JP2002506075A - Serine peptidase modulator - Google Patents

Abstract

  (57) [Summary] The present invention relates to serine peptidases and proteases, especially dipeptidyl peptidase IV, prolyl oligopeptidase (PO), dipeptidyl peptidase II (DPP II), fibroblast activation protein α (FAPα), lysosomal Pro-X carboxypeptidase. And compounds having modulating (inhibiting and promoting) activity of elastase. These new compounds can be used in the treatment of various disease states involving these peptidases.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates generally to serine peptidases and proteases, particularly dipeptidyl peptidase IV, prolyl oligopeptidase (PO), dipeptidyl peptidase II (DPP II), fibroblast activating protein α (FAPα), a novel regulator (inhibitor and promoter) of lysosomal Pro-X carboxypeptidase and elastase. Furthermore, the invention relates to the preparation and use of these compounds for the selective modulation (inhibition or promotion) of serine peptidases and proteases, and medicaments containing them. The terms “peptidase” and “protease” are used interchangeably.

BACKGROUND OF THE INVENTION [0002] Mast cell tryptase, elastase, trypsin-like enzymes, prolyl oligopeptidase, dipeptidyl peptidase II, dipeptidyl peptidase IV, such as serine peptidase / protease, granzyme, are used for blood coagulation, inflammation, It is involved in various processes that occur in the body, such as the immune response and general regulation of peptide hormone metabolism. Serine peptidase is physiologically necessary, and if the activity of serine peptidase is not controlled in the body, it is a serious obstacle to health.

[0003] Serine peptidases have been reported to be involved in various medical indications. Blood coagulation serine proteases play a major role, for example, in vascular coagulation and cerebral and coronary infarction. Chymotrypsin-like enzymes and plasmin are involved in tumor invasion, tissue remodeling and clot dissociation. Pancreatitis, emphysema, rheumatoid arthritis, inflammation, and adult respiratory distress symptoms can, in some cases, occur with uncontrolled proteolysis by serine proteases such as elastase.

[0004] Serine peptidases form a large group with many members divided into families and families. One member of the tribe SC is dipeptidyl peptidase.
IV (DPP IV, EC 3.4.14.5), a highly specific exopeptidase with serine-type metabolism of protease activity, at the penultimate site at the amino terminus of peptides containing proline or alanine. Dissociate the dipeptide. further,
Slow release of dipeptides of X-Gly or X-Ser type has been reported for some natural peptides. DPP IV is constitutively expressed on epithelial and endothelial cells of a variety of different tissues and is also found in body fluids. In the hematopoietic system, DPP IV has been identified as leukocyte antigen CD26.

[0005] Prolyl oligopeptidase (PO, EC 3.4.21.26) was discovered as an oxytoxin degrading enzyme in the human uterus. This enzyme has high specificity for proline residues and hydrolyzes peptide bonds on the carboxyl side. However, proline is not at the peptide amino terminus. This endopeptidase is DPPIV-like, has a serine-type metabolism, and is characterized by activity on oligopeptides. PO is a biologically active peptide (substance P, oxytoxin, vasopressin, gonadoriberin, bradykinin,
(Gonadriberine, bradykinin, neurotensin) cleaves the Pro-Xaa bond and appears to be involved in in vivo regulation of these effects. The role of PO in processes such as memory has been published (Yoshimoto T. and Ito K. in Handbook
of proteolytic enzymes, eds.Barrett et al., Academic Press, 1998, p. 2
72-374).

[0006] Fibroblast activation protein α (FAPα) has been discovered as a cell surface antigen on cultured normal fibroblasts. Its in vivo expression is very restricted in normal cells. On the other hand, activated tumor stromal fibroblasts found in certain carcinomas express high levels of FAPα. The biological role of FAPα expression has not been elucidated, but its function in tissue remodeling and repair is predicted (Rettig, FAPe in Barrett, supra, p. 385-389).

[0007] Dipeptidyl peptidase II (DPPII, EC 3.4.14.2) is an oligopeptide N-
When the termini are not substituted, release the N-terminal dipeptide from the oligopeptide. Preferred P1 residues are Ala and Pro. Serum DPPII is found in cancer patients, and very high levels of DPPII are present in human carcinoma cells. DPPII can be inhibited by classical (non-specific) inhibitors of serine-type peptidases (JKNcDonald in Barre
tt, supra, p. 408-411).

[0008] The clear potency of elastase is the release of soluble proteins from insoluble elastin fibers by proteolysis, called elastin degradation. Elastase belongs to the chymotrypsin family of leukocyte serine-type proteases. Human leukocyte elastase (EC 3.4.21.37) preferentially cleaves peptides with Val at P1, but also degrades peptides with Ala, Ser, and Cys at P1 and is thought to possess an extended substrate binding site. ing. The possible involvement of leukocyte elastase in inflammation could trigger research for the development of specific inhibitors. Furthermore, the etiology of emphysema, cystic fibrosis, and adult respiratory distress symptoms has been suggested.
(J. Bieth in Barrett, supra, p. 54-60; D. Farley et al. In Pharmaceutical E
nzymes, ed.A. Lauwers and S. Scharpe, Marcel Dekker, Inc., 1997, p.306-
326).

Lysosomal Pro-X carboxypeptidase (prolyl carboxypeptidase, angiotensinase C, EC 3.4.16.2) cleaves the C-terminal amino acid from a peptide having the general formula X-Pro-Y. In the formula, X is a blocking group, another protected amino acid or a peptide, and Y is an aromatic or aliphatic amino acid having a free carboxyl group. The enzyme has an acidic pH optimum (p) for small synthetic substrates.
H 5.0), but retains 50% of the maximum activity for large substrates at physiological pH. (Des-Arg9) -Bradykinin and Angiotensin II are lysosome Pro-X
A possible natural substrate for carboxypeptidases (Tan, F. and Erdos, E
in Barrett et al., supra, p.405-407).

[0010] These roles in various physiological processes are expected to be involved in the activity of serine peptidases. This involvement is promotion or inhibition. Various types of serine peptidase inhibitors have been published. For example, EP-764 151 of the present inventor. In particular, this application discloses compounds of the general formula Z-Xaa-Y '. In the formula, Z is a protecting group that is present or absent, Xaa is a dipeptide or an amino acid, and Y ′ can be a phosphonate such as diphenylphosphonate. According to further studies by the inventor, its toxicity is not acceptable and its strength / effect is not satisfactory.

US Pat. No. 5,543,396 to Powers et al. Relates to proline phosphonic acid derivatives. The phosphonate can be substituted with one or two phenyl groups. These phenyl groups are halogen, C ₁ -C ₆ alkyl, C ₁ -C ₆ perfluoroalkyl, C ₁ -C ₆ alkoxy, NO ₂ , CN, OH, CO0H, amino, C ₁ -C ₆ alkylamino, C ₂ -C _{1 2} dialkylamino, C ₁ -C ₆ acyl, C ₁ -C ₆ alkoxy -CO-, C ₁ -C ₆ alkyl -S- mono, di, may be trisubstituted. The inventor independently developed the same compound in the work that reached the present invention. However, due to the toxicity, stability, and efficacy of these compounds, these compounds are not optimal.

[0012] It is therefore an object of the present invention to provide a serine peptidase / proteinase that has the proper combination of inhibitory capacity, stability in plasma, bioavailability, time of action and simple synthesis. Furthermore, an object of the present invention is to provide a serine peptidase
/ Providing a compound having a promoting activity on protease. These two types of compounds of the present invention are referred to herein as "modulatory compounds." More specifically, the present invention provides compounds having a suitable combination for modulating the activity of DPP IV, PO, DPP II, FAPα, liposomal Pro-X carboxypeptidase and elastase.

Thus, in the research that reached the present invention, the effects of various functional groups on the selective regulation (inhibition or promotion) of phosphonates were investigated. Pyrrolylpyrrolidine diphenylphosphonate was synthesized and substituted on phenyl with hydroxy, methoxy, acylamino, sulfonylamino, ureyl, methoxycarbonyl and alkylaminocarbonyl. The phenyl ester was also substituted with other groups having excellent leaving group functions such as trichloroethyl and trifluoroethyl. The in vitro and in vivo inhibitory activity against serine peptidases such as DPP IV, the stability and properties of these compounds were tested.

As a result, the compounds of claim 1 are generally potent modulators, especially inhibitors of serine peptidases / proteases, especially DPP IV, DPP II, PO, FAPα, lysosomal Pro-X carboxypeptidase and elastase. I understood that. Claim 12
Are potent inhibitors of DPP IV and PO.

The compounds of the present invention are based on peptides. These peptides are composed of either naturally occurring amino acids or other amino acids. The C-terminal carboxy functional group is replaced with a phosphate group.

The compound of the present invention is represented by the following general formula I:

Embedded image In the formula, A is an (R2) or H or C ₁ -C ₆ or halogen alkyl (excluding perfluoroalkyl); phenyl group is mono R1 or R2 -, di - or tri - is substituted; X is a peptide- or amino acid-derived moiety; A and a phenyl group substituted with R1 optionally form a biphenyl diester; all R1 and R2 substituents independently of one another Selected from the group consisting of: a) C ₁ -C ₆ acylamino; b) aroylamino, which is alkyl at the o- and / or p- and / or m-position, especially C ₁ -C ₆ alkyl, and / or halogen C) C ₁ -C ₆ alkylsulfonylamino; d) arylsulfonylamino, which is alkyl at the o- and / or p- and / or m-position, especially C ₁ -C ₆ Optionally substituted with alkyl and / or halogen Has been replaced;

E) α-aminoacylamino, wherein aminoacyl is L, D or DL at the α-carbon atom
A blocked or unblocked α-amino acid residue of a side chain having a conformation, a group consisting of:
Selected from: alanine, methionine, methionine sulfoxide, arginine, homoarginine
, Phenylalanine, aspartic acid, proline, hydroxyproline, Aspa
Lagin, serine, cysteine, threonine, histidine, glycine, tyrosine,
Glutamic acid, pyroglutamic acid, tryptophan, glutamine, valine, nor
Valine, isoleucine, lysine, leucine, norleucine, thioproline, homo
Proline, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), 2,3-dihydroindole-2-carboxic acid, α-naphthylglycine, O-phenylglycine,
4-amidinophenylglycine, 4-phenylproline, 4-amidinophenylalani
, O-benzyl tyrosine, ω-acetyl lysine, α-aminobutyric acid, citrulline, e
Moscitrulline, ornithine, O-methylserine, O-ethylserine, S-methylcysteine
In, S-ethylcysteine, S-benzylcysteine, homoserine, 4-dehydrop
Lorin, penicillamine, β- (2-thienyl) alanine, NH_Two-CH (CH_TwoCHEt_Two) -COOH, α
-Aminoheptanoic acid, NH_Two-CH (CH_Two1-naphthyl) -COOH, NH_Two-CH (CH_Two(-2-naphthyl) -C
OOH, NH_Two-CH (CH_Two-Cyclohexyl) -COOH, NH_Two-CH [CH- (cyclohexyl)_Two] -COOH, NH_Two-CH (CH_Two-Cyclopentyl) -COOH, NH_Two-CH [CH- (cyclopentyl)_Two] -COOH, NH _Two -CH (CH_Two-Cyclobutyl) -COOH, NH_Two-CH [CH- (cyclobutyl)_Two] -COOH, NH_Two-CH (CH_Two -Cyclopropyl) -COOH, NH_Two-CH [CH- (cyclopropyl)_Two] -COOH, 5,5,5-triflu
Ololeucine, hexafluoroleucine, (S) -azetidine-2-carboxylic acid, (S) -pipecoric acid, (S) -oxazolidine-4-carboxylic acid, (R) -thiazolidine-4-carboxylic acid
Acid (L-thioproline), sarcosine;

F) a residue selected from the group consisting of 3-aminobenzoic acid, ε-aminocaproic acid, and β-alanine; g) Y-NH-CO-NH-; h) Y'O ₂ CCH (NHCo-Y ) -CH _2- ; i) Y'NHCO-; j) CH ₃ -O-CO-Y '-NH-CO-; k) CH ₃ -CH ₂ -O-CO-Y'-NH-CO-; Wherein Y is C ₁ -C ₆ alkyl, aryl or H, Y ′ is C ₁ -C ₆ alkyl, and includes pharmaceutically acceptable salts thereof.

In a specific embodiment of the invention, X is a moiety of the general formula (AA) p-aa-, wherein: p is 0, 1, 2, 3, 4 or 5 residues AA Which may be the same or different in one molecule; AA and aa are selected from one of the following: a) C ₁ -C ₆ alkyl or aryl optionally substituted in the α-position Or an aminocarboxylic acid having an aralkyl group; b) alanine, methionine, methionine sulfoxide, arginine, homoarginine, phenylalanine, aspartic acid, proline, hydroxyproline,
Asparagine, serine, cysteine, threonine, histidine, glycine, tyrosine, glutamic acid, pyroglutamic acid, tryptophan, glutamine, valine, norvaline, isoleucine, lysine, leucine, norleucine, thioproline, homoproline, 1,2,3,4-tetrahydroisoquinoline- 3-carboxylic acid (Tic), 2,3
-Dihydroindole-2-carboxylic acid, α-naphthylglycine, α-phenylglycine, 4-amidinophenylglycine, 4-phenylproline, 4-amidinophenylalanine, O-benzyltyrosine, ω-acetyllysine, α-baminobutyric acid , Citrulline, homocitrulline, ornithine, o-methylserine, o-ethylserine, S-methylcysteine, S-ethylcysteine, S-benzylcysteine, homoserine, 4-dehydroproline, penicillamine, 9- (2-thienyl) alanine, NH ₂ -CH (CH ₂ CHEt ₂ ) -COO
H, alpha-amino heptanoic acid, NH ₂ -CH (CH ₂ -l- _{naphthyl) -COOH, NH 2 -CH (CH} 2 -2- _{naphthyl) -COOH, NH 2 -CH (CH} 2 - cyclohexyl) -COOH , NH ₂ -CH [CH- (cyclohexyl) ₂ ] C
_{OOH, NH 2 -CH (CH 2} - _{cyclopentyl) -COOH, NH 2 -CH [CH-} ( _{cyclopentyl) 2] -COOH, NH 2 -CH} (CH 2 - cyclobutyl) -COOH, NH ₂ -CH [CH- (Cyclobutyl) ₂ ] -COOH, NH ₂ -CH
(CH ₂ - _{cyclopropyl) -COOH, NH 2 -CH [CH-} ( cyclopropyl) _2] -COOH, 5,5,5-trifluoro-leucine, hexafluoro leucine, (S) - azetidine-2-carboxylic acid , (
(S) -Pipecoric acid, (S) -oxazolidine-4-carboxylic acid, (R) -thiazolidine-4-carboxylic acid (L-thioproline), 3-aminobenzoic acid, sarcosine, ε-aminocaproic acid, β-alanine Wherein the α-amino residue is a blocked or unblocked side chain, and the α carbon element is L,
And has a D or DL conformation; and pharmaceutically acceptable salts thereof.

