CZ55199A3 - Stable, non-hygroscopic crystalline form of N-[N¡[N-(4¡(piperidin¡4¡yl)butanoyl]¡N¡ethylglycyl] derivatives - Google Patents

Abstract

A stable, non-hygroscopic crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl] -N-ethylglycyl] derivatives having antithrombic effects including inhibition of platelet aggregation and thrombus formation in mammals and is useful in preventing and treating thrombosis associated with disease conditions such as myocardial infarction, stroke, peripheral artery disease, and multiple intravascular coagulation.

Description

*> Stable, non-hygroscopic crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] derivative

Technical Field The invention relates to a non-hygroscopic stable crystalline form

N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexyl and 1-aminamide of Formula I

OH (1) which has antithrombic effects, including inhibiting platelet aggregation and thrombus formation in mammals, and is useful in preventing and treating thrombosis associated with disease states such as myocardial infarction, stroke, peripheral artery disease and multiple intravascular coagulation.

^b Furthermore, the invention relates to a process for preparing a crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl3 - (L) - * aspartyl] - (L) -3-cyclohexylalanine of formula (I), pharmaceutical compositions containing it, and intermediates for their preparation.

BACKGROUND OF THE INVENTION

Hemostasis, the biochemistry of blood coagulation i, is an extremely complex phenomenon in which normal whole blood and body tissue spontaneously stop bleeding from injured veins. Effective hemostasis requires combined vascular, platelet activity

9 9 9 9 999 9999

9 9 9999

9 9 * · 99999 9,999,999

999 ··· · ·

And plasma factors, as well as a control mechanism to prevent excessive clotting and thrombotic sequelae. Disorders, insufficiency or excessiveness of any of these three components may lead to hemorrhagic and thrombotic consequences.

Platelet adhesion, scattering and aggregation of extra-cellular matrices are central events in thrombus formation. These events are mediated by a family of adhesive glycoproteins, i.e. fibrinogen, fibronectin and Wi11ebrand factor. Fibrinogen is a contributing factor in platelet aggregation, while fibronectin promotes platelet attachment and scattering reactions, and von Willebrand factor is important in platelet binding and scattering on subendothelial matrices. The location of the fibrinogen, fibronectin, and Wilebrand factor binding sites was localized to the platelet membrane protein complex known as glycoprotein IIb / IIIa.

Adhesive glycoproteins, such as fibrinogen, do not associate with normally resting platelets. However, when the platelets are activated by an agonist such as thrombin or adenosine diphosphate, the platelets change their shape, probably to ensure the availability of GPIIb / IIa binding sites for fibrinogen. The compound of the invention blocks the fibrinogen receptor and thus has the above-mentioned antithrombic effects.

The presence of Arg-Gly-Asp (RGD) in fibrinogen, fibronectin and von Wi11ebrand factor has been found to be a prerequisite for their interaction with the cell surface receptor (Ruoslahti E., Pierschbacher, Cell 44, 517-518, 1986). Two other amino acid sequences also appear to be involved in the function of fibrinogen platelet assembly, namely the Gly-Pro-Arg sequence and the His-His-Leu-Gly-Gly-Ala-Lys-Gln-Alaf dodecapeptide sequence.

Gly-Asp-Val. Small synthetic peptides containing RGD or dodecapeptide have been shown to bind to the platelet GPIIb / IIIa receptor and competitively prevent the binding of fibrinogen, fibronectin and von i

As well as preventing aggregation of activated platelets (Plow et al. Proc. Natl. Acad. Sci. USA 82: 8057-8061, 1985; Ruggeri et al. Proc. Natl. Acad. Sci. USA. P. 5708-5126, 1986; Ginsberg et al., J. Biol. Chem., 260, pp. 3931-3936, 1985; and Gartner et al., J. Biol. Chem., 260, 11, pp. 89-94, 1987 ).

Tjoeng et al. (U.S. Pat. Nos. 5,037,808 and 4,879,313) disclose that indolyl compounds containing the asparagyl moieties of guanidinoalkanoyl and guanonidinoalkenoyl are inhibitors of platelet aggregation.

In U.S. Patent 4,992,463 (issued Feb. 12, 1991), Tjoeng et al. Discloses that a number of aryl and aralkyl mimetic compounds of the guanidinoalkyl peptide exhibit platelet aggregation inhibitory effects and specifically disclose a series of monomethoxyphenylpeptide mimetic and dimethoxyphenylpeptide mimetic compounds and mimetic alkyl biphenyl compounds. .

In U.S. Patent 4,857,508 (issued Aug. 15, 1989), Adams et al. Discloses that a number of guanidinoalkyl peptide derivatives containing terminal aralkyl substituents exhibit platelet aggregation inhibitory activity and specifically disclose a series of biphenyl and naphthyl O-methyltyrosine derivatives containing functionality. of the terminal amine.

Haverstick, D.M. et al. (Blood 66 (4), 946-952, 1985) discloses that a variety of synthetic peptides, including Arg-Gly-AspSer and Gly-Arg-Gly-Asp-Ser, are capable of inhibiting thrombin-induced platelet aggregation.

Plow, E.F. (Proc. Natl. Acad. Sci. USA 79: 3711-3715, 1982) discloses that the glycyl-L-prolyl-L-arginyl-Lproline tetrapeptide inhibits the binding of fibrinogen to human platelets.

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0 0 00 0000 • 0 0 0 0 0

French Patent Application No. 86/17507, filed

On December 15, 1986, tetrapeptide, penta-peptide and hexapeptide derivatives containing the sequence -Arg-Gly-Asp- are reported to be useful antithrombic agents.

U.S. Pat. No. 4,683,291 (granted Jun.

Zimmerman et al., discloses that a series of peptides, comprising six to forty amino acids, which contain the sequence -Arg-Gly-Asp-, is an inhibitor of platelet binding.

European Patent Application No. 0 319 506, published

On June 7, 1989, a number of tetrapeptide, pentapeptide and hexapeptide derivatives containing the sequence -Arg-Gly-Asp- are reported to be an inhibitor of platelet aggregation.

Cyclic peptide analogs containing a Gly-Asp moiety are disclosed in U.S. Pat. No. 5,023,233 as fibrinogen receptor antagonists.

»Peptides and pseudopeptides containing amino-, guanidino-, imidazaloyl and / or amidinoalkanoyl and alkenoyl moieties, are disclosed as antithrombic agents in U.S. patent applications pending 07 / 677,006, filed Mar. 28, 1991, no. No. 07/534 385, filed June 7, 1990, and 07/460 777 filed January 4, 1990, and U.S. Pat. No. 4,952,562 and International Patent Application No. PCT / US90 / 05448 filed Sep. 25, 1990, by the author of this invention.

Peptides and pseudopeptides containing amino-, guanidino-alkylbenzoyl and alkenylbenzoyl, phenylakanoyl and phenylakenoyl moieties are disclosed as antithrombic agents in U.S. patent application Ser. No. 07 / 475,043, filed Feb. 5, 1990, and International Application. No. PCT / US91 / O2471, filed Apr. 11, 1991, published

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9 9

9

99

9 9 9

No. 9 9 99 as an international application WO 92/13117 on Oct. 29, 1992 by the author of the present invention.

Platelet aggregation inhibitors include alkanoyl and substituted alkanoyl derivatives of azacycloalkylformyl lactic acid in U.S. Pat. No. 5,053,392, filed December 1, 1989, by the same author and relating to the same problem as the present invention.

N-substituted azacycloalkylcarbonyl cyclic aminoacylaspartic acid derivatives are disclosed as antithrombically active compounds in U.S. Pat. No. 5,064,814, filed Apr. 5, 1990, the author of the present invention. Azacycloalkylformylglycyl-aspartic acid derivatives are disclosed to be antithrombic in U.S. Pat. No. 5,051,405, filed Oct. 10, 1989, the author of this invention.

European Patent Application No. 0 479 481, published

On April 8, 1992, azacycloalkylalkanoylglycylasparagylamino acids are disclosed as fibrinogen receptor antagonists.

European Patent Application No. 0 478 362, published April 1, 1992, discloses azacycloalkylalkanoylpeptidyl-β-alanines as fibrinogen receptor antagonists.

PCT application WO95 / 102295 discloses azacycloalkylalkanoyl peptides and pseudopeptides of formula II

O

NH-CH-C-Z

CH ₂ COOH (II)

In particular N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) asparagyl] - (L) -cyclohexylalaninamide which inhibit platelet aggregation and thrombus formation in mammals and are useful in the prevention and treatment of thrombosis N- [N- [N- (4- (Piperidin-4-yl) butanoyl] N-ethylglycyl] - (L) -asparagyl] - (L) ) -β-cyclohexylalaninamide according to PCT WO95 / 1O295 is amorphous, hygroscopic and physically unstable as it binds water, PCT WO95 / 1O295 does not disclose a non-hygroscopic stable crystalline form of N- [N- [N- ( 4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexyl-laninamide.

PCT World Patent Publication No. WO95 / 102895 discloses that azacyclic and 1-canoyl peptides and pseudopeptides are generally prepared by solid or solution peptide synthesis procedures using starting materials and / or readily available intermediates from chemical supply companies such as Aldrich or Sigma (H. Paulsen, G. Merz, V. Weichart, S. id-Phase Synthesis of Glycopeptide Sequences [Solid Phase Synthesis of Glycopeptide Sequences] Angew. Chem. Int. Ed. Engl. 27, 1988; H. Mergler, R. Tanner, J. Gosteli and P. Grogg, Peptide Synthesis by A Combination of Solid-Phase and Solution Methods I. A New Vera Acid-Labile Anchor Group for Solid-Phase Synthesis of Fully Protected Fragments, [Peptide synthesis by combination of methods in a solid phase and in a solution (a new highly acid labile anchoring group for solid phase synthesis of fully protected fragments], Tetrahedron Letters 29, 40000 (1988)); Merrifield, R.B.Solid-Phase Peptide Synthesis after 25 Years: The Design and Synthesis of Antagonists of Glucagon [25]: Solid Phase Peptide Synthesis: Construction and Synthesis of Glucagon Antagonists], Macromol. Chem. Macromol. Symp. 19, p. 31 (1988). In addition, PCT Publication No. WO95 / 102295 states that the amorphous and hygroscopic form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) - aspartyl] - (L) -3-cyclohexylalaninamide was prepared

It has been disclosed by Scheme I. PCT Publication No. WQ95 / 10295 does not disclose the formation of tetraazacycloalkylalkanoyyl peptides and pseudopeptides, in particular N- [N- [N- (4- (piperidin-4-yl) butanoyl)]. -Nethylglycyl] - (L) -asparagyl} - (L) -3-cyclohexylalaninamide from a central di (pseudopeptide or peptide), wherein the N- and C-terminal ends of the central di (pseudopeptide or peptide) are both linked to pseudoamino acids and / or with amino acids to form tetraazacycloalkylalkanoyl peptides and pseudopeptides.

Scheme I θ Ζθ \ coupling θ P / 0 \

P _r NH-CH-C-OH + HN-CH-Cy-G & apos ►P ₁ -NH-CH-C-N-CH-LCY-G '

CH ₂ -C-OP ₂ E 'F ^Γ CH-J C — OP, F ^r

O

CH ₂ -C-OP ₂ F ii * removal of OF / O \ O from the temple of group ii ii i. . ii

-NH ₂ —CH — C — —N — CH— (Cy — G '+ P3N - ^ CH ₂ J — C — OH

CH ₂ -C-OP ₂ F ^Γ B ^P ii *

O η coupling - ^: -> ² ) deprotection

HN fCMj -C-NH-C- CH-N = CH- (C) - G '+ Fr-CH ^{<j (R 2)} m. ° ^P b H ₂ -c-op _2'R / -ΤΓ- ⁽⁰ ^ -ΟΗ

AND*

O ^A

1) coupling ² ) deprotection

RNM OOOE / 0 \ _(CH2) -C- = CH ₂ N-C-NH-CH-C - _N-CH-CH2 4cJ- ^BP e ^r COOH

WO 95/10295 does not relate to the formation of tetraazacycloalkylalkanoyl peptides and pseudopeptides or, in particular, N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -aspagyl] - (L) ) -p-cyclohexylalaninamide from the central di (pseudopeptide or peptide), wherein the N- and C-terminal ends of the central di (pseudopeptide or peptide) are always coupled with pseudoamino acids and / or amino acids to form

tetraazacycloalkylalkynoyl peptides and pseudopeptides.

SUMMARY OF THE INVENTION

The present invention provides a non-hygroscopic stable crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] (L) -asparagyl] - (L) -β-cyclohexyl and 1-ananamide of the formula AND,

OH (i) which according to the invention has antithrombic effects, including inhibition of platelet aggregation and thrombus formation in mammals, and is useful in preventing and treating thrombosis associated with disease states such as myocardial infarction, stroke, peripheral artery disease and multiple intravascular coagulation. In addition, the invention relates to a process for preparing a non-hygroscopic stable crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl-N-ethyl-glycyl] - (L) asparagyl] - (L) -3- cyclohexylalaninamide and pharmaceutical compositions containing it; and intermediates therefor.

The invention further relates to a process for the preparation of a tetra-azacycloalkyl-alkanoyl peptide or pseudopeptide derivative of the general formula

(Π)

II

- 9 - ::

Where 4 means

And a hydrogen atom

B is alkyl, cycloalkyl, cycloalkyl and alkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl or alkylaralkyl,

Group

COG

I \ CH

E ¹

E ¹ hydrogen atom,

E ² α-carbon side chain naturally occurring α-amino acids, hydrogen atom, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heterocyclyl, substituted heterocyclyl, heterocyclyl, substituted heterocyclyl, , or E ¹ and E ² together with the nitrogen and carbon atom to which they are attached form a 4-membered, 5-membered, 6-membered or 7-membered azacycloalkane ring,

G is OR ¹ or NR ¹ R ² ,

R ¹ and R ² are independently hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl or alkylaralkyl,

R is hydrogen, alkyl, aryl or aralkyl,

······ · · · · · ···

m number 1 to 5 - n number 0 to 6 a P number 1 to 4,

and in particular non-hygroscopic stable crystalline forms of N- [N- [N [4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) β-cyclohexylalaninamide.

The invention is explained in more detail by the following detailed description and the accompanying drawings.

Overview of pictures

Fig. 1 is a powder X-ray diffraction pattern of a sample of a non-hygroscopic crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -β -cyclohexylalaninamide prepared according to Example 13, Method A.

Figure 2 is a powder X-ray diffraction pattern of a sample of a non-hygroscopic crystalline form of N- [N- [N- (N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -β -cyclohexylalaninamide prepared according to Example 13, Method B (a).

Figure 3 is a powder X-ray diffraction pattern of a sample of a non-hygroscopic crystalline form of N- [N- [N- [N- (4-piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -β -cyclohexyl and 1-aminamide prepared according to Example 13, Method B (b).

Figure 4 is a powder X-ray diffraction pattern of a sample of the hygroscopic crystalline form of N- [N- [N- (N- (4-piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -β -cyclohexylalar ninamide prepared according to Example 14.

Fig. 5 is a powder X-ray diffraction pattern of a sample. Non-hygroscopic crystalline forms of N- [N- [N- (4-piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -3-cyclohexylala ninamide prepared according to Example 14.

