HK1022318A - Antithrombotic azacycloalkylalkanoyl peptides and pseudopeptides - Google Patents

Description

Antithrombotic azacycloalkyl alkanyl peptides and pseudopeptides

no marking

This application is a partially-filed co-pending U.S. application 08/476750 filed 6/7 in 1995, 08/476750 is a partially-filed co-pending U.S. application 08/628648 filed 10/17 in 1994, 08/628648 is a partially-filed co-pending U.S. application 08/138820 filed 10/15 in 1993, which has been abandoned. Background of the invention 1 field of the invention

The invention relates to N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl) -amino of formula (I)]- (L) -aspartyl]A non-hygroscopic stable crystalline form of- (L) - β -cyclohexylalaninamide. The compound has an antithrombotic activity and is useful as a drug,including inhibiting platelet aggregation and thrombosis in mammals, and can be used for preventing and treating thrombosis related to myocardial infarction, apoplexy, peripheral artery disease and disseminated intravascular coagulation.

In addition, the present invention relates to processes for the preparation of crystalline forms of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide, pharmaceutical compositions thereof, and intermediates thereof.

Furthermore, the invention relates to intermediates useful in the preparation of the tetraazacycloalkylalkanoyl peptide or pseudopeptide compounds.

Hemostasis, the biochemical process of blood coagulation, is a very complex phenomenon by which normal whole blood and body tissues spontaneously stop bleeding from damaged blood vessels. Effective hemostasis requires the combined activity of vascular, platelet and plasma factors and control mechanisms to prevent excessive coagulation. Defects, deficiencies, or excesses of any of these components can lead to bleeding or thrombotic consequences.

The adhesion, diffusion and aggregation of platelets on the extracellular matrix are important events in thrombosis. These events are regulated by a family of adhesive glycoproteins, fibrinogen, fibronectin, and von Willebrand (von Willebrand) factors. Fibrinogen is a cofactor for platelet aggregation, while fibronectin supports the adhesion and diffusion reactions of platelets, and von willebrand factor is very important for the adhesion and diffusion of platelets on the subendothelial matrix. The binding sites for fibrinogen, fibronectin and von willebrand factor are located on a complex of platelet membrane proteins called glycoprotein iib/iiia.

Adhesion glycoproteins such as fibrinogen do not bind to normal resting platelets. However, when platelets are activated by agonists such as thrombin and adenosine diphosphate, the shape of the platelets changes, possibly making the GPIIb/IIIa binding sites accessible to fibrinogen. The compounds of the present invention may block fibrinogen receptors and thus have the above-mentioned antithrombotic activity. 2. Reported progress

The presence of Arg-Gly-Asp (RGD) has been found to be essential for the interaction of fibrinogen, fibronectin and von Willebrand factor with Cell surface receptors (Ruoslahtie., Pierschbacher, Cell (Cell)1986,44, 517-18). Two other amino acid sequences also appear to be involved in the platelet adhesion function of fibrinogen, namely the Gly-Pro-Arg sequence and the dodecapeptide His-His-Leu-Gly-Gly-ALa-Lys-Gln-Ala-Gly-Asp-Val sequence. Small synthetic peptides containing RGD or dodecapeptide have been shown to bind to platelet GPIIb/IIIa receptors and competitively inhibit fibrinogen, fibronectin, and von Willebrand factor binding as well as inhibit aggregation of activated platelets (Plow et al, Proc. Natl.Acad.Sci.USA)1985,82,8057-61, Ruggeri et al, Proc. Sci.USA 1986,5708-12, Ginsberg et al, J.Biol.Chem.)1985,260,3931-36, and Gartner et al, J.Biol.Chem.260, 11, 891-94).

Tjoeng et al in U.S. Pat. Nos. 5037808 and 4879313 report that indolyl compounds containing guanidinoalkanoyl-and guanidinoalkenoyl-aspartyl moieties are inhibitors of platelet aggregation.

U.S. Pat. No. 4992463(Tjoeng et al), issued 2/12/1991, discloses a series of aryl and aralkyl guanidinoalkyl peptide analogs having platelet aggregation inhibitory activity, and specifically discloses a series of mono-and dimethoxyphenyl peptide analogs and diphenylalkyl peptide analogs.

U.S. Pat. No. 8/15 (Adams et al), 1989, discloses a series of guanidinoalkyl peptide derivatives having a terminal aralkyl substituent with platelet aggregation inhibitory activity, and specifically a series of O-methyltyrosine, biphenyl and naphthyl derivatives having a terminal amide functional group.

Haverstick, D.M. et al, in "Blood" (Blood)66(4),946-952(1985) disclose that various synthetic peptides, including arg-gly-asp-ser and gly-arg-gly-asp-ser, inhibit thrombin-induced platelet aggregation.

Plow.E.F., et al, in the Proc. Natl.Acad.Sci.USA, 79,3711-3715(1982), disclose that the tetrapeptide glycyl-L-prolyl-L-arginyl-L-proline inhibits the binding of fibrinogen to human platelets.

French patent application 86/17507 filed 12/15/1986 discloses tetrapeptides, pentapeptides and hexapeptides containing the sequence-arg-gly-asp-as being useful as antithrombotic agents.

U.S. Pat. No. 4683291(Zimmerman et al), issued on 28.7.7.1987, discloses a series of peptides consisting of 6-40 amino acids containing the-arg-gly-asp-sequence as platelet binding inhibitors.

European patent application 0319506, published on 7.6.1989, discloses a series of tetrapeptides, pentapeptides and hexapeptides containing the sequence-arg-gly-asp-as inhibitors of platelet aggregation.

U.S. patent 5023233 reports that cyclic peptide analogs containing a Gly-Asp moiety are antagonists of the fibrinogen receptor.

Peptides and pseudopeptides containing amino-, guanidino-, imidazolyl and/or amidinoalkanoyl and alkenoyl moieties are reported as antithrombotic agents in pending U.S. patent applications 07/677006, 07/534385 and 07/460777, filed 3-28, 1990, 6-7, 1990, and 1-4, 1990, and U.S. patent application 4952562 and international application PCT/US90/05448, filed 9-25, 1990, respectively, which are assigned to the same assignee as the present invention.

Peptides and pseudopeptides containing amino-and guanidino-alkyl-and alkenyl-benzoyl, phenylalkanoyl and phenylalkenoyl moieties are reported as antithrombotic agents in pending U.S. patent application 07/475043 filed on 5.2.1990 and international application PCT/US91/02471 filed on 11.4.1991 (published on 29.10.1992, international application No. WO92/13117), which are assigned to the same assignee as the present invention.

Alkanoyl and substituted alkanoyl azacycloalkylformyl aspartic acid derivatives are reported to be inhibitors of platelet aggregation in us patent 5053392 filed 12/1 1989, assigned to the same assignee as the present invention and having the same inventors as the present invention.

N-substituted azacycloalkylcarbonyl cyclic aminoacyl aspartic acid derivatives are reported as antithrombotic agents in U.S. Pat. No. 5064814 filed 4/5 1990, the inventor and assignee of which are the same as the present application. The same assignee as the present application, in U.S. patent 5051405 filed 10.10.1989, reported that azacycloalkylformylglycyl aspartic acid derivatives are antithrombotic agents.

European patent application 0479481, published 8.4.1992, discloses azacycloalkylalkanoyl glycyl aspartyl amino acids useful as fibrinogen receptor antagonists.

European patent application 0478362, published on 1.4.1992, discloses azacycloalkylalkanoyl peptidyl β -alanines as fibrinogen receptor antagonists.

PCT patent application publication No. WO95/10295 discloses azacycloalkylalkanoyl peptides and pseudopeptides of formula II,in particular N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] -amino]- (L) -aspartyl]- (L) - β -cyclohexylalaninamide which inhibits platelet aggregation and thrombosis in mammals and is useful for the prevention and treatment of thrombosis. N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aminoacyl prepared according to PCT patent application publication No. WO95/10295]- (L) -aspartyl]- (L) - β -cyclohexylalaninamide is amorphous, hygroscopic, and its physical properties are also unstable due to the absorption of moisture. PCT patent application publication No. WO95/10295 does not disclose N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl)]- (L) -aspartyl]A non-hygroscopic, stable crystalline form of- (L) - β -cyclohexylalaninamide.

PCT patent application publication No. WO95/10295 also discloses that azacycloalkyl alkanoyl peptides and pseudopeptides are prepared by conventional solid-phase or liquid-phase peptide synthesis methods using starting materials and/or intermediates readily available from chemical suppliers such as Aldrich or Sigma (H.Paulsen, G.Merz, V.Weichart, "solid phase synthesis of O-glycopeptide sequences", Angew.chem.int.Ed.Engl.27 (1988); H.Mergler, R.tanner, J.Gostii and P.Grogg, "peptide synthesis by a combination of solid and liquid phase methods I: a novel very acid-labile anchor for solid phase synthesis of fully protected fragments"; Tetrahedron Letters 29,4005 (1988); Merrifield, R.B., "solid phase peptide synthesis after 25"; design and synthesis of glucagon antagonists ", Markromol.19.1988). In addition, PCT patent application publication No. WO95/10295 discloses N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] -N- [ N- [ N- (4- (piperidin-4-yl) butanoyl ] -N-ethylglycyl ] amino]- (L) -aspartyl]The amorphous and hygroscopic form of- (L) - β -cyclohexylalaninamide is prepared from the C-terminal amino acid by sequential synthesis, see scheme I. Reaction scheme IPCT patent application publication No. WO95/10295 does not disclose a centrally located di (pseudopeptide or peptide)Formation of tetra-azacycloalkylalkanoyl peptides and pseudopeptides, or, in particular, N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl-]- (L) -aspartyl]- (L) - β -cyclohexylalaninamide whereby both the N-and C-termini of the central di (pseudopeptide or peptide) are coupled to pseudoamino acids and/or amino acids to form tetra-azacycloalkylalkanoyl peptides and pseudopeptides. Summary of The Invention

The invention relates to N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl) -amino of formula (I)]- (L) -aspartyl]A non-hygroscopic, stable crystalline form of- (L) - β -cyclohexylalaninamide. The compound has an antithrombotic activity and is useful as a drug,including inhibiting platelet aggregation and thrombus formation in mammals, and are useful in the prevention and treatment of thrombosis associated with myocardial infarction, stroke, peripheral arterial disease and disseminated intravascular coagulation. The invention also relates to N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl) -N]- (L) -aspartyl]Pharmaceutical compositions of non-hygroscopic, stable crystalline forms of (L) -beta-cyclohexylalaninamide and intermediates thereof.

The invention also relates to a process for the preparation of tetra-azacycloalkylalkanoyl peptide or pseudopeptide compounds of formula (II),wherein:

a is H;

b is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, arylalkyl, alkylaryl or alkylarylalkyl;

z is

E¹Is H;

E²is the alpha-carbon side chain of a natural alpha-amino acid, H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, aralkylA group, a substituted aralkyl group, a heterocyclic group, a substituted heterocyclic group, a heterocyclic alkyl group, a substituted heterocyclic alkyl group, or E¹And E²And connection E¹And E²Together with the carbon atom(s) to form a 4-, 5-, 6-or 7-membered azacycloalkane ring;

g is OR¹Or NR¹R²；

R¹And R²Independently of one another, H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, arylalkyl, alkylaryl or alkylarylalkyl;

r is H, alkyl, aryl or aralkyl;

m is 1 to 5;

n is 0 to 6;

p is 1 to 4; in particular, it is a non-hygroscopic, stable crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide.

The invention also relates to compounds of the formula:wherein

B is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, arylalkyl, alkylaryl or alkylarylalkyl;

E¹is H;

E²is an alpha-carbon side chain of a natural alpha-amino acid, H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substituted heterocyclylalkyl, or E¹And E²And connection E¹And E²Together with the carbon atom(s) to form a 4-, 5-, 6-or 7-membered azacycloalkane ring;

g is OR¹Or NR¹R²；

R¹And R²Independently of one another, H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, arylalkyl, alkylaryl or alkylarylalkyl;

p is 1 to 4;

P¹is an acid protecting group which is labile to hydrogenation;

P^2’is P²Or TFA. H-;

P²is an acid labile amino protecting group. Brief description of the drawings

FIG. 1 shows an X-ray powder diffraction pattern of a sample of a non-hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide prepared in example 13, method A.

FIG. 2 shows an X-ray powder diffraction pattern of a sample of a non-hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide prepared in example 13, method B, (a).

FIG. 3 shows an X-ray powder diffraction pattern of a sample of a non-hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide prepared in example 13, method B, (b).

FIG. 4 shows an X-ray powder diffraction pattern of a hygroscopic crystalline form sample of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide prepared in example 14.