In another embodiment of the present invention, X is M- (AA) p-aa-, wherein: p, AA and aa are as defined above; M is selected from: a ) -CONH optionally substituted_Two, -CSNH_Two, -SO_TwoNH_Two, Phenyl-SO_Two-, Phenyl-CH _Two SO_Two-, 2-furylacryloyl; and b) acetyl, adamantyloxycarbonyl, benzyloxycarbonyl, benzoyl, benzyl, t-butoxycarbonyl, t-butyl, 2,4-dinitrophenyl
, Formyl, fluorenylmethoxycarbonyl, 4-methoxybenzyl, tosyl,
Trifluoroacetyl, trityl, phthaloyl, phenylalkylcarbonyl,
2-Indanylacetyl, 2- (1,2,3,4-tetrahydronaphthyl) acetyl, 4- (4-benzylphenoxy) alkyl.

In a specific embodiment, X is AA-aa-, wherein aa is proline and AA is as defined above. In another embodiment, X is AA-aa-, wherein both AA and aa are proline. If aa is alanine, R1 and R2 may be further selected from the following: 1) H, _{halogen, NO 2, CN, OH,} COOH m) amino, C ₁ -C ₆ alkylamino, C ₂ -C _{1 2} dialkylamino Amino n) C ₁ -C ₆ acyl o) C ₁ -C ₆ alkoxy-CO- p) C ₁ -C ₆ alkyl-S-.

In the compound, aa is alanine, preferably at least AA bound to aa is proline or phenylalanine. Preferred examples of the compound are Phe-Ala-diphenylphosphonate or Pro-Ala-diphenylphosphonate and pharmaceutically acceptable salts thereof.

In the present invention, the compounds can be divided into three groups. In a preferred compound of the invention, the phosphate group is a diphenyl-phosphonate group (P
(Indicated by (OPh) 2), and is preferably substituted. The first group consists of the following general formula II:

Embedded image Wherein R1, R2 and X are as defined above (Group 1).

Alternatively, the compound is a 2,2 ′ biphenyl diester of α-aminoalkyl phosphoric acid having the general formula:

Embedded image Wherein the substituents R1, R2 and X are as defined above (Group 2).

A third group (Group 3) consists of compounds having the following general formula IV:

Embedded image Wherein X and R 1 are as defined above, and A is H or C ₁ -C ₆ alkyl or halogenalkyl, except for fluoroalkyl. Specific members of these three groups are described below.

For the serine protease cathepsin G, X is Cbz-Gly-Leu-Phe-, Z-Phe
-Pro-Phe, Suc-Val-Pro-Phe-. For prolyl oligopeptidase X can be selected from: Cbz-Gly-Gly-Pro-, Cbz-Pro-Pro-, Boc
-Val-Pro-Val-, MeO-Suc-Ala-Ala-Ala-Val-, MeO-Suc-Ala-Ala-Pro-Val-. DPP I
For V, X is as follows: Ala-Pro-, Pro-Pro-, Ala-Pip-, Phe-Pro-, Ile-Pro
-, Arg-Pro-, pF-Phe-Pro-, cyclohexylara-Pro-, Pro-azetidine, Phe-azetidine, Lys-Pro-, Lys-azetidine-. If the enzyme to be inhibited is human leukocyte elastase, X is Suc-Lys (Cbz) -Val-Pro-Val-, Z-Ala-Ala-Ala, Boc-Va
Selected from l-Pro-Val-. For GranTeam, X is Cbz- (4-amidinophenylalanine)-, Z-Met-, 3-phenylpropanoyl-Pro- (4-aminophenylalanine)-, Cbz-Thr- (4-amidinophenylglycine) -, Boc-D-Phe-Pro- (4-amidinophenylalanine)-. When the enzyme to be inhibited is thymotrypsin, X is Z-Phe-Pro-Phe-, Z-Phe-, Suc-Val-Pro-Phe-, MeO-Suc-Ala-Al
a-Pro-Phe-, MeO-Suc-Ala-Ala-Ala-Phe. For a trypsin-like serine protease, X is Cbz-Orn-, Cbz-Lys-Ala-, Cbz-Lys, Cbz-HomoLys-, C
bz- (4-amidinophenylalanine)-, Cbz- (4-amidinophenylglycine)-, Ph-
CH ₂ -SO ₂ -Gly-Pro- (4-amidinophenylglycine)-, 3- (2-furyl) acryloyl-
(4-amidinophenylglycine), Cbz-Lys- (4-amidinophenylglycine)-, Cbz
-Lys-Ala- (4-amidinophenylglycine)-, Cbz-Thr- (4-amidinophenylglycine)-, 3- (2-furyl) acryloyl- (4-amidinophenylalanine)-, Cbz-Ala-
(4-amidinophenylglycine)-, Cbz-Ala-Ala-Ala- (4-amidinophenylglycine)-, 2-phenoxybenzoyl-Pro- (4-amidinophenylglycine)-, 3-phenoxybenzoyl-Pro- ( 4-amidinophenylglycine)-, 3-phenylpropanoyl-Pro- (4-amidinophenylalanine)-, 3,3-diphenylpropanoyl-Pro- (4
-Amidinophenylglycine)-. For the S. aureus V8 protease, X can be either acetyl Glu- or acetyl Asp-.

R 1 and R 2 are selected from the group consisting of: 3-AcNH, 4-AcNH, 4-MeSO ₂ NH
_{, 3-H 2 NCONH, 3} -H 2 NCONH, 4- (N-Bz-Gly-NH), 4- (H-Gly-NH), 4 (H- (S) -Ala-NH),
4 - ((S) -Pyr- NH), 4 - ((2S) -MeO 2 CCH (NHAc) CH 2), 4-MeO 2 C, 4- (EtO 2 CCH 2 NHCO), 4-
_{(MeO 2 C (CH 2)} 2 NHCO), 4-CH 3 (CH 2) 2 NHCO-.

Particularly preferred compounds of group 1 (formula II) are: di (3-acetamidophenyl) 1- (benzyloxycarbonyl- (S) -prolyl) pyrrolidine-2 (R, S) -Phosphonate (10d); di (4-acetamidophenyl) 1- (benzyloxycarbonyl- (S) -prolyl) pyrrolidine-2 (R, S) -phosphonate (10e); 5-di ( 4-methylsulfonylaminophenyl) 1- (benzyloxy-carbonyl- (S)-
(Prolyl) pyrrolidine-2 (R, S) -phosphonate (10f);

Di (3-ureylphenyl) 1- (benzyloxycarbonyl- (S) -prolyl) pyrrolidine-2 (R, S) -phosphonate (10 g); di [4- (N-benzoylglycyl) Amino) phenyl] -l- (benzyl-oxycarbonyl-
(S) -Prolyl) pyrrolidine-2 (R, S) -phosphonate (10h); di [4- (N-benzyloxycarbonylglycylamino) phenyl] -1- (benzyloxycarbonyl- (S)- Prolyl) pyrrolidine-2 (R, S) -phosphonate (10i): di [4- (N-benzyloxycarbonyl- (S) -alanylamino) phenyl] -1- (benzyloxycarbonyl- (S)- Prolyl) pyrrolidine-2 (R, S) -phosphonate (10j); di [4-((S) -pyroglutamylamino) phenyl] -1- (benzyloxycarbonyl- (S)
-Prolyl) pyrrolidine-2 (R, S) -phosphonate (10k); di {4-[-(S)-(2-methoxycarbonyl-2-acetamido) ethyl] -phenyl} l- (benzyloxy Carbonyl- (S) -prolyl) pyrrolidine-2 (R, S) -phosphonate (101); di (4-methoxycarbonylphenyl) l- (tert-butyloxycarbonyl- (S) -prolyl) pyrrolidine- 2 (R, S) -phosphonate (10m); di {4-[(ethoxycarbonyl) methylaminocarbonyl] phenyl} 1- (benzyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (R, S ) -Phosphonate (lOn); di {4- [2- (methoxycarbonyl) ethylaminocarbonyl] phenyl) 1- (benzyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (R, S) -phosphonate Acid salt (10o); di [4- (n-propylaminocarbonyl) phenyl] 1- (benzyloxycarbonyl- (
(S) -Prolyl) -pyrrolidine-2 (R, S) -phosphonate (10p); di (3-acetamidophenyl) 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonate Acid hydrochloride (11d); di (4-acetamidophenyl) 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (11e); di (4-methylsulfonyl (Aminophenyl) 1-((S) -prolyl) -pyrrolidine-2 (R, s)-
Phosphonic acid / hydrochloride (11f); di (3-ureylphenyl) 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonic acid / hydrochloride (11 g); di [4- ( N-benzoylglycylamino) phenyl] -1-((S) -prolyl) -pyrrolidine-2
(R, S) -phosphonic acid hydrochloride (11h);

Di [4- (N-glycylamino) phenyl] -1-((S) -prolyl) -pyrrolidine-2 (R, S) -phosphonic acid trihydrochloride (11i); di (4- (S ) -Alanylaminophenyl) -1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonic acid trihydrochloride (11j); di (4- (S) -pyroglutamylaminophenyl) -l-((S) -prolyl) -pyrrolidine-2 (R, S
) -Phosphonic acid / hydrochloride (11k); di {4-[-(S)-(2-methoxycarbonyl-2-acetamido) ethyl] -phenyl} -1- (S) -prolyl) pyrrolidine-2-phosphone Acid hydrochloride (11l); di {4-[(ethoxycarbonyl) methylaminocarbonyl] phenyl} 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonate (11n); di { 4- [2- (methoxycarbonyl) ethylaminocarbonyl] phenyl} 1-((S) -prolyl) -pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (11o); di [4- (n- Propylaminocarbonyl) phenyl] 1-((S) -prolyl) -pyrrolidine-2
(R, S) -phosphonic acid hydrochloride (11p).

Compounds having DPP IV inhibitory activity include, for example, 2,2′-biphenyl 1-((s) -prolyl) -pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (19) It is a salt acceptable for.

Other specific compounds of Group 2 (Formula III) are: 2,2′-biphenyl 1- (benzyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (R
, S) -phosphonate (17a); 2,2′-biphenyl 1- (t-butyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (R
, S) -Phosphonate (17b).

Examples of compounds of group 3 (Formula IV) are 2- (2′-hydroxyphenyl) phenylmethyl 1- (S
) -Prolyl) -pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (18).

The advantage of the compounds of the present invention over Powers et al. (Supra) is that when the inhibitor is covalently linked to serine at the active center of the peptidase, it is derived from the compound of Power and from the compound of the present invention. By comparison of the leaving groups obtained. Information, Merck Index M
erck Index, 12th edition, by Merck & Co, Inc. USA (1996).

The leaving groups for Powers are, among others, 2-methylphenol (o-cresol), 3-methylphenol (m-cresol), 4-methylphenol (p-cresol). Compounds belonging to the cresol group are disinfectants. These compounds are toxic to humans.
Chronic toxicity due to oral or transdermal absorption causes digestive disorders, neuropathy, dizziness, rash, jaundice, oliguria, uremic. Another leaving group in the Powers compound is 4-hydroxybenzoic acid methyl ester (methylparaben, nipagin M), which is used as a preservative in foods and beverages and cosmetics, but often causes an allergic reaction. 1,4-benzenediol (hydroquinolone) is used as an antioxidant in photographic developers and reducing agents. At very low concentrations, there is no systemic toxicity. However, consumption of 1 g or more causes nausea, vomiting, shortness of breath, cyanosis, convulsions, and collapse. More than 5 g is the lethal dose.

On the other hand, the leaving group of the compound of the present invention is, for example, 4-hydroxyacetanilide (paracetamol, compound 11e), which is commonly used as a very safe antipyretic analgesic. The other leaving group is 4-hydroxyhippuric acid ethyl ester (compound 11n)
And no toxicity has been reported. Hippuric acid has been found to be a metabolite in human metabolism and a component in human urine.

From the above, it can be said that the compound of the present invention has no safety problem that hinders the development as a medicament, as compared with the compound of Powers. The compounds of the present invention can be used in the treatment of pathological conditions associated with excessive, impaired, imbalanced activity of the enzyme.

[0038] Information on the correlation of a particular serine protease activity with various disease states will enable one skilled in the art to clarify the therapeutic utility of the modulatory compounds of the present invention. Such diseases are exemplified below and support certain utilities.

Thus, the present invention relates to compounds for use as therapeutics. In particular,
The present invention relates to inflammation, vascular disease, organ-specific or systemic autoimmune disease (e.g., Graves' disease, multiple sclerosis, inflammatory bowel disease), joint disease, muscular disease, nervous system disease, obesity, benign And malignant cell transformation related diseases, spread of malignant cells, worse glucose tolerance, abnormal or delayed growth, rejection of foreign cells and tissues after transplantation, abnormal blood cell development, abnormal thrombus, pain And compounds used for the treatment and prevention of central nervous system diseases.

In support of the usefulness of the compound of the present invention in the above-mentioned indications, there is the following report showing a correlation between serine peptidase and medical indications. Inhibition of this enzyme can be used for treatment or prevention. Involvement of plasma prekallikrein or kallikrein in shock W. Colman i
n Barrett, A., et al., supra, pp. 147-153.

The use of the compound of the present invention is useful for the prevention and treatment of thrombosis and the combination therapy of acute myocardial infarction in the factor X (Vlasuk, GP in New Therapeutic Agents in Thrombosis
and Thrombolysis, Sasahara, A., Loscalzo, J., eds (1997), pages 261-283)
, Thrombin (Shafer, JA in New Therapeutic Agents in Thrombosis and Th
rombolysis, (supra) pages 143-157), factor VII-Tissue Factor (Sh
afer, JA in New Therapeutic Agents in Thrombosis and Thrombolysis, supra, pages 225-260).

[0042] Various serine peptidases are known to be involved in inflammation. In this case, elastase (Bank U. et al., In Cellular Peptidases in Immune Fun
ction and Diseases, Ansorge, S. & Langner, J., Plenum Press (1997), page
s 231-242, D. Farley et al. in Lauwers, A. and Scharpe, S. eds., supra),
Kallikrein (Chao, J. in Barrett et al., Supra, pages 97-101; Naidoo, Y.
& Bhoola, K. in The kinin System, Framer, SG, Academic Press (1998), p.
ages 187-197); and Erdos, EG & Skidgel, RA in The Kinin System, supra.
, pages 112-141), cathepsin G (Fled, HD et al., in Cellular Peptidases
in Immune Function and Diseases, supra, pages 223-230), DPP IV (Tanaka,
S. et al., Immunopharmacology 40 (1998), 21-26) and Granteam (Bertho
u, C. et al., Pathol. Biol. 46 (1998), 617-624).