Fig. 6 is a microcalorimetric graph of released energy as a function of time for three different experiments performed as described in Example 15. The experiments were performed to investigate the thermal activity of various crystalline forms of N- [N- [N- (4-piperidin-4-yl)]. (butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexyl and 1-aminamide, exposed to vapors of various solvents. Trace (A) in Figure 6 shows that if the hygroscopic N- [N- [N- (4-piperidin-4-yl) -butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) - β-cyclohexyl alaninamide, prepared according to Example 5 or 11, exposed to 80% relative humidity (saturated potassium chloride solution) for 30 hours at 40 ° C during which the exposed hygroscopic form of the compound is converted to a non-hygroscopic form, producing a strong exothermic reaction. Trace (B) in Figure 6 shows the non-exothermic reaction that occurs when the hygroscopic N- [N- [N- (N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) - aspartic acid 1] - (L) - cyclopexyl alaninamide, prepared according to Example 5 or 11, exposed to methanol vapor at 40 ° C (solvent other than the compound in which the compound is soluble) and thus methanol does not promote mobility within the crystals of this forms when converted to a non-hygroscopic form. Trace (C) in Figure 6 shows the non-exothermic conversion that occurs when the non-hygroscopic N- [N- [N- [N- (4-piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - ( The L-β-cyclohexyl and laninamide prepared according to Example 13 were exposed to 80% relative humidity at 40 ° C and thus the non-hygroscopic form of the compound is not subject to conversion under these conditions, thus being a stable form.

Fig. 7 is a microcalorimetric graph of released energy as a function of time for three different experiments performed as described in Example 15. The experiments monitor the thermal activity of the conversion of the hygroscopic crystalline form of N- [N- [N- (4-piperidin-4-yl)]. ) either • 9

-.

9 · 9 · 9,999 9,999,999

999,999 9 9

999 99 9 99 99 tanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -3-cyclohexylalaninamide to its non-hygroscopic form when exposed to vapor at 80% relative humidity at 40, 50 and 60 Deň: 32 ° C. the numbers indicate a conversion of approximately 24 hours at 40 ° C, 6.5 hours at 50 ° C and 3 hours at 60 ° C.

Fig. 8 is a microcalorimetric graph of released energy as a function of time for four different experiments performed as described in Example 15. The experiments monitor the thermal activity of the conversion of the hygroscopic crystalline form of N- [N- [N- (4- (piperidine-4-) yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -3-cyclohexylalaninamide to its non-hygroscopic form when exposed to 60 ° C to 65% relative humidity at 60 ° C, 75 The relative figures in Fig. 8 indicate that higher relative humidity produces faster conversion. Another feature is that conversion to the non-hygroscopic form of the compound occurs at 100% relative humidity at temperature. 60 ° C without liquefaction, which occurs in the hygroscopic form at room temperature, based on these results, it is expected that the conversion rate to the non-hygroscopic form is considerably faster than the liquefaction rate measurement of hygroscopic form at 60 ° C.

Figure 9 is a comparison of percent weight gain versus percent relative humidity for the hygroscopic ( _- ) and non-hygroscopic (Q) forms of N- [N- [N- (4-piperidin-4-yl) -butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexylalaninamide at 25 ° C found in experiment 16. Figure 9 shows that the hygroscopic form absorbs more water than the non-hygroscopic form as the relative humidity increases and more significantly at higher relative humidity than 60%. In addition, Figure 9 shows that the hygroscopic form of the compound does not absorb moisture to its original moisture percentage, while the non-hygroscopic form of the compound does not absorb moisture to its original moisture content.

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The abbreviations and expressions used here have the following meanings:

BOC tert-butoxycarbones CBZ benzyloxycarbonyl Gly glycine Asp aspartic acid Obz 1 benzyloxy TFA trifluoroacetic acid Cha β-cyclohexylalanine EtOAc ethyl acetate DMF dimethylformamide DCC dicyclohexylcarbodiimide HOBt 1-hydroxybenzotriazol TBTU (2-1H-benzotriazol-1-yl) -1,1,3,3-tetrafluoronium tetrafluoroborate Dl deionized water PNP p-ni trophenol PFP pentafluorophenol DCU dicyclohexylurea NMM N-methylmorpholine MTBE methyl-tert-butyl ether games RH relative humidity THF tetrahydrofuran PipBu 4-Piperidine-butyric acid PipBuen (4-Piperidine) butylidenylcarboxylic acid of formula

NH f

co ₂ h

The term patient always refers to a human or other mammal.

· · · · · ·> · ·

The term "pharmaceutically acceptable salt" refers to a salt of the parent compound of formula (I) which is relatively harmless to the patient when used in a therapeutic dose, while the beneficial pharmaceutical properties of the parent compound of formula (I) do not interfere with the side effects attributed to the salt counterion. The pharmaceutically acceptable salt comprises a zwitterion or an internal salt of a compound of Formula I.

The term alkyl always refers to a straight-chain or branched-chain aliphatic hydrocarbon group having 1 to 20 carbon atoms. By branching it is meant that a lower alkyl group, for example a methyl, ethyl or propyl group, is bonded to a linear alkyl chain. Preferred straight-chain or straight-chain alkyl groups are lower alkyl groups, which are alkyl groups having 1 to 10 carbon atoms. The most preferred alkyl groups are alkyl groups having 1 to 6 carbon atoms.

The term cycloalkyl always means a saturated carbocyclic group having one or more rings having 3 to 10 carbon atoms. Preferred cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and decahydronaphthyl.

The term cycloalkylalkyl refers to an alkyl group substituted by a cycloalkyl group. Preferred cycloalkylalkyl groups include cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, decahydronaphth-1-ylmethyl and decahydronaphth-2-ylmethyl.

The term "alkylcycloalkyl" refers to a cycloalkyl group substituted with an alkyl group. Examples are 1-, 2-, 3- or 4-methylcyclohexyl or ethylcyclohexyl.

I · AAAA A

A · A

A A A i i:

AAA 4 A

AA 4

The term alkylcycloalkylalkyl refers to an alkyl group substituted with an alkylcycloalkyl group. Examples are 1-, 2-, 3- or 4-methylcyclohexylmethyl or ethylcyclohexylmethyl or 1-, 2-, 3- or 4-methylcyclohexylethyl or ethylcyclohexylethyl.

The term azacycloalkane refers to a saturated aliphatic ring containing a nitrogen atom. Preferred azacyclicane groups include pyrrolidine and piperidine groups.

The term naturally occurring α-amino acid always refers to glycine, alanine, valine, leucine, isoleucine, lilac, threonine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, proline, hydroxyproline, aspartic acid, asparagine, glutamine, glutamic acid. histidine, arginine, ornithine, and lysine.

The term α-carbon side chain of naturally occurring α-amino acid always refers to a moiety that substitutes for the α-carbon atom of naturally occurring α-amino acid. For example, the isopropyl, methyl and carboxymethyl groups for valine, alanine and aspartic acid are mentioned as the α-carbon side chain of the naturally occurring α-amino acid.

By amino protecting group is meant an easily removable group known to protect the amino group from undesired reactions during the preparation and which is selectively separable. The use of amino protecting groups is well known in the art to protect the amino group from unwanted reactions during preparation (for example, TH Green and PGM Wuts, Protective Groups in Organic Synthesis, 2nd Edition, John Walley & Sons, New Yourk, 1991) . Preferred amino protecting groups are f

formyl, acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, o-

• »| 4-4 Nitrocinnamoyl, picolinoyl, acylisothiocyanate, aminocaproyl and benzoyl groups 4-nitrocinnamoyl, picolinoyl, acylisothiocyanate, aminocaproyl and benzoyl groups and acyloxy groups such as methoxycarbonyl, 9-fluorenylmethoxycarbonyl, 22,2-trifluoroethoxycarbonyl, 2-trimethylsilyl-ethoxycarbonyl, vinyloxycarbonyl, allyloxycarbonyl, tert-butyloxycarbonyl, (B) -benzyloxycarbonyl, (B) nitrobenzyloxycarbonyl and 2,4-dichlorobenzyloxycarbonyl.

By acid labile amino protecting group is meant an amino protecting group as defined above, which is readily removed by acid while remaining relatively stable to the action of other agents. A preferred acid-labile amino protecting group is a tert-butyloxycarbonyl (BOB) group.

The term hydrogenation-labile amino-protecting group refers to an amino-protecting group as defined above, which is readily removed by hydrogenation while remaining relatively stable to the action of other agents. A preferred amine-labile hydrogenation hydrogenation is benzyloxycarbonyl (CBZ).

The term acid protecting group refers to an easily removable group known to protect the carboxylic acid group (-COOH) from undesired reactions during the preparation and which is selectively separable. The use of carboxylic acid protecting groups is well known in the art for protecting a carboxylic acid group from unwanted reactions during preparation (exemplified by TH Green and PGM Wuts, Protective Groups in Organic Synthesis, 2nd Edition, John Walley & Sons, New Yourk). (1991). Preferred groups for protecting the carboxylic acid group include methoxymethyl, tetrahydropyranyl, benzyloxymethyl, substituted and unsubstituted phenyl.

9 9 ϊ

9,2-cyl, 2,2,2-trichloroethyl, tert-butynyl, cinnamyl, substituted and unsubstituted benzyl and trimethylsilyl, and proportions of amides and hydrazides such as Ν, Ν-dimethyl, 7-nitroindolyl, hydrazide and N-phenylhydrazide.

The term hydrogenation-labile acid-protecting group refers to an acid-protecting group, as defined above, which is readily removed by hydrogenation while remaining relatively stable to the action of other agents. A preferred acid-labile hydrogenation hydrogenation is benzyl.

The term aryl in each case denotes a phenyl or naphthyl group substituted by one or more aryl group substituents which are the same or different and, for example, an aryl group represents an alkyl, alkenyl, alkynyl, aryl, arylalkyl, hydroxyl, alkoxy, aryloxy, aralkoxy group. hydroxyalkyl, acyl, formyl, carboxy, alkenyl, aroyl, halo, trifluoromethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamino, aralkylamoyl, dialkylcarbamoyl, alkylcarbamyl, alkylcarbamyl, alkylcarbamyl Aryl, arylsulfinyl, aralkylsulfonyl, aralkylsulfinyl or a group of the formula -NR _and Rb, where R a and R b are independently hydrogen, alkyl, aryl or aralkyl.

The term "aralkyl" refers to an alkyl group substituted with an aryl group. Preferred aralkyl groups include benzyl, naphth-1-ylmethyl, naphthylmethyl and phenethyl.

»1

The term "substituted aralkyl" refers to an aralkyl group substituted in the aryl moiety by one or more aryl group substituents.

The term heterocyclyl refers to an approximately 4- to 15-membered monocyclic or multicyclic ring system wherein one or more of the ring atoms is other than a carbon atom, such as nitrogen, oxygen or sulfur. Preferred heterocyclyl groups include pyridyl, pyrimidyl and pyrrolidyl.

The term "substituted heterocyclyl" refers to a heterocyclyl group substituted with one or more aryl group substituents.

The term heterocyclylalkyl and substituted heterocyclylalkyl refers to an alkyl group substituted by a heterocyclyl group or a substituted heterocyclyl group.

By hygroscopicity is meant sorption of the amount or condition of water sufficient to influence the physical or chemical properties of the substance (Eds. J. Swarbrick and J.C. Boylan, Encyclopedia of Pharmaceutical Technology, Vol. 10, p. 33).

The preferred compounds prepared by the present invention is a compound of formula II wherein E ² is hydrogen, alkyl, hydroxymethyl, 1-hydroxyethyl, mercaptomethyl, 2-methylthioethyl, carboxymethyl, 2karboxyethylovou, 4-aminobutyl, 3-guanidinopropyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substituted heterocyclylalkyl, or E ¹ . ² together with the nitrogen and carbon atom to which they are attached form a 4-membered, 5-membered, 6-membered or 7-membered azacycloalkane ring, with the proviso that the heterocyclylalkyl group is other than the indol-3-ylmethyl group.

A more preferred compound prepared according to the invention is a compound of formula II wherein E ² is hydrogen, alkyl, hydroxymethyl, 1-hydroxyethyl, mercaptomethyl, 2-methylthioethyl, carboxymethyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, or E ¹ and E ² together with the nitrogen and carbon atom to which they are attached form a 4-membered, 5-membered, 6-membered or a 7-membered azacycloalkane ring.

Even more preferred compound prepared according to the invention is a compound of formula II wherein E ² is hydrogen, alkyl, hydroxymethyl, 1-hydroxyethyl, mercaptomethyl, 2-methylthioethyl, carboxymethyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl , cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, or E ¹ and E ² together with the nitrogen and carbon atom to which they are attached form a 4-membered, 5-membered, 6-membered or 7-membered azacycloalkane ring.

Another more preferred compound prepared by the process of the invention is a compound of formula II wherein B is an alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl or alkylcycloalkylalkyl group.

F

Particularly preferred according to the invention are the compounds of the general formula IIIa

• 9 •

9 4 • 9 9 rt · ♦

Γϋ

N _{CH2) -C- _N-CH2) -C-NH-CH-C-NH-CH ₂ CH CL ^P J COOH (IIa) in which represents alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl and alkylaralkyl, hydrogen, alkyl, cycloalkyl, cycloalkyl, alkyl, cycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, alkylaryl and alkylaralkyl, OR ¹ or NR ¹ R ² ,

R @ ¹ and R @ ² independently of one another are hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl or alkylaralkyl, number 1 to 5, number 2 to 6 and number 1 or 2.

Particularly preferred in accordance with the invention are compounds of formula (IIa) wherein the group is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl or alkylcycloalkylalkyl, hydrogen, alkyl, cycloalkyl, cyclo. · »Φ φ · φ · · φ · · · · · | | | | | | | |

alkylalkyl, alkylcycloalkyl or alkylcycloalkylakyl, the number 3 and the number 3 or 4

Particularly more preferred are compounds of formula (IIa) wherein they are

B is an alkyl group,

J is alkyl, cycloalkyl, cycloalkylalkyl,

R ^l and R ² are independently hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, or alkylcycloalkylalkyl, m whose 1 to 3, number 3, or 4 and p denotes a number first

A particularly preferred compound of the invention is a compound of formula IIIa, which is N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) - β-cyclohexylalaninamide.

The invention also relates to a process for preparing a stable, non-hygroscopic crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -3- According to the invention, this form of the compound is capable of developing a stable composition of the compound of the invention Stable non-hygroscopic crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) - asparagyl] - (L) -p-cyclohexylalaninamines22 and a a a * * a a a a a a a a a a a a a a a a a a a a a a a a Also has a high melting point and does not tend to absorb water, and the stable form also has a unique and unexpected resistance to humidity and temperature even at higher temperatures. Conversion of a compound to a compound, such as those used in transportation, manufacture of dosage forms, or long-term shipping or long-term storage. Abile form does not lead to material loss or purity and does not adversely affect particle properties.

The invention encompasses all possible combinations of the preferred compounds, particularly preferred and particularly preferred embodiments of the above.

The compound of the invention is useful in the form of a free base or acid, a double salt, or in the form of a pharmaceutically acceptable salt. The invention includes all these embodiments.

