; X NHR, CH 2 NHR, NHNH 2 wherein R H, a linear or branched (Cl-C 4 )alkyl, a linear or branched (C 1 -C 4 )acyl, Y CH 2 NH 2 COOH, CONH 2 with the exclusion of the compounds according to formula wherein n 2, m 5, X NHR with R H, a linear or branched (C 1 -C 4 )alkyl, a 10 linear or branched (C 1 -C4)acyl, Y COOH, CONH 2 or a pharmacologically acceptable salt thereof, when used as an antiplatelet, antithrombotic agent, in human or veterinary medicine.

2. A compound of formula according to Claim 1 15 wherein: n 2, m 5, X CH 2 NH 2 Y COOH; n 2, m 5, X NHNH 2 Y COOH; n 2, m 5, X NH 2 Y CH 2 NH 2 .n 3, m 5, X= NH 2 X COOH; n 3, m 5, X CH 2 NH 2 Y COOH; n 3, m 5, X NH 2 Y CH 2 NH 2 n 2, m 4, X NH 2 Y COOH; n 2, m 6, X NH 2 Y COOH; n 2, m 7, X NH 2 Y COOH; or a pharmacologically acceptable salt thereof, when used as an antiplatelet, antithrombotic agent in human or .Rq4 veterinary medicine. 26

3. The compound R,S-cis-2-(6'-carboxyhexyl)-cis-3-n- hexyl-cyclohexylamine, or a pharmacologically acceptable salt thereof, when used as an antiplatelet, antithrombotic agent, in human or veterinary medicine.

4. The compound R,S-trans-2-(6'-carboxyhexyl)-cis-3- n-hexyl-cyclohexylamine, or a pharmacologically acceptable salt thereof, when used as an antiplatelet, antithrombotic agent, in human or veterinary medicine.

A pharmaceutical composition having antiplatelet, antithrombotic activity containing, as its active ingredient, a compound as defined in any one of Claims 1 to 4 together with a pharmacologically acceptable carrier, when used as an antiplatelet, antithrombotic agent in human or veterinary medicine.

6. A pharmaceutical composition according to Claim wherein the said compound is R,S-trans-2-(6'-carboxy- hexyl)-cis-3-n-hexyl-cyclohexamine or R,S-cis-2-(6'- carboxyhexyl)-cis-3-n-hexyl-cyclohexamine together with a pharmacologically acceptable carrier, when used as an S. 20 antiplatelet, antithrombotic agent in human or veterinary medicine.

7. A method of treatment of a thrombotic condition, comprising the step of administering to a human or animal in need of such treatment an effective amount of a compound 25 as defined in any one of Claims 1 to 4.

8. A method according to Claim 7 wherein said compound is R,S-trans-2-(6'-carboxyhexyl)-cis-3-n-hexyl- cyclohexamine or R,S-cis-2-(6'-carboxyhexyl)-cis-3-n- hexyl-cyclohexamine. S 30

9. A process for preparing R,S-cis-2-(6'-carboxy- hexyl)-cis-3-n-hexyl-cyclohexylamine wherein the compound obtained by addition of an equivalent of p-toluenesulfonic acid to a mixture containing all the stereoisomers of 2-(6'-carbomethoxyhexyl)-3-n-hexyl-cyclohexylamine is crystallized with ethyl ether, whereby the R,S-cis-2- (6'-carbomethoxyhexyl)-cis-3-n-hexyl-cyclohexylamine p-toluenesulfonate thus obtained is treated with a base thereby yielding R,S-cis-2-(6'-carboxyhexyl)-cis-3-n- hexyl-cyclohexylamine. 27 A process for preparing R,S-trans-2-(6'-carboxy- hexyl)-cis-3-n-hexyl-cyclohexylamine wherein 2-(6'-carboxy- hexyl)-3-n-hexyl-cyclohexanone is reacted with lithium-tri- sec-butylboronhydride in THF at -70 0 C thereby yielding a mixture of R,S-cis-2-(6'carboxyhexyl)-cis-3-n-hexyl- cyclohexanol and R,S-cis-2-(6'-carboxyhexyl)-trans-3-n- hexyl-cyclohexanol, and the thus-obtained mixture is then treated with methanol and p-toluenesulfonic acid and chromatographed on silica gel using a 7:3 mixture of hexane: ethyl acetate as the eluent, whereby R,S-cis-2-(6'- carbomethoxyhexyl)-cis-3-n-hexyl-cyclohexanol is separated which, after subsequent transformation into the corresponding mesilate and after substitution with sodium azide and reduction with a metal catalyst and hydrogen and subsequent saponification yields R,S-trans-2-(6'- carboxyhexyl)-cis-3-n-hexyl-cyclohexylamine. DATED THIS 21ST DAY OF MAY 1992 IBI ISTITUTO BIOCHIMICO ITALIANO GIOVANNI LORENZINI S.p.A. S 20 By Its Patent Attorneys: GRIFFITH HACK CO n Fellows Institute of Patent Attorneys of Australia i r INTERNATIONAL SEARCH REPORT International Aoolication No PCT/IT 89/00061 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symools aoply, indicate ill) According to International Patent Classification (IPC) or to both National Clastificaton and IPC A T~-K 'TI557. C 07 C 229/46 II. FIELDS SEARCHED Minimum Documentation Searched Classification Syster I Classificatlon Symbols IPC 5 A 61 K 31/00 I Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched 1 III. DOCUMENTS CONSIDERED TO BE'RELEVANT' Category I Citation of Document, with Indication, where appropriate, of the relevant passage t Relevant to Claim No. t X IT, A, 1060366 (ISTITUTO BIOCHIMICO 1,4,7,8 ITALIANO SAS)