DPP IV (Cheng, HC et al. J. Biol. Chem. 273 (1998) 24207-24215), PO
(Goossens et al., Eur. J. Clin. Chem. Clin. Biochem. 34 (1996), 17-22; a
nd Ishino, T. et al., J. Biochem. 123 (1998), 540-545) and urokinase are involved in tumor development and metastasis.

Holst, JJ & Deacon, C., have published a possible type 2 inhibition of DPP IV in diabetes type 2, ie, glucose intolerance (Diabetes 47 (1998), 1663-1670). Elastase plays an important role in autoimmune diseases such as rheumatoid arthritis (Nomohara, S. et al., Clin. Rheumatol. 16 (1997), 133-140; and Shing
uh, Y. et al., Eur. J. Pharmacol. 337 (1997), 63-71; Barrett, supra, Lauw.
ers & Scharpe, supra).

The involvement of DPP IV in transplant rejection has been reported (Korom, S. et al., Transplan
tation 63 (1997), 1495-1500). Similarly, the involvement of DPP IV is known to
Abnormal growth (Bai, P. & Chang, LL, J. Pharm. Pharmacol. 47 (1995), 698-701;
and Martin, R. et al., Biochim.Biophys.Acta 1164 (1993), 252-260), hypertension and pre-eclampsia (Neudeck, H. et 21., J. Reproductive Immunology 37 (1
997), 449-458). The latter two adaptations also correlate with PO (Chappe
ll, MC et al., Braz. J. Med. Biol. Res. 31 (1998), 1205-1212; and Umem.
ura, K. et al., Br. J. Clin. Pharmacol. 43 (1997), 613-618).

PO is further characterized by muscular dystrophy (Kar, N. & Pearson, C., Clin. Chim. Acta 11
1 (1981), 271-273) and behavioral neurochemical disorders (Toide, K. et al., Reviews in
Neurosciences 9 (1998), 17-29).

DPP IV is a very versatile enzyme due to its localization and different substrates, pain (Shane, R. et al., Brain Res. 815 (1999), 278-286), Obesity (Pederson,
R. et al., Di abetes 47 (1998) 1253; Flint, A. et al., J. Clin. Investig
ation 101 (1998), 515-520), tissue repair (Drucker, D. et al., Am. J. Physiol
276 (1999), G79-91).

The present invention includes derivatives wherein the N-terminal amino acid side chain has been modified without losing reactivity with the active site. Examples of such modifications include incorporation of radioactive labels, such as incorporation of iodine ¹²⁵ into tyrosine, and extension of side chains for attachment of biotin or fluorophore. To improve the half-life in circulation, the peptide bond between two amino acids may be replaced with a non-hydrolyzable bond. The incorporation of radioactive labels is useful in diagnostic methods using modulatory compounds.

For example, methods in which labeled compounds are used in diagnosis and research include fluorescence, radioassay, imaging, in situ histochemical and cytochemical staining, and are described below.

According to a further aspect, the invention relates to the use of a compound for the manufacture of a pharmaceutical composition for modulating (inhibiting or enhancing) the activity of a serine protease. Such pharmaceutical compositions are specifically intended for treatment and prevention of the above conditions.

The present invention further provides pharmaceutical formulations containing one or more compounds of the present invention and suitable excipients, carriers, and diluents. Such pharmaceutical preparations are specifically intended for treatment and prevention of the above conditions.

Excipients, carriers, and diluents that can be used are well-known, for example, Remington's
s Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985)
It is described in. Preservatives, stabilizers, dyes and fragrances are used in pharmaceutical compositions. For example, sodium benzoate, sorbic acid, p-hydroxybenzoic acid can be used as a preservative. In addition, antioxidants and suspending agents can be used.

The compounds of the present invention are formulated and used in tablets, capsules and elixirs for oral administration, as suppositories for rectal administration, as sterile solutions or suspensions for injection, and for topical and buccal administration. Is as a spray or a galenic preparation, and as a spray for nasal administration. If desired, absorption enhancing preparations (eg, liposomes) and other suitable delivery systems can be used. The quantity of active substance in a dosage unit can vary between 0.001 mg and 1 g.

The compounds or pharmaceutical compositions of the present invention can be used alone or in combination with each other, or with other therapeutic or diagnostic agents. Such other therapeutic and diagnostic agents may be in the same dosage form as the compounds of the present invention or in different dosage forms. The dosage of the compounds of the present invention may vary widely depending on the desired effect and therapeutic application.

The present invention further relates to a method for in vitro inhibition or promotion (modulation) of protease activity by a suitable concentration of a compound of the present invention. This method is particularly useful when inactivation of the peptide by a protease is performed in a peptide assay prior to its measurement. The compounds of the present invention can be used in such assays to inhibit the denaturation of peptide substrates by enzymes.

The compounds of the present invention are useful in methods for in vitro (ex vivo) inhibition of protease activity, for example, methods for treating transplanted cells or tissues in vitro to eliminate rejection at the recipient. is there.

Further, the compound of the present invention is useful in a method for inhibiting or promoting (modulating) protease activity in vivo by administering an appropriate amount of the compound to a living body.

Such modulation (inhibition or promotion) can be used for pharmacotherapy of diseases related to the following conditions: inflammation, organ-specific or systemic autoimmune diseases, non-malignant disorders of leukocytes and / or immunoglobulins, after transplantation Cell or tissue rejection, tissue destruction and bone degenerative diseases, neuroendocrine dysfunction, glucose intolerance, obesity, functional gastrointestinal disorders, abnormal or delayed growth, thrombosis and bleeding, vascular and cardiopulmonary disorders, Neurodegeneration and affective disorders, pain, neoplasm-related diseases.

As mentioned above, the present invention also relates to the diagnostic use of the compounds. The particular use of labeled inhibitors or enhancers is in the same type of application as the labeled monoclonal antibody. For example, fluorescence and emission assays, cytofluorimetry, fluorescence-activated cell sorting, and the like. The principles of such methods can be found in immunochemistry textbooks.
For example, Coligan, J. et al., Current Protocols in Immunology, vol. 1, 2 &
3, Wiley, 1998.

The compounds of the present invention can also be used as affinity ligands for analytical or preparative purposes. In cytochemistry and histochemistry, labeled modulators can be used to directly see the intracellular distribution of the target protease. Labels are fluorescence in fluorescence microscopy, radioactivity in autoradiography, and electron density in electron microscopy. Target structures are whole cells, cells fixed on slides or sections of solid tissue. A useful modification of these techniques is the use of an indirect (sandwich) assay using specific high affinity interactions between biotin and avidin (Coligan et al.,
Supra).

In order to visualize or therapeutically target tumors that express high amounts of the target protease, a suitable isotopically labeled modulator is injected. The tumor is then visualized by radioscintigraphy after removing excess modulator from the circulation. Principles of imaging (A. Bamias & AAE Penetos (1995)) and monoclonal antibodies, their production, engineering and clinical applications (MA Ritter and MM Ladyman, eds., Cambridge University)
Press, Cambridge, pp. 222-246).

The modulator of the present invention is suitable for the above-mentioned diagnostic applications, and is used, for example, for imaging and histochemical staining of protease and peptidase, respectively. This is due to the fact that the modulator forms a covalent long-lasting adduct with the protease or peptidase. Modulators are considered not to be large, and thus may enter tissues more easily than antibodies or the like. In the case of DPP IV, the modulator was found to recognize only the enzymatically active DPP IV by nature. Formulations of compounds for use in diagnostic applications are also part of the invention.

The localization of DPP IV by means of monoclonal antibodies and enzymatic activity is diagnostically useful and can be applied to clinical material in the case of thyroid follicular tumor and papillary thyroid carcinoma (Kotani, T. et al. , Int. J. Exp. Path. 73, 215-222 (1992); Kotani
, T. et al., J. Path. 168, 41-45 (1992). Similarly, compounds of the present invention, which form stable adducts with serine proteases such as DPP TV, are used as a means for diagnosing certain disease states and monitoring their progress and treatment (Kurktschiev, D. et al.
et al., Clin. Exp. Immunol. (1999) 115, 144-146).

The present invention relates not only to the claimed compounds, but also to the pharmaceutically acceptable salts thereof. The compounds of the present invention may also be pure diastereoisomers or racemic mixtures.

Hereinafter, the present invention will be described in more detail. The examples disclose the synthesis of the modulatory compounds of the invention and the inhibitory and promoting (modulating) activity of the compounds on the catalytic activity of human DPP IV, PO, DPP II, elastase.

Serine protease activity can interfere with enzymatic assays for other substrates, degrading the substrate used in the test to produce a false positive result (when the dye substrate is degraded) or a false negative result ( (When the peptide substrate is denatured). The inhibitors of the invention can be used to inactivate contaminating serine proteases or peptidases prior to performing the assay.

This application relates to compounds that can inhibit or enhance the activity of serine proteases or peptidases. The general terms “modulate” and “modulate” are used when referring to a group of compounds because the activity of the individual compounds in the group of compounds may be in conflict (ie, inhibition or enhancement). Individual compounds have inhibitory or promoting activity on individual serine peptidases and proteases. A prerequisite of the invention is that the claimed compounds are active against serine peptidases or serine proteases, ie have modulating (either inhibition or promotion) activity.

The terms “modulator”, “inhibitor”, “enhancer”, “compound”, “derivative”, “modulator compound” are used interchangeably.

The invention will be further clarified by reference to the following examples. The examples are provided for illustrative purposes only and do not limit the invention. According to the examples, the previously reported diphenyl 1- (S) -prolyl-pyrrolidine-2- (
(R, S) -phosphonate (5) is dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5)
Shows how it is used as an indicator compound for the development of potent irreversible inhibitors. The synthesis of diaryl 1- (S) -prolyl pyrrolidine-2- (R, S) -phosphonates with various substituents on the aryl ring started from the corresponding phosphite. Excellent correlation was observed between the electronic properties of the substituents and the inhibitory activity and stability. This correlation was most pronounced for the 4-acetylamino-substituted derivative (11e) with high stability and high strength. This compound was non-cytotoxic in human peripheral blood mononuclear cells and had favorable properties in vivo. Therefore, di (4-acetamidophenyl) 1- (S) -prolyl-pyrrolidine-2- (R, S) -phosphonate (11e) is considered a major improvement and further studies on the biological function of the enzyme Would be a DPP IV inhibitor for and would have therapeutic value in its inhibition.

In the examples, reference is made to the following reaction formulas and figures. Reaction formula I

Embedded image _{Reagents: i) PCl 3; ii)} HC1; iii) HOAc, 90 0 C, 2 h; iv) HCl / EtOAc (1 M); v) H 2, Pd / C 8 - about 11: R1 = a) H ; b) 4-OMe; c) 4-OAc (4-OH for 11); d)
3-NHAc; e) 4-NHAc; f) 4-NHSO ₂ Me; g). 3-NHCONH ₂ ; h) 4- (N-Bz-Gly-NH)
; i) 4- (NZ-Gly-NH) [11 for 4- (H-GlY-NH)]; j) 4- (NZ- (S) -Ala-NH) [11
About 4- (H- (S) -Ala-NH)]; k) 4-((S) -Pyr-NH); 1) 4-[(2S) -MeO ₂ CCH (NHA
c) CH ₂ ]; m) 4-COOMe; n) 4- (CONHCH ₂ COOEt); o) 4- [CONH (CH ₂ ) ₂ COOMe]; p)
4- (CONH (CH ₂ ) ₂ CH ₃ ).

Reaction Scheme II

Embedded image Reagents: i); ii) AcOH, Et ₃ N; iii) 7, HOAc, 90 ° C., 2 h; iv) H ₂ , Pd / C; v
) HCl / EtOAc (1 M).

FIG. Correlation between Hammett constant and log k _calc . The excellent correlation (log k = 4.6504δ + 2.5472, r ² = 0.82) shows that the Hammett constant and lo
It was recognized between gk _calc . The most pronounced divergence is the difference between the unsubstituted group (5) and the 4-acetylamino (11e). FIG. Correlation between Hammett constant and log half-life. A good correlation (log t _1/2 = -2.2138δ + 2.3043, r ² = 0.77) was observed between the Hammett constant and log t _1/2 . FIG. Correlation between log k _calc and log half-life. Figure 4. Plasma DPP IV activity in rabbits treated with 11n. "R7" and "R8" are rabbit numbers 7 and 8. Figure 5. Plasma DPP IV activity in rabbits treated subcutaneously on days 0-5 with 11n. "
“R1”, “R2”, “R3”, “R4” indicate each rabbit.

An excellent correlation (log t _1/2 = -0.4051 (log k) + 3.2899, r ² = 0.75) was observed between log half-life and log k. Compounds 11i, llj, 18, 19 were omitted. Compound l
li and llj had significantly shorter half-lives than expected. Other metabolism besides phosphonate hydrolysis is expected (see, description). Compounds 18 and 19 are not directly comparable to other diaryl phosphonates. .

EXAMPLES Example 1 Synthesis of a Series of Protected and Unprotected Prolyl Pyrrolidine Diaryl Phosphonates A series of prolyl pyrrolidine diaryl phosphonates were synthesized using 4-aminobutyraldehyde diethyl acetal. N-protected proline was first coupled and activated with isobutyl chloroformate as a mixed anhydride. Reference (Belyaev, A. et al. A Ne
w Synthetic Method for Proline Diphenyl Phosphonates. Tetrahedron Lett.
1995, 36, 3755-3758). The acetal 6 is hydrolyzed with HCl and the crude aldehyde 7 is treated with the corresponding triaryl phosphite 9 in acetic acid to give a diastereoisomeric mixture of the protected prolyl pyrrolidine diaryl phosphonate 10 (B
elyaev et al., (1995), supra) (reaction formula 1). Deprotection by standard methods provided final compound 11.

The production of substituted phenylphosphonates required the production of phenols that were not commercially available. 4-Methylsulfonylaminophenol 8f was prepared from the corresponding sulfonyl chloride and 4-aminophenol. Similarly, 4-acylaminophenol 8h-k was prepared by the mixed anhydride method by condensation of the corresponding carboxylic acid with 4-aminophenol. N-acetyl-L-tyrosine methyl ester (81) was prepared by an existing method (Jackson, EL Op-Toluenesulfonyl-L-tyrosine, its
N-acetyl and N-benzoil Derivatives. J. Am. Chem. Soc. 1952, 74, 837-838
,reference). The condensation of glycine ethyl ester with 4-hydroxybenzoic acid using a diphenylphosphoryl azide compound (DPPA) gave the glycine derivative 8n. The 4-hydroxybenzoic acid amides 8o and 8p were synthesized by coupling 4-acetoxybenzoic acid with the corresponding amine using the mixed anhydride method, followed by mild alkaline hydrolysis of the phenyl esters 12 and 13 (Buechi, G .; Weinreb, SM T
otal Syntheses of Aflatoxins M1 and G1. and an Improved Synthesis of Afl
atoxin B1. J. Am. Chem. Soc. 1971, 93, 746-752). The triaryl phosphite 9 was then synthesized from the corresponding substituted phenol 8 and phosphorus trichloride.