When a compound of the invention is substituted with a basic group, an acid addition salt is prepared which is a simple and more convenient form for use. In practice, the use of salt is more common than the free bases. The acid that can be used to prepare the acid addition salt is preferably an acid that forms a pharmaceutically acceptable salt with the free base, i.e. a salt whose anion is non-toxic to the patient in a pharmaceutical salt dose, thus favorably inhibiting platelet aggregation and thrombus formation. as a free principle and has no side effects attributed to the anion. Although pharmaceutically acceptable salts of the free bases are preferred, all acid addition salts are useful as the free base form when a salt as such is desirable only as an intermediate, for example, when the salt is prepared solely for purification and identification purposes or when it is used as an intermediate for the preparation of a pharmaceutically acceptable salt in ion exchange.

F

Pharmaceutically acceptable salts within the scope of the invention are, for example, derived from acids from the group consisting of mineral acids such as hydrochloric acid, sulfuric acid, and the like. Phosphoric and sulphamic acids and organic acids, such as acetic acid, citric acid. , lactic, tartaric, malonic, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclohexylsulfamic and quinic acids. Corresponding acid addition salts include hydrohalides such as hydrochloride and hydrobromide, sulfate, nitrate, sulfamate, acetate, citrate, lactate, tartrate, malonate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate.

The acid addition salts of the compounds of the invention can be prepared by reacting the free base with a suitable acid by methods known per se or modified. For example, the acid addition salt of the compounds of the invention may be prepared by dissolving the free base in an aqueous alcoholic solution or other suitable solvent containing the appropriate acid and isolating the salt by evaporating the solution or reacting the free base and acid in an organic solvent. concentrating the solution.

The free compounds of the invention can be regenerated from the salts using or adapting methods known per se. For example, the parent compounds of the invention may be regenerated from their acid addition salts with an alkali treatment such as aqueous sodium bicarbonate solution or aqueous ammoniacal solution.

When a compound of the invention is substituted with an acidic group, a base addition salt is prepared which is a simple and more convenient form for use. In practice, the use of salt is more common than the free acid. The base which can be used to prepare the base addition salt is preferably a base which forms a pharmaceutically acceptable salt with the free acid, i.e. a salt whose canion is non-toxic to the patient in a pharmaceutical salt dose, thus favorably inhibiting platelet aggregation and thrombus formation free principle and has no side effects,

9 9 9 9 9 9 · «· · · · · ^ύ ♦ • 9 · 9 ·« ·· »» · «» «· 9 9 9

99 9 9 9 9 * e

I 9 »· • · 9 to be attributed to the canon. Pharmaceutically acceptable salts include, for example, alkali metal and alkaline earth metal salts. Pharmaceutically acceptable salts within the scope of the invention are those derived, for example, from the following bases: sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonium, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N, N'-dibenzylethylenediamine, chlorprocain, diethanolamine, procain, N-benzylphenethylamine, diethylamine, piperazine, tris (hydroxymethyl) aminomethane and tetrachloride ammonium hydroxide.

The metal salts of the compounds of the invention may be prepared by reacting the free acid compound with a base selected from, for example, a hydride, hydroxide, carbonate and the like reactive compound of the selected metal by known or modified methods in an aqueous or organic solvent. The aqueous solvent used may be water or a mixture of water and an organic solvent, preferably an alcohol such as methanol or ethanol, a ketone such as acetone, an aliphatic ether such as tetrahydrofuran or an ester such as ethyl acetate. Such reactions are generally carried out at room temperature, but can also be carried out with heating.

The amine salts of the compounds of the invention can be prepared by reacting the free acid compound with an amine in an aqueous or organic solvent. The aqueous solvent used may be water or a mixture of water and an organic solvent, preferably an alcohol such as methanol or ethanol, an ether such as tetrahydrofuran or a nitrile such as acetonitrile or a ketone such as acetone. The ammonium salts of the compounds in acid form can be prepared in a similar manner.

salts

The free compounds according to the invention can be regenerated or adapted by themselves from known methods. Na25 · · ► ► ► ► ► C C C C C C 9 9 9 9 9 9 9

For example, the parent compounds of the invention can be regenerated from their acid addition salts with an acid treatment, e.g. for example hydrochloric acid.

Although useful as active compounds of the invention as such, salts of the compounds of the invention are useful for the purification of the compounds to be prepared, using, for example, different solubility of salts and parent compound, by-products and / or starting materials by a person skilled in the art.

The compounds of the invention may contain asymmetric centers. These asymmetric centers may be independently in the R or S configuration. It will be apparent to those skilled in the art that certain compounds of Formula I may exhibit geometric isomerism. Geometric isomers include cis and trans forms of compounds of the invention having alkenyl moieties. The invention includes individual geometric isomers and stereoisomers, and mixtures thereof.

Such isomers may be separated from the mixtures by the use or adaptation of methods known per se, such as chromatographic methods and recrystallization, or may be prepared separately from suitable isomers or from intermediates thereof, for example by use or adaptation of the methods described above.

U.S. Patent Application Serial Nos. 08/138 820 and 08/476 750 disclose methods for preparing an amorphous compound of Formula II, and in particular an amorphous compound of Formula I.

A novel process for the preparation of a compound of formula II and f

In particular, the crystalline compounds of formula I according to the invention are illustrated by Scheme II, wherein B, E ¹ , E ² , G, R, m, n and p are as defined above and P ¹ is a labile group when hydrogenated. · · · * • »· pin protecting acid group such as benzyl, P ² labile amino protecting group, for example a tert-butoxycarbonyl (BOC), and P ³ in the hydrogenation labile amine protecting group such as benzyloxycarbonyl (CBZ.

Scheme II

O

P ² -N- (CH ₂ ) _p -COH i

(B)

O,

II ⁺ H ₂ N-CH-COH ₂ CH ₂ P ¹ as coupling agent

P ² -N- (CH ₂ ) p B

TFA HN- _(CH2) pi

(B)

P ² -N- (CH ₂ ) _p coupling reagents

E ² O

II II _N-CH-CH2 CG _CO2 P ¹

TFA

O

1L

-omJÍ

HN-CHI coupling agent p3 ·, z

N *

O

II ¹¹ - ^ - HN - CH - CO ₂ H CH ₂ CO ₂ P ¹

E ¹ E ² OII ii-N-CH-C-G

CH ₂ CO ₂ P ¹ I - ( ^Η > ² ) p3

NO

II

- (ch ₂ - | ~ c-oh

- o OE ¹ E ² O

ICH ₂ A-C-N- _{(CH2) p} -IL-HN-CH - CH-N-C-G 'ni | ³ CH ₂ CO ₂ P ¹ hydrogenation (eg gas or chemical transfer, fer)

SS

LOOOE ¹ E ² O

-f-CHD-C-N- _{(CH2) p HN} _CH -1L-Π-N-OH, -C -G 'n' I ^B CHoCOoH>

• »99 99 ·»

0 9 9 9 '»· 9 ·· * ·· tf

9 99

9 9

9 ·

999 999

During the preparation of the compounds of formula (I) or intermediates thereof, it may be desirable or necessary to avoid a cross-linking reaction between chemically active substituents on naturally occurring amino acids or pseudoamino acids. Such substituents may be protected by known protecting groups, which may subsequently be removed or may optionally be left using known methods to achieve the desired product or intermediate (e.g., TH Green Protective Groups in Organic Synthesis, Walley New York, 1981). deprotection and deprotection may also be necessary or desirable to prevent conversion or removal of existing substituents or to allow subsequent reaction to achieve the desired product.

The process of Scheme II is an exemplary method of preparing a compound of Formula II, but it is noted that the compound of Formula I can also be prepared in this manner using appropriate starting materials. When preparing compounds of formula I as described in Scheme II, R B is ethyl, E ¹ is hydrogen, E ² cyk1ohexylmethy1ovou group G amino, R is hydrogen, m is 3 and n is 3, p is 1, P ¹ is benzyl, P ² tert-butoxycarbonyl (BOC) and P ³ benzyloxycarbonyl (CBZ).

Alternatively, a compound of formula I according to the invention as prepared in Scheme II, with the výjinkou that a compound of general formula III, _P 3

OH (ΠΙ) where P ^{3 of} formula IV has the meaning given above, instead of a compound of the general formula

(IV) wherein R represents a hydrogen atom, m number 3, n P ³ represents a benzyloxycarbonyl group of the intermediate of formula V

HN · Number 3 and p number (CBZ), for obtaining

O-oh-IL

AND

E ¹

I • N ² OI II • CH-C-G _{CH2 CO2} P ¹ and

me (V) wherein B represents ethyl, E ¹ hydrogen, E ² cyclohexylmethyl, G amino, o 1, P ¹ benzyl and P ³ benzyloxycarbonyl (CBZ).

The process of Scheme II is a five step process for preparing compounds of the invention starting with the formation of the central dipeptide intermediate of the invention of Formula VI

Ώ

HN - CH - CO ₂ H i.

B CH ² CO ² P ¹ (vi) wherein B, p, P ¹ and P ² are as defined above. For the preparation of a compound of formula I, the central dipeptide intermediate of the invention is BOC-N (Et) Gly- (L) -Asp (OBz1) -OH. The central dipeptide intermediate is prepared without protecting the free carboxylic acid moiety.

In Step 2 of Scheme II, coupling can be performed to give the central dipeptide either in dichloromethane or ethyl acetate in the presence or absence of tetrahydrofuran as a cosolvent and in the presence of an organic base such as N-methylmorpholine, at about room temperature. up to about 40 ° C. Activation of amino acid or pseudoamino acid of general formula

O

P ² -N- (CH ₂ ) _p - COH B where B, P ² and p are as defined above, for the coupling may be carried out using non-isolated active esters with p-nitrophenol, pantafluorophenol and N-hydroxysuccinimide via dicyclohexylcarbodiimide. The coupling time is 1 to 20 hours, depending on the amino acid or pseudoamino acids to be coupled, the activating agent, the solvent and the temperature. The central dipeptide product of step 1 is not isolated. The reaction mixture is generally washed in step 1 with water or dilute aqueous acid (e.g. aqueous hydrochloric acid) and used directly without drying in step 2. In the case where active phenol-based esters are used, the central peptide is extracted into an alkaline aqueous medium from the reaction mixture and then re-extracted after acidifying the aqueous solution back into the organic solvent; the solution is reacted directly as in step 2.

The dipeptide intermediate of formula (VI) is used to prepare the tripeptide intermediate of formula (VII)

P ² -N—

Ž o

-1L u ΜΗΝ — CH I

-NE ² OI II CH — C — G

CH ₂ CO ₂ P ¹ (Vil) wherein Β, E ¹ , E ² , F, G, P ¹ and p are as defined above and wherein P ² 'is P ² or TFA.H-. When P ² 'represents a TFA group. dissociation of trifluorooc99 9 99 9 9

9

9 9 9

9,999 9,999 99 9

9 9 ··

9 99 99

- 30 • 9 • 9 'i f

acetic acid (TFA) on FsCCCU- and H ⁺ , wherein H + protonates the terminal amine in the compound of formula VII, i.e. the formation of the trifluoroacetate salt of formula VIIa

CF3CO2

H2N- _{(CH2) r} B

HN — CH - “- N1

CH ₂ CO ₂ P ¹

E ² OI II CH — C — G (Villa)

For the preparation of a compound of formula (I), the tripeptide intermediate of the invention is P ²⁵ -N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH 2.

In step 2, coupling of the amino acid or pseudo amino acid to the central dipeptide can be carried out either in dichloromethane or in a mixture of ethyl acetate and dimethylformamide or tetrahydrofuran at about room temperature or below room temperature. Activation of the central dipeptide of formula p2. _N - (CH 2) |

AND

B fi

HN-CH-CO ₂ H ₂ CO ₂ p ¹ where B, P ¹ , P ² and p are as defined above, for coupling may be carried out using non-isolated active esters of pentafluorophenol or N-hydroxysuccinimide via dicyclohexylcarbodiimide. Activation can also be accomplished using isopropyl chloroformate. The coupling time is 1 to 20 hours, depending on the amino acids or pseudoamino acids to be coupled, the activating agent, the solvent and the temperature. The tripeptide product is not isolated. When the tripeptide is not isolated, the reaction mixture is washed with an aqueous organic base such as an aqueous solution of N-methylmorpholine and an aqueous acid (e.g., aqueous hydrochloric acid)

ΑΑ · A

AAA

A AAA

AAA

AA

A A

A A A A

AAA

A A

and allowed to react as it is in step 3 after washing with water and without drying.

In step 3 of Scheme II, the protecting group, for example the BOC group, is removed from the tripeptide obtained in step 2, for example, using a solution of trifluoroacetic acid in dichloromethane or using a mixture of hydrogen bromide in acetic acid and ethyl acetate. The reaction can be carried out at about room temperature and requires about one hour (for a method based on hydrogen bromide) and about two hours (for a process based on trifluoroacetic acid). The acid salt of the tripeptide produced is isolated by filtering off the crystalline solid either directly from the reaction mixture (in the case of a hydrogen bromide method) or after partially removing the solvent by distillation and adding the non-polar solvent to the residue.

Another method of the invention is characterized as one chained process for the rapid and simple preparation of TFA.HN (Et) Gly (L) -Asp- (OBzl) - (L) -Cha-NH ₂ from BOC-N (Et) Gly-OH, a one-pot reaction involving the first two coupling steps of Scheme II and trifluoroacetic acid treatment. Uniquely, TFA.HN (Et) Gly- (L) -Asp- (OBz1) - (L) -Cha-NH2 is obtained as directly crystallizes from the chain reaction solution. This chain process eliminates three corresponding separate reaction steps according to Scheme 2 and solves the problem of preparation in a simple, time and cost saving, environmentally friendly manner.

Scheme II illustrates the reverse order of construction of a polypeptide starting from a nitrogen protected amino acid and gradually added to the carboxyl terminus, as opposed to the usual order where the polypeptide is constructed by sequential amidation at the C-terminal protected ammonium terminus. The reverse synthesis process of the invention requires protection of the nitrogen atom only on the first amino acid to allow

-.

* Use from this point onwards sequential amino acids without a protecting group at either the amino or acid terminus (except for side chain functional groups). The reverse synthesis process also rationalizes the production of compounds of formula II, and in particular of compounds of formula I, by allowing the use of a continuous process as opposed to the continuous process for peptide chemistry in the solution phase. The new approach reduces costs by removing amino acid-protected amino acid procurement. No special equipment, reagents or analytical methods are required.

Another method of the invention is a reproducible process for the preparation of a non-hygroscopic stable crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -pcyclohexylalaninamide solid state conversion from the hygroscopic crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl-3-N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexylalaninamide, prepared by according to Scheme II and referred to as alternative reaction steps.

The hygroscopic crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -fi-cyclohexyl-laninamide is physically unstable and is converted by humidity and temperatures to a highly stable, non-hygroscopic crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -f-cyclohexylalaninamide.

General conditions of the invention for the conversion from the hygroscopic crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl) N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexylalaninamide to the highly stable non-hygroscopic crystalline form of N- [N- [N- (4f (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -p-cyclohexylalaninamide is influenced under static and dynamic conditions.

4 4 · 44 444

4 4 4 4 4 5

4444 4444

4 4 4 4 4

444 44 4 ·· • 4 4 4

444 · 44 ·

The static method of the invention is described as static conversion since it involves exposing the hygroscopic crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - ( L) 3-3-cyclohexylalaninamide in a stationary vessel, such as vials or bowls under certain conditions of exposure to temperature and humidity in a chamber under controlled conditions.This static conversion is performed at a temperature of about 20 to about 80 ° C, more preferably about 40 to about 80 ° C. And at a relative humidity of about 40% to 100%, preferably 65% to 80%.