FIG. 5 shows an X-ray powder diffraction pattern of a non-hygroscopic crystalline form sample of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide, prepared according to example 14.

FIG. 6 shows an isothermal trace calorimetry plot of the output power as a function of time for three different experiments performed as described in experiment 15. This experiment monitored the thermal activity of different crystalline forms of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide when exposed to various solvent vapors. Line (a) in figure 6 shows that when hygroscopic N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide prepared according to example 5 or 11 is exposed to 80% RH (saturated potassium chloride solution) at 40 ℃ for more than 30 hours, a strong exothermic event occurs, during which the hygroscopic form of the compound is converted to the non-hygroscopic form of the compound. Line (B) in figure 6 shows that when hygroscopic N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide prepared according to example 5 or 11 is exposed to methanol vapor (a solvent other than water that can dissolve the compound) at 40 ℃, no exothermic conversion event occurs, and therefore, methanol does not cause the conversion of the crystals of the hygroscopic form to the non-hygroscopic form. Line (C) of FIG. 6 shows that when the non-hygroscopic form of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide prepared according to example 13 is exposed to 40 deg.C/80% RH, no exothermic transformation event occurs, and therefore, the non-hygroscopic form of the compound does not undergo a form transformation, that is, is a stable form, under these conditions.

FIG. 7 shows an isothermal trace calorimetry plot of the output power as a function of time for three different experiments performed as described in experiment 15. This experiment monitored the thermal activity of converting the hygroscopic crystalline form of N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide to its non-hygroscopic form when exposed to 80% RH at 40 ℃,50 ℃ and 60 ℃. The graph shows that at 40 ℃ the transition occurs at about 24 hours, at 50 ℃ at 6.5 hours and at 60 ℃ at 3 hours.

FIG. 8 shows an isothermal trace calorimetry plot of output power as a function of time for four different experiments performed as described in experiment 15. This experiment monitored the thermal activity of converting the hygroscopic crystalline form of N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide to its non-hygroscopic form when exposed to 65%, 75%, 80% and 100% RH at 60 ℃. A significant feature of fig. 8 is that higher relative humidity causes a faster transition. Another significant feature is that the transition to the non-hygroscopic form of the compound at 60 ℃ at 100% RH does not occur as liquefaction of the hygroscopic form occurs at room temperature. Based on these results, the transition rate to the non-hygroscopic form at 60 ℃ is expected to be much higher than the liquefaction rate of the hygroscopic form.

FIG. 9 shows a comparison of the hygroscopic (■) and non-hygroscopic (●) forms of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide plotted as a function of the description of experiment 16 for% weight gain versus% RH at 25 ℃. Figure 9 shows that as RH is increased, the hygroscopic form absorbs more water than the non-hygroscopic form, and is more pronounced at relative humidities greater than 60%. In addition, figure 9 also shows that the hygroscopic form of the compound does not desorb to its original weight% while the hygroscopic form of the compound desorbs to its original weight%. Detailed Description

Unless stated to the contrary, the following terms used throughout the specification are to be understood as having the following meanings:

abbreviations used herein include: BOC (tert-butyloxycarbonyl), CBZ (benzyloxycarbonyl), Gly (glycine), Asp (aspartic acid), Obzl (benzyloxy), TFA (trifluoroacetic acid), Cha (. beta. -cyclohexylalanine), EtOAc (ethyl acetate), DMF (dimethylformamide), DCC (dicyclohexylcarbodiimide), HOBT (hydroxybenzotriazole), TBTU ((2-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate), DI (deionized water), PNP (p-nitrophenol), PFP (pentafluorophenol), DCU (dicyclohexylurea), NMM (N-methylmorpholine), MTBE (methyl tert-butyl ether), RH (relative humidity), THF (tetrahydrofuran), PipBu (4-piperidinebutanoic acid), PipBu (4- (piperidin-4-ylidene) -butanoic acid) is a compound of the formula:

"patient" includes humans and other mammals.

By "pharmaceutically acceptable salt" is meant a salt form of the parent compound of formula i which is relatively harmless to the patient when the salt is used in therapeutic amounts, so that the beneficial pharmaceutical properties of the parent compound of formula i are not affected by side effects of the counter-ions of the salt form. Pharmaceutically acceptable salts also include the zwitterions or inner salts of the compounds of formula I.

"alkyl" refers to a saturated aliphatic hydrocarbon group which may be straight or branched and contains from about 1 to about 20 carbon atoms in the chain. Branched means that a lower alkyl group such as methyl, ethyl or propyl is attached to the linear alkyl chain. Preferred straight or branched chain alkyl groups are "lower alkyl" groups containing from 1 to about 10 carbon atoms. Most preferred lower alkyl groups contain 1 to about 6 carbon atoms.

"cycloalkyl" refers to a saturated carbocyclic group containing one or more rings and containing from about 3 to about 10 carbon atoms. Preferred cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and decahydronaphthalene.

"cycloalkylalkyl" refers to an alkyl group substituted with a cycloalkyl group. Preferred cycloalkylalkyl groups include cyclopentylmethyl, cyclohexylmethyl, cyclohexylethyl, decahydronaphthalen-1-ylmethyl, and decahydronaphthalen-2-ylmethyl.

"Alkylcycloalkyl" refers to a cycloalkyl group substituted with an alkyl group. Examples of alkylcycloalkyl include 1-, 2-, 3-, or 4-methyl or ethylcyclohexyl.

"Alkylcycloalkylalkyl" refers to an alkyl group substituted with an alkylcycloalkyl group. Examples of alkylcycloalkylalkyl groups include 1-, 2-, 3-or 4-methyl or ethylcyclohexylmethyl or 1-, 2-, 3-or 4-methyl or ethylcyclohexylethyl.

"Azacycloalkane" refers to a saturated aliphatic ring containing a nitrogen atom. Preferred azacycloalkanes include pyrrolidine and piperidine.

"native α -amino acids" refer to glycine, alanine, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, methionine, proline, hydroxyproline, aspartic acid, asparagine, glutamine, glutamic acid, histidine, arginine, ornithine and lysine.

The "alpha-carbon side chain of a natural alpha-amino acid" refers to a moiety that replaces the alpha-carbon of a natural alpha-amino acid. Examples of the α -carbon side chain of a natural α -amino acid include isopropyl, methyl and carboxymethyl groups of valine, alanine and aspartic acid.

The term "amino protecting group" refers to an easily removable group known in the art for preventing an adverse reaction of an amino group during synthesis and which can be selectively removed. The use of amino protecting groups is known in the art for the prevention of adverse reactions of amino groups during synthesis, and many of said protecting groups are known, see, e.g., t.h. greene and p.g. m.wuts, protecting groups in organic synthesis, 2 nd edition, John Wiley & Sons, New York (1991), which is incorporated herein by reference. Preferred amino protecting groups are acyl groups including formyl, acetyl, chloroacetyl, trichloroacetyl, o-nitrophenylacetyl, o-nitrophenoxyacetyl, trifluoroacetyl, acetoacetyl, 4-chlorobutyryl, isobutyryl, o-nitrocinnamoyl, picolinoyl, acylisothiocyanate, aminocaproyl, benzoyl, and the like; and acyloxy groups including methoxycarbonyl, 9-fluorenylmethoxycarbonyl, 2,2, 2-trifluoroethoxycarbonyl, 2-trimethylsilylethoxycarbonyl, vinyloxycarbonyl, allyloxycarbonyl, tert-Butyloxycarbonyl (BOC), 1-dimethylpropyleneoxycarbonyl, benzyloxycarbonyl (CBZ), p-nitrobenzyloxycarbonyl, 2, 4-dichlorobenzyloxycarbonyl and the like.

The term "acid labile amino protecting group" refers to an amino protecting group as defined above that is readily removed by treatment with an acid and is relatively stable to other reagents. A preferred acid-labile amino protecting group is tert-Butyloxycarbonyl (BOC).

The term "hydrogenation-labile amino protecting group" refers to an amino protecting group as defined above that is readily removed by hydrogenation and is relatively stable to other reagents. A preferred hydrogenation-labile amino protecting group is benzyloxycarbonyl (CBZ).

The term "acid protecting group" refers to an easily removable group known in the art for preventing a carboxylic acid (-COOH) group from undergoing an adverse reaction during synthesis and which can be selectively removed. The use of carboxylic acid protecting groups is well known in the art, and many of these protecting groups are known, see, e.g., t.h.greene and p.g.m.wuts, protecting groups in organic synthesis, 2 nd edition, John Wiley & Sons, New York (1991), which is incorporated herein by reference. Examples of carboxylic acid protecting groups include esters such as methoxymethyl, methylthiomethyl, tetrahydropyranyl, benzyloxymethyl, substituted and unsubstituted phenacyl, 2,2, 2-trichloroethyl, t-butyl, cinnamyl, substituted and unsubstituted benzyl, trimethylsilyl, and the like, as well as amides and hydrazides including N, N-dimethyl, 7-nitroindolyl, hydrazide, N-phenylhydrazide, and the like.

The term "hydrogenation labile acid protecting group" refers to an acid protecting group as defined above that is readily removed by hydrogenation and is relatively stable to other reagents. A preferred hydrogenation-labile amino protecting group is benzyl.

"aryl" refers to phenyl or naphthyl.

"substituted aryl" refers to phenyl or naphthyl substituted with one or more aryl substituents, which may be the same or different, wherein "aryl substituent" includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxy, alkoxy, aryloxy, aralkoxy, hydroxyalkyl, acyl, formyl, carboxy, alkenoyl, aroyl, halogen, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, arylcarbamoyl, aralkylcarbamoyl, alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl, aralkylsulfonylAralkyl sulfinyl group or-NR_aR_bWherein R is_aAnd R_bIndependently of one another, hydrogen, alkyl, aryl or aralkyl.

"aralkyl" refers to an alkyl group substituted with an aryl group. Preferred aralkyl groups include benzyl, naphthalen-1-ylmethyl, naphthalen-2-ylmethyl, and phenethyl.

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

"heterocycle" refers to about 4-to about 15-membered monocyclic or polycyclic ring systems in which one or more of the atoms in the ring is an element other than carbon, such as nitrogen, oxygen, or sulfur. Preferred heterocycles include pyridyl, pyrimidinyl and pyrrolidinyl.

"substituted heterocycle" refers to a heterocyclic group substituted with one or more aryl substituents.

"Heterocyclylalkyl" and "substituted heterocyclylalkyl" refer to alkyl groups substituted with a heterocyclic group and a substituted heterocyclic group, respectively.

"hygroscopicity" refers to sorption, and refers to the quantity or state of water available sufficient to affect the physical or chemical properties of a substance (eds. j. swarbrick and j.c. boylan, Encyclopedia of Pharmaceutical Technology, vol.10, page 33). Preferred embodiments

Preferred compounds prepared according to the invention are those of formula II, wherein E2 is hydrogen, alkyl, hydroxymethyl, 1-hydroxyethyl, mercaptomethyl, 2-methylthioethyl, carboxymethyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substituted heterocyclylalkyl, or E2 is¹And E²And connection E¹And E²Taken together, form a 4-, 5-, 6-, or 7-membered azacycloalkane ring, provided that the heterocyclylalkyl group is not indol-3-ylmethyl.

More preferred compounds prepared according to the invention are those of formula II wherein E2 is hydrogen, alkyl, hydroxymethyl, 1-hydroxyethyl, mercaptomethyl, 2-methylthioethyl, carboxymethyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, or E¹And E²And connection E¹And E²Together with the carbon atom(s) to form a 4-, 5-, 6-or 7-membered azacycloalkane ring.

More preferred compounds prepared according to the invention are those of formula II, wherein E²Is hydrogen, alkyl, hydroxymethyl, 1-hydroxyethyl, mercaptomethyl, 2-methylthioethyl, carboxymethyl, 2-carboxyethyl, 4-aminobutyl, 3-guanidinopropyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, or, E¹And E²And connection E¹And E²Together with the carbon atom(s) to form a 4-, 5-, 6-or 7-membered azacycloalkane ring.

More preferred compounds prepared according to the invention are those of formula II wherein B is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl or alkylcycloalkylalkyl.

A particular embodiment prepared according to the invention is a compound described by formula IIa:wherein:

b is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, arylalkyl, alkylaryl or alkylarylalkyl;

j is H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, arylalkyl, or substituted arylalkyl;

l is OR¹Or NR¹R²；

R¹And R²Independently of one another, H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, arylalkyl, alkylaryl or alkylarylalkyl;

m is 1 to 5;

n is 2 to 6;

p is 1 or 2.

A more preferred embodiment prepared according to the invention is a compound described by formula IIa, wherein

B is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl or alkylcycloalkylalkyl;

j is H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, or alkylcycloalkylalkyl;

m is 3;

n is 3 or 4.