The 2,2′-biphenylacetyl phosphite (16) and the aldehyde 7a or 7b were reacted in acetic acid to produce cyclic N-protected 2,2′-biphenyl derivatives 17a and 17b (reaction Equation 2). The benzyloxycarbonyl (Z) protection was removed from the phosphonate 17a by hydrogenolysis of a methanol solution of Pd / C catalyst, and the dioxaphosphepane was ring-opened to give the mixed methyl aryl ester 18. Deprotection of the Boc-derivative 17b with HCl / EtOAC gave the free 2,2'-biphenylphosphonate 19.

Generally using this method, a mixture of diastereoisomers of phosphonate 10 was obtained. To obtain the pure diastereoisomer 5, it was necessary to introduce trityl protection after removal of the Z-protection. The readily isolated isomers (10a (S, R) and 10a (S, S)) were thus obtained. In our view, the most active isomer has the R configuration at the carbon atom site adjacent to the phosphorus atom. This was confirmed by comparing the activity of (S) -Ile- (R) -ProP (OPh) 2 with the relative fluidity, optical activity, ¹ H-NMR spectrum, and biological activity of TLC. went. The configuration of this related compound was determined by X-ray crystallography (Belyaev et al., (1995), supra).

(Experiment) (General) 4-methoxyphenol (8b), 1- (3-hydroxy-phenyl) urea (8 g), methyl-4
-Hydroxybenzoate (8m), triphenyl phosphite (9a), 2,2'-biphenol (14), L-proline, L-tyrosine, 4-aminophenol, glycine, L-pyroglutamic acid , 4-Hydroxybenzoic acid, glycine ethyl ester hydrochloride, β-alanine and all common chemicals were purchased from Acros Chimica NV, Belgium. 3-acetamidophenol (8d), 4-amino-butyraldehyde diethyl acetal, PCl _3, diphenylphosphoryl azide (DPPA) and 4-acetoxybenzoic acid, Sigma-Aldrich Chemie BV, were obtained from Belgium. 4-acetamidophenol (paracetamol) (8e) was obtained from Sterling Organics Ltd., England. The purity of all newly synthesized compounds was determined by TLC, ¹ H-NMR, ¹³ C-NMR or
Inspected by MS. The final product was checked by TLC, ¹ H-NMR, ¹³ C-NMR, FAB-MS and / or basic analysis.

Using a mixture of EtOAc-petroleum ether, EtOAc-MeOH or n-BuOH-AcOH-H ₂ O (4: 1: 1) as eluent, silica gel-coated POLYGRAM® SIL G / UV254 (Machinery -Nagel G
mbH, Germany). Silica gel H, 5-40 μm (F
luka, Switzerland) was used for preparative vacuum column chromatography. NMR spectra were recorded on a Varian EM360L or Brucker Avance DRX 400 spectrometer. Mass spectra were recorded on a VG 70-SEQ spectrometer. Optical rotations were measured on a Perkin-Elmer 241 polarimeter. Melting points were measured with a digital melting point meter (electrothermal type) and were not corrected. Using Z-Cl and (Boc) ₂ O, respectively, Z- and B
The oc-protected amino acids were prepared by standard methods (Bodanszky, M. & Bodanszky, A. In T
he Practice of Peptide Synthesis, 2nd ed .; Springer-Verlag: Berlin Heide
lberg, 1994; pp11-18 and 28-29). β-alanine methyl ester was synthesized according to a known method by treating an amino acid with a methanolic HCl solution (Bodanszky & Bodanszk
y (1994), supra). A method for producing 4- (Z- (S) propyl) aminobutyraldehyde diethyl acetal (6a) has already been published (Belyaev et al., (1995), supra).
2-Chloro (dibenzo [d; f] -1,3,2-dioxaphosphepane) (15) was prepared by known methods (Veriznikov, LV & Kirpichnikov, PA Synthesis of the o, o'-Biph
enylphosphinic Acid Esters. Zh. Obshch. Khim. 1967, 37, 1355-1358).

4- (Boc- (S) prolyl) aminobutyraldehyde diethyl acetal (6b) Boc- (S) -proline (10 mmol, 2.14 mL) in dry CHCl_ThreeEt in solution_ThreeN (10 mmol, 1.4 m
L) at −10 ° C. with stirring, then isobutyl chloroformate (10 mmol, 1.31
mL) was added. After 20 minutes, 4-aminobutyraldehyde diethyl acetal (10 mmo
l, 1.73 mL) was added and the mixture was stirred overnight between -10 ° C and room temperature. Chlorophor
Was removed, the residue was partitioned between EtOAc (100 mL) and water (100 mL), and the organic layer was dried over MgSO 4._Four And purified by column chromatography. Yield 2.96 g (83%): oil;¹H-NMR (CDCl_Three) δ (ppm) 1.0-2.4 (m, 23H, 3- and 4-CH_Two (Pro), CH_TwoCH_Two, C (CH_Three) 3, CH_Three), 2.9-3.9 (m, 9H, 5-CH_Two, NCH_Two, OCH _Two And (EtO)_TwoCH), 4.25 (m, 1H, 2-CH), 4.5 (t, 1H, NH).

4-Methylsulfonylaminophenol (8f) A suspension of 4-aminophenol (200 mmol, 21.8 g) in MeOH (250 mL) was added to CH ₃ SO ₂ Cl (10
0 mmol, 7.75 mL) at 10-15 ° C with stirring. The resulting solution was stirred at room temperature for 1 hour and evaporated. The residue was suspended in 1N HCl (250 mL), the solid was filtered, washed with water, and dried over NaOH in vacuo. Yield 13.6 g (8196): mp 165-166.C; ¹ H-NMR (DMSO) δ (ppm) 2.85 (s, 3H, CH ₃ S), 6.70 (d, 2H _arom ), 7.10 (d, 2H _arom ), 8.70 (s, ¹ H, OH), 8.85 (s, ¹ H, N
H).

Synthesis of 4-acylaminophosphenol 8h-k (General Method) To a solution of carboxylic acid (100 mmol) in DMF (100 mL) was added Et ₃ N (100 mmol, 14 mL) at −10 ° C. Then, isobutyl chloroformate (100 mmol, 13 mL) was added. Same after 30 minutes-
At 10 ° C, 4-aminophenol (115 mmol, 12.5 mL) was added and the mixture was stirred overnight (between -10 ° C and room temperature). After dilution with 1N HCl (500 mL), the precipitate was collected by filtration, washed on the filter with water, NaHCO ₃ solution, water, and finally ether.
The product was dried and, if necessary, recrystallized from a suitable solvent.

N2-benzoyl-N- (4-hydroxyphenyl) glycinamide (8h) Recrystallized from EtOH-acetone. Yield 35%: mp 244-245 ° C; ¹ H-NMR (DMSO) δ (ppm)
4.05 (d, 2H, CH ₂ CO), 6.5-8.0 (m, 9H _arom ), 8.7 (m, 1H, NH), 9.15 (s, 1H,
OH), 9.75 (s, 1H, NH).

N2-benzoyloxycarbonyl-N- (4-hydroxyphenyl) glycinamide (8i) Yield 44%: mp 184-185 ° C .; ¹ H-NMR (DMSO) σ (ppm) 3.80 (d, 2H, CH ₂ CO), 6.5-
7.5 (m, 10H, 9H _arom and NH), 8.85 (s, 1H, OH), 9.40 (s, 1H, NH).

N2-benzoyloxycarbonyl-N- (4-hydroxyphenyl)-(S) -alanine amide (8j) yield 73%: mp 97-100 ° C .;¹H-NMR (DMSO) δ (ppm) 1.36 (d, 3H, CH_Three), 4.29 (m,
1H, CH of L-Ala), 5.00 (s, 2H, CH_Two), 6.00 (m, 1H, NH), 6.35-7.38 (m, 9H, H _arom ) 8.69 (br s,¹H, OH), 9.24 (br s,¹H, NHCO).

N- (4-hydroxyphenyl)-(S) -pyroglutamylamide (8k) Yield 64%: mp 303-305 ° C dec;¹H-NMR (DMSO) σ (ppm) 1.7-2.7 (m, 4H, CH_TwoCH _Two ), 4.2 (m, 1H, CHCO), 6.65 (d, 2H_arom), 7.40 (d, 2H_arom), 7.8 (s, 1H, N
H), 9.1 (br s, 1H, OH), 9.7 (s, 1H, NH).

N- (4-hydroxybenzoyl) glycine ethyl ester (8n) To a solution of 4-hydroxybenzoic acid (100 mmol, 13.8 g) in DMF (71 mL) was added H-Gly-OEt.HCl (
100 mmol, 14 g) and then DPPA (100 mmol, 21.6 mL) and Et ₃ N (200 mmol,
28 mL) was added at 0 ° C. with stirring. The mixture was stirred overnight (between 0 ° C. and room temperature) and the Et ₃ N.HCl was filtered off. The filtrate was evaporated to half of the original volume. The residue was mixed with 5% NaHCO ₃ (200 mL) and extracted with EtOAc (500 mL). The organic layer was washed with brine, dried over MgSO _4, and evaporated to crystals. The mixture was diluted with ether (100 mL) and the crystals were collected. Yield 9 g (40%): mp 205-209 ° C; ¹ H-NMR (DMSO) σ (ppm) 1.25 (t, 3H, CH ₃ ), 4.
05 (m, 4H, CO ₂ CH ₂ and COCH ₂ N), 6.8 (d, 2H _arom ), 7.75 (d, 2H _arom ), 8.3
5 (tr, 1H, NH), 9.7 (s, 1H, OH).

Synthesis of 4-hydroxybenzoic acid amides 8o and 8p (General method) Et ₃ N (100 mmol, 18 mmol) was added to a solution of 4-acetoxybenzoic acid (100 mmol, 18 g) in THF (250 mL).
14 mL) was added, followed by isobutyl chloroformate (100 mmol, 13 mL) at −10 ° C. with stirring. After 15 minutes, the corresponding amine (free base or HCl salt) was added, followed by Et ₃ N (100 mmol, 14 mL) for the hydrochloride salt. The mixture was stirred overnight (between -10 ° C and room temperature), the solids were removed by filtration, the filtrate was evaporated and the residue crystallized from an ether-hexane mixture. The thus obtained 4-acetoxy benzoic acid amide 12 and 13 (70 mmol) was dissolved in MeOH (240 mL), NaHCO ₃ saturated solution after adding H ₂ 0 to (90 mL) to (120 mL) added. An excess of NaHCO ₃ was precipitated. The heterogeneous mixture was stirred at room temperature for 4 hours, the methanol was removed under vacuum and the residue was treated with EtOAc (25
0 mL), the organic layer was washed with 1N HCl, brine, then dried over MgSO ₄ and evaporated. The residue crystallized from ether was purified on a silica gel column.

N- (4-acetoxybenzoyl) -β-alanine methyl ester (12). Yield 69%: mp 84-88 ^° C .; ¹ H-NMR (CDCl ₃ ) δ (ppm) 2.35 (s, 3H, COCH ₃ ), 2.6 (
_{t, 2H, CO 2 CH 2} ), 3.7 (m, 5H, NCH 2 and _{CO 2 CH 3), 7.15 (} m, 3H, 2H arom Contact and NH), 7.75 (d, 2H arom).

Nn-propyl-4-acetoxybenzoylamide (13) Yield 73%: mp 94-98 ^° C .; ¹ H-NMR (CDCl ₃ ) σ (ppm) 0.9 (t, 3H, CH ₃ ), 1.6 ( m,
_{2H, CH 2), 2.25 (} s, 3H, COCH 3), 3.4 (m, 2H, NCH 2), 7.1 (m, 3H, 2H arom and NH), 7.8 (d, 2H arom).

N- (4-hydroxybenzoyl) -β-alanine methyl ester (8o) Yield 65%: mp 124-126.C; ¹ H-NMR (DMSO) σ (ppm) 2.55 (t, 2H, CO ₂ CH ₂ ), 3.
7 (m, 5H, NCH ₂ and CO ₂ CH ₃ ), 6.9 (m, 3H, 2H _arom and NH), 7.55 (d, 2
H _arom ), 8.7 (br s, 1H, OH).

Nn-propyl-4-hydroxybenzoylamide (8p). Yield 78%: oil; ¹ H-NMR (DMSO) σ (ppm) 1.1 (t, 3H, CH ₃ ), 1.55 (m, 2H,
CH ₂ ), 3.3 (m, 2H, NCH ₂ ), 6.9 (m, 3H, 2H _arom and NH), 7.65 (d, 2H _arom )
, 8.5 (br s, 1H, OH); MS (FAB +) m / z 180 (M + H) ⁺ .

Synthesis of Triaryl Phosphite 9 (General Method) Dry MF (30 mL) of the corresponding phenol (90 mmol) and Et ₃ N (90 mmol, 12.6 mL)
To the stirring solution, a solution of PCl ₃ (30 mmol, 2.62 mL) in dry CHCl ₃ (10 mL) was added dropwise at 0 ° C. The mixture was stirred overnight (between 0 ° C. and room temperature), diluted with CHCl ₃ (500 mL),
The final product solution was washed with water (2 x 500 mL). The organic layer was dried over MgSO _4, and evaporated. The residue was finally purified by column chromatography, using either EtOAc-methanol or an EtOAc-petroleum ether mixture as eluent, along with insolubles in both layers.

Tri (3-acetamidophenyl) phosphite (9d). Yield: 42%: solid; ¹ H-NMR (DMSO) σ (ppm) 2.1 (s, 9H, COCH ₃ ), 6.7 -7.7 (m,
12H _arom ), 9.7 (s, 3H, NH).

Tri (4-acetamidophenyl) phosphite (9e) yield 37%: solid; ¹ H-NMR (DMSO) σ (ppm) 2.1 (s, 9H, COCH ₃ ), 6.9-7.7 (dd
, 12H _arom ), 9.2 (s, 3H, NH).

Tri (4-methylsulfonylaminophenyl) phosphite (9f). Yield 60%: solid; ¹ H-NMR (DMSO) δ (ppm) 2.95 (s, 9H, SO ₂ CH ₃ ), 7.2 (m, 1
2H _arom ), 9.15 (s, 3H, NH).