The dynamic process of the invention is described as dynamic conversion since it involves exposing the hygroscopic crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - ( L) -p-cyclohexylalaninamide by incubation under temperature and humidity conditions as in the static model but with stirring including inversion of the hygroscopic crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl]] - (L) asparagyl] - (L) -β-cyclohexylalaninamide in a rotary evaporator flask or in a cylindrical vessel (in a moisture oven) with vane stirring.

The following example illustrates the invention in more detail, but does not limit the invention in any way. Percentages and parts are by weight unless otherwise indicated.

Unless otherwise stated, mass spectral analysis values mean the results of low resolution fast atom bombardment performed on a VG 70SE with calculated (M + H) ⁺ values. Nuclear resonance magnetic spectra values are obtained on a Brucker ACF 300 instrument in D2O. Flash chromatography was performed on silica gel. High performance liquid chromatography (HPLC) is performed on C-18 reverse phase columns with 8 to 15 µm particles.

«· · · · · · · · · · · · · · · · · · · · · · ·

Unless otherwise noted, powder X-ray diffraction patterns are obtained using a Siemens D5OOO diffractometer with a copper radiation source (1.8 kW, 45 kV and 40 mA). Samples are ground prior to measurement to eliminate the effect of product particle size on peak intensities. Approximately 60 mg of sample is added to a sample holder of 1.5 x 1 cm and readings are made in the 4 to 40 ° 2 theta (2 Θ) range of 0.04 ° and a total exposure of one second per stage.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Preparation of BOC-N (Et) Gly- (L) -Asp (OBzl) -0H (Step 1, Scheme II)

A 3-neck round bottom flask was charged with 51 g (0.25 mol) of BOC-N (Et) Gly-OH, 35 g (0.25 mol) of PNP, 400 ml of ethyl acetate and 100 ml of dimethylformamide. The mixture was stirred until dissolved and cooled to 4-6 ° C. A solution of 51.5 g (0.25 mol) of dicyclohexylcarbodiimide in 125 ml of ethyl acetate is added dropwise over 10 minutes, maintaining the temperature at 5 ° C to about 8 ° C. When all dicyclohexylcarbodiimide was added, the cooling bath was removed and the mixture was stirred for 1.5 hours while warming to room temperature (20-22 ° C). A solid DCU precipitate formed. Formation of the PNP ester is detected by analytical HPLC (disappearance of BOC-N (Et) Gly-OH). The reaction mixture was filtered and the remainder of the DCU was washed with 2-50 mL portions of ethyl acetate and the wash was added to the filtrate. The DCU is thrown away.

To the stirred, diluted solution was added 67 g (0.3 mL)

mol) H 2 N- (L) -Asp (OBz) -0H as a suspension in 150 mL (138 g, 1.36 mol) NMM. The mixture is heated to 38-40 ° C and maintained at 41 hours, the time at which

44

4 4 4 4 4 5

443 4 44 4

444444 444 ···

4 4 4 4

44 44

9 · 4 analytical HPLC shows complete consumption of BOC-N (Et) Gly-OPNP. The reaction mixture was cooled to 25 ° C and unreacted H 2 N (L) -Asp (OBz) -OH was filtered off. The solution was cooled and filtered again to give an additional 1.2 g (21.7 g; 11.2 g representing an added 20% excess and 10.5 g (0.047 mol) representing unreacted material).

The filtered solution was extracted in a 2 L Squibb funnel with one portion of 500 mL deionized water, followed by two 250 mL portions. The combined aqueous solution was extracted with three 300 mL portions of 1: 1 MTBE / EtOAc to remove residual PNP (analytical HPLC shows only the remaining traces), cooled to 5 ° C and acidified from pH 8.9 to pH 1.79 dropwise 150 ml concentrated hydrochloric acid. The acidified aqueous solution shows no desired product. The ethyl acetate extracts were combined, dried over magnesium sulfate, filtered and concentrated on a rotary evaporator at 35 ° C. The resulting pale orange oil is pumped at 35 ° C to maximize removal of residual solvent and give 85.68 g of BOC-N (Et) -Gly- (L) Asp (OBz) -0H as an oil (21.3 mmol, 85, 5% yield, uncorrected for residual solvent).

Characteristics:

NMR (250 Hz): 7.3 ppm (s), 5.1 ppm (s), 3.3 ppm (dq), 3.0 (dq), 1.4 ppm (s), 1.1 (t )

MS: M = 408; M + 1 = 409 found

HPLC: 90, 79A% (3.87A% ρ-nitropheno1, uncorrected for e)

Analysis: C20H28N2O7,

Η, N; Found 57.54, calculated 58.81

Example 2

Preparation of BOC-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH 2 (Step 2 of Scheme II)

Method A: The Isopropyl Chloroformate Method. &Lt; tb &gt; ______________________________________ &lt; tb &gt; ______________________________________ &lt; tb &gt;

-.

• ·

One equivalent of BOC-N (Et) Gly- (L) -Asp (OBzl) -0H was dissolved in ethyl acetate (6-8 volumes; 1: 6.5 w / v) and maintained at -15 to 0 ° C. . Add NMM (1 equivalent) while maintaining a temperature of -15 to about 0 C. Isopropyl chloroformate (1 to 1.1 equivalents) is added to the protected isopeptide solution at -15 to 0 °. The reaction mixture is maintained at -15 to about 0 ° C for 2 to 5 minutes. To a cold dipeptide solution maintained at -15 to about 0 ° C was added a solution of H 2 N- (L) -Cha-NH 2 (1 equivalent) in THF (10 volumes; 1:10 w / v). The reaction was monitored by controls (HPLC) after 15 minutes, after one hour and two, 2 hours to assess the completion of the reaction. (The reaction is complete when the amount of peptide observed is less than 10% per area by HPLC analysis).

The BOC-tripeptide product precipitates directly from the reaction solution and is filtered from the reaction mixture, washed with ethyl acetate (2 x 1 volume / w / v) and dried under vacuum. The usual yield is> 60% of theory, with a purity of> 9OA%, <1A% of the epimeric diastereomer of aspartic acid.

Resuspension in ethyl acetate yields a final yield of approximately 60% of the theory of BOC-N (Et) Gly- (L) -Asp (OBzl) - (L) -ChaNH2 with improved purity of> 95A% while reducing the diastereoisomer content to <0.5% .

A specific example of the isopropyl chloroformate method, following the general procedure of Method A and using 4.55 g (8.1 mmol) of BOC-N (Et) Gly- (L) -Asp (OBz1) -0H, provides the amount of BOC-N prepared ( Et) Gly- (L) -Asp (OBz1) - (L) -Cha-NH 2 3.26 g (97.9 A% pure 0.3A% diastereomer) in 70% theoretical yield.

F

Method B: Method using pentafluorophenoldicyclohexylcarbodiimide complex

I · 4 · 4 · 4 · 4 ·· ··

Dissolve pentafluorophenol (PFP, 2.9 equivalents) and dicyclohexylcarbodiimide (1 equivalent) (5 volumes, 1: 5 w / v) at room temperature in ethyl acetate and cool to -15 to 0 ° C. Dissolve one equivalent of BOC-N (Et) -Gly- (L) -Asp (OBz) -OH in ethyl acetate (6 volumes, 1: 6 w / v) and mix with one equivalent of H 2 N- (L) -Cha- NH 2, which was previously dissolved in dimethylformamide (10 vol, 1:10 w / v). A dipeptide / H 2 N (L) -Cha-NH 2 solution is added dropwise to the PFP solution and dicyclohexylcarbodiimide and the temperature is maintained at -15 to 0 ° C. The reaction mixture is maintained at 15-22 ° C for 5-16 hours and control samples (HPLC) are taken after 1, 2, 3, 4, and 16 hours to assess the completion of the reaction. (The reaction is complete when the amount of dipeptide observed is less than 2% per area by HPLC analysis).

The reaction mixture was filtered and the filter cake (DCU) was washed with ethyl acetate (2 x 0.5 volumes, w / v). The filtrate was treated with water (10 vol, 1:10 w / v) and the aqueous layer was removed. The ethyl acetate layer was washed with water (1x, 5 volumes, 1: 5 w / v). The ethyl acetate layer was cooled to precipitate the product, which was washed with ethyl acetate (2 x 0.4 volumes; 1: 0.4 w / v). The isolated molar gains are> 60% in the usual purity of> 90A%, with 1-4A% epimeric aspartic acid diastereomers.

Resuspension in ethyl acetate yields a final yield of approximately 60% of the theory of BOC-N (Et) Gly- (L) -Asp (OBzl) - (L) -ChaNH2 with improved purity of > 99A% by reducing the diastereoisomer to < 0.5%.

A specific example of the method using pentafluorophenoldicyclohexylcarbodiimide complex, following the general procedure of Method B and using 10 g (24.5 mmol) of BOC-N (Et) Gly (L) -Asp (OBz1) -OH, provides the amount of BOC-N (Et) prepared. Gly (L) -Asp (OBzl) - (L) -Cha-NH2 8.15 g (99% pure, 0.49% diastereomer) in 59% of theory.

-?

• · ···· ··

I · · · · · · · · · · · ·

Method C: Method based on the use of the hydroxybenzotriazole (HOBT) / 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) system

One equivalent of BOCN (Et) -Gly- (L) -sp (OBzl) -OH (9-10 volumes; 1:10 w / v) was dissolved in dimethylformamide and maintained at room temperature. To this solution was added 12 N - (L) -Cha-NH 2, (one equivalent) and hydroxybenzotriazole (HOBT, 1 equivalent). The resulting solution was cooled to about 0 to 10 ° C and NMM (1 to 1.1 equivalents) was added. The coupling reagent, TBTU (1-1.1 equivalents) was dissolved in dimethylformamide (4-5 volumes, 1: 5 w / v) and added to the protected dipeptide solution at a temperature of about 0-10 ° C. The solution was stirred at about 10 ° C to about 25 ° C for about 3 hours until HPLC indicated complete reaction (less than 2% starting material per area). To the stirred mixture of 5% aqueous sodium chloride solution (about 4 volumes per reaction volume) was added the reaction mixture and ethyl acetate (about 2 volumes per reaction volume). The phases were separated and the aqueous phase was extracted with another portion of ethyl acetate (approximately 1.5 volumes per reaction volume). The organic phases were combined and washed successively with 0.5 N aqueous citric acid (about 0.6 to 0.7 volumes per volume of organic phase), 10% aqueous sodium bicarbonate solution (twice, about 0.6 to 0.7 volumes). per volume of organic phase) and 25% sodium chloride solution (about 0.3 to 0.4 volumes per volume of organic phase). The resulting organic phase is concentrated to about a quarter to half the volume under reduced pressure at about 30 to 50 ° C and an equal volume of heptane is added to this warm solution. The mixture is stirred and cooled to 0 ° C to about 20 ° C to precipitate the desired tripeptide. This solid was filtered off and treated with ethyl acetate and heptane and dried. The usual yield is> 60% f

theory of usual purity> 95.7A% epimeric aspartic diasteromer <2A%.

• · ·······································

As a specific example of a method based on the use of the HOBT / TBTU system, following general method C, 10 g (24.5 mmol) of BOC-N (Et) -Gly- (L) -Asp (OBz1) -0H were used and then Preparation of 9.3 g of BOC-N (Et) Gly- (L) -Asp- (OBz1) - (L) -Cha-NH2 in a theoretical yield of 67.7% (96, IA% pure, 1.77A% diastereomer at Asp).

Mass Spectrum: M 560.7 calculated; M + 1 561 found melting point 182.17 ° C (DSC) ¹ HNMR (delta vs TMS, D 6 DMSO); (dt, 2H), 1.15 (m, 2H), 1.06-1.75 (m, 6H), 2.65 (m, 1H), (d, 2H), 4.2 (S, 1H) ), 4.66 (d, 1H (d, 1H), 7.36 (s, 5H), 7.88 (dd)

0.89 (m, 1 H), 0.94 (m, 1 H), 1.0

1.3 (m, 4H); 1.36 (d, 9H);

2.85 m (1H), 3.18 (m, 2H), 3.75, 5.08 (s, 2H), 7.02 (s, 1H), 7.18 1H), 8.24 (dd) , 1H).

Example 3

Preparation of TFA-N (Et) -Gly- (L) -Asp (OBzl) - (L) -Cha-NH 2 (Step 3, Scheme II)

BOC-N (Et) Gly- (L) -Asp (OBz1) - (L) -Cha-NH 2 was dissolved in dichloromethane (about 1:12 by weight) and TFA was added to the solution at room temperature. The solution was stirred until HPLC indicated complete reaction (three to five hours). The solution is concentrated to approximately half the volume at 40-45 ° C. MTBE (approximately 1:10 w / v against B0C-N (Et) Gly- (L) -Asp (OBz1) - (L) Cha-NH2)) is added to this warm solution while maintaining the temperature > 40 ° C. The mixture was cooled slowly to about 5 ° C and stirred for one hour to ensure complete crystallization. The resulting solid was filtered off and cooled with MTBE. The solid was dried under reduced pressure and analyzed for TFA. N (Et) Gly- (L) -Asp (OBz1) - (L) -Cha-NH 2 (HPLC w / w test). The yield is generally almost quantitative, purity > 95A%.

F

Mass spectrum: M 460 (free base) calculated; M + 1 461 found

Analysis: C 26 H 37 N 4 O 7 F 3 N, H, F, C 54.35 calculated 53.82 found ·

9999 9 99 9 • 9 9 · 9 99 9

9999 99 99 999 99 99 1 HNMR (m, 1 H) (s, 2H) (s, 1 H) (delta vs TMS, D ⁶ DMSO): 0.9 (m, 2H), 1.16 (t, 6H) 1.5, 1.5 to 1.8 (m, 6H), 2.65 (dd, 1H), 2.9 (m, 3H), 3.7, 3.9 (m, 2H), 4 2 (m, 1H), 4.75 (m, 1H), 5.1 (s, 2H), 7.0, 7.15 (s, 1H), 7.2 (s, 5H), 8, 13 (d, 1H), 8.7-8.8 (m, 3H) ¹³ CNMR (protruding signals, delta vs 25.49, 25.68, 25.96, 31.66, 33.07, 41.88 , 47.02, 49.40, 50.47, 65.71, 165.10, 169.34, 173.79

TMS (D6 DMSO): 33.36, 36.25,

127,81-128,34,

10.76, 38.59,

135,82,

Specific examples of deprotection are given in Table A.

Table A

Lab

Example

Amount of reaction scale (BOC-N (Et) Gly- (L) -Asp (OBsl) (L) -Cha-NH 3)

Yield & Purity A% (TFA.N (Et) Gly (L) -Asp (OBzl) - (L) -Cha-NH 3)

7.5 g (13.3 mmol)

6.53 g (11.6 mmol)

7.4 g (12.9 mmol) 97% yield 98.8 A% pure

6.4 (11.1 mmol) 97% yield 98.47A% pure

Example 4

Preparation of CBZ-PipBu-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH 3 (Step 4, Scheme II)

Prepare a suspension of approximately equimolar amounts of (TFA.N (Et) Gly- (L) -Asp (OBsl) - (L) -Cha-NH 3, CBZ-PipBu and TBTU in ethyl acetate, dimethylformamide, and water (v / v = 100 = 8 = 4, approximately 114 vol / wt against TFA.N (Et) Gly- (L) -Asp (OBsl) 41-9 (L) -Cha-NH 2, which suspension is cooled to 0-10 C. and about 3-4 equivalents of NMM are added and the mixture is allowed to warm to room temperature and stirred until complete by HPLC (one to three hours, during which time a solution appears). The organic phase was washed with two additional portions of water, the combined aqueous washings were back extracted with ethyl acetate, and the combined organic phases were washed with 25% aqueous sodium chloride solution. phase is concentrated under reduced pressure to approximately half the volume and MTBE (ob The mixture was allowed to crystallize (several hours) and the solid was dried under reduced pressure. The content of CBZ-PipBu-N (Et) Gly- (L) -Asp (OBz1) - (L) -ChaNH 2 was analyzed by HPLC, mass / weight test. The yield is generally> 80% of theory, purity> 95A%.