A further preferred embodiment of the preparation according to the invention are compounds described by the formula IIa, in which

B is an alkyl group;

j is alkyl, cycloalkyl or cycloalkylalkyl;

R¹and R²Independently of one another, H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl or alkylcycloalkylalkyl;

m is 3;

n is 3 or 4;

p is 1.

Another further preferred embodiment which is prepared according to the invention is a compound as described by formula IIa which is N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide,

another embodiment of the present invention is the formation of stable, non-hygroscopic crystalline forms of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide. This form of the compound can be made into a stable formulation of the compound according to the present invention. The stable, non-hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide also has a high melting point and no tendency to absorb water. The stable form also exhibits unique, unexpectedly good stability to humidity and temperature at temperatures higher than normal encountered during shipping, dosage form manufacture or long term shipping or storage. These characteristics also facilitate the manufacture of the dosage form. The conversion to the stable form also does not cause a loss of the substance or its purity, nor does it adversely affect its particle properties.

A preferred embodiment of the present invention are compounds of formula VII, wherein B is alkyl; e¹Is H; e²Is cycloalkylalkyl; g is NR¹R²；R¹And R²Is H; p is 1.

Another preferred embodiment of the present invention are compounds of formula VII wherein B is ethyl; e¹Is H; e²Is a cyclohexylmethyl group; g is NR¹R²；R¹And R²Is H; p is 1.

Another preferred embodiment of the present invention are compounds of formula VII wherein B is ethyl; e¹Is H; e²Is a cyclohexylmethyl group; g is NR¹R²；R¹And R²Is H; p is 1; p¹Is benzyl; p^2’Is TFA. H-.

Another preferred embodiment of the present invention are compounds of formula VII wherein B is ethyl; e¹Is H; e²Is a cyclohexylmethyl group; g is NR¹R²；R¹And R²Is H; p is 1; p¹Is benzyl; p²Is tert-butyloxycarbonyl.

It is to be understood that the invention includes all combinations of preferred compounds, preferred embodiments and specific embodiments defined herein.

The compounds of the present invention may be used in the form of the free base or acid, its zwitterionic salts or its pharmaceutically acceptable salts. All of these forms are included within the scope of the present invention.

If a compound of the invention is substituted with a basic group, acid addition salts may be formed, which are in a more convenient form for use; in practice, the use of the salt form corresponds to the use of the free base form. Acids which may be used in the preparation of acid addition salts preferably include those which, when mixed with the free base, form pharmaceutically acceptable salts, i.e. salts whose anions are non-toxic to the patient at a pharmaceutical dose of the salt, so that the beneficial platelet aggregation and thrombosis inhibiting effect of the free base is not affected by side effects of the anions. Although pharmaceutically acceptable salts of the basic compounds are preferred, all acid addition salts can be used as the source of the free base form, even though a particular salt is only suitable as an intermediate product per se, e.g., when the salt is formed for purposes of purification and identification only, or as an intermediate in the preparation of a pharmaceutically acceptable salt by ion exchange procedures. Pharmaceutically acceptable salts within the scope of the present invention are salts derived from the following acids: inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid; and organic acids such as acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, quinic acid, and the like. Corresponding acid addition salts include: hydrohalic acid salts such as hydrochloride and hydrobromide, sulfate, phosphate, nitrate, sulfamate, acetate, citrate, lactate, tartrate, malonate, oxalate, salicylate, propionate, succinate, fumarate, maleate, methylene-bis- β -hydroxynaphthenate, gentisate, methanesulfonate, isethionate and ditoluoyltartrate methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinic acid salts, and the like.

According to another characteristic of the invention, the acid addition salts of the compounds of the invention are obtained by reacting the free base with a suitable acid, using known methods. For example, the acid addition salts of the compounds of the invention may be prepared by dissolving the free base in an aqueous or aqueous-alcoholic solution or other suitable solvent containing the appropriate acid and isolating the salt by evaporating the solution, or by reacting the free base and the acid in an organic solvent, in which case the salt may be isolated directly or may be obtained by concentrating the solution.

The acid addition salts of the compounds of the present invention can be regenerated from the salts by known methods. For example, the parent compounds of the invention may be regenerated from their acid addition salts by treatment with a base such as aqueous sodium bicarbonate or aqueous ammonia.

If a compound of the invention is substituted with an acidic group, base addition salts may be formed, which are in a more convenient form to use; in practice, the use of the salt form corresponds to the use of the free acid form. Bases which can be used for the preparation of base addition salts preferably include those which, when mixed with the free acid, form pharmaceutically acceptable salts, i.e. salts whose cations are non-toxic to the animal body at pharmaceutical doses of the salt, so that the beneficial platelet aggregation and thrombosis inhibiting effect of the free acid is not affected by side effects of the cations. Pharmaceutically acceptable salts include, for example, alkali metal salts and alkaline earth metal salts, with pharmaceutically acceptable salts within the scope of the present invention being salts derived from: sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N' -dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris (hydroxymethyl) -aminomethane, tetramethylammonium hydroxide, and the like.

The metal salts of the compounds of the present invention may be prepared by contacting a hydride, hydroxide, carbonate or similar reactive compound of the selected metal in an aqueous or organic solvent with the free acid form of the compound. 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. The reaction is usually carried out at room temperature, if desired, under heating.

The ammonium salts of the compounds of the present invention can be prepared by contacting the amine with the free acid form of the compound in an aqueous or organic solvent. Suitable aqueous solvents include water and mixtures of water with alcohols such as methanol or ethanol, ethers such as tetrahydrofuran, nitriles such as acetonitrile, or ketones such as acetone. The amino acid salts can be prepared in a similar manner.

Base addition salts of the compounds of the invention can be regenerated from the salts by known methods. For example, the parent compound of the invention may be regenerated from its base addition salt by treatment with an acid such as hydrochloric acid.

Salts of the compounds of the invention may be used for the purpose of purifying the compounds, in addition to their own use as active compounds, for example by using the differences in solubility between the salt and the parent compound, by-products and/or starting materials, by methods well known to those skilled in the art.

The compounds of the invention may contain asymmetric centers. These asymmetric centers may be independently of each other in the R or S configuration. It will be apparent to those skilled in the art that certain compounds of formula I may have geometric isomers. Geometric isomers include cis and trans forms of the compounds of the present invention with an alkenyl moiety. The present invention includes individual geometric and stereoisomers as well as mixtures thereof.

The isomers may be separated from their mixtures by known methods, such as chromatography and recrystallization techniques, or may be prepared separately from the appropriate isomers of their intermediates by the methods described herein.

The preparation of amorphous form II compounds, particularly amorphous form I compounds, is described in U.S. patent applications 08/138820 and 08/476750, which are incorporated herein by reference.

The synthetic procedure shown in scheme II describes a novel process of the invention for the preparation of compounds of formula II, in particular the crystalline compounds of formula I of the invention, wherein B, E¹、E²G, R, m, n and P are as defined above, P¹Is an acid protecting group which is labile to hydrogenation, e.g. benzyl, P²Is an acid-labile amino protecting group such as tert-Butyloxycarbonyl (BOC), P³Is an amino protecting group which is unstable to hydrogenation, such as benzyloxycarbonyl (CBZ). Reaction scheme II

It may also be desirable to prevent cross-reactivity between chemically active substituents present on natural or pseudo amino acids during the preparation of compounds of formula ii or intermediates thereof. These substituents may be protected with conventional protecting groups which may then be removed or retained as required by known methods to give the desired product or intermediate (see, e.g., Greene, "protecting groups in organic synthesis", Wiley, New York, 1981). It may also be desirable to selectively protect or deprotect existing protecting groups for conversion or removal, or to allow subsequent reactions to produce the final desired product.

The process of scheme II is illustrated by the preparation of the compound of formula II, but it will be appreciated that the compound of formula I is prepared using suitable starting materials. In the preparation of compounds of formula I according to scheme II, B is ethyl, E is H, F is cyclohexylmethyl, G is NH₂R is H, m is 3, n is 3, P is 1, P¹Is benzyl, P²Is BOC, P³Is CBZ.

According to the invention, a further process for the preparation of compounds of the formula I is analogous to that of scheme II, except that a compound of the formula III (in which P is present)³As defined above)Instead of the compound of the formula IV,wherein R is H, m is 3, n is 3,p is 1, P³Is CBZ to yield an intermediate of formula v,wherein B is ethyl, E¹Is H, E²Is cyclohexylmethyl, G is NH₂P is 1, P¹Is benzyl, P³Is CBZ.

Scheme II illustrates a five-step process for preparing the compounds of the invention, first by forming the central dipeptide intermediate of formula VI of the invention,wherein B, P, P²And P¹As defined above. In the case of the preparation of the compounds of formula I, the central dipeptide intermediate of the invention is BOC-N (Et) -Gly- (L) -Asp- (OBzl) -OH. The central dipeptide intermediate is prepared without protection of the free carboxyl moiety.

In step 2 of scheme II, the coupling to form the central dipeptide can be carried out in a mixture of dichloromethane or ethyl acetate (with or without DMF as a co-solvent) and an organic base such as NMM at a temperature of from room temperature to about 40 ℃. Activation of amino acids or pseudo-amino acids of the formula for coupling may be employedThe formation of the active ester without isolation is accomplished by the action of dicyclohexylcarbodiimide using p-nitrophenol, pentafluorophenol and N-hydroxy-succinimide. The time of the coupling reaction is about 1 to 20 hours depending on the amino acid or pseudo amino acid to be coupled, the activator, the solvent and the temperature. The central dipeptide product of step 1 need not be isolated. The reaction mixture of step 1 is typically washed with water or dilute aqueous acid (e.g., hydrochloric acid) and then used directly in step 2 without drying. When using active esters based on phenol, the central dipeptide product is extracted from the reaction mixture into an aqueous alkaline solution and then from the acidified aqueous solution into an organic solvent, which solution is then directly subjected to the reaction of step 2.

The dipeptide intermediate of formula VI is used to prepare the tripeptide intermediate of formula VII of the present invention,b, E therein¹、E²G, P and P¹As defined above, P^2’Is P²Or TFA. H-. When P is present^2’When is TFA. H-, the "-" symbol indicates that TFA is cleaved to form F₃CCO₂ ^-And H⁺In which H is⁺Protonating the amine at the terminus of the compound of formula VII, i.e., forming the TFA salt of formula VIIa.In the preparation of the compounds of formula I, the tripeptide intermediate of the present invention is P^2’-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂。

In step 2, the coupling reaction of the amino acid or pseudo-amino acid with the central dipeptide may be carried out in dichloromethane or a mixture of ethyl acetate and DMF or THF at room temperature, which is lower than room temperature. Activation of the Central dipeptide of the formulaThis can be done with pentafluorophenol or N-hydroxy-succinimide by the action of dicyclohexylcarbodiimide to form an active ester without isolation. The activation reaction can also be carried out with isopropyl chloride. The reaction time varies depending on the amino acid or pseudo amino acid to be coupled, the activator, the solvent and the temperature, and is about 1 to 20 hours. The tripeptide product may not be isolated. When the tripeptide intermediate is not isolated, the reaction mixture is washed with an aqueous solution of an organic base such as aqueous N-methylmorpholine and an aqueous acid such as hydrochloric acid, and the reaction is carried out directly by the process of step 3 without drying after washing with water.

In step 3, scheme II, the protecting group, e.g. BOC, may be removed from the tripeptide product of step 2 by using trifluoroacetic acid in dichloromethane or by using a mixture of HBr in acetic acid and ethyl acetate. The reaction can be carried out at room temperature and takes about 1 hour (HBr method) and about 2 hours (TFA method), respectively. The tripeptide acid addition salt product is isolated from the reaction mixture by filtration as a crystalline solid either directly (HBr method) or after evaporation of part of the solvent and addition of a non-polar solvent to the residue.

Another process of the invention can be described as the rapid and simple preparation of TFA. H-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH from BOC-N (Et) Gly-OH₂The single-ring process of (a), which is a "one-pot" reaction comprising the first two steps of scheme II and treatment with TFA. As a result, surprisingly, TFA-H-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH was obtained₂Since it crystallizes directly from the catenated reaction solution. The interlinking method avoids corresponding three-step discontinuous reaction in a reaction route II, establishes a simple, time-saving and economic synthesis method, and can be used for industrial production.

Scheme II shows that the polypeptide is constructed in the reverse order, starting with the N-protected amino acid and then sequentially added at the carboxy terminus, which is the reverse of the conventional order in which the polypeptide is constructed by sequential amidation at the amino terminus of the protected C-terminal amino acid. The reverse synthesis of the present invention requires only protection of the nitrogen of the first amino acid, from which point onwards amino acids can be used which are unprotected at either the amine or acid terminus (with the exception of side chain functional groups). The reverse synthesis process also allows for the in-line production of compounds of formula II, particularly compounds of formula I, since flow-type production techniques can be used rather than batch-type techniques typically required for liquid phase peptide chemistry. This new method eliminates the need to purchase amino acids protected at the amine terminus, thereby reducing production costs. No special equipment, reagents or analytical methods are required.