Tri (3-ureylphenyl) phosphite (9 g) Yield 30%: solid; ¹ H-NMR (DMSO) δ (ppm) 5.85 (m, 6H, CONH ₂ ), 6.5-7.5 ( m
, 12H _arom ), 8.65 (s, 3H, NH).

Tri [4- (N-benzoylglycylamino) phenyl] phosphite (9h) Yield 90%: mp 210.C (dec.); ¹ H-NNR (DMSO) δ (ppm) 4.1 ( d, 6H, COCH ₂ N),
6.9-8.0 (m, 27H _arom ), 8.5 (m, 3H, NH), 9.9 (s, 3H, NH).

Tri [4- (N-benzyloxycarbonylglycylamino) phenyl] phosphite (9
i) Yield 76%: mp 173-C (dec.); ¹ H-NMR (DMSO) δ (ppm) 3.85 (d, 6H, COCH ₂ N)
, 5.05 (s, 6H, CH ₂ Ph), 6.5-8.0 (m, 30H, NH and _Harom ), 9.8 (br s, 3H,
NH).

Tri [4- (N-benzyloxycarbonyl- (S) -alanylamino) phenyl] phosphite (9j) Yield: 85%: solid; ¹ H-NMR (DNSO) δ (ppm) 1.42 ( d, 9H, CH ₃ ), 4.29 (m, 3H
, CH), 5.01 (s, 6H, CH ₂ Ph), 6.45-7.15 (d, 3H, NH), 7.25 (m, 27H, _Harom ),
9.76 (br s, 3H, NH).

Tri [4-((S) -pyroglutamylamino) phenyl] phosphite (9k) Yield 31%: solid; ¹ H-NMR (DMSO) δ (ppm) 1.8-2.8 (m, 12H , CH ₂ CH ₂ ), 4.25
(m, 3H, COCHN), 7.1 (d, 6H _arom ), 7.65 (d, 6H _arom ), 8.25 (s, 3H, NH), 10.
3 (s, 3H, NH).

Tri {4-[-(S)-(2-methoxycarbonyl-2-acetamido) ethyl] phenyl diphosphite (91) Yield 73%: solid; ¹ H-NMR (DMSO) δ ( ppm) 2.0 (s, 9H, COCH ₃ ), 3.1 (d, 6H,
ArCH ₂ ), 3.7 (s, 9H, CO ₂ CH ₃ ), 4.8 (m, 3H, COCHN), 6.6 (d, 3H, NH), 7.1 (
br s, 12H _arom ).

Tri (4-methoxycarbonylphenyl) phosphite (9m) Yield: 57%: oil; ¹ H-NMR (CDCl ₃ ) δ (ppm) 3.95 (s, 9H, CO ₂ CH ₃ ), 6.65 -8.
05 (m, 12H _arom ).

Tri {4-[(ethoxycarbonyl) methylaminocarbonyl] phenyl diphosphite
(9n) Yield 70%: solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.3 (t, 9H, CH ₃ ), 3.9-4.3 (m,
12H, COCH ₂ N and _{OCH 2), 6.7-8.0 (m,} 15H, NH and H _arom).

Tri {4- [2- (methoxycarbonyl) ethylaminocarbonyl] phenyl} phosphite (9o) Yield 71%: solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 2.65 (t, 6H, CO ₂ CH ₂ ), 3.7 (m, 1
5H, NCH ₂ and _{CO 2 CH 3), 7.1 (} d, 6H arom), 7.9 (m, 9H, H arom and NH).

Tri [4- (n-propylaminocarbonyl) phenyl] phosphite (9p) Yield 54%: mp 207.C; ¹ H-NMR (DMSO) δ (ppm) 1.1 (t, 9H, CH ₃ ), 1.6 (m, 6
_{H, CH 2), 3.25 (} m, 6H, CH 2 N), 7.15 (d, 6H arom), 8.1 (m, 9H, NH and H _{ar om).}

Synthesis of N-Boc- or NZ-protected diaryl 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonate 10. (General method) Diethyl acetal 6a or 6b (10 mmol) was added to THF (60 mL) and 0.5N HCl (15 mL).
(30 mmol, 30 mL) and dissolved with stirring at room temperature. After 2 hours, ether (90 mL) was added with stirring and the mixture was neutralized with an excess of solid NaHCO ₃ (ca. 5 g). The organic layer was separated, dried over MgSO _4, and evaporated. The residue (crude aldehyde 7a or 7b was dissolved in acetic acid (30 mL) with the corresponding triaryl phosphite (9.10 mmol) and the final product solution was stirred at 85-90 ° C. for 1.5-2 hours After cooling to room temperature, the acetic acid was evaporated, the residue was dissolved in chloroform (250 mL), water (200 mL), saturated Na
Washed with HCO ₃ solution (100 mL) and water (100 mL). The organic layer was dried over MgSO ₄ , evaporated and the residue was purified by column chromatography using an ethyl acetate-petroleum ether or ethyl acetate-methanol mixture as eluent.

Di (3-acetamidophenyl) 1- (benzyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (R, S) -phosphonate (10d) Yield 31%: solid: ¹ H -NMR (CDCl ₃ ) δ (ppm) 1.0-2.7 (m, 8H, 3-CH ₂ and 4
-CH ₂ ), 2.1 (s, 6H, CH ₃ CO), 4.0-3.2 (m, 4H, 5-CH ₂ ), 4.3-5.1 (m, 2H, 2-CH)
, 5.1 (br s, 2H, CH ₂ Ph), 6.6-7.7 (m, 13H _arom ), 9.1 (s, 2H, NH); NS (FAB +
) m / z 649 (M + H) ⁺ .

Di (4-acetamidophenyl) 1- (benzyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (R, S) -phosphonate (10e). Yield 60%: solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.6-2.7 (m, 8H, 3-CH ₂ and 4
-CH ₂ ), 2.1 (s, 6H, CH ₃ CO), 3.3-3.8 (m, 4H, 5-CH ₂ ), 4.3-5.1 (m, 2H, 2-CH)
, 5.1 (br s, 2H, CH ₂ Ph), 6.8-7.5 (m, 13H _arom ), 8.8 (s, 2H, NH); MS (FAB +
) m / z 649 (M + H) ⁺ .

Di (4-methylsulfonylaminophenyl) 1- (benzyloxycarbonyl- (S) -prolyl) pyrrolidine-2 (R, S) -phosphonate (10f) yield 66%: solid; ¹ H- NMR (CDCl ₃ ) δ (ppm) 1.2-2.7 (m, 8H, 3-CH ₂ and 4
-CH ₂ ), 2.8 (s, 6H, CH ₃ SO ₂ ), 3.3-3.8 (m, 4H, 5-CH ₂ ), 4.3-5.2 (m, 4H, 2-CH
And CH ₂ Ph), 6.8-7.4 (m, 13H _arom ), 8.0 (s, 2H, NH); MS (FAB +) m / z 721
(M + H) ⁺ .

Di (3-ureylphenyl) 1- (benzyloxycarbonyl- (S) -prolyl) -pyrrolidin-2 (R, S) -phosphonate (10 g) Yield 8%: solid; ¹ H -NMR (CDCl ₃ ) δ (ppm) 1.3-2.4 (m, 8H, 3-CH ₂ and 4-
CH ₂ ), 2.8-3.8 (m, 4H, 5-CH ₂ ), 4.3-5.2 (m, 4H, 2-CH and CH ₂ Ph), 5.6 (m
, 4H, NH ₂ ), 6.8-7.5 (m, 13H _arom ), 8.65 (br s, 2H, NH); MS (FAB +) m / z 65
1 (M + H) ⁺ .

Di [4- (N-benzoylglycylamino) phenyl] -1- (benzyloxycarbonyl- (
(S) -pyrrolyl) pyrrolidine-2 (R, S) -phosphonate (10h) yield 47%: solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.2-2.7 (m, 8H, 3-CH ₂ and 4
-CH ₂ ), 3.2-3.8 (m, 4H, 5-CH ₂ ), 4.0 (m, 4H, NCH ₂ CO), 4.4-5.0 (m, 2H, 2-CH
), 5.1 (br s, 2H, CH ₂ Ph), 6.6-8.3 (m, 25H, 23H _arom and NH), 9.5 (br s
, 2H, NH); MS (FAB +) m / z 887 (M + H) ⁺ .

Di [4- (N-benzyloxycarbonylglycylamino) phenyl] -1- (benzyloxycarbonyl- (S) -pyrrolidine-2 (R, S) -phosphonate (10i) Yield 19%: Solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.2-2.6 (m, 8H, 3-CH ₂ and 4
-CH ₂ ), 3.2-4.1 (m, 8H, 5-CH ₂ and COCH ₂ N), 4.3-5.2 (m, 8H, 2-CH and
CH ₂ Ph), 6.1 (m, 2H, NH), 6.7-7.6 (m, 23H _arom ), 8.95 (br s, 2H, NN); NS
(FAB +) m / z 947 (M + H) ⁺ .

Di [4- (N-benzyloxycarbonyl- (S) -alanylamino) phenyl] -1- (benzyloxycarbonyl- (S) -prolyl) pyrrolidine-2 (R, S) -phosphonate ( 10j) Yield 40% .mp: 200-203-C; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.35 (d, 6H, CH ₃ ), 1.
65-2.35 (m, 8H, 3- CH 2 and _{4-CH 2), 2.95-3.95 (} m, 4H, 5-CH 2), 4.0-4.65
(m, 4H, 2-CM, CH), 5.00 (s, 6H, CH ₂ Ph3, 6.20 (br, 2H, NH), 7.04 (s, 23H,
_Harom ) 9.25 (br, 2H, NH); MS (FAB +) m / z 975 (M + H) ⁺ .

Di [4-((S) -pyroglutamylamino) phenyl] -1- (benzyloxycarbonyl- (S)
-Prolyl) pyrrolidine-2 (R, S) -phosphonic acid (10k) Yield 25%: mp 170.C (dec.); ¹ H-NMR (CDCl ₃ ) (ppm) 1.2-2.9 (m, 16H, 3 -CH ₂ and 4-CH ₂ ), 3.55 (m, 4H, 5-CH ₂ ), 4.1 (m, 2H, 5-CH Pyr), 4.3-5.2 (m, 4
H, 2-CH and CH ₂ Ph), 6.7-8.1 (m, 15H, NH and 13H _arom ), 9.55 (br s,
2H, NH); MS (FAB +) m / z 787 (M + H) ⁺ .

Di {4-[-(S)-(2-methoxycarbonyl-2-acetamido) ethyl] phenyl} 1- (benzyloxycarbonyl- (S) -prolyl) pyrrolidine-2 (R, S) -phosphone Acid (10 l) yield 57%: solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.3-2.7 (m, 8H, 3-CH ₂ and 4
-CH ₂ ), 1.9 (s, 6H, COCH ₃ ), 3.1 (d, 4H, CH ₂ Ar), 3.7 (s, 6H, COOCH ₃ ), 3.2-
3.9 (m, 4H, 5-CH ₂ ), 4.5-5.0 (m, 4H, 2-CH), 5.1 (br s, 2H, CH ₂ Ph), 6.1 (d
, 2H, NH), 7.0 (s, 8H _arom ), 7.3 (br s, 5H, C ₆ H ₅ ); MS (FAB +) m / z 821 (M +
H) ⁺ .

Di {4-[(ethoxycarbonyl) methylaminocarbonyl] phenyl} 1- (benzyloxycarbonyl- (S) -pyrrolyl) -pyrrolidine-2 (R, S) -phosphonate (10n) Yield 32% : Solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.2-2.7 (m, 8H, 3-CH ₂ and 4
-CH ₂ ), 1.3 (t, 6H, CH ₃ ), 3.2-3.9 (m, 4H, 5-CH ₂ ), 4.0 (m, 4H, NCH ₂ CO), 4.
2 (q, 4H, OCH ₂ ), 4.3-5.1 (m, 2H, 2-CH), 5.1 (br s, 2H, CHzPh), 6.9-7.8 (
m, 15H, 13H _arom and NH); MS (FAB +) m / z 793 (M + H) ⁺ .

Di {4- [2- (methoxycarbonyl) ethylaminocarbonyl] phenyl} 1- (benzyl
(Oxycarbonyl- (S) -pyrrolyl) -pyrrolidine-2 (R, S) -phosphonate (10o) yield 22%: solid;¹H-NMR (CDCl_Three) δ (ppm) 1.2-2.8 (m, 8H, 3-CH_Two And 4
-CH_Two), 2.65 (t, 4H, CH_TwoCO_Two), 3.2-3.8 (m, 8H, 5-CH_Two And CH_TwoN), 3.75 (
s, 6H, CO_TwoCH_Three), 4.3-5.2 (m, 4H, 2-CH and CH_TwoPh), 6.7-7.9 (m, 15H, 13H _arom And NH); MS (FAB +) m / z 793 (M + H)⁺.

Di [4- (n-propylaminocarbonyl) phenyl] 1- (benzyloxycarbonyl- (
(S) -Prolyl) -pyrrolidine-2 (R, S) -phosphonate (l0p) yield 4.3%: solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 0.85 (t, 6H, CH ₃ ), 1.3-2.8 (m
, 12H, 3-CH ₂ , 4-CH ₂ and CH ₂ ), 3.1-3.9 (m, 8H, 5-CH ₂ and CH ₂ N), 4.5
(m, ¹ H, 2-CH Pro), 4.7-5.2 (m, 3H, CH ₂ Ph and 2-CH ProP), 6.9-8.1 (m,
15H, 13H _arom and NH); MS (FAB +) m / z 705 (M + H) ⁺ .

Diaryl 1-((S) -prolyl) pyrrolidine-2-phosphonic acid hydrochloride (11) (Method A) Diaryl 1- (t-butyloxycarbonyl- (S) -prolyl) -pyrrolidine-2- Phosphonate (10) or diaryl 1- (trityl- (S) -propyl) pyrrolidine-2-phosphonate (10.5 mmol) was dissolved in 1 M HCl in EtOAc (25 mL) and the solution was dissolved. Stirred at room temperature for 2 hours. Dry ether (30 mL) was added and the mixture was kept in the refrigerator overnight. If the product crystallized, the crystals were collected by filtration; if not, the oil was dispersed in dry ether. The resulting material was dried in vacuo over NaOH granules.

(Method B) Diaryl 1-((S) -benzyloxycarbonylprolyl) -pyrrolidine-2-phosphonate (10,2 mmol) was treated with a solution of Pd / C in methanol (50 mL) for 5 to 5 minutes. Hydrogenated for 6 hours. The catalyst was removed by celite filtration and the filtrate was acidified with 1M HCl / EtOAc (2.2 mL),
Evaporated and the residue was precipitated in methanol (5 mL) with dry ether. The final product was dried in vacuo over NaOH granules.