A specific example of preparation, following the general procedure of Step 4, provides 7.25 g of TFA. N (Et) Gly- (L) -Asp (OBzl) - (L) Cha-NH 2 7.9 g of CBZ-PipBu-N (Et) Gly- (L) -Asp (OBz1) - (L) -Cha-NH 2 (> 99A% pure 0.08A% diastereomer at Asp), theoretical yield 84% of theory.

Mass spectrum: M 747 Calcd; M + 1 748 found

Analysis: C 41 H 57 N 5 O 5 H, N, C 65.84 calculated 65.38 found melting point 101.6 C (DSC) ¹ H NMR (delta vs TMS, CDCl ₃ ): 0.88 (m, 1H), 0.98 (m 1 H) 1.13 (2H), 1.23 (m, 6H), 1.4 (m, 1H), 1.62-1.76 (m, 8H), 1.86 (qd, 1H), 2.35 (t, 1H), 2.74 (dd, 2H), 3.25 (dd, 1H), 3.47 (q, 2H), 3.7 (d, 1H), 3.84 (d) 1 H), 4.15 (ds, 2H), 4.5 (qd, 1H), 4.68 (dt, 1H), 5.07 (d, 1H), 5.14 (bd, 2H), 5 16 (d, 1H), 7.28-7.39, (m, 10H), 7.57 (dd, 1H).

¹³ C NMR (delta vs TMS, CDC1 ₃₎ :( protruding peaks) 66.93 (both benzyl carbons _f), 127.78 to 128.64 (both phenyl rings), 155.249, 170.00, 170.24, 171, 69, 174.27, 175.21 (all carbonyl carbons).

- 42. ** · φ φ φ φ

Example 5

Example of a hygroscopic crystalline form of N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexyl-alaninamide (Step 5, Scheme II)

Prepare a mixture containing CBZ-PipBu-N (Et) Gly- (L) Asp (OBzl) - (L) -Cha-NH 2, ammonium format and 10% palladium on carbon in a 20: 1 alcohol / water system (10: 1) v / v vs. CBZ-PipBu-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH 2). The mixture was warmed to 40-50 ° C and stirred until complete by HPLC (1-2 hours). The mixture was cooled to room temperature and filtered to remove the catalyst. Heat the resulting solution to 40-50 ° C and add acetone (approximately volume vs. filtered solution), allow the solution to cool to 35-40 ° C. N- [N [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] (L) -3-cyclohexyl-alaninamide is seeded into the mixture and the hygroscopic form of N- is crystallized therefrom. [N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] (L) -asparagyl] - (L) -β-cyclohexyl-alaninamide upon cooling to room temperature (several hours). The solids were collected by filtration under nitrogen and washed with acetone. The solids were dried under reduced pressure and analyzed for the crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl) N-ethylglycyl] - (L) -asparagyl] - (L) - β-cyclohexyl-alaninamide (HPLC wt / wt). The yield is usually> 85% of theory, purity> 95A%.

A specific example of preparation following the general procedure of Step 5 gives 5 g of CBZ-PipBu-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH2 3.1 g of hygroscopic crystalline N- [N] form. [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] (L) -3-cyclohexylalaninamide as a white solid (purity 99.6A%) in a stoichiometric yield of 89, 4% of theory.

Other compounds prepared according to Examples 1-5 but using the appropriate starting materials are: N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] valine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] D-valine,

N- [N- [N- (3- (piperidin-4-yl) propanoyl) -N-ethylglycyl] aspartyl] valine,

N- [N- [N- (5- (piperidin-4-yl) pentanoyl) -N-ethylglycyl] asparagyl] valine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] L-.alpha.-cyclocohexylglycine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] norleucine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] asparagyl] L-α- (2,2-dimethyl) prop-3-yl-gycin,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] L-p-decahydronaphth-1-yl-alanine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] asparagyl] L-a-2-cyclohexylethyl) glycine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] phenylalanine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] -L-p-naphth-1-yl-alanine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] -L-p-naphth-2-yl-alanine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl ΙΕ- β-cyclohexylalanine ethyl ester,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] -L-p-cis-decahydronaphth-2-ylalanine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] alpha-aminocyclohexanecarboxylic acid, f

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] -β-cyclohexyl-D-alanine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] aspartyl] ·

- 44 .’-.

> ·· · ···. Β-decahydronaphth-1-yl-alanine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] asparagyl] β-cyclohexylalanine ethylamide,

Ν- [Ν- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] -asparagyl-β-cyclooctylalanine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] asparagyl] β-cyclohexylmethyl and lanidinide,

Ν- [Ν- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] asparagyl] β-adamant-1-ylalanine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] asparagyl] [beta] - (1,2,3,4) -tetrahydronaphth-5-ylalanine,

Ν- [Ν- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] -asparagyl] β- (4-cyclohexyl) cyclohexylalanine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] asparagyl] beta-cycloheptylalanine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] asparagyl] beta-cyclooctylalaninamide,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] -asparagyl-cyclohexylpropylglycine,

N- [N- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] asparagyl] β-cyclooctylmethylalanine,

Ν- [Ν- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] asparagyl] β-cyclopentylalanine and

Ν- [Ν- [N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl] -asparagyl-β-cyclohexylmethyl-alanine-ethyl ester.

Example 6

Preparation of 4-N-CBZ-piperidone

A mixture of 40 kg of N- (benzyloxycarbonyl) succinimide and 26 kg (175 mol) of 4-piperidone hydrochloric acid.H2O in 38.8 kg of water and f

kg of tetrahydrofuran is stirred at 15 + 5 ° C for 2.5 hours until dissolution is complete (approximately 15 minutes). NMM (22.8 kg) is added to the stirred (exothermic) mixture while maintaining the temperature.

- 45. ° C or lower. The bath was stirred at 15 + 5 ° C for 2.5 hours until the reaction was complete by HPLC. The mixture was diluted to 115.2 kg MTBE and 38.8 kg water and stirred at 20 + 5 ° C for 5 minutes. Stirring is complete, the layers are allowed to separate and the aqueous (lower) layer is discarded and discarded. The organic layer was washed with 2 x 129.6 kg of water (stirred for 5 minutes, the phases were separated and the aqueous (lower) layer was discarded and discarded. The organic layer was washed with 5.2 kg of sodium chloride 46.8 kg of water (with stirring) minutes, the phases are separated and the aqueous (lower) layer is discarded and discarded The organic phase is treated with 11.5 kg of magnesium sulfate with stirring for one hour, then filtered and the reactor is rinsed with 8 kg of MTBE (the solution is filtered and combined The total water content of the filtrate is 0.52%) The volume of the mixture is reduced by half by distillation under reduced pressure at 30 ° C. Vacuum is evacuated with nitrogen and the residue is cooled to 20 ° C (water content of the residue). Dilute the residue with 57.6 kg MTBE, then reduce the volume again in half at 30 ° C under vacuum (residual water content: 0.25%) .This is repeated 5 times. The reactor was diluted with 28.8 kg of MTBE and stirred for 5 minutes after which the contents were determined water and 4-N-CBZ-piperidone content (water 0.05%; w / w of 4-N-CBZ-piperidone: 22.66% by weight, 35.36 kg, 155 mol, 88.6% stoichiometric yield) .

Example 7

Preparation of PipBu

A solution of 53.5 kg of 3-carboxypropyltriphenylphosphonium bromide in 230.1 kg of 1,2-dimethoxyethane is prepared while bubbling nitrogen and stirring. Potassium tert-butoxide / tetrahydrofuran system (20 wt%, 141.8 kg of solution) was added over 35 min at rt.

maintaining a temperature of 24 to 28 ° C. The mixture was stirred at this temperature for half an hour until completion of the reaction by HPLC. The stirred mixture is cooled to 10 + 2 ° C, then 96.45 kg is added

- 46. * - ·.

(titer: 1.15 molar eq.vs.) of 4-CBZ-piperidone in MTBE over 40 minutes, leaving the temperature at 12 + 2 ° C. The mixture was stirred at this temperature for 10 minutes, then heated to 20 + 2 ^e C and stirred for two hours at this temperature. A solution of 22.5 kg of concentrated aqueous hydrochloric acid in 245.6 kg of water was added to the stirred mixture while maintaining a temperature of 20 + 2 ° C; the final pH is 0.5. The mixture is extracted with stirring 214.0 kg of methyl tert-butyl ether. Stirring is complete, the phases are allowed to separate and the aqueous (bottom) layer is removed and discarded. The organic phase was washed with 133.75 kg of water (stirred for 5 minutes, separated, the aqueous layer (bottom) was removed and then discarded then washed with 10.7 kg of 50% sodium hydroxide solution in 126 kg of water (stirred for 10 minutes, layers). The aqueous layer was extracted with 2 x 123.05 kg of ethyl acetate (stirred for 5 minutes, the layers were separated and the organic (upper) layer was discarded.) 13.1 was added to the stirred aqueous layer. kg of concentrated aqueous hydrochloric acid to pH 2.5 to 3.5 (final pH 2.82), then the mixture is extracted with 123.05 kg of ethyl acetate (stirred for five minutes and the aqueous layer (bottom) discarded and discarded). of ethyl acetate was washed with 133.75 kg of water (stirred for five minutes, the layers were separated and the aqueous layer (bottom) was removed and discarded, then the CBZ-PipBuen content (w / w) was determined (total weight: 194.86 kg, 17 , 05% CBZ-PipBuen [33.22 kg, 108 mol, 87.9% stoichiometric yield).

A solution of PipBuen in ethyl acetate along with 6.6 kg of 5% palladium on carbon (wt. 50% water) was added to the stainless steel pressure vessel with stirring and the mixture was heated to 55 + 2 ° C. Potassium format (38.2 kg) dissolved in 66.4 kg of water was added to the mixture while maintaining a temperature of 55 + 2 ° C (about 30 minutes). The mixture was stirred at 55 ± 2 ° C for two hours and the reaction was terminated (HPLC). 6.6 kg of Celite and 33.2 kg of water were added to the reactor, stirred and filtered. The reactor is rinsed with 33.2 kg of water, filtered and added to the main filtrate. Pour the filtrate into a new container, cool to 20 to 25 ° C.

The layers were allowed to separate and the organic layer was removed and discarded. The aqueous layer was acidified with 52.1 kg of concentrated aqueous hydrochloric acid to a pH of 2-3 (final pH 2.82), then extracted 4 x 129.5 kg of methylene chloride (stirred After 5 minutes, the layers were separated, discarded and the organic layer discarded. The aqueous phase is adjusted to pH 6.1 with stirring by the addition of 17.85 kg of aqueous 50% sodium hydroxide solution. The mixture was filtered to give 224 kg of a solution containing 17.6 kg (103 mol) of 4- (3'-carboxypropyl) piperidine.

Example 8

Preparation of CBZ-PipB

A solution of 224 kg of 4- (3'-carboxypropyl) piperidine in aqueous sodium hydroxide solution is mixed with 55.3 kg of tetrahydrofuran and the mixture is cooled to 8 + 2 ° C with stirring. NMM (20.9 kg) was added while maintaining the temperature below 10 ° C. After the addition was complete, the temperature was adjusted to 8 + 2 C and 25.7 kg of 1- (benzyloxycarbonyl) succinimide dissolved in 49.8 kg of tetrahydrofuran was added over 1 hour while maintaining the temperature below 15 ° C. The reaction was complete (according to HPLC) after three hours at 10-15 ° C. Concentrated aqueous hydrochloric acid (29.9 kg) was added to adjust the pH to 2.5-3.5 (final pH 3.3), then 61.4 kg MTBE was added to the mixture and stirred for 5 minutes. Stirring is stopped and the layers are allowed to separate and the aqueous (bottom) layer is discarded. The MTBE layer was washed with 3 portions of 83.1 kg water (stirring for 10 minutes, 5 minutes and 5 minutes); the aqueous phase is allowed to separate and in any case discarded. Without stirring, 8.3 kg of 50% aqueous sodium hydroxide in 95.7 kg of water are added and after the addition is complete the mixture is stirred for about 5 minutes. Stirring is complete, the phases are allowed to separate and the inorganic (upper) layer is separated and discarded. The aqueous layer was returned to the reactor and extracted with 2 x 38.4 kg of methyl tert-butyl ether (stirred for five minutes, the layers were separated and the organic upper layers were removed / discarded). This operation was repeated using 18.5 kg of methyl tert-butyl ether. The aqueous layer returned to the reactor was acidified to pH 2.5-3.5 (final pH 3.37) with 9.9 kg of concentrated aqueous hydrochloric acid. The mixture was extracted with 76.4 kg of methyl tert-butyl ether (stirred for five minutes, the layers were separated and the aqueous (lower) layer was removed / discarded). The organic layer was washed (with stirring for 5 minutes) with a solution of 1.1 kg of sodium bicarbonate in 12.4 kg of water (stirred for 5 minutes, the layers were separated and the aqueous (lower) layer was removed / discarded) and then 41.5 kg of water ( Stir for five minutes, separate the layers and remove the aqueous (bottom) layer. A vacuum is generated in the reactor and the volatile solvents are removed at 55 ° C until the distillate flows out. Toluene (32.4 kg) was added and the mixture was distilled at ambient pressure until the distillate had run off, increasing the temperature to 90-95 ° C. The mixture was then cooled to 30-35 ° C, heptane (56.85 kg) was added (in two phases) to the reactor, and the mixture was heated to 90-95 ° C (one phase). The mixture was then recooled to 38-42 ° C. CBZ-PipBu seed crystals were added and the product crystallized from the mixture within 1 hour. The solid was filtered off and washed with 19.35 kg of toluene / heptane 1: 2 and then 33.4 kg of heptane. The filter cake was dried under vacuum at 40 ° C (0.13% loss on drying analysis) to give 22.4 kg (72.96 mol, 42% stoichiometric yield based on 4-piperidone) of CBZ-PipBu.

Example 9

Preparation of CBZ-PipBuen

To a suspension of 82 g of 3-carboxypropyltriphenylphosphonium bromide in 407 ml of 1,2-diethoxyethane at 14 ° C was added 220 g of 20% by weight of potassium tert-butoxide in tetrahydrofuran over a period of 25 minutes while maintaining the reaction mixture at 24-28 ° C. The mixture was stirred for one hour, cooled to 10 ° C, and a solution of 52.5 g of 4-N was added over 30 minutes with constant cooling. · ··· · * i

CBZ-piperidone in 246 mL of tert-butyl methyl ether. After the addition was complete, the mixture was stirred at 12 ° C for 10 minutes, warmed to 20 ° C and stirred for a further 30 minutes. The reaction mixture is treated with 410 ml of 1N aqueous hydrochloric acid for 10 minutes, diluted with 328 ml of tert-butyl methyl ether and the phases are separated. The organic phase is washed with 205 ml of water, then 210 ml of 1N aqueous sodium hydroxide solution. The sodium hydroxide layer containing the product was collected separately, washed three times with 189 g of ethyl acetate, acidified to pH 3> 48 with concentrated hydrochloric acid and extracted with 189 ml of ethyl acetate. The ethyl acetate layer was separated, washed with 211 mL of water, dried for 30 min with 10 g magnesium sulfate, filtered and concentrated in vacuo. The oily residue (50.7 g) crystallized from toluene / heptane to give a total of 29.46 g (yield: 50.9% of theory about 95% A pure) of CBZ-PipBuen.