Another process of the present invention is the reproducible formation of stable, non-hygroscopic crystals of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide by a novel solid state transition process from hygroscopic crystals of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide produced by the process described in scheme II and the indicated alternative reaction steps.

The physical properties of the hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide are unstable and, upon exposure to conditions of humidity and temperature, convert to the non-hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide, which is highly stable.

According to the present invention, the general conditions for the transition from the hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide to the non-hygroscopic crystalline form of highly stable N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide are accomplished under static and dynamic conditions.

The static process of the present invention refers to a static transformation in that the process comprises exposing a hygroscopic crystalline form of N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide placed in an immobile container such as a vial or tray to specific temperature and humidity conditions in a controlled ambient chamber. This "static" transition is carried out at a temperature of about 20 ℃ to 80 ℃, more preferably about 40 ℃ to 80 ℃, and a relative humidity of about 40% to 100% RH, preferably about 65% to 80% RH.

The dynamic process of the present invention refers to a dynamic transformation in that the process comprises exposing a hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide to the same conditions of humidity and temperature as in a static model, and with stirring, comprising tumbling the hygroscopic crystalline form of N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide in a rotary evaporator or stirring with a stirring blade in a cylindrical container (in a humidity oven).

The following examples are intended to illustrate the present invention and are not intended to limit the scope thereof.

If not stated otherwise, the reported mass spectrometry data are low resolution fast atom bombardment mass spectra performed on VG 70SE, "Calculations"value of (M + H)⁺. Nuclear Magnetic Resonance (NMR) spectroscopic data at D with Brucker ACF 300₂Obtained in O. Flash chromatography was performed on silica gel. High Performance Liquid Chromatography (HPLC) was performed on a C-18 reverse phase column with a particle size of 8-15. mu.m.

Unless stated to the contrary, the reported x-ray powder diffraction patterns were obtained by scanning powder samples with a Siemens D5000 diffractometer (1.8kW,45kV,40mA) with a Cu radiation source. Prior to the measurement, the sample was ground to eliminate the influence of particle size on the peak intensity. Approximately 60mg of sample was loaded into a 1.5X 1cm sample holder and scanned in a range of 3-40 deg. 2 theta, steps of 0.04 deg., with each step being irradiated for a total of 1 second. Example 1 preparation of BOC-N (Et) Gly- (L) -Asp (OBzl) -OH

(step 1 of reaction scheme II)

To a 1 liter three-necked round bottom flask was added 51g (0.25mol) BOC-N (Et) Gly-OH, 35g (0.25mol) PNP, 400mL ethyl acetate, and 100mL DMF. The mixture was stirred to form a solution and cooled to 4-6 ℃. A solution of 51.5g (0.25mol) DCC in 125mL of ethyl acetate was added dropwise over 10 minutes while maintaining the temperature at about 5 deg.C to about 8 deg.C. After all the DCC was added, the cooling bath was removed and the mixture was warmed to room temperature (20-22 deg.C) and stirred for 1.5 hours. During this time, a solid precipitate of DCU formed. Complete formation of the PNP ester was determined by analytical HPLC (disappearance of BOC-N (Et) Gly-OH). The reaction mixture was filtered and the DCU residue was washed with 2-50mL ethyl acetate and the washings were added to the filtrate. The DCU was discarded.

To the stirred filtrate was added 67g (0.3mol) of H₂A slurry of N- (L) -Asp- (OBzl) -OH in 150mL (138g,1.36mol) NMM. The mixture was heated to 38-40 ℃ and held at that temperature for 41 hours, at which point the analytical HLPC showed complete consumption of BOC-N (Et) Gly-OPNP. The reaction mixture was cooled to 25 ℃ and unreacted H was filtered off₂N- (L) -Asp- (OBzl) -OH. The solution was cooled and filtered to give again 1.2g (21.7 g recovered, 11.2g was added in 20% excess, 10.5g (0.047mol) was unreacted material).

The filtrate was extracted with 500mL of deionized water in a 2-liter Squibb funnel, thenExtract with 2X 250 mL. The combined aqueous solutions were extracted with 3X 300mL of 1: 1 MTBE/EtOAc to remove residual PNP (HPLC analysis indicated only trace amounts of residue), then cooled to 5 ℃ and acidified by dropwise addition of 150mL concentrated HCl from pH8.9 to pH 1.79. The acidified aqueous solution was extracted with 2X 200mL of ethyl acetate. HPLC analysis of the aqueous solution showed no residual desired product. The ethyl acetate extracts were combined, dried over magnesium sulfate, filtered and concentrated by rotary evaporator at 35 ℃. The resulting pale orange oil was vacuumed at 35 ℃ to remove as much residual solvent as possible to give 85.68g of BOC-N (Et) Gly- (L) -Asp (0Bzl) -OH as an oil (21.3mol, 85.5% yield, uncorrected for residual solvent). And (3) identification: NMR (250MHz) 7.3ppm(s),5.1ppm(s),3.3ppm (dq),3.0ppm (dq),1.4ppm(s),1.1ppm (t) MS: M = 408; m +1_{Observed value}=409HPLC:90.79 a% (3.87A% p-nitrophenol, not corrected for e) elemental analysis: c₂₀H₂₈N₂O₇:H,N；C_{Measured value}57.54,C_{Calculated value}58.81 example 2 BOC-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH₂Preparation of

(step 2 of scheme II) method A: isopropyl chloride process

1 equivalent of BOC-N (Et) Gly- (L) -Asp (OBzl) -OH is dissolved in ethyl acetate (6-8 vol; 1: 6.5 wt: vol) and the temperature is maintained between-15 ℃ and 0 ℃. NMM (1 eq) was added and the temperature was maintained at about-15 ℃ to about 0 ℃. Isopropyl chloride (1-1.1 equiv.) is added to the protected dipeptide solution at a temperature of about-15 ℃ to 0 ℃. The reaction solution is maintained at a temperature of about-15 ℃ to about 0 ℃ for 2-5 minutes. Adding H to a dipeptide solution maintained at about-15 ℃ to about 0 ℃₂N-(L)-Cha-NH₂(1 equiv.) of THF (10 vol, 1: 10 w: vol). The reaction was monitored with a process control (HPLC) sample obtained at 15 minutes, 1 hour and 2 hours to assess whether the reaction was complete. (completion of the reaction is indicated when the area of the observed dipeptide in HPLC analysis is less than 10%).

The BOC-tripeptide product precipitated directly from the reaction solution, and the precipitate was filtered off from the reaction mixture, washed with ethyl acetate (2 times, 1 vol, w: vol) and then dried in vacuo. The general yield is more than 60 percent, and the purity is more than 90A percent; aspartic acid epimers of < 1A% are usually observed.

A slurry of about 60% BOC-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH was obtained in a final yield of about 60% from ethyl acetate₂And increased purity to > 95A% while decreasing diastereoisomers to < 0.5%.

As a specific example of isopropyl chloroformate, when the general procedure of example A is followed and 4.55g (8.1mmol) BOC-N (Et) Gly- (L) -Asp (OBzl) -OH is used, then BOC-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH is prepared₂The amount of (D) was 3.26g (purity 97.9A%, 0.3A diastereomer), 70% of theory yield. The method B comprises the following steps: Pentafluorophenol-DCC complex method

Pentafluorophenol (PFP,2.9 equiv.) and DCC (1 equiv.) were dissolved in ethyl acetate (5 vol; 1: 5 wt: vol) at room temperature and cooled to-15 deg.C to 0 deg.C. 1 equivalent of BOC-N (Et) Gly- (L) -Asp (OBzl) -OH was dissolved in ethyl acetate (6 vol, 1: 6 w: vol) and mixed with 1 equivalent of H previously dissolved in DMF (10 vol, 1: 10 w: vol)₂N-(L)-Cha-NH₂And (4) mixing. dipeptide/H₂N-(L)-Cha-NH₂The solution was added dropwise to a solution of PFP and DCC, maintaining the temperature at-15 ℃ to 0 ℃. The reaction solution was maintained at 15-22 ℃ for 5-16 hours, and whether the reaction was completed was evaluated using a process control (HPLC) sample obtained at 1,2,3,4 and 16 hours. (completion of the reaction is indicated when the observed amount of dipeptide is less than 2% area in HPLC analysis).

The reaction mixture was filtered and the filter cake (DCU) was washed with ethyl acetate (2X 0.5 vol; weight: vol). The filtrate was treated with water (10 vol; 1: 10 w: vol) and the aqueous layer was removed. The ethyl acetate layer was washed with water (1 time, 5 vol; 1: 5 w: vol). The ethyl acetate layer was cooled until the product precipitated, which was filtered off and washed with ethyl acetate (2X 0.4 vol; 1: 0.4 w/v). The isolated molar yield is more than 60%, and the general purity is more than 90A%; contains 1-4A% of aspartic acid diastereoisomer.

A slurry was formed with ethyl acetate, from which BOC-N (Et) Gly- (L) -Asp (0Bzl) - (L) -Cha-NH was obtained in a final yield of about 60%₂And increased purity to > 99A% while decreasing diastereoisomers to < 0.5%.

As a specific example of the pentafluorophenol-DCC complex method, when the general method of example B is followed and 10g (24.5mmol) of BOC-N (Et) Gly- (L) -Asp (OBzl) -OH is used, then BOC-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH is prepared₂The amount of (D) was 8.15g (purity 99A%, 0.49A diastereomer), 59% of theory yield. The method C comprises the following steps: hydroxybenzotriazole (HOBT)/2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium tetrafluoroborate (TBTU) process

1 equivalent of BOC-N (Et) Gly- (L) -Asp (OBzl) -OH was dissolved in DMF (9-10 vol, 1: 10 wt: wt) and kept at room temperature. Adding H to the solution₂N-(L)-Cha-NH₂(1 equivalent) and hydroxybenzotriazole (HOBT,1 equivalent). The resulting solution was cooled to about 0-10 ℃ and then NMM (1-1.1 equivalents) was added. The coupling agent TBTU (1-1.1 equiv.) is dissolved in DMF (4-5 vol, 1: 5 wt: wt) and added to the protected dipeptide solution at 0 ℃ to about 10 ℃. The solution was stirred at about 10-25 ℃ for about 3 hours until HPLC analysis indicated the reaction was complete (area of starting material less than 2%). The reaction mixture was added to a stirred mixture of 5% aqueous sodium chloride solution (about 4 times the volume of the reaction solution) and ethyl acetate (about 2 times the volume of the reaction solution). The aqueous phase was extracted with additional ethyl acetate (about 1.5 times the volume of the reaction solution). The organic phases were combined and washed successively with 0.5N aqueous citric acid (about 0.6 to 0.7 times the volume of the organic phase), 10% aqueous sodium bicarbonate (twice each about 0.6 to 0.7 times the volume of the organic phase) and 25% aqueous sodium chloride (about 0.3 to 0.4 times the volume of the organic phase). The resulting organic phase was concentrated to about 1/4 to 1/2 volumes under reduced pressure at about 30-50 ℃ and then the same volume of heptane was added to the warm solution. The mixture is stirred and cooled to about 0 ℃ to about 20 ℃ to precipitate the desired tripeptide. The solid was filtered off, washed with a mixture of ethyl acetate and heptane and dried. The general yield is more than 60 percent, and the general yield isThe purity is more than 95.7A%; the content of aspartic acid epimers is < 2A%.