Diphenyl 1-((S) -prolyl) pyrrolidine-2 (R) -phosphonic acid hydrochloride (11a (S, R)) A. Yield 81%: mp 175-177.C; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.6-2.8 (m, 8H, 3-C
H ₂ and 4-CH ₂ ), 3.3-3.9 (m, 4H, 5-CH ₂ ), 4.8 (m, 1H, 2-CH), 5.0 (m, 1H,
2-CH), 7.2 (m, 5H _arom ), ₇₃ (m, 5H _arom ), 8.1 (br s, 1H, N + H), 10.2 (br
s, 1H, N + H); ¹³ C-NMR (CDCl ₃ ) δ (ppm) 24.1 (4-CH ₂ ), 24.6 (4-CH ₂ ), 26.1 (3
-CH ₂ ), 28.8 (3-CH ₂ ), 46.2 (5-CH ₂ ), 47.1 (5-CH ₂ ), 54.5 (d, 2-CH-P, 1J (C-
P) = 162 Hz), 59.1 (2-CH), 120.3, 125.2, 129.7, 150.3 (C _arom ), 168.2 (CO
^{); [Α] D 20 =} -100.4 0, (C1, CHCl 3);. Analysis _{(C 21 H 25 N 2 O} 4 P 1.25 HC1) C, H, N.

Diphenyl 1-((S) -prolyl) pyrrolidine-2 (S) -phosphonic acid hydrochloride (11a (S, S)) A. Yield 88%: solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.7-2.6 (m, 8H, 3-CH 2 and _{4-CH 2), 3.2-3.9 (} m, 4H, 5-CH 2), 4.75 (m, 1H, 2-CH), 4.95 (m, 0.7H, 2-
CH), 5.4 (m, 0.3H, 2-CH), 6.9-7.4 (m, 10H _arom ), 8.6 ( _brs , 1H, N + H), 10.
3 (br s, 0.3H, N + H), 10.6 (br s, 0.7H, N + H); 13C-NMR (CDCl ₃ ) δ (ppm) 22
.1, 24.0 (4-CH ₂ ), 23.5, 23.9 (4-CH ₂ ), 26.4, 27.7 (3-CH ₂ ), 28.5, 29.1 (3-
CH ₂ ), 45.6, 46.2 (5-CH ₂ ), 46.6, 47.1 (5-CH ₂ ), 54.4, 55.3 (d, 2-CH-P, 1J
(CP) = 160 Hz), 58.3, 58.7 (2-CH), 120.1, 125.0, 129.4, 149.5 (C _arom ),
167.3, 168.4 (CO); [α] D ²⁰ = 25.9. (C1, CHCl ₃ ); MS (FAB +) m / z 401 (M + H) ⁺ .

Di (3-acetamidophenyl) 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (lld). B. Yield 83%: solid; ¹ H -NMR (DMSO) δ (ppm) 1.6-2.6 (m, 8H, 3-CH ₂ and
4-CH ₂ ), 2.1 (s, 6H, COCH ₃ ), 3.3-3.8 (m, 4H, 5-CH ₂ ), 4.7 (m, 1H, 2-CH),
4.9 (m, 1H, 2-CH), 6.7-7.6 (m, 8H _arom ), 8.5 (m, 1H, N + H), 9.7 (m, 1H, N +
H), 9.9 (m, 2H , CONHPh);. Analysis _{(C 25 H 31 N 4 O} 6 P 1.5 HCl 0.5 H 2 O) C, H, N.

Di (4-acetamidophenyl) 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (11e) B. Yield 90%: solid; ¹ H- NMR (DMSO) δ (ppm) 1.6-2.6 (m, 8H, 3-CH 2 and
4-CH ₂ ), 2.1 (s, 6H, COCH ₃ ), 3.3-3.8 (m, 4H, 5-CH ₂ ), 4.6 (m, 1H, 2-CH),
4.9 (m; 1H, 2-CH), 7.1 (m, 4H _arom ), 7.6 (m, 4H _arom ), 8.7 (m, 1H, N + H), 1
0.0 (m, 2H, CONHPh), 10.2 (m, 1H, N + H); 31P-NMR (DMSO) (ppm) 17.48, 17.6
2;. Analysis _{(C 25 H 31 N 4 O} 6 P HC1 2 H 2 O) C, H, N.

Di (4-methylsulfonylaminophenyl) 1-((S) -pyrrolyl) -pyrrolidine-2 (R, S)-
Phosphonic acid / hydrochloride (11f) B. Yield 80%: solid; ¹ H-NMR (DMSO) δ (ppm) 1.55-2.5 (m, 8H, 3-CH ₂ and 4-CH ₂ ), 2.95 (s, 6H, SO ₂ CH ₃ ), 3.3-3.8 (m, 4H, 5-CH ₂ ), 4.55 (m, 1H, 2-
CH), 4.85 (m, 1H, 2-CH), 7.15 (m, 8H _arom ), 8.6 (br s, 1H, N + H), 9.79 (s,
2H, (br s ¹ H N + H); Analysis. (C ₂₃ H ₃₁ N ₄ O ₈ PS ₂ HCl) C, H, N.

Di (3-ureylphenyl) 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (llg) B. Yield 90%: solid;¹H-NMR (CD30D) δ (ppm) 1.4-2.5 (m, 8H, 3-CH_Two And 4-CH_Two), 3.3-3.8 (m, 4H, 5-CH_Two), 4.5 (m, 1H, 2-CH), 4.8 (m, 1H, 2-CH),
 6.4-7.2 (m, 6H_arom), 7.4 (m, 2H_arom); MS (FAB +) m / z 517 (M + H)⁺; Analysis (C _{twenty three} H₂₉N₆O₆P HC1 2.3 H_TwoO) C, H, N.

Di [4- (N-benzoylglycylamino) phenyl] -1-((S) -prolyl) pyrrolidine-2 (
(R, S) -phosphonic acid / hydrochloride (11h) B. Yield 87%; solid: ¹ H-NMR (DMSO) δ (ppm) 1.6-2.6 (m, 8H, 3-CH ₂ and 4- CH ₂ ), 3.2-3.6 (m, 4H, 5-CH ₂ ), 4.2 (d, 4H, NCH ₂ CO), 4.5-4.9 (m, 2H,
2-CH), 6.9-7.9 (m, 20H, 18H _arom and NH), 8.5 (m, 1H, N + H), 10.0 (s, 2
(H, NH), 10.2 (m, 1H, N + H); MS (FAB +) m / z 753 (M + H) ⁺ ; analysis. (C ₃₉ H _{4 1} N ₆ O ₈ PH
_{C1 2 H 2 O) C,} H, N.

Di [4- (N-glycylamino) phenyl ll-((s) -prolyl) pyrrolidine-2 (R, S) -phosphonic acid trihydrochloride (11i) B. Yield 81%: solid; ^{1 H-NMR (DMSO) δ} (ppm) 1.55-2.5 (m, 8H, 3-CH 2 and _{4-CH 2), 3.3-3.8 (} m, 4H, 5-CH 2), 3.8 (s, 4H, COCH ₂ N), 4.55 (m, 1H, 2-C
H), 4.85 (m, 1H, 2-CH), 7.15 (m, 4H _arom ), 7.65 (m, 4H _arom ), 8 35 (br s,
6H, N + H), 8.5 (br s, 1H, N + H), 10.35 (br s, 1H, N + H), 11.0 (s, 2H, NH);
Analysis. (C ₂₅ H ₃₃ N ₆ O ₆ P 3 HC1 2.5 H ₂ O) C, H, N.

Di (4- (S) -alanylaminophenyl) -1-((S) -pyrrolyl) pyrrolidine-2 (R, S) -phosphonic acid trihydrochloride (11j). B. Yield 70 %; Solid; ¹ H-NMR (D ²⁰ ) δ (ppm) 1.49 (d, 6H, CH ₃ ), 1.65-2.60
(m, 8H, 3-CH 2 and _{4-CH 2), 3.23-3.75 (} m, 4H, 5-CH 2), 4.12 (m, 2H, CH of Ala), 7.05 (m, 4H , H arom) , 7.35 (m, 4H, _Harom ); analysis (C ₂₇ H ₃₇ N ₆ O ₆ P 3 HC1
_{0.5 H 2 O) C, H} ; N: calc, 12.16; found, 11.73.

Di (4- (S) -pyroglutamylaminophenyl) -1-((S) -prolyl) pyrrolidine-2 (R, S)
-Phosphonic acid / hydrochloride (11k) B. Yield 95%: solid; ¹ H-NMR (DMSO) δ (ppm) 1.5-2.4 (m, 16H, 3-CH ₂ and 4-CH ₂ ), 3.1-4.2 (m, 6H, 5-CH ₂ and 5-CH), 4.5 (m, 1H, 2-CH Pro), 4.
85 (m, 1H, 2-CH ProP), 7.1 (m, 4H _arom ), 7.65 (m, 4H _arom ), 7.9 (s, 2H, NH
), 8.6 (m, 1H, N + H), 10.1 (m, 1H, N + H), 10.3 (s, 2H, NH); Analysis (C ₃₁ H ₃₇ N ₆ O ₈ P 1.5 HC1 3 H ₂ O ) C, H, N.

Di {4-[-(s)-(2-methoxycarbonyl-2-acetamido) ethyl] phenyl} 1-((S) -prolyl) pyrrolidine-2-phosphonic acid hydrochloride (11l) Yield 85%: solid; ¹ H-NMR (DMSO) δ (ppm) 1.6-2.6 (m, 8H, 3-CH ₂ and
4-CH ₂ ), 1.9 (s, 6H, COCH ₃ ), 3.0 (d, 4H, CH ₂ Ar), 3.2-3.8 (m, 4H, 5-CH ₂ ),
3.7 (s, 6H, COOCH ₃ ), 4.4-4.9 (m, 4H, 2-CH), 7.1 (m, 8H _arom ), 8.2 (d, 2H
, AcNH), 8.7 (m, 1H, N + H), 10.4 (m, 1H, N + H); MS (FAB +) m / z 687 (M + H) ⁺ ;
Analysis _{(C 33 H 43 N 4 0} 10 P 1.5 HC1 2 H 2 O) C, N; H: calc, 6.29; found, 5.81.

Di {4-[(ethoxycarbonyl) methylaminocarbonyl] phenyl} 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonate (11n) B. Yield 86%: solid ; ¹ H-NMR (DMSO) δ (ppm) 1.3 (t, 6H, CH ₃ ), 1.4-2.6 (m
, 8H, 3-CH ₂ and 4-CH ₂ ), 3.2-3.8 (m, 4H, 5-CH ₂ ), 4.0 (m, 4H, NCH ₂ CO),
4.2 (q, 4H, OCH ₂ ), 4.6 (m, 1H, 2-CH), 4.9 (m, 1H, 2-CH), 7.3 (m, 4H _arom )
, 7.9 (m, 4H _arom ), 8.7 (m, 4H, N + H and NH); MS (FAB +) m / z 659 (M + H) ⁺ ;
_{Analysis. (C 31 H 39 N 4} O 10 P HC1 1.5 H 2 O) C, H, N.

Di {4- [2- (methoxycarbonyl) ethylaminocarbonyl] phenyl} 1-((S) -prolyl) -pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (11o) Yield 79%: solid; ¹ H-NMR (DMSO) δ (ppm) 1.2-2.8 (m, 8H, 3-CH ₂ and
4-CH ₂ ), 2.65 (t, 4H, CH ₂ COO), 3.2-3.9 (m, 8H, 5-CH ₂ and CH ₂ N), 3.8 (
s, 6H, COOCH ₃ ), 4.7 (m, 1H, 2-CH), 4.9 (m, 1H, 2-CH), 7.55 (m, 4H _arom ),
8.15 (m, 4H _arom ), 8.55 (m, 4H, N + H and NH).

Di [4- (n-propylaminocarbonyl) phenyl] 1-((S) -propyl) -pyrrolidine-2
(R, S) -phosphonic acid hydrochloride (11p) B. Yield 80%: solid; ¹ H-NMR (CD30D) δ (ppm) 0.95 (t, 6H, CH ₃ ), 1.6 (m,
_{4H, CH 2), 1.7-2.6 (} m, 8H, 3-CH 2 and _{4-CH 2), 3.2-3.9 (} m, 8H, 5-CH 2 Contact and _{CH 2 N), 4.65 (m} , 1H, 2-CH Pro), 5.1 ( m, 1H, 2-CH ProP), 7.3 (m, 4H ar om), 7.85 (m, 4H arom); 13C-NMR (CD30D) δ (ppm) 11.7 (CH 3) , 23.5 (CH ₂ ),
25.0 (4-CH ₂ ), 25.8 (4-CH ₂ ), 27.3 (3-CH ₂ ), 29.7 (3-CH ₂ ), 42.8 (NCH ₂ ), 47.
4 (5-CH ₂ ), 48.3 (5-CH ₂ ), 55.9 (d, 2-CH-P, 1J (CP) = 161 Hz), 60.6 (2-CH
), 121.4, 130.4, 133.1, 153.7 (C _arom ), 168.8 (CO), 169.2 (CO).

2,2′-biphenylacetyl phosphite (16). A solution of chloroanhydride 15 (0.152 mol, 38 g) in dry THF (50 mL) was added to AcOH (0.152 m
mol, 9.1 mL) and Et ₃ N (0.152 mol, 21.3 mL) in dry THF (100 mL) were added dropwise and stirred at 0 ° C. The cooling was stopped and the mixture was stirred at room temperature for 3 hours. The solid was filtered off and the filtrate was evaporated. The crude product was used in the next step without further purification. Yield 40 g (96%): viscous oil; ¹ H-NMR (CDCl ₃ ) δ (ppm) 2.1 (s, 3H, CH ₃ CO),
6.9-7.5 (m, 8H, _Harom ).

2,2′-biphenyl 1- (benzyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (R
, S) -Phosphonate (17a) Prepared with acetal 6a and phosphite 16 according to the general procedure for diaryl phosphonates. Yield 27%: solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.2-2.9
(m, D 8H, 3-CH ₂ and 4-CH ₂ ), 3.1-4.0 (m, 4H, 5-CH ₂ ), 4.55 (m, 1H, 2-CH
Pro), 4.85 (m, 1H, 2-CH ProP), 5.2 (br s, 2H, CH ₂ Ph), 7.1-7.85 (m, 15H _{a rom} ); MS (EI) m / z (relative intensity) 532 (M, 40), 397 (20), 300 (26), 232 (29),
215 (41), 204 (45), 168 (42), 160 (74), 91 (100), 70 (48).