Mass spectrum: M 303 calculated, M + 1 304 found ¹ H NMR (δ vs TMS, CDCl 3): 2.2 (t, 2H), 2.25 (t, 2H), 2.35 (m, 4H), 3 45 (m, 4H), 5.15 (s, 2H), 5.2 (m, 1H), 7.33 (2.5H).

¹³ CNMR (delta vs. TMS, CDCl 3) 22.43, 28.2, 34.26, 35.66 44.88, 45.74, 67.20, 122.02, 127.83, 127.95 128.45 , 128.69, 128.90,

136.17, 136.72, 155.34, 178.39

Example 10

Preparation of CBZ-PipBuen-N (Et) Gly- (L) -Asp (OBz1) - (L) -Cha-NH2 Alternative Step Scheme II)

CBZ-PipBuen (70 g, 0.23 mol) and dimethylformamide (230 mL) were added to a double-jacketed flask and stirred with cooling to 0 ° C, then TBTU (74.9 g, 0.23 mol) was added in one portion. The temperature was maintained at 0 ° C and DIPEA (61.9 g, 0.61 mol) was added. After <45 minutes, TFA-N (Et) Gly- (L) -Asp (OBzl) - (L) Cha-NH 2 (138.7 g, 0.24 mol) was added as a solution in dimethylformamide (230 mL). The pH was adjusted to 7-8 with DIPEA (45 mL) and the mixture was allowed to reach room temperature. Two

50. «.

• · ·· ·

The hours are complete (according to HPLC). The mixture was poured into water (2.5 L) and extracted with ethyl acetate (1 L). The aqueous phase was extracted back with ethyl acetate (0.3 L). Combine the organic layers, wash with aqueous citric acid (5% w / w, 2 L), wash with aqueous sodium bicarbonate (5% w / w, 2x 1 L), and wash with water (2 L). The ethyl acetate layer was poured into a 2 L flask and heptane (500 mL) was added with stirring to induce crystallization. The solid was aspirated on a Buchner funnel, washed with ethyl acetate / heptane (2: 1, 11 vol) and dried to constant weight to give CBZ-PipBuen-N (Et) Gly- (L) -Asp (OBzl). - (L) -Cha-NHa (143.2 g, 0.19 mol), 83% yield).

Mass spectrum: M 745.91 calculated; M + 1: 746 found Analysis: C 41 H 55 N 5 O 7 C: calculated 66.02 found 65.53, 3, N.

1 HNMR (δ vs TMS, CDCl 3): 0.86 (qd, 1H), 0.98 (qd, 1H), 1.16 (t, 2H), 1.24 (dt, 6H), 1.37 (m) (1H), 1.64-1.78 (m, 4H),

1.86 (qd, 1H), 2.2 (bd, 4H), 2.35 (m, 1H), 2.4 (m, 2H), 2.74 (dd, 1H) 3.07 (m, 4H), 3.52 (d, 1H), 3.85 (d, 1H), 4.12 (q, 1H), 4.49 (qd, 1H), 4.68 (dt, 1H), 5, 07 (d, 1H), 5.14 (s, 1H), 5.16 (d, 1H), 5.22 (t, 2H), 6.45 (s, 1H), 7.28 (d, 1H) 7.26 (s, 5H), 7.35 (s, 5H), 7.56 (d, 1H).

¹³ CNMR (delta vs. TMS, CDCl 3): 14.15, 22.68, 24.95, 25.61,

26.03, 26.45, 28.20, 31.71, 32.89, 33.80, 33.89, 34.00, 35.63, 38.37, 44.79, 45.13, 45, 65, 50.23, 51.34, 60.40, 66.87, 67.06, 76.50, 77.13, 77.77, 122.46, 126.88, 127.80, 128.60,

135.15, 155.19, 170.11, 170.20, 171.61, 173.76, 175.35

Example 11

Preparation of the hygroscopic crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexyl-alaninamide (Alternative Step 5, Scheme II)

· · · ·>> • • • • • • • • • • • •

Add a CBZ-PipBuen-N (Et) Gly (L) -Asp (OBzl) - (L) -Cha-NH 2 (14 g, 0.19 mol), ammonium formate (61 g, 0, 96 mol) and 10% palladium on carbon (50% wet type degusa, 28 g). Ethanol (200 proof, 1260 ml), isopropanol (70 ml) and water (DI, 70 g) were added. The mixture was warmed to 40-50 ° C and stirred until completion of the reaction by HPLC (five hours). The mixture was allowed to cool to room temperature, filtered and the catalyst was removed. The resulting solution was heated to 40-50 ° C and acetone (about the same volume as the filtered solution) was added and the mixture was cooled to 35-40 ° C. N- [N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexylalaninamide is seeded into the mixture and the hygroscopic form of N- is crystallized. [N- [N (4-Piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) β-cyclohexylalaninamide, cooled to room temperature (several hours). The solid is aspirated on a Buchner funnel under nitrogen, the filter cake is washed with acetone and air dried to constant weight to give N [N- [N- (4-piperidin-4-yl) butanoyl) -N- ethylglycyl] - (L) -asparyl] - (L) -? -cyclohexylalaninamide (84.3 g, 0.16 mol), yield 84.8% of theory,> 95A%).

Example 12

Sequential Preparation of TFA-N (Et (Gly) - (L) -Asp (OBz1) - (L) ChaNH2 (Alternative Stages 1 to 3 of Scheme II)

A 500 ml flask equipped with a thermometer was charged with BOC-N (Et) -Gly (20.3 g, 0.1 mol), N-hydroxysuccinimide (11.5 g, 0.1 mol) and dichloromethane (200 mL). ml). The mixture was stirred at moderate speed and dicyclohexylcarbodiimide (20.6 g, 0.1 mol) was added all at once to the resulting solution as a solid. The solution was stirred for one hour under a slightly exothermic reaction (the temperature was raised from 20 to 28 ° C) and the DCU precipitated. The resulting suspension is vacuum filtered on a Buchner funnel equipped with filter paper.

• · · · · · · • • • · • •

Whatman # 1. The filter cake was washed with dichloromethane (2 x 25 mL). Return the filtrates to the original 500 ml flask and then add (L) Asp (OBzl) (22.3 g, 0.1 mol), NMM (33.8 ml, 0.3 mol) and dimethylformamide (80 g, 1.01 mol). After stirring at room temperature for 2 hours, formation of BOC-N (Et (Gly- (L) Asp (OBzl)) is complete (according to HPLC) .The reaction mixture is poured into an extraction funnel containing ice water (100 mml). 36%, 25 mL) to pH 1. The layers were separated and the dichloromethane layer was washed with ice water (100 mL) and the phases were separated (pH 3-4) and the dichloromethane layer was returned to the original 500 mL flask, to which successively NH 2 - (L) -Cha - NH 2 (17 g, 01 mol), Nhydroxysuccinimide (11.5 g, 0.1 mol) and dicyclohexylcarbodiimide (20.6 g, 0.1 mol) are added simultaneously in After stirring at room temperature for 2 hours, formation of BOCN (Et) Gly- (L) Asp (OBzl) (L) -Cha-NH 2 is complete (according to HPLC) and the DCU is vacuum filtered on a Buchner funnel equipped with Whatman filter paper. # 1 The filter cake was washed with deionized water (200 mL) containing N-methylmorpholine (15 mL, and pH 8-9) The phases were separated and the dichloromethane layer was washed again with water (DI 2 x 150 mL). The dichloromethane phase was washed with 150 ml of 1N hydrochloric acid (pH 1). The phases were separated and the dichloromethane layer was washed again with deionized water (200 mL, pH 3). Dichloromethane solution of BOCN (Et) Gly- (L) Asp (OBzl) - (L) -Cha-NH 2 was returned for purification to a 500 mL flask and TFA (100 mL) was added. After stirring at room temperature for 2 hours, formation of TFA.HN (Et) Gly- (L) Asp (OBz1) - (L) Cha-NH 2 is complete (according to HPLC). The reaction mixture was distilled under vacuum to remove dichloromethane and most of the TFA, then an inoculant and MTBE (500 mL) were added to initiate crystallization. The mixture was vacuum filtered on a Buchner funnel equipped with Whatman # 1 filter paper. The filter cake was washed with MTBE (2 x 25 mL) and air dried to give TFA.HN (Et) Gly- (L) Asp (OBzl) (L) -Cha-NH 2 (46.8 g, 81.5%). ) as a white solid (> 97A% pure, <0.2A% D-Asp diast.).

• · «··

Example 13

Preparation of stable non-hygroscopic crystalline form

N- [N- [N- (4-piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) aspartyl] - (L) -p-cyclohexylalaninamide

Method A - static conversion

The hygroscopic crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -3-cyclohexyl-laninamide (7.45 kg) was ground in hammer mill. The resulting solid 7.35 kg is placed on a drying pan (90x28 cm) and the pan is covered with perforated aluminum foil. The dish is then sealed into a moisture oven (LUNARIRE Humidity Cabinet model No. CEO 941W-3). The furnace is kept sealed for the duration of the conversion process, except for sampling for analysis. The furnace is set at 40% relative humidity (RH) and 60 ° C for 1 hour. The humidity furnace is then set to 80% RH / 60 ° C with a 12 hour delay. The sample is removed after 18 hours at 80 RH / 60 ° C and the conversion to a non-hygroscopic crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl) -Nethylglycyl] - (L) is assessed by X-ray powder diffraction. -aspartyl] - (L) -β-cyclohexylalaninamide. The moisture oven is resealed and set to 40% RH / 60'C with a 2 hour delay. The oven is returned to ambient conditions and the pan is then removed from the oven to give a non-hygroscopic, crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) aspartyl ] - (L) -3-Cyclohexylalaninamide (7.2 kg, yield 96.6% of theory). The conversion is confirmed by an X-ray pattern. diffraction (FIG. 1). X-ray powder diffraction is also shown in the table as a function of increasing order of diffraction angle (2 theta) corresponding to crystal interlayer distance (d) in nanometers read per second (Cps) and relative intensity peak (in%) (Table I, N stands for number).

• 0 0 »c 0 • i <

• 00

0 00 0

0 0 · 1 · »

0 «0 0 0 1

000 · 00 0

00000 «000 000 0 0 0 ·»

0 ♦ 0 0 · 0

Table I

-N- 2Θ —d— —Cps—

11 12

21 22

5.065

6,323

7,518

8,163

8.780 10,383 11 351 12,596 13,858 15,191 16,476 16,745 17,980 18,572 18,799 19,147 19,619 20,200 20,466 20,870 21.625 22,088 22,840 23,947 24,569 25,608 27,015 27,837 27,967 29,255 29,689 30,665 31,318 31,894 33,370

33.562

33,919

840 35,789

940

36.780 37.042 37.959 39.017

1.74314 1.9672 1.17489 1.08222 1.00633 0.851-x 0.77886 0.70218 0.63852 0.58274 0.53759 0.52901 0.49294 0.47735 0.47165 0.46315 0.45211 0.43924 0.43360 0 , 42528 0.41061 • 0.40210 0.38903 0.37129 0.36203 0.34757 0.32978 0.32022 0.31877 0.30502 0.30066 0.29130 0.28538 0.28036 0.26829

0.26679

0.26407

0.25730

0.25069

0.24967

0.24416

0.24249

0.23684

0.23066

86.00 248.00 221.00 496.00 155.00 218.00 112.00 999.00 316.00 1338.00 481.00 556.00 679.00 1079.00 1230.00 1229.00 1380, 00 1246,00 1478,00 1088,00 584,00 891,00 613,00 597,00 680,00 506,00 1100,00, 00 420,00 400,00 536,00 603,00 518,00 451,00 533,00 518/00

552 /) 0 581.00 561.00 559.00 560.00 740.00 736.00 683 00 643.00

5.82 16,78

14.95 33.56 10.49 14.75 7.58 67.59 21.38

90.53

32.54 37.62

45.95 73.00 83.22 83.15 93.37

84.30 100.00 73, Art

39.51 60.28 41.47 40 39 46.01 34.24

74.42

28.42 27.06 36.27 '

40.80 35,05

30.51 36.06 35.05

37.35

39.31 38 96

37.82 37.89 5007

49.80 46.21 43.50

Method B. Dynamic conditions

a. Conversion of the mold

Hygroscopic crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) - β-cyclohexyl 55 Alaninamide (50 g) is placed in a 400 ml graduated cylinder (height) and the alanine amide (50 g) is placed in a 400 ml graduated cylinder (height). 6 cm) on a circular washer and equipped with a mechanical stirrer. The instrument is placed in a humidity controlled oven (LUNARIRE Humidity Cabinet Model No. CEO 941W-3). The stirrer speed is set at 275 rpm and a temperature of 60 ° C and a relative humidity of 40% is established within 30 minutes. The compound is maintained under these conditions for one hour, after which the conditions change to 80% RH / 60 C.C over 45 minutes. The compound is maintained under these conditions for 16 hours before the conditions are again adjusted to 40% RH / 60 ° C for 3.2 to 5 hours. The compound is allowed to cool to room temperature (4 cm high), then removed from the graduated cylinder to give a non-hygroscopic crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexylalaninamide (yield> 95% of theory). The conversion was confirmed by the X-ray diffraction pattern (Fig. 2). X-ray powder diffraction is also shown in the table as a function of increasing order of diffraction angle (2 theta) corresponding to crystal interlayer distance (d) in nanometers read per second (Cps) and relative intensity peak (in%) (Table II, N stands for number).