As a specific example of the HOBT/TBTU method, when it is carried out according to this general method, using 10g (24.5mmol) of BOC-N (Et) Gly- (L) -Asp (OBzl) -OH, 9.3g of BOC-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH are obtained₂(purity 96.1A%, 1.77A% diastereomer of Asp), 67.7% theoretical yield. Mass spectrum: m_{Calculated value}560.7；M+1_{Observed value}561mp182.17(DSC)¹H NMR (. delta.vs TMS, D6 DMSO) 0.89m (1H); 0.94, m (1H); 1.0, dt (2H); 1.15, m (2H); 1.06-1.3.m (4H); 1.36, d (9H); 1.4-1.74, m (6H); 2.65, m (1H); 2.85, m (1H); 3.18, m (2H); 3.75, d (2H); 4.2, s (1H); 4.66, d (1H); 5.08, s (2H); 7.02, s (1H); 7.18, d (1H); 7.36, s (5H); 7.88, dd (1H); 8.24, dd (1H). Example 3 TFA-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH₂Preparation of

(step 3 of reaction scheme II)

Adding BOC-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH₂Dissolved in dichloromethane (ca. 1: 12w/w) and TFA was added to the solution at room temperature. It was stirred until the reaction was complete (3-5 hours) as indicated by HPLC. The solution was concentrated to about 1/2 volumes at 40-45 ℃. To the warm solution was added MTBE (vs BOC-N (Et) Gly- (L) -Asp (OBzl)) - (L) -Cha-NH₂About 1: 10w/w) while maintaining a temperature > 40 ℃. The mixture was slowly cooled to about 5 ℃ and stirred for 1 hour to ensure complete crystallization. The resulting solid was filtered and washed with cold MTBE. The solid was dried under reduced pressure and analyzed for TFA.N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH₂Content (HPLC w/w analysis). The yield is usually nearly quantitative, with a purity of > 95A%. Mass spectrum: m_{Calculated value}: 460 (free base); m +1_{Observed value}: 461 elemental analysis: c₂₆H₃₇N₄O₇F₃H, N, F, C54.35, found 53.82¹H NMR(δvs TMS,D⁶DMSO):0.9,m(2H)；1.15,t(6H)；1.5,m(1H)；1.5-1.8,m(6H)；2.65dd(1H)；2.9m(3H)；3.7,s,(2H)；3.9,m(2H)；4.2,m(1H)；4.75,m(1H)；5.1,s(2H)；7.0,s(1H)；7.15,s(1H):7.2,s(5H)；8.13,d(1H)；8.7-8.8,m(3H)。¹³CNMR (significant Signal. delta. vs. TMS, D6 DMSO) 10.76,25.49,25.68,25.96,31.66,33.07,33.36,36.25,38.59,41.88,47.02,49.40,50.47,65.71,127.81-128.34,135.82,165.10,169.34,173.79

Specific examples of deprotection are shown in table a.

TABLE A

Examples of laboratories	Reaction Scale (BOC-N (Et) Gly- (L) -Asp (OBzl)) - (L) -Cha-NH2)	Yield and A% purity (TFA. N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH2)
			Example 1	7.5g(13.3mmol)	7.4g (12.9mmol) 97% yield; 98.8A% purity
Example 2	6.53g(11.6mmol)	6.4g (11.1mmol) 96% yield; 98.44A% purity

Example 4 CBZ-PipBu-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH₂Preparation of (step 4 of scheme II)

Preparation of approximately equimolar amounts of TFA.N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH₂CBZ-PipBu and TBTU in ethyl acetate, DMF and water (100: 8: 4 v/v; vs TFA. N (Et) Gly- (L) -Asp (OBzl)) - (L) -Cha-NH₂About 11: 1 total volume/weight). The suspension is cooled to 0-10 ℃ and about 3-4 equivalents of NMM are added. The mixture was warmed to room temperature and stirred until the reaction was complete by HPLC (1-3 hours during which time a solution formed). Adding water (most preferably2-3 times of the initial amount of water added) and liquid separation. The aqueous phase was retained and the organic phase was washed twice with water. The combined aqueous washes were extracted with ethyl acetate and the combined organic phases were washed with 25% aqueous sodium chloride solution. The organic phase was concentrated under pressure to about 1/2 volumes and MTBE was added (about 1/2v/v relative to the volume of the solution). The mixture was allowed to crystallize (several hours) and the solid was collected by filtration and rinsed with a cold mixture of ethyl acetate and MTBE. The solid was dried under reduced pressure. Determination of CBZ-PipBu-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH by HPLC w/w analysis₂The content of (a). The yield is usually > 80% and the purity > 95A%.

As a specific example of the above preparation method, when it is carried out according to this general method, 7.25g (24.5mmol) of TFA.N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH is used₂7.9g of CBZ-PipBu-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH were obtained₂(purity > 99A%, 0.08A% of diastereomer of Asp), 84% of theoretical yield. Elemental analysis: c₄₁H₅₇N₅O₈H, N, C,65.84, found, 65.38 mass spectrum: m_{Calculated value}747；M+1_{Observed value}748mp 101.6(DSC)¹H NMR(δvs TMS,CDCl₃):0.88m(1H)；0.98,m(1H)；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(1H)；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)¹³C NMR (. delta.CDCl vs. TMS)₃): [ significant peak]66.93 (two benzylic carbons), 127.78-128.64 (two phenyl rings), 155.249,170.00,170.24,171.69,174.27,175.21 (all carbonyl carbons). Example 5N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl)]- (L) -aspartyl]Preparation of hygroscopic crystalline form of- (L) -beta-cyclohexylalaninamide (step 5 of scheme II)

Preparation of CBZ-PipBu-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH₂Ammonium formate and 10% Pd/C in 20: 1 alcohol/water (vs CBZ-PipBu-N (Et) Gly- (L) -Asp (OBzl)) - (L) -Cha-NH₂10: 1 v/w). Heating the mixture to 40-50 ℃ and then stirred until HPLC indicated completion (1-2 hours). The mixture was cooled to room temperature and the catalyst was filtered off. The resulting solution was heated to 40-50 ℃ and acetone (approximately equal volume to the filtrate) was added and the solution was cooled to 35-40 ℃. Adding N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl) -N to the mixture]- (L) -aspartyl](ii) seed crystals of (L) -beta-cyclohexylalaninamide which crystallize N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] amide upon cooling to room temperature]- (L) -aspartyl]Hygroscopic crystalline forms of (L) -beta-cyclohexylalaninamide (several hours). The solid was collected by filtration under nitrogen and rinsed with acetone. The solid was dried under reduced pressure and analyzed for N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl)]- (L) -aspartyl]Content of hygroscopic crystals of (L) -beta-cyclohexylalaninamide (HPLC w/w analysis). The yield is usually > 85% and the purity > 95A%.

As a specific example of the above preparation method, when it is carried out according to the general method of step 5, 5g of CBZ-PipBu-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH is used₂3.1g of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl) as a white solid was obtained]- (L) -aspartyl]Hygroscopic crystalline form of- (L) - β -cyclohexylalaninamide (purity 99.6A%), 89.4% of stoichiometric yield.

The following compounds were prepared according to the above examples 1-5 using the appropriate starting materials:

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) propionyl) -N-ethylglycyl ] aspartyl ] valine,

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

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -L-alpha-cyclohexylglycine,

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

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -L-alpha- (2, 2-dimethyl) propan-3-ylglycine,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -L-beta-decahydronaphthalen-1-ylalanine,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -L-alpha- (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-beta-naphthalen-1-ylalanine,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -L-beta-naphthalen-2-ylalanine,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -L-beta-cyclohexylalanine, ethyl ester

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

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

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

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -beta-decahydronaphthalen-1-ylalanine,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -beta-cyclohexylalanine-N-ethylamide,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -beta-cyclooctylalanine,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -beta-cyclohexylmethyl alaninamide,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -beta-adamantan-1-ylalanine,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -beta- (1,2,3,4) -tetrahydronaphthalen-5-ylalanine,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -beta- (4-cyclohexyl) cyclohexylalanine,

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

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -beta-cyclooctylacrylamide,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -alpha-cyclohexylpropylglycine,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -beta-cyclooctylmethylalanine,

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -beta-cyclopentylalanine, and

n- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] aspartyl ] -beta-cyclohexylmethyl alanine ethyl ester. EXAMPLE 64 preparation of N-CBZ-piperidone

40Kg of N-benzyloxycarbonyloxy succinimide and 26Kg (175mol) of 4-piperidone HCl. H₂A mixture of O in 38.8Kg of water and 88Kg of THF was stirred at 15 ℃. + -. 5 ℃ until dissolution (about 15 minutes). To the stirred mixture was added NMM (22.8Kg) (exothermic) while keeping the temperature at or below 20 ℃. Subjecting the mixture to a temperature of 20 ℃ +/-5 ℃Stir for 2.5 hours, at which point HPLC indicated the reaction was complete. The mixture was diluted with 115.2Kg of MTBE and 38.8Kg of water and stirred at 20 ℃. + -. 5 ℃ for 5 minutes. Stirring was stopped, the layers separated and the aqueous layer (lower layer) was separated and discarded. The organic layer was washed with 2X 129.6Kg of water (stirred for 5 minutes, separated and the aqueous phase discarded [ lower layer]). The organic layer was washed with 5.2Kg of sodium chloride in 46.8Kg of aqueous solution (stirred for 5 minutes, separated and the aqueous phase discarded [ lower layer)]). The organic layer was treated with 11.5Kg magnesium sulfate, stirred for 1 hour, and the mixture was filtered. The reactor was flushed with 8Kg of MTBE (filtered, combined with the main filtrate; total filtrate water content: 0.52%). The volume of the mixture was reduced by half by distillation under reduced pressure at 30 ℃. Nitrogen was charged to the vacuum and the residue was cooled to 20 deg.C (water content in the residue: 0.43%). The residue was diluted with 57.6Kg of MTBE and the volume of the mixture was again reduced by half by distillation at 30 ℃ under reduced pressure. Nitrogen was charged to the vacuum and the residue was cooled to 20 deg.C (water content in the residue: 0.25%). This was repeated 5 times. The final residue was diluted with 28.8Kg of MTBE and stirred for 5 minutes, then analyzed for water content and 4-N-CBZ-piperidone content (water: 0.05%, 4-N-CBZ-piperidone w/w analysis: 22.66% by weight, 35.36Kg,155mol, 88.6% stoichiometric yield). Example 7 preparation of PipBu

A solution of 53.5Kg of 3-carboxypropyltriphenylphosphonium bromide in 230.1Kg of 1, 2-dimethoxyethane was prepared under a stream of nitrogen and with stirring. Potassium tert-butoxide/THF (20% by weight of the solution 141.8 Kg) was added over 35 minutes while maintaining the temperature at 24-28 ℃. The mixture was stirred at this temperature for 0.5 h, at which point HPLC indicated the reaction was complete. The stirred mixture was cooled to 10 ℃. + -. 2 ℃ and then 96.45Kg (titer: 1.15 molar equivalents) of a solution of 4-CBZ-piperidone in MTBE was added to the mixture over 40 minutes, maintaining the temperature of the reaction solution at 12 ℃. + -. 2 ℃. The mixture was stirred at this temperature for 10 minutes, then heated to 20 ℃. + -. 2 ℃ and stirred at this temperature for 2 hours. To the stirred mixture was added 22.5Kg of concentrated hydrochloric acid in 245.6Kg of water while maintaining the temperature at 20 ℃. + -. 2 ℃; the final pH was 0.5. The mixture was extracted with 214.0Kg of methyl tert-butyl ether with stirring. Stirring was stopped, the layers were separated and the aqueous layer (lower layer) was discarded. The organic phase was washed with 133.75Kg of water (5 min stirring, separation and removal of the lower aqueous layer) and then with 10.7Kg of 50% sodium hydroxide in 126.8Kg of aqueous solution (10 min stirring, separation and removal of the upper organic layer). The aqueous layer was extracted with 2X 123.05Kg of ethyl acetate (stirred for 5 minutes, separated and the organic [ upper ] layer discarded). 13.1Kg of concentrated hydrochloric acid was added to the stirred aqueous solution to pH2.5-3.5 (final: 2.82). The mixture was then extracted with 123.05Kg of ethyl acetate (stirring for 5 minutes, separating and discarding the aqueous [ lower ] layer). The ethyl acetate solution was washed with 133.75Kg of water (stirring for 5 minutes, separating and discarding the aqueous [ lower ] layer) and then analyzed for the content of (w/w) CBZ-PipBuen (total weight: 194.86Kg, 17.05% CBZ-PipBuen [33.22Kg,108mol ], 87.9% stoichiometric yield).