2,2′-biphenyl 1- (t-butyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (
(R, S) -Phosphonate (17b) Prepared with acetal 6b and phosphite 16 according to the general procedure for diaryl phosphonates. Yield 24%: solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.3-2.
55 (m, 8H, 3- CH 2 and _{4-CH 2), 1.5 (} s, 9H, CH 3), 3.35-3.9 (m, 4H, 5-CH 2), 4.55 (m, 1H, 2-CH ), 4.9 (m, 1H, 2-CH), 6.95-7.75 (m, 8H, _Harom ); MS
(FAB +) m / z 499 (M + H) ⁺ .

2- (2′-hydroxyphenyl) phenylmethyl 1- (S) -prolyl) -pyrrolidine-2 (R, S
) -Phosphonic acid hydrochloride (18) Prepared from Z-protected derivative 17e using method "B" for diarylphosphonates.
Built. Yield 92%: solid;¹H-NMR (CDCl_Three) δ (ppm) 1.5-2.6 (m, 8H, 3-CH_Two And 4
-CH_Two), 3.1-3.9 (m, 7H, 5-CH_Two And OCH_Three), 4.4-4.8 (m, 2H, 2-CH Pro and
And 2-CH ProP), 6.85 (br s, 1H, POOH), 7.1-7.6 (m, 8H_arom), 8.4 (m, 1H, N
+ H), 9.6 (m, 1H, N + H); 13C-NMR (CDCl_Three) δ (ppm) 25.7 (4-CH_Two), 26.1 (4-CH _Two ), 28.2 (3-CH_Two), 30.5 (3-CH_Two), 48.1 (5-CH_Two), 48.6 (5-CH_Two), 55.6 (OCH_Three),
 55.6 (2-CH-P, 1J (C-P) = 142 Hz), 60.8 (2-CH), 117.8, 121.0, 126.3, 130
.1, 132.5, 133.8, 149 7, 156 2 (C_arom), 168.9 (CO); MS (FAB +) m / z 431 (M
+ H)⁺; Analysis. (C_{twenty two}H₂₇N_TwoO_FiveP HC1 H_TwoO) C, H; N: calc, 5.78; found, 6.26.

2.2′-Biphenyl 1-((S) -Prolyl) pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (19) Boc-protection using method “A” for diarylphosphonates Prepared from derivative 17b. Yield 81%: solid; ¹ H-NMR (CDCl ₃ ) δ (ppm) 1.3-2.55 (m, 8H, 3-CH ₂ and
_{4-CH 2), 3.35-3.75 (} m, 4H, 5-CH 2), 4.75 (m, 2H, 2-CH), 7.0-7.5 (m, 8H, H a rom), 10.9 (m, 2H, N + H); 13C-NMR (CDCl ₃ ) and (ppm) 24.4 (4-CH ₂ ), 24.9
(4-CH ₂ ), 26.7 (3-CH ₂ ), 28.7 (3-CH ₂ ), 46.6 (5-CH ₂ ), 51.9 (5-CH ₂ ), 53.4 (
2-CH-P, 13 (CP) = 151 Hz), 59.2 (2-CH), 116.9, 120.9, 121.7, 121.9, 125
.8, 126.6, 128.4, 129.0, 130.3, 131.5, 147.9 (C _arom ), 167.8 (CO); MS (FA
(B +) m / z 399 (M + H) ⁺ ; analysis (C ₂₁ H ₂₃ N ₂ O ₄ P 1.4 HC1 1.1 H ₂ O) C, H; N: calculated,
5.97; found, 5.43.

Example 2 Synthesis of a Series of Diarylphosphonates with Alanine as Residue aa Diphenylalanine phosphonate was prepared using Oleksyszyn et al., Synthesis 1979, 985
Synthesized according to the principle described in -986. In this method, acetaldehyde was used to make the alanine analog diphenyl 1-benzyloxycarbonylaminoethanephosphonate. After deprotection, diphenyl 1-aminoacetanphosphonate can be combined with various amino acids: glycine, L-alanine, L-valine, L-isoleucine, L-phenylalanine and L-proline using a mixed anhydride method. Was.

(Experiment) Production of diphenylalanine phosphonate diphenyl 1- (N-benzyloxycarbonyl) aminoethanephosphonate (X) triphenyl phosphate (100 mmol, 31.03 g), acetaldehyde (150 mmol, 6 .
61 g-freshly distilled), benzyl carbamate (100 mmol, 15.10 g) and glacial acetic acid (
15 ml) of the mixture was stirred for about 30 minutes until the exothermic reaction disappeared.

The mixture was heated at 80-85 ° C. for 1 hour and the volatile products were removed by heating on a boiling water bath under reduced pressure in a rotary evaporator. The oily residue was dissolved in methanol (180 ml) and left overnight at -10 ° C for crystallization. The crystalline ester was collected by filtration, redissolved in a small amount of warm chloroform (30-40 ml), and recrystallized by adding 4 volumes of methanol. Yield: 46%; mp: 117-118.C IR (KBr): 3260 (NH); 3050, 3020 (CHar); 296
0, 2920 (CHalif); 1720 (C = O); 1240 (P = O); 1200 (PO); 740, 690 (C ₆ H ₅ ); ¹ H-NMR (CDCl ₃ , 60 MHz): 1.55 ( 3H, dd, J _PH = 17.5 Hz, J _HH = 7 Hz, CH ₃ ); (lH,
m, CH); 5.10 (2H, s, OCH,); 5.37 (lH, br, NH); 6.93-7.23 (lOH, m, Har);
7.23-7.37 (5H, m, Har).

Diphenyl 1-aminoethanephosphonic acid / hydrobromide (Y) Compound X (10 mmol, 4.11 g) was dissolved in a 45% solution of hydrogen bromide in acetic acid (5 ml).
After 1 hour at room temperature, solvents and volatile products were removed under reduced pressure on a boiling water bath. The oily residue was dispersed in dry ether. The crystals were filtered and dried in vacuo over sodium hydroxide to give pure hydrobromide. Yield: 95%; mp: 148.5.C; IR (KBr): 3100-2700 (NH 3 +); 1230 (P = O); 1200
(P-0); 760, 680 (C ₆ H ₅ ); ¹ H-NMR (CDCl ₃ + d6-DMSO, 60 MHz): 1.77 (3H, dd
, J _PH = 17.0 Hz, J _HH = 7 Hz, CH ₃ ); 4.05 (lH, m, CH); 6.97-7.30 (10H, m,
Har)

Diphenyl 1 (R, S)-[N-benzyloxycarbonyl-L-phenylalanyl] -aminoethanephosphonate, diphenyl 1 (R, S)-[N-benzyl-oxycarbonyl-L-propyl [Loryl] aminoethanephosphonate (T, Q) General procedure for mixed anhydrides that bind to isobutyl chloroformate. Dissolve the Z-protected amino acid (5 mmol) in dry tetrahydrofuran (25 ml) and add sodium chloride in an ice bath. And cooled to -150 ° C. N-methylmorpholine (5 mmol, 0.51 g) and isobutyl chloroformate (5 mmol, 0.68 g) were added to the solution and stirring was continued for 10 minutes. A solution of Y (5 mmol, 1.79 g) and triethylamine (5 mmol, 0.51 g) in dimethylformamide (10 ml) was added dropwise to the stirred mixture, and the temperature was maintained at -100 ° C or lower.

After one hour in a cold water bath, the reaction mixture was warmed to room temperature. The hydrochloride of N-methylmorpholine and triethylamine was collected by filtration and washed with tetrahydrofuran.
The combined filtrate and washings were concentrated on a rotary evaporator. The residue was treated with ethyl acetate (7
5 ml) and water (25 ml), and the organic phase is dissolved in 5% sodium hydrogen carbonate solution (25 ml).
, 0.5N hydrogen chloride solution (25 ml) and saturated sodium chloride solution (25 ml), dried over sodium sulfate, and evaporated to dryness under reduced pressure.

T: yield: 84%; oil; IR (KBr): 3280 (NH); 3060 (Char); 2960, 2930, 2880
(CHalif); 1720-1660 (C = O); 1250 (P = 0); 1180 (P-0); 760,685 (C 6 H 5). ¹ H-NMR (CDCl ₃ , 60 MHz): 1.33 (3H, dd, J _pH = 17.5 Hz, J _HH = 7.OHz, CH ₃ ); 2.80-
3.10 (2H, m, αH); 4.27-4.73 (2H, m, αH, CH); 5.05 (2H, m, CH ₂ ); 5.53 (2H,
br, NH); 6.87-7.30 (20H, m, Har).

Q: Yield: 79%; oil; IR (KBr): 3280 (NH); 3060 (CHar); 2960, 2930, 2880 (
CHalif); 1720-1660 (C = 0); 1260 (P = 0); 1180 (P-0); 760,685 (C ₆ H ₅ ) ¹ H-NMR (CDCl ₃ , 60 MHz): 1.43 (3H, dd, J _pH = 18.0Hz, J _HH = 7.0Hz, CH ₃ ); 1.75-2
.10 (4H, m, pH, yH); 3.35-3.70 (2H, m, δH); 4.25-4.50 (1H, m, αH); 4.85 (1
H, m, CH); 5.13 (2H, s, CH 2); 7.10-7.4-0 (15H, m, Har).

Diphenyl 1 (R, S) -L-phenylalanylaminoethanephosphonate, Diphenyl 1 (R, S) -L-prolylaminoethanephosphonate (K, L) Z-group by hydrogenation General method for removal Z-protected dipeptide phosphonates T and Q (1.5 mmol) were dissolved in dry methanol (20 ml) and acetic acid (0.7 ml). After adding palladium on charcoal (10%, 0.25 g), the air above the solution was replaced with nitrogen and hydrogen was bubbled through the solution for 3 hours. The flask was flushed with nitrogen for 10 minutes before removing the solvent under reduced pressure. The residue was dissolved in dry chloroform (15 ml) and a 0.5 N solution of hydrogen chloride in ethyl acetate was gently added to the stirring solution. Removal of the solvent under reduced pressure and co-evaporation with 0.5 N hydrogen chloride in ethyl acetate (10 ml) gave a white foam. This was dispersed in dry ether. After 2 days at −10 ° C., the crystals were filtered off and the hygroscopic product was stored in a desiccator.

K: Yield: 80%; oil; IR: 3240 (NH); 3100-2800 (NH ₃ ⁺ ); 1690 (C = 0); 1250 (P = 0
); 1180 (P-0) ; 750,680 (C 6 H 5). ¹ H-NMR (CDCl ₃ , 250 MHz): 1.15-1.50 (3H, dd, J _pH = 17 Hz, J _HH = 6.7 Hz, CH ₃ ); 3.
16,3.43 (2H, m, βH); 44.46-4.58; 4.46-4.70 (3H, m, αH, CH, NH); 6.98-7.3
4 (15H, m, Har) ; 8.30-8.52 (2H, -br, NH 2).

L: yield 75%; oil; IR: 3240 (NH): 3100-2800 (NH ₃ ⁺ ); 1690 (C = 0); 1250 (P = O)
; 1180 (P-0); 760,680 (C 6 H 5). ¹ H-NMR (CDCl ₃ , d6-DMSO, 250 MHz): 1.37-1.65 (3H, m, CH ₃ ); 1.77-2.03 (4H, m
, βH, yH); 3.38-3.51 (2H, m, δH); 4.31 (1H, m, NH); 4.55 (1H, m, αH); 4.
77-4.88 (1H, m, CH); 7.13-7.31 (10H, m, Har).

Example 3 DPP IV Inhibition and Stability of Inhibitors Previous studies on the role of P-2 amino acids in dipeptide diphenylphosphonate (Lambeir, AM et al. Dipeptide-derived Diphenyl Phosphonat)
e Esters: Mechanism-based Inhibitors of Dipeptidyl Peptidase IV. Biochim
In Biophys. Acta 1996, 1290, 76-82) we have shown that proline at this position is one of the most potent inhibitors. A major advantage of 5, which is a mixture of diastereoisomers of 11a, is that it is more stable in human citrated plasma than other dipeptide derivatives. The stability of 5 in plasma equals the stability in buffer, whereas the stability of other mixtures in plasma is lower compared to buffer. This reflects the relative stability of the Pro-Pro amino acid sequence to the proteolytic action, indicating that the diminished activity of 5 is primarily due to phosphonate hydrolysis.

In order to increase the inhibitory activity while maintaining stability, various substituents were introduced on the phenyl ring acting as leaving groups. The effects of electron donating or electron withdrawing substituents on enzyme inhibition and stability were investigated as shown in Table 1. All compounds are irreversible inhibitors of DPP IV, probably due to the formation of phosphonated serine in the active site of the enzyme. The inactivity constant was calculated from the experimental value of the IC ₅₀ and showed considerable agreement with the measured inactivity constant to the extent available.

A large correlation was observed between the electron-withdrawing property of the substituent and its activity (FIG. 1). The introduction of the electron donating substituent (4-OH, 11c) reduces the strength and, on the contrary, the electron withdrawing substituent (
The introduction of 4-methoxycarbonyl, 11m) increases the strength.

Quite unexpectedly, compounds 11e (4-acetylaminophenyl) and 11f
For (4-methylsulfonylaminophenyl), divergence was observed in the correlation between Hammett's constant and inhibitory activity. Compounds (11e) and (11f) both strongly inhibited DPP IV. These compounds have similar Hamm's as compared to the diastereoisomeric mixture5.
It has an ett constant, but nevertheless is almost 100 times more powerful.

The introduction of another acylamino substituent was also studied. 4-Glycylamino (11i) and 4-alanylamino (11j) -substituted diphenylphosphonates have comparable activity to 4-acetylamino (11e) -substitutes, but have much lower plasma stability.

The half-life of the inhibitor also appears to correlate with Hammett's constant (FIG. 2). Compounds with similar electronic properties (eg, 5 and 11e) are found to have similar stability in plasma. The O-methyl derivative (18) is almost as potent and stable as 5.

From the correlation between electronic properties and inhibitory activity and between electronic properties and stability,
There is an inverse correlation between inhibitory activity and stability (Figure 3). This means that more active compounds are more unstable at the same time. Therefore, the higher potency (IC ₅₀ = 0.4 μM) and equivalent stability (t _1/2 = 3 μl) of paracetamol-substituted phenylphosphonates (11e)
20 min) is an important improvement for unsubstituted 5.

(Experiment) DPP IV was purified from human semen plasma by an established method (De Meester et al.
, J. Immunol. Meth. 189, 99-105, (1996)). Gly-Pro-p-nitroanilide (Sigm
a) as a chromogenic substrate, using a microtiter plate reader Spectramax 3
Enzyme activity was measured at 37 ° C. by 40 (Molecular Devices). The reaction was monitored at 405 nm and the absorbance unit was set between 0 and 0.25 as the initial rate. The reaction compound is 2
Include mM substrate, approximately 1 mU DPP IV, 40 mM TRIS-HCl buffer (pH 8.3) and appropriate amount of inhibitor (range 0 and 10 mM) in a total volume of 0.2 ml. Activity measurements were repeated twice in daily work.