10 July 1982 see page 10; claims, especially claims 10,14 cited in the application X EP, A, 0218953 (ISTITUTO BIOCHIMICO 1,4,7,8 ITALIANO SpA) 22 April 1987 see pages 1-3 cited in the application Y WO,A, 88/10252 (ISTITUTO BIOCHIMICO 1,4,7,8 ITALIANO SpA) 29 December 1988 see abstract; pages 6-11 cited in the application Y The Merck Manual of Diagnosis and Therapy, 1,4,7,8 edition, 1987, edited by R. Berkow et al., publ. by Merck Sharp Dohme Research Laboratories, (Rahway, US), pages 2513-2516 see the whole document, especially table 284-1 Special categories of cited documents: e later document published after the International filing date document defining the general late of the art which is not r priority date and not In conflict with the application but considered to be of particular relevance cited to understand the principle or theory underlying the invention earlier document but published an or ater the International E arlinr dtoument but publshed on or fter the nternational document of particular relevance: the claimed invention lig dt cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or Involve an Inventive lstp which is cited to establish the publication date of another Y document of particular relevance; the claimed invention ctation or other special reason (as specified) cannot be considered to involve an Inventive step when the document referring to an oral discloure. use, exhibition or document is combined with one or more other such docu- other means ment., Cuch combination being obvious to a person skilled document published prior to the International filing date but In the art. later then the priority date claimed document member of the sme patent family IV. CERTIFICATION Dale ol the Actual Completion of the international Search Date of Mailing of this International Search Report 27th November 1989 1 04. nternational Searching Authority Signature of Authorized Officer EUROPEAN PATENT OFFICE st PEIS Form PCTIISA210 (second sheet) (January 196) International ApplicationNo. PCT/IT 89/00061 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET V.OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND UNSEARCHABLE This International search report has not been established In respect of certain claims under Article 17(2) for the following reasons: 1.g:laim numbers5'...6.. because they relate to subject matter not required to be searched by this Authority, namely: See PCT Rule 39.1(iv): Methods for treatment of the human or animal body by means of surgery or therapy, as well as diagnostic methods. 2. Claim because they relate to parts of the international application that do not comply with the prescribed require- ments to such an extent that no meaningful International search can be carried out, aspectically: 3Q Claim becau they are dependent clim and are not drafted In eoordance with the seond and tird sentence of PCT Rule 6.4(a). VI.[ OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING This International Searching Authority found multiple Inventions In this International appllcation as follows: Please refer to Form PCT/ISA/206 dated 19th December 1989 As all required additional search fees were timely paid by the applicant, this International search report covers all searchabl claims of the International application. 2.M As only some of the required additional search fees were timely paid by the applicant, this International search report covers only those claims of the International application for which fees were paid, specifically claims: 3. No required additional search fees were timely paid by the applicant. Consequently, this International search report is restricted to the invention first mentioned in the claims; It is covered by claim numbers: 1,4,7,8 As all searchable claims could be searched without effort Justifying an additional fee, the International Searching Authority did not invite payment of any additional fee. Remark on Protest Q The additional search fees were accompanied by applicant's protast. No protest accompanied the payment of additional search fees. Form PCTlISA210 (supplemental heet (January 19IS) 1 J :i u ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. IT 8900061 SA 31252 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 27/03/90 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date IT-A- 1060366 None EP-A- 0218953 22-04-87 US-A- 4816472 28-03-89 WO-A- 8810252 29-12-88 AU-A- 1291188 19-01-89 EP-A- 0319576 14-06-89 ZA-A- 8801451 23-08-88 For mor details about this annex see Oficial Journal of the European Patent Offi, No. 12/82 For more details about this annex :see Official Journal of the European Patent Office, No. 12182