An ethyl acetate solution of PipBuen was added to a stainless steel pressure box with 6.6Kg of 5% Pd/C (50% water, wt.%) under stirring, and the mixture was then heated to 55 ℃ ± 2 ℃. Potassium formate (38.2Kg) dissolved in 66.4Kg of water was added while maintaining the temperature of the reaction mixture at 55 ℃. + -. 2 ℃ (about 30 minutes). The mixture was stirred at 55 ℃. + -. 2 ℃ for 2 hours, at which point the reaction was complete (HPLC). 6.6Kg of diatomaceous earth and 33.2Kg of water were added to the reactor, and the mixture was stirred and then filtered. The reactor was rinsed with 33.2Kg of water (filtered, added to the main filtrate). Placing the filtrate in a new container, cooling to 20-25 deg.C, separating, and removing organic layer. The aqueous layer was acidified with 52.1Kg concentrated HCl to pH2-3 (final 2.82) and then extracted with 4X 129.5Kg dichloromethane (stirred for 5 min, separated, discarded organic [ lower ] layer). The aqueous phase is adjusted to pH6.1 by adding, with stirring, 17.85Kg of 50% aqueous sodium hydroxide solution. The mixture was filtered to give 224Kg of solution containing 17.6Kg (103mol) of 4- (3' -carboxypropyl) piperidine. EXAMPLE 8 preparation of CBZ-PipBu

224Kg of a solution of 4- (3' -carboxypropyl) piperidine in aqueous sodium hydroxide solution was mixed with 55.3Kg of THF, and the mixture was then cooled to 8 ℃. + -. 2 ℃ with stirring. NMM (20.9Kg) was added while maintaining the temperature < 10 ℃. After the end of the addition, the temperature was adjusted to 8 ℃. + -. 2 ℃ and then 25.7Kg of 1- (benzyloxycarbonyl) succinimide dissolved in 49.8Kg of THF were added over 1 hour, while keeping the temperature at < 15 ℃. The reaction was complete after 3 hours at 10-15 ℃ (analytical HPLC). Concentrated hydrochloric acid (29.9Kg) was added to adjust the pH to 2.5-3.5 (final 3.3), then 61.4Kg MTBE was added and the mixture was stirred for 5 minutes. Stirring was stopped, the layers were separated and the water (lower) layer was separated (discarded). The MTBE layer was washed with 3X 83.1Kg of water (stirred for 10 minutes, 5 minutes and 5 minutes, respectively); the water layer was divided equally and discarded each time. Without stirring, 8.3Kg of 50% sodium hydroxide solution in 95.7Kg of water was added and, after the end of the addition, the mixture was stirred for about 5 minutes. Stirring was stopped, the layers were separated and the organic (upper) layer was separated and discarded. The aqueous layer was poured back into the reactor and extracted with 2X 38.4Kg of methyl tert-butyl ether (stirred for 5 minutes, separated, discarded organic [ upper ] layer). This operation was repeated with 18.5Kg of methyl tert-butyl ether. The aqueous layer was poured back into the reactor and acidified to pH2.5-3.5 (final 3.37) with 9.9Kg of concentrated HCl. The mixture was extracted with 76.4Kg of methyl tert-butyl ether (stirred for 5 minutes, separated, and the lower [ water ] layer discarded). The organic layer was washed with a solution of 1.1Kg of sodium bicarbonate in 12.4Kg of water (stirred for 5 minutes, separated, the sub-aqueous layer discarded), and then with 41.5Kg of water (stirred for 5 minutes, separated, the sub-aqueous layer discarded). The reactor was placed under reduced pressure and the volatile solvent was distilled off at 55 ℃ until no more distillate flowed out. Toluene (32.4Kg) was added and the mixture was distilled at normal pressure until no more distillate flowed out, at which point the bath temperature rose to 90-95 ℃. The mixture was then cooled to 30-35 ℃, heptane (56.85Kg) (two phases) was added to the reactor, the mixture was heated to 90-95 ℃ (one phase) and then cooled again to 38-42 ℃. CBZ-PipBu seeds were added and the product crystallized from the mixture within 1 hour. The solid was collected by filtration and washed successively with 19.35Kg of 1: 2 toluene/heptane and 33.4Kg of heptane. The filter cake was dried under vacuum (0.13% loss in drying analysis) at 40 ℃ to give 22.4Kg (72.96mol, 42% stoichiometric yield based on 4-piperidone) of CBZ-PipBu. EXAMPLE 9 preparation of CBZ-PipBuen

To a suspension of 82g of 3-carboxypropyltriphenylphosphonium bromide in 407mL of 1, 2-diethoxyethane at 14 ℃ over the course of 25 minutes, 220g of a 20% by weight solution of potassium tert-butoxide in tetrahydrofuran were added while maintaining the trans-formThe temperature of the mixture should be between 24 and 28 ℃. The mixture was stirred for 1 hour, cooled to 10 ℃ and then 52.5g of 4-N-CBZ-piperidone in 246mL of methyl tert-butyl ether were added over 30 minutes with cooling. After the addition was complete, the mixture was stirred at 12 ℃ for 10 minutes, then warmed to 20 ℃ and stirred for 30 minutes. The reaction mixture was treated with 410mL of 1N hydrochloric acid for 10 minutes, diluted with 328mL of methyl t-butyl ether, and then separated. The organic phase was washed with 205mL of water and then with 210mL of 1N aqueous sodium hydroxide solution. The sodium hydroxide layer containing the product was separated, washed with 3X 189g of ethyl acetate, acidified to pH3.48 with concentrated hydrochloric acid and extracted with 189mL of ethyl acetate. The ethyl acetate layer was separated, washed with 211mL of water, then dried over 10g of magnesium sulfate for 30 minutes, filtered, and concentrated in vacuo. The oily residue (50.7g) was crystallized from toluene/heptane to give 29.46g of CBZ-PipBuen (50.9% yield, ca. 95A% pure). Mass spectrum: m_{Calculated value}：303,M+1_{Observed value}：304¹H NMR:(δvs TMS,CDCl₃)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)。¹³C NMR(δvs TMS,CDCl₃)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 CBZ-PipBuen-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH₂Preparation (alternative step 4 of scheme II)

CBZ-PipBuen (70g,0.23mol) and DMF (230mL) were added to a 1 liter jacketed flask, cooled to 0 ℃ with stirring, and then TBTU (74.9g,0.23mol) was added immediately. The temperature was maintained at 0 ℃ and DIPEA (61.9g,0.61mol) was started. After 45 min, TFA.N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH was added₂(138.7g,0.24mol) in DMF (230 mL). DIPEA (45mL) was added to adjust the pH to 7-8 and the mixture was warmed to room temperature. After 2 hours, the reaction was complete (HPLC analysis). The reaction was quenched in water (2.5 l) and extracted with ethyl acetate (1 l). The aqueous phase was extracted with ethyl acetate (0.3 l). The organic layers were combined and washed successively with aqueous citric acid (5% w/w, 2X 1 l), aqueous sodium bicarbonate (5% w/w, 2X 1 l) and water (2 l). The ethyl acetate layer was transferred to a 2L flask, and heptane (500mL) was added with stirring to initiate the formation of a noduleAnd (4) crystallizing. The solid was collected by suction filtration on a Buchner funnel, washed with ethyl acetate/heptane (2: 1v/v,1 liter) and dried to constant weight to give CBZ-PipBuen-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH₂(143.2g,0.19mol, 83% yield). Elemental analysis: c₄₁H₅₅N₅O₇C: calcd 66.02 found 65.53, H, N. Mass spectrum: m_{Calculated value}745.91；M+1_{Observed value}746¹H NMR(δvs TMS,CDCl₃):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)；2.2bd(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)¹³C NMR(δvs TMS,CDCl₃):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 11N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl)]- (L) -aspartyl]Preparation of hygroscopic crystalline forms of (L) -beta-cyclohexylalaninamide (alternative step 5 of scheme II)

CBZ-PipBuen-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH₂(140g,0.19mol), ammonium formate (61g,0.96mol) and 10% Pd/C (50% water, type degussa, 28g) were charged to a 5 liter jacketed flask. Ethanol (200 proof, 1260mL), isopropanol (70mL) and water (deionized water, 70g) were added. The mixture was heated to 40-50 ℃ and stirred until the reaction was complete as indicated by HPLC (5 h). The mixture was cooled to elevated temperature and the catalyst was filtered off. The resulting solution was heated to 40-50 ℃ and acetone (about the same volume as the filtrate) was added and the solution was cooled to 35-40 ℃. Adding N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl) -N to the mixture]- (L) -aspartyl](ii) seed crystals of (L) -beta-cyclohexylalaninamide from which N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] amide precipitates while cooling to room temperature (several hours)]- (L) -aspartyl]-(Crystalline hygroscopic form of L) - β -cyclohexylalaninamide. The solid was collected by suction filtration through a Buchner funnel under nitrogen, the filter cake was washed with acetone and air dried to constant weight to give N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] -N- [ N- [ N- (4-piperidin-4-yl) butanoyl ] -N-ethylglycyl ] amino]- (L) -aspartyl]- (L) - β -cyclohexylalaninamide (84.3g,0.16mol, 84.8% yield, > 95A%). Example 12 TFA-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH₂Preparation of (step 1-3 of scheme II)

A500 mL flask equipped with a thermometer was charged with BOC-N (Et) Gly (20.3g,0.1mol), N-hydroxysuccinimide (11.5g,0.1mol), and methylene chloride (200 mL). The mixture was stirred at the appropriate speed and DCC (20.6g,0.1mol) solid was added to the resulting solution in one portion. The solution was stirred for 1 hour during which time a slight exotherm (temperature rising from 20 ℃ to 28 ℃) and precipitation of DCU was observed. The resulting suspension was vacuum filtered through a Buchner funnel fitted with Whatman filter paper No. 1. The filter cake was washed with dichloromethane (2X 25 mL). The filtrate was poured back into the original 500mL flask, followed by the sequential addition of (L) Asp (OBzl) (22.3g,0.1mol), NMM (33.8mL,0.3mol) and DMF (80g,1.01 mol). After stirring at room temperature for 2 hours, the reaction to form BOC-N (Et) Gly- (L) -Asp (OBzl) is complete (HPLC monitoring). The reaction mixture was poured into an extraction funnel containing ice water (100 mL). The mixture was acidified to pH1 with hydrochloric acid (36%, 25 mL). The layers were separated and the dichloromethane layer was washed with ice water (100mL) and separated (aqueous pH 3-4). The dichloromethane layer was poured back into the original 500mL flask and NH was added sequentially as a solid in one portion₂-(L)-Cha-NH₂(17g,0.1mol), N-hydroxysuccinimide (11.5g,0.1mol) and DCC (20.6g,0.1 mol). After stirring for 2 hours at room temperature, BOC-N (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH was formed₂After the reaction (HPLC monitoring), DCU was filtered off in vacuo using a Buchner funnel fitted with a Whatman filter No. 1. The filter cake was washed with dichloromethane (2X 25 mL). The filtrate was transferred to an extraction funnel and washed with deionized water (200mL) containing N-methylmorpholine (15mL, pH 8-9). The layers were separated and the dichloromethane layer was washed again with water (deionized water, 2X 150 mL). The dichloromethane layer was washed with 150mL of 1N hydrochloric acid (pH 1). The layers were separated and the dichloromethane layer was washed with deionized water (200mL, pH 3). Will be provided withBOC-N(Et)Gly-(L)-Asp(OBzl)-(L)-Cha-NH₂The dichloromethane solution was poured back into a clean 500mL flask, followed by addition of TFA (100 mL). After stirring for 2 hours at room temperature, TFA, HN (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH is formed₂The reaction was complete (HPLC monitoring). The reaction mixture was distilled under vacuum to remove dichloromethane and most of the TFA, followed by addition of MTBE (500mL) and seed crystals to initiate crystallization of the product. The mixture was vacuum filtered through a Buchner funnel fitted with Whatman filter No. 1. The filter cake was washed with MTBE (2X 25mL) and air dried to give TFA.HN (Et) Gly- (L) -Asp (OBzl) - (L) -Cha-NH as a white solid₂(46.8g, 81.5% yield) (> 97A% pure, < 0.2A% D-Asp diastereomer). Example 13N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl)]- (L) -aspartyl]Preparation of stable non hygroscopic crystalline form of- (L) - β -cyclohexylalaninamide process a: static transition

Hygroscopic crystalline forms of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide (7.45Kg) were milled with a hammer mill. The 7.35Kg of solid obtained was placed in a stainless steel desiccator tray (90X 28cm) and the tray was covered with perforated aluminum foil. The trays were then sealed in a Humidity oven (LUNAIRE Humidity cabin CEO 941W-3); the oven was kept sealed throughout the form conversion process except for sampling for analysis. The oven was adjusted to 40% RH and 60 ℃ and held at this level for 1 hour. The humidity oven was then adjusted to 80% RH/60 ℃ and held at this level for 12 hours. After 18 hours at 80% RH/60 deg.C, samples were taken and examined by X-ray powder diffraction analysis for conversion to the non-hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide. The humidity oven was sealed again and adjusted to 40% RH/60 ℃ for 2 hours. The oven was brought to ambient conditions and the trays were removed from the oven to give the non-hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide (7.2Kg, 96.6% yield). The transformation was confirmed by X-ray powder diffractogram (fig. 1). X-ray powder diffraction was also tabulated (Table 1) with the in-plane distance (d) of the crystal expressed in units of angstroms (), the count per second (Cps), and the relative peak intensity (%) increasing in the order of diffraction angle (2. theta.). TABLE 1-N-2 θ … d … … Cps … …% … 15.06517.431486.005.8226.32313.9672248.0016.7837.51811.7489221.0014.9548.16310.8222496.0033.5658.78010.0633155.0010.49610.3838.5125218.0014.75711.3517.7886112.007.58812.5967.0218999.0067.59913.8586.3852316.0021.381015.1915.82741338.0090.531116.4765.3759481.0032.541216.7455.2901556.0037.621317.9804.9294679.0045.951418.5724.77351079.0073.001518.7994.71651230.0083.221619.1474.63151229.0083.151719.6194.52111380.0093.371820.2004.39241246.0084.301920.4664.33601478.00100.002020.8704.25281088.0073.612121.6254.1061584.0039.512222.0884.0210891.0060.282322.8403.8903613.0041.472423.9473.7129597.0040.392524.5693.6203680.0046.012625.6083.4757506.0034.242727.0153.29781100.0074.422827.8373.2022420.0028.422927.9673.1877400.0027.063029.2553.0502536.0036.273129.6893.0066603.0040.803230.6652.9130518.0035.053331.3182.8538451.0030.513431.8942.8036533.0036.063533.3702.6829518.0035.053633.5622.6679552.0037.353733.9192.6407581.0039.313834.8402.5730561.0037.963935.7892.5069559.0037.824035.9402.4967560.0037.894136.7802.4416740.0050.074237.0422.4249736.0049.804337.9592.3684683.0046.214439.0172.3066643.0043.50 method B: dynamic transformation a. form transformation

A hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide (50g) was placed in a 400mL measuring cylinder (height 6cm) with a mechanical stirrer on a ring-shaped holder. The device was placed in a Humidity controlled oven (LUNAIRE Humidity cabin CEO 941W-3). The stirring was set at 275rpm and the temperature and RH were adjusted to 60 ℃ and 40% respectively in 30 minutes. The compound was kept under these conditions for 1 hour, and then the conditions were changed to 80% RH/60 ℃ within 45 minutes. The compound was held under these conditions for 16 hours and then the oven was reset to 40% RH/60 ℃ and held for 3.25 hours. The compound was then brought to ambient conditions (bed height 4cm) and removed from the cylinder to give the non-hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide (> 95% yield). The transformation was confirmed by X-ray powder diffraction analysis (fig. 2). X-ray powder diffraction was also tabulated (Table 2) in the order of increasing diffraction angle (2. theta.) as the in-plane distance (d) of the crystal expressed in units of angstroms (), counts per second (Cps), and relative peak intensity (%). TABLE 2-N-2 θ … d … … Cps … …% … 15.18617.0268196.008.4326.37113.8615722.0031.0737.57011.6689516.0022.2048.23210.73231094.0047.0758.81710.0206257.0011.06610.4288.4761365.0015.71711.3777.7714129.005.55811.6007.6223117.005.55912.6676.98281805.0077.671013.9136.3599551.0023.711114.3986.1468178.007.661215.2265.8442285.0098.321316.5385.3557861.0037.051416.7735.2814929.0039.971518.0194.91901132.0048.711618.6724.74831871.0080.511718.8154.71252052.0088.301819.2044.61782071.0089.111919.6544.51322226.0095.782020.2374.38451939.0083.432120.5234.32402324.00100.002220.9344.24001656.0071.262321.6914.0938923.0039.722422.1434.01121411.0060.712522.9103.8786994.0042.772624.0073.7037964.0041.482724.6423.6097991.0042.642825.6423.6097991.0042.642927.0703.29131687.0072.593027.8553.2002688.0029.603129.4973.0258843.0036.273229.4973.0013878.0037.783330.7512.9051809.0034.813431.9162.8017821.0035.333533.9822.6360882.0037.953635.2002.5475865.0037.223736.0012.4926841.0036.193836.9272.43221106.0047.593938.3892.3429968.0041.65 b

A hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide (370g) was charged to a 2L rotary evaporator flask. The flask was placed on a rotary evaporator (Heidolph UV 2002) and lowered into a pre-heated (58 ℃) water bath (Heidolph MR 2002). The apparatus was placed under a vacuum of 60 mbar using a vacuum pump (Divatrion DV1) and the vacuum was broken in a controlled manner by passing moist air generated in a further heated aqueous flask. The introduction of humid air was controlled by a humidity control device (Vausalo Humiditique and Temperature changer) so as to obtain an RH of 79% (130-. The rotary evaporator was then rotated at 145-160 rpm for 5 hours while maintaining the heating bath at about 60 ℃ and the RH inside the vessel at 71-79%. The vacuum was then broken with nitrogen, the vessel and its contents were cooled to room temperature, and the product was removed to give the non-hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide. Batch 317g N of hygroscopic crystalline form of N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide was treated in the same manner to obtain a non-hygroscopic crystalline form of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide. The transformation was confirmed by X-ray powder diffraction analysis (fig. 3). Two times together gave 667g N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide in a non-hygroscopic crystalline form (97% overall yield). The transformation was confirmed by X-ray powder diffraction analysis (fig. 3). X-ray powder diffraction was also tabulated (Table 3) with the in-plane distance (d) of the crystal expressed in units of angstroms (), the count per second (Cps), and the relative peak intensity (%) increasing in the order of diffraction angle (2. theta.). TABLE 3-N-2 θ … d … … Cps … …% … 15.12417.2309180.0010.1726.32813.9565408.0023.0537.57411.6623305.0017.2348.19110.7851556.0031.4158.79710.0432166.009.38610.3988.5004244.0013.79712.6287.00401198.0067.68813.8716.3791353.0019.94915.2185.81721543.0087.181015.7235.6317187.0010.561116.5385.3558589.0033.281216.7515.2882621.0035.081318.0244.9175869.0049.101418.6404.75631156.0065.311518.8094.71411241.0070.111619.1914.62101521.0085.931719.6594.51201413.0079.831820.8654.40641303.0073.621920.4954.32991770.00100.002020.8654.25391120.0063.282121.6164.1077683.0038.592222.1134.0166919.0051.922322.9503.8719697.0039.382424.1173.6871659.0037.232524.6183.6132716.0040.452625.6443.4709662.0037.402726.2973.3862486.0027.462827.0523.29341270.0071.752927.9603.1885518.0029.273029.6403.0115705./0039.383130.7442.9058695.0039.273233.4652.6755697.0039.383333.8402.6467764.0043.163435.8122.5053736.0041.583536.8112.4396858.0048.473637.0762.4228919.0051.923738.1852.354987049.153839.6222.2728882.0049.83X-ray powder diffraction patterns of samples of hygroscopic crystalline forms of example 14N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide and transformed non-hygroscopic crystalline forms thereof

Samples of hygroscopic crystals of N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide prepared in example 5 or 11 and samples of the corresponding non-hygroscopic crystalline form thereof converted in accordance with the procedure of example 13 were taken. The X-ray powder diffraction patterns of the hygroscopic crystalline form and the non-hygroscopic crystalline form are shown in fig. 4 and 5, respectively. X-ray powder diffraction of the hygroscopic crystalline form and the non-hygroscopic crystalline form were also tabulated in order of increasing diffraction angle (2 θ) for in-plane distance (d) of the crystal expressed in unit angstrom (a), count per second (Cps), and relative peak intensity (%) (see tables 4 and 5, respectively). TABLE 4-N-2 theta … d … … Cps … …% … 15.07317.40371487.0086.5026.45113.6905447.0026.0037.83711.2712411.0023.9148.49110.4049602.0035.0259.6999.111993.005.41610.4888.4278421.0024.49711.5707.642392.005.35812.5507.0474411.0023.91913.5766.5168760.0044.211015.3275.7763606.0035.251115.7905.6080456.0026.531216.1795.4739346.0020.131316.7705.2824938.0054.571417.0855.1856685.0039.851517.7504.9927924.0053.751618.1514.8835741.0043.111718.5044.7909593.0034.501819.3234.5897930.0054.101919.7144.4996792.0046.072020.5454.31941719.00100.002121.3884.1510897.0052.182222.3813.9691373.0021.702322.8703.8852258.0015.012423.6403.7604563.0032.752523.8413.7292680.0039.562624.0483.6976623.0036.242724.7463.5949338.0019.662825.2003.5311366.0021.292925.7923.4513590.0034.323026.2663.3901731.0042.523126.9593.3045555.0032.293227.4263.2494769.0044.743327.9673.1876528.0030.723429.0203.0744771.0044.853529.9222.9837491.0028.563630.9702.8851384.0022.343731.5522.8332510.0029.673833.3382.6854627.0036.473934.8382.5731520.0030.254035.8732.5012653.0037.994136.1072.4855639.0037.174237.1622.4174683.0039.734338.5092.3359775.0045.084439.7012.2684784.0045.61 TABLE 5-N-2 theta … d … … Cps … …% … 15.15217.1371123.007.3426.38613.8287483.0028.8437.58011.6540389.0023.2248.22510.7410752.0044.9058.80110.0390180.0010.75610.4088.4928276.0016.48712.6606.98631399.0083.52813.9146.3594391.0023.34915.2515.80471675.00100.001016.5415.3548608.0036.301116.7715.2818652.0038.931218.0474.9112775.0046.271318.6764.74721078.0064.361418.9024.69101099.0065.611519.1824.62311151.0068.721619.6974.50351164.0069.491720.2404.38381049.0062.631820.5684.31471403.0083.761929.9334.24031024.0061.132021.6844.0951569.0033.972122.1224.0150746.0044.542222.9703.8685564.0033.672324.0803.6927546.0032.602424.2183.6720556.0033.192524.6943.6023618.0036.902625.6803.4662510.0030.452726.4003.3732403.0024.062827.1053.28711093.0065.252927.9293.1920450.0026.873029.3603.0395555.0033.133129.7243.0031595.0035.523230.3402.9435429.0025.613330.6932.9105552.0032.963431.3532.8507476.0028.423531.8222.8098531.0031.703632.0062.7940545.0032.543732.8852.7213485.0028.963833.5082.6722547.0032.663934.0402.6316606.0036.184034.8392.5730580.0034.634135.9982.4928596.0035.584236.6802.4480629.0037.554336.9482.4309727.0043.404437.1972.4152703.0041.974539.6022.2739697.0041.61 EXAMPLE 15 isothermal microcalorimetry experiments for N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) -beta-cyclohexylalaninamide in hygroscopic and non-hygroscopic crystalline forms

Using a thermometer^Thermal Activity Monitor (TAM) on N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl)]- (L) -aspartyl]- (L) -beta-cyclohexylalaninamideThe hygroscopic and non-hygroscopic crystalline forms of (a) were subjected to isothermal microcalorimetry experiments. The solid state transition of different crystalline forms is studied by exposing them to different humidity or solvent vapor at different temperatures. The following salt solutions were used to create the different humidities: KCl (80% RH), NaCl (75% RH) and NaBr (65% RH). Approximately 100mg of each crystalline form of the compound was weighed into a TAM glass ampoule and a micro-hygrostat containing either a saturated salt solution (containing excess solids) or an organic solvent was placed in the ampoule. The ampoule is sealed, allowed to equilibrate to ambient temperature, and then lowered to the measurement position for TAM. The same system, replacing the sample to be tested with washed sea sand, was placed on the reference side. The output power (. mu.W) was measured as a function of time (FIGS. 6 to 8). Example 16N- [ N- [ N- (4- (piperidin-4-yl) butanoyl) -N-ethylglycyl)]- (L) -aspartyl]Hygroscopic isotherms of hygroscopic and non-hygroscopic crystalline forms of (L) -beta-cyclohexylalaninamide

The hygroscopic isotherms of the hygroscopic and non-hygroscopic crystalline forms of N- [ N- [ N- (4- (piperidin-4-yl) butyryl) -N-ethylglycyl ] - (L) -aspartyl ] - (L) - β -cyclohexylalaninamide were determined on a VTI MB300G moisture balance. The experiment can be performed in two ways, one by performing the steps of increasing and decreasing% RH on about 15mg of the crystalline form to be tested and determining (at each equilibration step) the weight increase as a function of% RH (fig. 9), and the other by subjecting the crystalline form to be tested to a constant humidity and determining the weight increase as a function of time.

The compounds of formula II exhibit valuable pharmacological activity and can therefore be incorporated into pharmaceutical compositions and used for the treatment of patients suffering from specific diseases.

The invention also relates to methods of treating patients suffering from diseases that can be alleviated or prevented by inhibiting the binding of fibrinogen to activated platelets and other adhesive glycoproteins involved in platelet aggregation and blood coagulation by administering inhibitors of platelet aggregation. In addition, the present invention relates to methods for preventing or treating thrombosis associated with myocardial infarction, stroke, peripheral arterial disease, and disseminated intravascular coagulation in humans and other mammals.

Reference herein to treatment is to be understood to include prophylactic treatment as well as treatment of established diseases.