Table II —Cps ......% -

-Ν- 20 —d—

1 5,186 1,70268 2 6,371 1,38615 3 7,570 1.16689 4 8,232 8,817 1.07323 5 1,00206 6 10,428 0.84761 7 11,377 0.77714 8 11,600 12,667 0.76223 9 0.69828 10 13,913 14,398 0.63599 11 0.61468 12 15,226 16,538 0.58440 13 0.53557 14 16,773 0.52814 15 Dec 18,019 r 18,672 0.49190 16 0.47483 17 18 815 19 204 0.47125 18 0.46178

196.00 8.43

722.00 31.07

516.00 22.20

1094.00 47.07

257.00 11 06

365.00 15 71

129.00 5.55

117.00 5.55

1805.00 77.67

551.00 23.71

178.00 7.66

2285.00 98.32

861.00 37 05

929.00 39.97

1132.00 48.71

1871,00 80,51

2052,00 88,30

2071,00 89 11 β c • «4 4 ♦ β« 4 ® ® ® ® ® ® ® β ® β β 9 9 99 9 999

9

9 9 9

21 22

19.654 0.45132

20.237 0.43845

20,523 0,43240

20.934 0.42400

21,691 0,40938

22.143 0.40112

22.910 0.38786

24.007 0.37037

24.642 0.36097

25.642 0.36097

27.070 0.32913

27.855 0.32002

29.497 0.30258

29,497 0,30013

30.751 0.29051

31.916 0.28017

33.982 0.26360

35,200 0.25475

36,001 0,24926

36,927 0,24322

38.389 0.23429

2226,00 1939,00 2324,00 1656,00 923,00 1411,00 994,00 964 00 991,00 991,00 1687,00 688 00 843 00 878,00 809,00 821,00 882,00 865, 00 841.00 1106.00 968 00

95.78

83.43

100.00

71.26 39.72 60.71

42.77 41.48 42 64

42.64

72.59

29.60

36.27

37.78 34 81 35.33 37.95 37.22 36.19 47.59

41.65

b. Conversion of the mold

The hygroscopic crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -p-cyclohexylalaninamide (370 g) was added to a 2 L rotary flask. evaporator flasks. The flask was placed in a rotary evaporator (Heidolph UV 2002) and lowered into a preheated (58 ° C) bath (Heidolph MR 2002). The vacuum pump is adjusted to a pressure of 6000 Pa (Divatrion DVI), then the vacuum is removed in a controlled manner to create a humid environment in a separate, heated flask containing water. The supply of the humid environment is controlled by a Vausalo Humiditique and Temperature Traumettor to achieve an RH of 79% inside the device (internal pressure 13000 to 18000 Pa). The rotary evaporator vessel is then rotated for 145 hours at 145 to 160 rpm, maintaining the heating bath at about 60 ° C and maintaining the RH at 71 to 79%. The vacuum was removed with nitrogen, the vessel and its contents were allowed to cool to ambient temperature and the product was removed to give a non-hygroscopic crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexylalaninamide. A second batch of 317 g of the hygroscopic crystalline form N [N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -p- cyclohexylalaninamide to give a non-hygroscopic crystalline form of N- [N- [N-4- (piperidin-4-yl) butanoyl) -N-ethyl57aA]

A A A A A A A A A A A A A A AA A A A 4

4 4 glycyl] - (L) -asparagyl] - (L) -β-cyclohexylalaninamide. The conversion is confirmed by an X-ray pattern. diffraction (Fig. 3). The two batches together yield 667 g of a non-hygroscopic crystalline form of N- [N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -pcyclohexylalaninamide (total yield 97). % of theory). The conversion was confirmed by the X-ray diffraction pattern (FIG. 3). X-ray powder diffraction is also shown in the table as a function of increasing order of diffraction angle (2 theta) corresponding to crystal interlayer distance (d) in nanometers read per second (Cps) and relative intensity peak (in%) (Table III, N stands for number). ).

Table III

-N- 2Θ - d— —Cps— -% -

11 12

21 22

5,124 6,328 7,574 8,191 8,797 10,398 12,628 13,871 15,218 15,723 16,538 16,751 18,024 18,640 18.809 19 191 19,659 20,865 20,495 20,865 21,616 22,113 22 950

117 24,618

644

297

052 27 960 29.640, 30744 33 465 33 840 35.812 36.811 37.076 38.185 39.622

1.72309

1.39565

1,16623

1,07851

1,00432

0.85004

0.70040

0.63791

0.58172

0.56317

0.53558

0.52882

0.49175

0.47563

0.47141

0.46210

0.45120

0.44064

0.43299

0.42539

0.41077

0.40166

0.38719

0.36871

0.36132

0.34709

0.33862

0.32934

0.31885

0.30115

0.29058

0,26755 '

0.26467

O, 25053

0.24396

0.24228

0.23549

0.22728

180.00 408.00 305.00 556.00 166.00 244.00 1198 00 353 00 1543.00 187.00 589 00 621.00 869.00 1156.00 1241 00 1521 00 1413.00 1303.00 1770.00 1120, 00 683,00 919,00 697,00 659,00 716,00 662,00 486,00 1270,00 518,00 705/00 695,00 697,00 76400 736,00 858,00 919,00 870 '882 , 00

10.17 23,05

17.23 31,41

9.38 13.79 67-68 19.94

87.18 10.56 33Í28 35.08

49.10 65,31

70.11 85,93

79.83 73.62 100.00 63.28 38.59 51 92

39.38

37.23

40.45 37; 40

27.46 71.75 29 27 39 38 3927 39 38 43 16 41.58

48.47 51.92 49.15

49.83 »· · 4 · 9 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

Example 14

X-ray powder diffraction graphs of a sample of N- [N- [N- (N- (4-piperidin-4-yl) butanoyl) - N-ethylglycyl] - (L) -asparagyl] - (L) -? -Cyclohexylalaninamide in hygroscopic crystalline form crystalline frorm

A sample of the hygroscopic crystalline form of N- [N [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] (L) -p-cyclohexylalaninamide was prepared as in Example 5 or 11. and converted to the corresponding non-hygroscopic crystalline form by the method of Example 13. The X-ray powder diffraction pattern of the hygroscopic crystalline form is shown in Figure 4 and the non-hygroscopic crystalline form is shown in Figure 5. 2 theta) of the corresponding crystal interlayer distance (d) in nanometers read per second (Cps) and the relative intensity peak in% (Tables IV and V, N denotes a number).

Table IV

-N- 2Θ d— —Cps— -% -

11 12

21 22

5.073

6,451

7,837

8.491

9,699

10,488

11,570

12,550

13,576

15,327

15,790

16,179

16,770

17.085

17,750

18,151

18,504 ^f

19,323

19.714

20,545

21.388

22,381

1.74037 1.36905 1.12712 1.04049 0.91119 0.84278 0.76423 0.70474 0.65168 0.57763 0.56080 0.54739 0.52824

0.51856

0.49927

0.48835

0.47909

0.45897

0.44996

0.43194

0.41510

0.39691

1487,00

447,00

411,00

602,00

00

421,00

92.00

411,00

760,00

606,00

456.00

346,00

938.00

685,00

924.00

741,00

593,00

930.00

792,00

1719,00

897.00

373,00

86.50

26,00

23.91

35.02

5.41

24.49 5.35 23.91 44.21 35.25 26.53 20.13 54.57 39.85 53.75 43 11

34.50 54.10 46.07 100.00 52.18 21.70

W · 9 · 0 · 0 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· · · 9 ·

22187 23.640 23.841 24048 24.746 25 200

792

266 26.959 27.426 27.967 29.020 29.922 30970 31.552 33.338 34.838 35.873 36.107 37.162 38 _r 509 39.701

0.38852

0.37604

0.37292

0.36976

0.35949

0.35311

0.34513

0.33901

0.33045

0.32494

0.31876

0.30744

0.29837

0.28851

0.28332

0.26854

0.25731

0.25012

0.24855

0.24174

0.23359

0.22684

258I00 563.00 680.00 623.00 338.00 366.00 590.00 731.00 555.00 769.00 528 00 771 00 491.00 384.00 510.00 627.00 520.00 653.00 639 00 683.00 775.00 784.00

01 32,75

39.56

36.24

19.66 21.29 34.32 42.52 32 29 44.74 3072 44.85

28.56 22,34

29.67 36.47

30.25 37.99 37 17 39.73 45.08 45.61

Table V

-N1

11 12

21 22

5,152 6,386 7,580 8,225 8,801 10,408 12,660 13,914 15,251 16,541 16,771 18,047 18,676 18,902 19,182 19,697 20,240 20 568 29.933 21,684 22,122 22,970 24 080 24,218

694

680

400 _f

105 27.929 29.360 29.724 30.340 30.693 —d— 1.71371 1.38287 1.16540

1.07410

1,00390

0.84928

0.69863

0.63594

0.58047

0.53548

0.52818

0.49112

0.47472

0.46910

0.46231

0.45035

0.43838

0.43147

0.42403

0.40951

0.40150

0, 3§685

0.36927

0.36720

0.36023

0.34662

0.33732

0.32871

0.31920

0.30395

0.30031

0.29435

0.29105 — Cps— 123.00 483.00 389 00 752.00 180.00 276.00

1399,00

391,00

1675,00

608,00

652.00

775.00

1078.00

1099,00 1151,00 1164100 1049,00 1403,00 1024/00 569,00 746j00 564,00 546,00 556,00 618,00 510,00 403,00 1093,00 450,00 555,00 595,00 429.00 552.00 -% -

7.34 28.84 23.22

44.90

10.75

16.48

83.52

23.34 100.00 36.30 38.93 46.27 64.36 65.61 68 72

69.49 62.63

83.76

61.13 33 97 44.54 33.67

32.60 33,19

36.90 30.45 2406 65 25 26.87

33.13

35.52

25.61 32.96 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

31.‘35 3 31.822 32.006 32 885 33.508 34.040 34.839 35.998 36.680 36.948 37.197 39.602

0.28507

0.28098

0.27940

0.27213

0.26722

0.26316

0.25730

0.24928

0.24480

0.24309

0.24152

0.22739

476 (00

531,00

545,00

485,00

547,00

606,00

580,00

596,00

629,00

727,00

703.00

697,00

28.42

31.70

32.54

28.96 32.66 36.18 34.63 35.58

37.55 43.40

41.97 41.61

Example 15

Isothermal microcalorimetric tests of the hygroscopic and non-hygroscopopic crystalline forms of N- [N- [N- [N- (4-piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -p-cyclohexylalaninamide

Isothermal microcalorimetric tests of the hygroscopic and non-hygroscopic crystalline forms of N- [N- [N- (N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexylalaninamide are performed on Thermometric® Thermal Activity Monitor (TAM). Solid state conversions of different crystalline forms are studied by exposing the forms to different moisture or solvent vapors and different temperatures. The saturated solutions used to achieve different humidity are: potassium chloride (80% RH), sodium chloride (75% RH) and sodium bromide (65% RH). Approximately 100 mg amounts are weighed in a TAM glass vial and a microhygrostat containing a saturated saline solution (with excess solid) or organic solvent is added to the vial. The vial is sealed, leveled to the test temperature, and lowered to the TAM measuring position. The same system containing washed sea sand instead of the test form shall be placed on the reference side. The power (gW) is measured as a function of time (Figs. 6 to 8).

F

Example 16

Isothermy moisture sorption hygroscopic and non-hygroscopic

9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

9

9 99 99 crystalline forms of N- [N- [N- (4-piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexylalaninamide

Moisture sorption isotherms of the hygroscopic and non-hygroscopic crystalline forms of N- [N- [N- (4-piperidin-4-yl) butanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -4-cyclohexylalaninamide are obtained on moisture content VTI MB3OOG. The experiments were carried out either by exposing 15 mg of the respective crystalline form to an environment with increasing and decreasing relative humidity and monitoring the increase in weight (at each equilibrium stage) as a function of the relative humidity percentage (Fig. 9), or constant humidity and monitoring weight gain over time.

The compounds of formula II exhibit useful pharmacological effects and are therefore incorporated into pharmacological compositions and are used to treat patients suffering from certain pathological conditions.

Vanaliz also relates to a method of treating patients suffering from conditions that may be alleviated by preventive administration of an inhibitor of platelet aggregation by preventing binding of fibrinogen to activated platelets and other adhesive glycoproteins involved in blood aggregation and clotting. In addition, the invention relates to a method of preventing or treating thrombosis associated with certain disease states such as myocardial infarction, stroke, external artery disease, and multiple intravascular coagulation in humans and other mammals.

By treatment there is always meant both prophylactic therapy and treatment to improve the condition.

The invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable amount of at least one compound of formula I together with a pharmaceutically acceptable carrier and

an excipient.

In practice, the compounds of the invention may be administered by any suitable route, for example, topically, by inhalation, parenterally, rectally or orally, but preferably orally.

The compounds of formula (II) are presented in forms suitable for administration by the most suitable route, and the invention also relates to pharmaceutical compositions comprising at least one compound of the invention which is suitable for use in human and veterinary medicine. These compositions may be prepared by methods known per se using one or more pharmacologically acceptable excipients or excipients. Adjuvants include, but are not limited to, diluents, sterile aqueous media, and various non-toxic organic solvents. The compositions may be administered in the form of tablets, pills, capsules, granules, powders, aqueous solutions or suspensions, injectable solutions, elixirs, and syrups, and may contain one or more agents selected from the group consisting of sweetening, flavoring, coloring, stabilizing or preserving agents. obtaining pharmaceutically acceptable compositions.

The choice of carrier and the active ingredient content of the carrier is generally determined according to the solubility and chemical properties of the product, according to the particular mode of administration according to the practice of pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate, and disintegrating agents such as starch, alginic acids and certain complex silica gels combined with lubricants such as magnesium stearate, sodium lauryl sulfate and talc may be used to prepare tablets. Lactose and high molecular weight polyethylene glycols are preferably used to prepare the capsules. If aqueous suspensions are used, f

they may contain emulsifiers or suspending agents. Like other materials, diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol can be used.

Vj ···· «« · «·« · · · · · · 9 9 9 ······

9

9,999 9 9 9

glycerol and chloroform mixtures thereof.

For parenteral administration, emulsions, suspensions or solutions of the compounds of the invention in vegetable oil, for example sesame oil, peanut oil, olive oil or a water-organic solution such as water and propylene glycol, injectable organic esters such as ethyl oleate, as well as as sterile aqueous solutions of pharmaceutically acceptable salts. The salt solutions of the products of the invention are also suitable for administration by intramuscular or subcutaneous injection. Aqueous solutions, also including solutions of salts in pure distilled water, may also be used for intravenous administration provided that their pH is appropriately adjusted, that they are properly buffered and isotonicly treated with sufficient glucose or sodium chloride and that they are sterilized by heat, radiation and / or microfiance.

Gels (based on water or alcohol), creams or ointments containing the compounds of the invention are suitable for external administration. The compounds of the invention may also be incorporated into gels or matrices for application as patches that allow the controlled release of the compounds by the transdermal barrier.

Solid form preparations for rectal administration include suppositories formulated by known methods containing at least one compound of Formula II.

The percentage of active ingredient in the compositions of the invention may be varied to form a proportion of the appropriate dose. Alternatively, multiple doses may be administered simultaneously. The dose employed can be determined by a physician or a qualified medical person and is dependent upon the desired therapeutic effect, the route of administration and duration of treatment, and the condition of the patient. The dosing regimen of the method of the invention guarantees maximum therapeutic response until improvement and then a minimum effective level that provides relief. Ob64 may be an oral dose of 0.1 mg / kg to about 100 mg / kg, preferably about 0.1 mg / kg to 20 mg / kg, and most preferably about 1 mg / kg to 20 mg / kg and i.v. a dose of about 0.1 pg / kg to about 100 pg / kg, preferably about 0.1 mg / kg to 50 mg / kg. In each individual case, dosages are determined in accordance with determining factors for the patient being treated, such as age, general health, and other characteristics that may affect the efficacy of the composition of the invention.

In addition, the compound of formula (II) may be administered as often as necessary to achieve the desired therapeutic effect. Some patients may respond quickly to a higher or lower dose and may find significantly weaker maintenance doses appropriate. In other patients, prolonged treatment with 1 to 4 doses per day, preferably once or twice daily, may be necessary, depending on the physiological requirements of each individual patient. Typically, the active product may be administered orally 1 to 4 times per day. However, in other patients it may be necessary not to prescribe more than one to two doses per day.