The invention also includes pharmaceutical compositions comprising a pharmaceutically acceptable amount of at least one compound of formula i and a pharmaceutically acceptable carrier or excipient.

In practice, the compounds or compositions of the invention for use in therapy may be administered by any suitable means, for example topically, by inhalation, parenterally, rectally or orally, but preferably by oral administration.

The compounds of formula ii may be provided in a form which allows administration by the optimal route, and the invention also relates to pharmaceutical compositions suitable for use in humans or veterinary medicine containing at least one compound of the invention. These compositions may be prepared according to conventional methods using one or more pharmaceutically acceptable adjuvants or excipients. The adjuvants include diluents, sterile aqueous media, and various non-toxic organic solvents. The compositions may be in the form of tablets, pills, capsules, granules, powders, aqueous solutions or suspensions, injectable solutions, elixirs, syrups, and the like, and may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents, stabilizing agents, or preserving agents in order to obtain pharmaceutically acceptable formulations.

The choice of the carrier and the amount of active ingredient in the carrier will generally be determined by the solubility and chemical nature of the product, the particular mode of administration and the conditions which require attention in pharmaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as starch, alginic acid and certain complex silica gels and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc may be used to prepare tablets. For the preparation of capsules, lactose and high molecular weight polyethylene glycols are preferably used. When aqueous suspensions are used, they may contain emulsifying agents or agents which assist in suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or combinations thereof may also be used.

For parenteral administration, emulsions, suspensions or solutions of the compounds of the invention in vegetable oils such as castor oil, arachis oil or olive oil, or in aqueous-organic solutions such as water and propylene glycol, injectable organic esters such as ethyl oleate, and sterile aqueous solutions of pharmaceutically acceptable salts may be employed. The salt solutions of the products of the invention can also be administered by intramuscular or subcutaneous injection. Aqueous solutions, including solutions of salts in purified distilled water, can also be used for intravenous administration, provided that the pH is appropriately adjusted, properly buffered and made isotonic with sufficient amounts of glucose or sodium chloride, and sterilized by heat, radiation and/or microfiltration.

For topical administration, gels (aqueous or alcoholic), creams or ointments containing the compounds of the invention may be used. The compounds of the present invention may also be incorporated into the matrix of a gel or patch so that the compounds may be controlled released across the skin barrier.

Solid compositions for rectal administration include suppositories containing at least one compound of formula ii formulated according to known methods.

The percentage of active ingredient in the compositions of the invention may vary provided that it constitutes a fraction of the appropriate dosage. It will be apparent that multiple unit dosage forms may be administered at about the same time. The dosage employed can be determined by a physician or qualified medical practitioner and will depend upon the desired therapeutic effect, the route of administration, and the duration of treatment, as well as the condition of the patient. The dosage regimen for carrying out the method of the invention is first a dosage which ensures maximum therapeutic response until the condition is ameliorated, and then the lowest effective amount which will alleviate the condition. In general, oral doses may range from about 0.1mg/kg to about 100mg/kg, preferably from about 0.1mg/kg to about 20mg/kg, most preferably from about 1mg/kg to about 20mg/kg, and intravenous doses may range from about 0.1 μ g/kg to about 100 μ g/kg, preferably from about 0.1mg/kg to about 50 mg/kg. For each particular case, the dosage will be determined based on the particular factors such as age, weight, general health, and other characteristics of the patient to be treated which may affect the efficacy of the compounds of the present invention.

In addition, the compounds of formula II may be administered at a desired frequency to achieve the desired therapeutic effect. Some patients may respond rapidly to higher or lower doses and may require very small maintenance doses. For other patients, long-term treatment may be required at 1-4 oral doses per day, preferably 1-2 times per day, depending on the physiological needs of the particular patient. Of course, for other patients, it may be necessary to prescribe no more than one or two doses per day.

The compounds of formula ii exhibit significant pharmacological activity when tested according to literature and it is believed that these test results correlate with pharmacological activity in humans and other mammals. The following in vitro and in vivo pharmacological test results are typical of the compounds of formula II.

The inhibitory activity of the compounds of formula ii on fibrinogen-mediated platelet aggregation, fibrinogen-to-thrombin activated platelet binding, and inhibition of ADP-induced ex vivo platelet aggregation was evaluated in the following pharmacological tests, the results of which correlate with the in vivo inhibitory properties of the compounds of formula ii.

The platelet aggregation assay is based on the description in "blood", 66(4),946-952 (1985). The fibrinogen binding assay is based on Ruggeri, Z.M. et al, Proc. Natl.Acad.Sci.USA, 83, 5708-. The test for inhibition of ADP-induced ex vivo platelet aggregation is based on Zucker, "platelet aggregation by photoelectric Methods", an enzymatic method (Methods in Enzymology),169,117-133 (1989). Platelet aggregation assay preparation of immobilized activated platelets

Platelets were isolated from human platelet concentrates using gel filtration techniques described by Magueie.G.A et al, J.Biol.Chem.254, 5357-5363(1979) and Rggeri, Z.M. et al, J.Clin.invest. 72,1-12 (1983). Mixing platelets at 2 × 10⁸The cells/mL were suspended in a modified calcium-free Tyrode's buffer containing 127mM sodium chloride, 2mM magnesium chloride, 0.42mM Na₂HPO₄,11.9mM NaHCO₃2.9mM KCl,5.5mM glucose, 10mM HEPES at pH 7.35, and 0.35% Human Serum Albumin (HSA). These washed platelets were activated by the addition of human a-thrombin at a final concentration of 2 units/mL followed by the addition of thrombin inhibitor I-2581 at a concentration of 40. mu.M. Paraformaldehyde is added to the activated platelets to a final concentration of 0.50% and then incubated at room temperature for 30 minutes. The immobilized activated platelets were then collected by centrifugation at 650Xg for 15 minutes. The platelet pellet was washed 4 times with Tyrode's-0.35% HSA buffer as described above and then resuspended to 2 × 10 with the same buffer⁸Concentration of cells/mL. Platelet aggregation assay

The fixed activated platelets were incubated with the selected dose of test compound to detect platelet aggregation inhibition for 1 minute and aggregation induced by the addition of human fibrinogen to a final concentration of 250 μ g/ml. Platelet aggregation was recorded using a platelet aggregation mapper, PAP-4. The degree of inhibition of agglutination was expressed as a percentage of the observed rate of agglutination in the absence of the inhibitor. The IC of each compound was then calculated₅₀The amount of inhibitor required to reduce the rate of aggregation by 50% (see, e.g., Plow, E.F., et al, Proc. Natl. Acad. Sci. USA 82,8057-8061 (1985)). Fibrinogen binding assay

Plasma fractions were washed from platelets using an albumin density gradient technique (J.C. 76,1950, 1958(1985)) modified by Walsh, P.N., et al, Br.J.Haematol.281-296(1977), Trapai-Lombardo, V.et al. In each experiment, a mixture of platelets in modified Tyrode's buffer (Rggeri, Z.M., et al, J. Clin. Res., 72,1-12(1983)) was stimulated with human a-thrombin at 22-25 ℃ for 10 minutes (3.125X 10)¹¹Platelets/liter and thrombin 0.1 NIH units/mL). Then a 25-fold excess (unit/unit) of hirudin is added¹²⁵I-labeled fibrinogen and test compound were added 5 minutes prior to the test compound. After addition of all these substances, the final platelet count in the mixture was 1X 10¹¹And L. After further incubation at 22-25 ℃ for 30 minutes, 50. mu.l of the mixture was centrifuged at 12000Xg in 300. mu.L of 20% sucrose solutionBound and free ligands were separated for 4 minutes. The platelet deposit is then separated from the rest of the mixture to determine the platelet-bound radioactivity. Nonspecific binding was determined in a mixture containing excess unlabeled ligand. When the binding curves were analyzed by Scatchard analysis, non-specific binding was obtained as a fit parameter from the binding isotherms using a computer program (Musson. P.J. Methods Enzymol. 92, 542-. To determine the concentration (IC) required for each compound to inhibit 50% of fibrinogen binding to thrombin-activated platelets₅₀) For each compound, 6 or more concentrations were tested,¹²⁵i-labeled fibrinogen was maintained at 0.176. mu. mol/L (60. mu.g/mL). IC was determined by plotting the log of residual fibrinogen binding versus sample compound concentration₅₀. Inhibition experiment method for ADP-induced in vitro platelet aggregation

Control blood samples were taken 5-10 minutes before the test compound was administered to the crossbred dogs weighing 10-20 kg. The compounds are administered intragastrically via a water feeding tube, or orally via a gelatin capsule. After administration, blood samples (5mL) were taken at 30 minute intervals for 3 hours, and then sampled at 6,12 and 24 hours. Each blood sample was obtained by venipuncture of the cephalic vein and collected directly into a plastic syringe containing 3.8% trisodium citrate in an amount of 1 part trisodium citrate per 9 parts blood. In vitro canine platelet aggregation

The blood samples were centrifuged at 1000rpm for 10 minutes to obtain platelet-rich plasma (PRP). After removal of the PRP, the sample was centrifuged at 2000rpm for an additional 10 minutes to obtain platelet-poor plasma (PPP). Platelets in PRP were counted using a Coulter counter (Coulter Electronics, Hialeah, FL). If the concentration of platelets in PRP is greater than 300000 platelets/. mu.L, then dilution of PRP with PPP adjusts the platelet count to 300000 platelets/. mu.L. An aliquot of PRP (250 μ L) was then placed in a quartz glass cuvette (7.25 × 55mm, Bio/Data corp. horsham, PA). Epinephrine (final concentration of 1 μ M) was then added to the PRP, which was incubated at 37 ℃ for 1 minute. The platelet aggregation stimulator, ADP, was then added to PRP at a final concentration of 10 μ M. Platelet Aggregation was monitored spectrophotometrically using a light transmission aggregometer (Bio/Data Platelet Aggregation Profiler, model PAP-4, Bio/Data Corp. Horshim, Pa.). To determine the compounds, the rate of change (slope) of light transmittance and maximum light transmittance (maximum agglutination) was recorded in duplicate. Platelet aggregation data are reported as the percent reduction in slope or maximal aggregation (mean ± SEM) compared to data obtained from a control PRP prepared from a blood sample obtained prior to administration of the test compound.

The compounds of formula ii show significant activity in the aforementioned assays and are believed to be useful in the prevention and treatment of thrombosis associated with certain diseases. Their activity in humans can be predicted from antithrombotic activity in ex vivo canine platelet aggregation assays (see, e.g., Catalfamo, j.l., and Dodds, w.jean, "isolation of platelets from laboratory animals", methods in enzymology, 169, part a, 27 (1989)). The results obtained from the tests carried out on the compound of formula II by the above method are shown in Table 6 below. Also shown in this table are the results of comparative experiments with 4-4 (piperidinyl) butyrylglycosyl aspartyl tryptophan, a compound disclosed in European patent application publication No. 0479481. TABLE 6

Inhibitory Effect of ADP-induced inhibition of Ex vivo platelet aggregation of immobilized platelet aggregation dose The% inhibition of Ex vivo platelet aggregation after oral administration (mg/kg) was

(IC₅₀mu.M) for

1h 3h 6h 12h 24h 0.047553 < 20 of the compounds of example 150.09751001001009850 EPA' 481

Those skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned. The compounds, compositions, and methods described herein are representative of preferred embodiments or are intended to be illustrative, and are not intended to limit the scope of the invention.

Claims (5)

1. A compound of the formula VII,wherein

B is alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, arylalkyl, alkylaryl or alkylarylalkyl;

E¹is H;

E²is an alpha-carbon side chain of a natural alpha-amino acid, H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, substituted aryl, arylalkyl,Substituted aralkyl, heterocyclyl, substituted heterocyclyl, heterocyclylalkyl, substituted heterocyclylalkyl, or E¹And E²And connection E¹And E²Together with the carbon atom(s) to form a 4-, 5-, 6-or 7-membered azacycloalkane ring;

g is OR¹Or NR¹R²；

R¹And R²Independently of one another, H, alkyl, cycloalkyl, cycloalkylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl, aryl, arylalkyl, alkylaryl or alkylarylalkyl;

p is 1 to 4;

P¹is an acid protecting group which is labile to hydrogenation;

P^2’is P²Or TFA. H-;

P²is an acid labile amino protecting group.

2. The compound of claim 1, wherein

B is an alkyl group;

E¹is H;

E²is cycloalkylalkyl;

g is NR¹R²；

R¹And R²Is H;

p is 1.

3. The compound of claim 2, wherein

B is ethyl;

E²is a cyclohexylmethyl group.

4. The compound of claim 3, wherein

P¹Is benzyl;

P^2’is TFA. H-.

5. The compound of claim 3, wherein

P¹Is benzyl;

P²is tert-butyloxycarbonyl.