The compound of formula (II) exhibits significant pharmacological effects according to the tests described in the literature, and the test results are believed to correlate with pharmacological activity in humans and other mammals. The following in vitro and in vivo pharmacological test results are typical of the compound of Formula II.

The following pharmacological assays evaluate the inhibitory effect of the compound of formula II on fibrinogen-mediated platelet aggregation, the binding of fibrinogen to thrombin-stimulated platelets and the inhibition of ADP-induced platelet aggregation and the results of these tests correlate with the inhibitory properties of the compound of formula II in vivo.

The platelet aggregation test is based on the test described • 4 4 44 4 · 4 44

4444 444 4444 • 4 4 4 4 4 4 · 4 4 4 • 4444 4444 4444 444

444,444,444,444,444,444 44 in the literature (Blood 66 (4), 946-952, 1985). The fibrinogen challenge assay is essentially that described by Z. M. Ruggeri et al., (Proc. Natl. Acad. Sci. USA 83: 5708-5712, 1986) and E. F. Plow et al., Proc. Nati. Acad. Sci. USA 82: 8057-8061 (1985). The ex-vivo ADP-induced platelet aggregation inhibition assay is based on the assay described by Zucker, Platelet Aggregation Measured by the Photoeletric Method Methods in Enzymology 169, pp. 117-133 (1989).

Platelet aggregation test

Preparation of fixed activated platelets

Plates are isolated from human platelet concentrates by gel filtration techniques as described by GA Margueri et al. (J. Biol. Chem. 254: 5357-5363, 1979) and ZM Ruggeri et al., (J. Clin. Invest. 72: 1-12, 1983). Plates are suspended at 2x10 ⁸ cells / ml in modified calcium-free Tyrodes buffer containing 127 mM sodium chloride, 2 mM magnesium chloride, 0.42 mM sodium phosphate dibasic, 11.9 mM sodium bicarbonate, 2.9 mM potassium chloride, 5.5 mM glucose, 10 mM HEPES at pH 7.35 and 0.35% human albumin serum (HSA). These washed plates are activated by the addition of human α-thrombin at a final concentration of 2 units / ml, followed by a thrombin inhibitor 1-2581 at a final concentration of 40 μΜ. To the activated plates, paraformaldehyde was added at a final concentration of 0.50% and incubated for 30 minutes at room temperature. Fixed activated plates are obtained by centrifugation at 650xg for 15 minutes. Platelet pellets were washed four times with the indicated Tyrodes buffer - 0.35% HSA and resuspended at 2x10 ⁸ cells / ml in the same buffer.

Platelet aggregation test

Fixed activated platelets are incubated with a selected dose of the test compound to prevent platelet aggregation after 9

The aggregation was initiated by the addition of human fibrinogen at a final concentration of 250 pg / ml. Model ΡΑΡ-4 platelet aggregation profiler is used to record platelet aggregation. The extent of inhibition of aggregation is expressed as a percentage of the observed level of aggregation in the absence of inhibitor. The IC 50, which is the amount of inhibitor required to reduce the aggregation rate by 50%, is then calculated for each compound (for example, E. F. Plow et al., Proc. Natl. Acad. Sci. USA 82, 8057-8061, 1985).

Fibrinogen binding assay

Platelets are deprived of plasma residues by the albumin density gradient technique described by Ρ. N. Walsh et al. (Br. J. Haematol. Pp. 281-296, 1977), as modified by V. TrapaniLombardo et al., (J. Clin. Invest. 76, 1950-1958, 1985). In each assay, the plates are stimulated in Tyrode's buffer (ZM Ruggeri et al., J. Clin. Invest. 72, pp. 1-12, 1983) with human α-thrombin for 10 minutes at 22-25 ° C (3.125 x ^{10 11).} platelets per liter of thrombin at 0.1 NIH units / ml). Hirudin is then added in a 25-fold excess (unit / unit) five minutes before the isotope ^12S J labeled fibrinogen and test compound are added. After these additives, the final number of platelets in the mixture is 1x10 ¹¹ / liter. After incubation for an additional 30 minutes at 22 to 25 ° C, bound and free ligand are separated by centrifuging 50 μΐ of the mixture to 300 μΐ of 20% sucrose at 12000 xg for 4 minutes. The platelet pellet is separated from the remainder of the platelet bound radioactivity mixture. Non-specific binding is measured in mixtures containing an excess of unlabeled ligand. When the binding curves are analyzed by Scatchard analysis, non-specific binding as a parameter is derived from the binding isotherm using a computer program (PJ Munson, Methods. Enzynol. 92, pp. 542-576 (1983)).

of each compound required to inhibit 50% of the fibrinogen binding to thrombin-stimulated platelets (IC 50), each compound is tested at at least six concentrations of labeled fibrinogen ¹²⁵ J held at 0.176 pmol / l (60 gg / ml). The IC 50 value is derived from plotting residual fibrinogen binding as a function of the logarithm of the concentration of compounds in the sample.

Inhibition of ADP-induced platelet aggregation ex-vivo

Experimental protocol

Control blood samples are obtained 5 to 10 minutes before administration of the test compound to 10-20 kg mongrel dogs. The compound is administered intragastrically by an aqueous dose or orally in a gelatin capsule. Blood samples (5 ml) are taken at 30-minute intervals for three hours and then at 6, 12 and 24 hours post-dose. Each blood sample is obtained from a brain vein and collected directly into a plastic syringe containing one part 3.8% trisodium citrate per 9 parts blood.

Ex-vivo canine platelet aggregation

Blood samples are centrifuged for 10 minutes at 1000 rpm to obtain platelet-rich plasma (PPR). After removal of the PRP, the sample is centrifuged for an additional 10 minutes at 2000 rpm to obtain platelet-poor plasma (PPP). The number of platelets in the PRP is determined using a Coulter counter (Counter Electronic, Hialeah, FL). If the concentration of platelets in the PRP is greater than 300,000 platelets / μΐ, dilute the PRP with PPP to adjust the platelet count to 300,000 platelets / μΐ. The PRP aliquots (250 μΐ) are then placed in siliconized glass cuvettes (7.25x55 mm, Bio / Data Corp., Horsham, PA). Epinephrine (final concentration 1 μΜ) is added to the PRP, which is incubated for one minute at 37 C. C. Platelet aggregation stimulator, ADP to a final concentration of 10 μΜ is added to the PRP. Platelet aggregation is monitored spectrophotometrically using a translucent light aggregometer (Bio / Data Plated Aggregation Profiler, model PAP-4, Bio / Data Corp., Horsham, PA). For compound testing, record 4

· Double rate of change (slope) of translucency and maximum translucency (maximum aggregation). Platelet aggregation data is expressed as percent decrease (mean + SEM) in slope or the maximum aggregation is compared to values obtained from control PRP prepared from blood samples obtained prior to test compound administration.

The compound of formula II exhibits significant activity in the above assays and is believed to be useful in the prevention and treatment of thromosis associated with certain disease states. Antithrombic activity in an ex vivo platelet aggregation assay is a prerequisite for such activity in humans (e.g., J.L. Catalfamo and W. Jean Dodds, Isolation of Platelets from Laboratory Animals Methods Enzymol. 169, A, 27, 1989). The results of the tests of the compound of formula II by the above methods are summarized in Table VI. The same table also shows the comparative test results for 4,4- (piperidyl) butanoylglycylasparagyltryptophan, the compound of EP 0 479 481. (In the following table, the test results of the compound of Example 15 in the first row and comparing test results of said compound according to EP 0 479 481).

Table VI

Inhibition of aggregation Fixed plate dose (IC 50 μΜ) (mg / kg)

Inhibition of ADP-induced platelet aggregation ex-vivo percent inhibition of ex vivo platelet aggregation after oral administration

0,097

0.047 hours

1 3 6 12 24 ÍOO 100 ALIGN! 100 ALIGN! 98 50 53 <20

· · · · · · · · · · · · · · · · · · · · · · · · · · · ·

The above examples are well understood by those skilled in the art and it is recalled that it is not intended to limit the invention in any sense.

Industrial applicability

A stable, non-hygroscopic crystalline form of N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] derivative having antithrombic effects, including inhibiting platelet aggregation and thrombus formation in mammals for the manufacture of pharmaceutical compositions for prevention and treating thrombosis associated with disease states such as myocardial infarction, stroke, peripheral artery disease and multiple intravascular coagulation.

Claims (24)

1. N- (N-tert-butoxycarbonyl-N-ethylglycyl) (L) -aspartic acid β-benzyl ester. 2. (L) -β-Cyclohexyl and 1-ananamide N- [N- [N- [4- [N-benzyloxycarbonylpiperidin-4-yl] butanoyl] -N-ethylglycyl] - (L) -asparagyl] -benzyl ester 3. 4- (4-Piperidine) butylidenylcarboxylic acid. 4. (L) - N -cyclohexylalaninamide N- [N- [N- [3- [N-benzyloxycarbonyl-4-piperidine) propylidenylcarbonyl] -N-ethylglycyl] - (L) -asparagyl] -β-benzyl ester. 5. Non-hygroscopic crystalline N- [N- [N- (4- (piperidin-4-yl) butanoyl] -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexylalaninamide or a pharmaceutically acceptable salt thereof. 6. A pharmaceutical composition comprising N- (N-tert-butoxycarbonyl-1-methylglycyl) - (L) -aspartic acid β-benzyl ester according to claim 1 and a pharmaceutical carrier. 7. A process for the preparation of a non-hygroscopic crystalline N- [N [N- [4- (piperidin-4-ylbutanoyl) -N-ethylglycyl] - (L) -asparagyl] (L) -3-cyclohexylalaninamide, comprising: the hygroscopic crystalline N- [N- [N- (4- (piperidin-4-ylbutanoyl) -N-ethylglycyl] - (L) -asparagyl] - (L) -β-cyclohexylalaninamide is treated with an environment of about 40 to 100% 20 to 80 ° C. F 8. The method of claim 7, wherein the environment is treated with about 65-80% real humidity. 9 0 9 9 0 9 · 9. The method of claim 7, wherein the environment is treated with about 40-80% reactive humidity. 10. The method of claim 7, wherein the process is performed under static conditions. 11. The method of claim 7, wherein the process is performed under dynamic conditions. 12. A process for preparing a salt of a compound of formula OO Ε ¹ E ² O TFA · HN- (CH ₂ ) _P —IL-hn — u — N CH — C — G ^B CH ₂ CO ₂ P ¹ where means B is alkyl, cycloalkyl, cycloalkylalkyl, alkyl 1 cycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl or alkylaralkyl, E ¹ hydrogen atom, E ² α-carbon side chain naturally occurring α-amino acids, hydrogen atom, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heterocyclyl, substituted heterocyclylallyl heterocyclylalkyl, or E ¹ and E ² together with the nitrogen and carbon atom to which they are attached form a 4-membered, 5-membered, 6-membered or 7-membered azacycloalkane ring, 9 9 G is OR ¹ or NR ¹ R ² , R ¹ and R ² are independently hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alky1cykloa1ky1ovou, alkylene cycloalkyl alkylene nominal one, aryl, aralkyl, alkylaryl or alkylaralkyl, p is 1-4, A P * acid protecting group, characterized in that a compound of formula (I) is coupled O P ² -N- (CH ₂ ) _p -COH B where P ^{2 is} an acid labile amino protecting group, the first intermediate of formula P ² -N- (CH ₂ ) _p · AND (B) HN - CH - CO ₂ H CH ₂ CO ₂ P ¹ is fused with a compound of formula Ε ¹ E ² O I I II HN-CH-C-G 'and the second intermediate of the general formula O HN — CH B1-CH-C-G CH ₂ CO ₂ P ¹ · ** ♦ ♦ · · «« «« « _{2 2 2 2 2 2} 9 9 · 99 99 I is reacted with trifluoroacetic acid to remove the protecting group P ^second 13. The method according to claim 12 characterized in that when P ¹ is a hydrogenation labile protecting group, an acidic group. 14. A compound of formula HN — CHI -NCH ₂ CO ₂ P ¹ E ² O I // CH-C-G where is P ³ amino protecting group, B an alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl or alkylaralkyl group, P * acid protecting group, E ¹ hydrogen atom, E ² α-carbon side chain naturally occurring α-amino acids, hydrogen atom, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heterocyclyl, heterocyclyl, substituted heterocyclyl or E ¹ and E ² together with the nitrogen and carbon atom to which they are attached form a 4-membered, 5-membered, 6-membered or 7-membered azacycloalkane ring; · Ur · e e e e 9 9 9 9 9 9 9 ς ς 9 9 9 9 G is OR ¹ or NR ¹ R ² , R @ ¹ and R @ ² independently of one another are hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl or alkylaralkyl, p numbers 1-4. The compound of claim 14, wherein P ¹ is an acid labile labile group when hydrogenated and P ³ is an amino labile labile group for hydrogenation. The compound of claim 15, wherein is B is alkyl, cycloalkyl, cycloalkylalkyl, alkyl 1 cycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl or alkylaralkyl, E ¹ hydrogen atom, E ² is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, aralkyl or substituted aralkyl, L is OR ¹ or NR ^X R ² , R ¹ and R ² are independently hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl or alkylaralkyl af p number 1 or 2. 17. A compound according to claim 16, wherein * 9 is > B is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl or alkylcycloalkylalkyl, E ² is hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl or alkylcycloalkylalkyl. The compound of claim 17, wherein is B is an alkyl group, E ² an alkyl, cycloalkyl or cycloalkylalkyl group, R ^l and R ² are independently hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, or alkylcycloalkylalkyl, p is the first The compound of claim 18, wherein is P ³ benzyloxycarbonyl group, B is ethyl, P ¹ benzyl group, E ² cyclohexylmethyl, G is NH2. 20 May A process for the preparation of a compound of the general formula CH-C-G ΦΦΦ Φ Φ Φ Φ φ φφφφ φ φ Φ «Φ ΦΦ ΦΦ Φ Φ Φ φ φ Φ φ ΦΦΦ ΦΦΦ ΦΦΦ · · φ Φ Φ · 4 «where means Ρ ³ amino protecting group, B an alkyl, cycloalkyl, cycloalkylalkyl, alkyl 1 cycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl or alkylaralkyl group, P 'acid protecting group, E ¹ hydrogen atom, E ² α-carbon side chain naturally occurring α-amino acids, hydrogen atom, alkyl, cycloalkyl, cycloalkylakyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heterocyclyl, substituted heterocyclylallyl heterocyclylalkyl, or E ¹ and E ² together with the nitrogen and carbon atom to which they are attached form a 4-membered, 5-membered, 6-membered or 7-membered azacycloalkane ring, G is OR ¹ or NR 11 ^; R ² , R @ ¹ and R @ ² independently of one another are hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, aralkyl, alkylaryl or alkylaralkyl; acids of general formula 77 • 4 • 4 4 4 • 44 444 4 4 4 • 4 »4 4 · with the tripeptide of the general formula θ 8 í ^{£ 20} HN- (UH _{2) p} II HN CH -U-N-CH-C-G ^B CH ₂ CO ₂ R ^1, or with its acid addition salt, wherein the symbols have the above meanings. 21. The method according to claim 20, characterized in that P ¹ is the hydrogenation labile acid protecting group and P ³ in the hydrogenation labile amine protecting group. A compound of the formula wherein is P ³ amino protecting group. A compound according to claim 22, wherein P ³ is an amino-labile protecting group during hydrogenation. The compound of claim 23, wherein P ³ is a benzyloxycarbonyl group.