IC₅₀The titer is defined as 15 minutes of enzymatic precipitation at 37 ° C prior to addition of the substituents.
This is the concentration of inhibitor required to reduce DPP IV activity by 50% after cuvulation. Inhibition in DMSO or phosphate buffer (pH 7.4), depending on compound solubility.
Harmful stock solutions (100 mM) were prepared and stored at -20 ° C. The stock solution was diluted with 50 mM TRIS-HCl buffer (pH 8.3) if necessary just before the experiment. Modification described in this specification
The compound completely inactivates DPP IV according to the provisional first order kinetics, ₅₀ The titer is inversely related to the inert second-order rate constant (Lambeir et al., (1996), supra). single
The activity after incubation with the inhibitor (vi) for the pure temporary primary inactivation process
The sex varies according to the inhibitor concentration (i) as follows: vi = vo * e-k.o.t;
Where vo is activity without inhibitor, k is the second order rate constant of inactivity, and t is time. i =
I c₅₀ Since, by definition, vi = 1 / 2vo, from the equation, k = 1n2 / (ti * IC₅₀)When
Become. Where ti = 15 min. Table 1 shows the calculated values of k.

Table 1. Strength and stability of DPP IV inhibitors

[Table 1]

The determination of the IC ₅₀ value was performed after 15 minutes of pre-incubation at 37 ° C. with DPP IV. The values shown are the mean ± standard deviation measured separately. Evaluation of functional stability in plasma, was graduated the reciprocal apparent an IC ₅₀ value with respect to time by a simple exponential. Half-life was expressed as ± standard deviation.

For the inhibitors (5, 11a, 11b, 11d, 11e, 11h, 11n), the inactivation rate constants were as described previously (Lambeir et al., (1996), supra) Time series measurements of inhibition Decided from.

[0164] The functional stability of the inhibitors was determined by using 1 mM diluted compounds in citrated human plasma at 37 ° C.
In 0 to 300 minutes, it was evaluated in inhibitory potency measured at 3-4 time points (valence apparent IC _50). Taking the reciprocal of the apparent IC ₅₀ value against time by a single index scale gives the half-life reported in Table 1.

Example 4 Inhibition of Prolyl Oligopeptidase (PO) by Protected Prolyl Pyrrolidine Diarylphosphonate

The inhibitory potencies of various compounds of the present invention were evaluated. Table 2 shows the results.

Table 2. Compound 10 strength as PO inhibitor. IC ₅₀ values are expressed in μM.

[Table 2]

(Experiment) Prolyl oligopeptidase was purified from human platelets, and Z-Gly-Pro-AMC (4.
The 4 mM) as substrate, k containing 1 mM EDTA, 1 mM dithiothreitol Torr and 1 mM NaN ₃
-Enzyme activity was measured using 100 mM phosphate buffer (pH 7.5). 2 at 37 ° C
A 0 minute incubation was performed. The reaction was stopped by adding 5 volumes of 1.5 M acetic acid. Fluorescence was measured at 370 and 440 nm as excitation and emission wavelengths, respectively. Inhibitors were added at concentrations ranging from 1 μM to 1 mM.

Example 5 Inhibition of Dipeptidyl Peptidase II by a Dipeptide-Derivative Diarylphosphonate Having an ala at the Site aa Compounds Pro-Alap (OPh) 2 and Phe-Alap (OPh) 2 inhibit DPP II in vitro. Inhibited. When IC ₅₀ is calculated valence, Pro-Alap (OPh) 2 is 1.5 mM, Phe-Alap (OPh ) 2 is 0.8 mM
Met.

(Experimental Results) Dimeptidyl peptidase II was semi-purified from rabbit kidney and Lys-Ala-4-Me
O-2-NA 1.4M (Sigma, L-2270) 100 mM acetate buffer (pH 5.5, containing 2 mM EDTA) was hydrolyzed and its activity was measured. After incubation at 37 ° C. for 20 minutes, the reaction was stopped by adding a 10-fold excess of sodium acetate (pH 3.6). The fluorescence of the formed 4-MeO-2-NA was measured at 340 and 425 nm, respectively, as the excitation and emission wavelengths. In order to determine the IC ₅₀ valent were tested compounds in a series of concentrations of 1 μM~5 mM range during the incubation.

Example 6 In Vitro Cytotoxicity and Efficacy of Pyrrolylpyrrolidine Diarylphosphonate in Human Peripheral Blood Mononuclear Cells (PBMC) Based on the inhibitory potency, plasma stability and synthesis efficiency, compounds 11e and 11n Was selected for further in vitro and in vivo studies. Both compounds were evaluated in human peripheral blood mononuclear cells (PBMCs) using concentrations up to 100 μM in freshly isolated mononuclear cells or hemagglutinin-stimulated blasts. Did not show any injury. In such circumstances, the cell lysate and supernatant
More than 90% of DPP IV activity was inhibited. This result contrasts with the result obtained for the active dialtereoisomer of unsubstituted diphenylphosphonate (11a). In 11a
Satisfactory inhibition of DPP IV activity could not be achieved without cytotoxicity to PBMC culture medium. Thus, compounds 11e and 11n are promising tools to advance cellular studies. The irreversible mechanism of inhibition can overcome the very limited stability of compounds in organisms.

Experiments Peripheral blood mononuclear cells were isolated from buffy coat (obtained from a blood transfusion center in Antwerp). After dilution by a factor of 4 in phosphate buffered saline (PBS), cells were Ficoll-Hy
Place in paque gradient (Pharmacia, Upsala, Sweden) at 550 xg for 20 minutes at room temperature
And centrifuged. The middle layer was collected and washed three times in RPMI-1640. Finally, remove the cells 1
0% heat-inactivated fetal bovine serum and antibiotics (penicillin / streptomycin) (
Gibco) was reprecipitated in a 1 × 10 ⁶ cells / mL RPMI-1640 solution. Use these freshly isolated cells immediately for inhibitor studies, or
g / mL hemagglutinin (Murex diagnostics) was used to stimulate for 3 days at 37 ° C. in a 5% CO ₂ humidified incubator. Inhibitors (a solution stored in phosphate buffer at -80 ° C directly diluted in RPMI-1640) or vehicle alone at different concentrations were added to cells (5 × 10 ⁶ / test) . After overnight incubation at 37 ° C., aliquots were removed and tested for cytotoxicity by the 0.4% trypan blue exclusion test. The remaining cells were washed three times in PBS and the final cell pellet was washed with 1% v / v Triton X-100.
And 100 KIU / mL aprotinin (Bayer) in 200 μL PBS and centrifuged at 20000 × g for 10 minutes. The supernatant was used immediately for the yeast assay, and protein measurements were made with the Braford micro assay. The specific activity (u / g protein) was compared, and the inhibition rate (%) was determined with respect to a control sample containing no inhibitor.

Example 7 Inhibition of DPP IV In Vivo A single intravenous injection of 5 (0.3-5 mg / kg) in rabbits resulted in 80 DPP IV activity in plasma.
It has already been reported that a full recovery took more than 20 days, with more than 20% decline (De Mee
ster et al., Biochem. Pharmacol. 54, 173-179 (1997)).

In the case of rats, intravenous injection of a dose per body weight performed for comparison gave DPP IV
Could not be sufficiently inhibited. The reason is that reasonable inhibition cannot be reached without severe systemic toxicity. However, when 11a was administered in combination with subcutaneous and peritoneal injections, DPP IV plasma could be reduced to 10% or less of the pretreatment value. Treatment with only a mixture of diastereoisomers of 11a (5) not only significantly prolonged transplant acceptance for alloantigens, but also sometimes caused systemic toxicity, and more often local ulceration, and resulted in 11e and 11n rabbits, rats and rats. In vivo tests in mice were stimulated.

In rabbits, a higher in vivo potency of 11n compared to 11e was also observed.
IC ₅₀ valent inhibition of rabbit plasma DPP IV by single intravenous injection in the case of 11n 20 μg / kg
It was about 0.2 mg / kg for 11e. 11n 0 in this chemical
A single intravenous injection of .2 mg / kg can produce> 90% plasma DPP I in at least 24 hours without side effects
V activity was inhibited (see FIG. 4).

In rats, 11e as well as 11n also maintained DPP IV plasma activity at a pre-treatment value of less than 10% by subcutaneous injection of 50 mg / kg daily (first dose 100 mg / kg). . There was no acute systemic or local toxicity. FIG. 5 shows residual plasma DPP IV activity in rats treated subcutaneously 0-5 times daily with 11e.

In the rat, another different route of administration was attempted with compound 11n. In vivo DPP IV inhibition was obtained by oral, intraperitoneal and rectal administration as well as subcutaneous administration. The intraperitoneal route has minimal efficiency, i.e., less than 50% inhibition after 24 hours, while another route has even lower DPP IV activity one day after single administration (initial titer is less than 35%)
). The dose was 50 mg / kg for all routes.

In mice, pharmacologically useful inhibition was only obtained with 11n, but not with 11a and 11e. Elucidation at the molecular level of the large heterogeneity in efficiency remains.

(Experimental Results) Male New Zealand white rabbits (2.5-3.5 kg), Wistar rats (250-350 g), and Swiss mice (23-36 g) were environmentally controlled for 7 days. Standard feed was provided ad libitum. Test compounds were dissolved in phosphate buffer 50 mM (pH 7.4), concentrations ranged from 10-100 mg / mL, stored in aliquots at -80 ° C, and thawed before use. For rabbits, the test compound or test reagent alone was gently injected once intravenously into the peripheral vein of the ear. Blood was sampled from the central artery of the ear. Rats and mice were injected subcutaneously or intraperitoneally. The rats were anesthetized (Forene), and a sample blood was collected from the femoral vein by puncturing the skin after incision. Mice were anesthetized with pentobarbital and punctured in the orbit and bled. After coagulation, the sample blood was centrifuged (3000 xg, 10 min) and the resulting serum was stored at -80 ° C until testing for DPP IV activity.

According to the above examples, all compounds tested are irreversible inhibitors. Place
A correlation was found between the electron withdrawing properties of the substituents and the inhibition of DPP IV. Methoxycarboni
And substituted derivatives of alkylaminocarbonyl₅₀Titer is approximately 20 nM and the inactivation rate constant is approximately 3000 M^-1s^-1Is the most potent inhibitor. A similar correlation was observed between the electron donating properties of the substituents and plasma stability. The most powerful
Inhibitors are at the same time the most unstable compounds. The notable exception is 4-acetyl
Luminophenylphosphonate (11e, t_1/2= 320min).
In addition, it has a higher potency than expected (IC₅₀= 0.4 μM, k = 1900 M^-1s ^-1 ). Therefore, there is a need for in vitro and in vivo studies on this compound with very potent 11n. These inhibitors are used in human peripheral blood mononuclear
It does not show cytotoxicity in cells even at concentrations up to 100 μM. For rabbits, IC of 11e and 11n in inhibition of plasma DPP IV upon single intravenous injection₅₀Price is
, Were approximately 0.2 mg / kg and less than 20 μg / kg, respectively. These compounds are
Exhibits acute systemic or local toxicity as observed with unsubstituted compound 5
Never.

Since the stability of 11e is higher than 11n, di (4-acetamidophenyl) -1- (S) -prolylpyrrolidine-2- (R, S) -phosphonate (11e) was superior. It is an improved compound. The advantage of this compound compared to a pyrrolidine-2-nitrile reversible inhibitor is a long-lasting irreversible inhibition. In addition, the compounds are more stable than frequently used boric acid inhibitors.

[Brief description of the drawings]

FIG. 1 is a correlation between Hammett's constant and log k _calc .

FIG. 2 is a correlation between Hammett's constant and log half-life.

FIG. 3 shows the correlation between log k _calc and log half-life.

FIG. 4 shows plasma DPP IV activity in rabbits treated with 11n. "R7" and "R8" are rabbit numbers 7 and 8. .

FIG. 5 shows plasma in rabbits treated subcutaneously with 11n from day 0 to day 5.
DPP IV activity. “R1”, “R2”, “R3”, “R4” indicate each rabbit.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 7/02 A61P 7/02 9/00 9/00 11/00 11/00 19/08 19/08 25 / 00 25/00 25/04 25/04 29/00 29/00 35/00 35/00 37/02 37/02 37/06 37/06 43/00 111 43/00 111 C07F 9/572 C07F 9 / 572 Z (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG) , ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR , HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW Alexandre Arcadicvich Beriev Portugal, P-4480 Mindelo-Villa do Conde, Lua Particular en Henheiro Alberto Martins Mezquita 224 (72) Inventor Anne-Marie Virginie Rene Lambert, Belgium-Bee-3001 Heverleigh, Spa Lendreaf No. 35 (72) Inventor Ken Jan Ludfix Augustin Belgium, Be-2322 Mindelhout, Heike No. 2 (72) Inventor Asiel Jean-Marie Emmel Belgium, Bé-9830 Sint-Martens-Latem, De Knock No. 2 (72) Inventor Philip Josef Annie Gohens Belgium, Bée-1910 Kampenhout, Van Bering-Hernlan No. 3 (72) Inventor Dirk, France, Hendricks, Belgium-2360 Aalto Cellar, Zwarwenlan 9th F-term (reference) 4C086 AA01 AA02 AA03 DA38 MA01 MA04 NA14 ZA01 ZA08 ZA18 ZA36 ZA53 ZA54 ZA59 ZA66 ZA70 ZA96 ZB02 ZBZ ZB Z ZB Z 4H050 AA03 AB20 AB21 AB26

Claims (34)

[Claims] (1) The present invention has a regulatory activity on serine protease, and has the following
General formula:Wherein A is R2 or H or C₁-C₆Or a halogenalkyl (excluding perfluoroalkyl); the phenyl group is mono-, di- or tri-substituted by R1 or R2; X is a peptide- or amino acid-derived moiety; A substituted phenyl group optionally forms a biphenyl diester; all R1 and R2 substituents are each independently selected from the group consisting of:
: A) C₁-C₆Acylamino; b) aroylamino, which is alkyl at the o- and / or p- and / or m-position, especially C₁-C₆Optionally substituted with alkyl and / or halogen; c) C₁-C₆Alkylsulfonylamino; d) arylsulfonylamino, which is alkyl at the o- and / or p- and / or m-position, especially C₁-C₆Optionally substituted with alkyl and / or halogen; e) α-aminoacylamino, wherein aminoacyl is L, D or DL at the α-carbon atom.
A blocked or unblocked α-amino acid residue of a side chain having a conformation, a group consisting of:
Selected from: alanine, methionine, methionine sulfoxide, arginine, homoarginine
, Phenylalanine, aspartic acid, proline, hydroxyproline, Aspa
Lagin, serine, cysteine, threonine, histidine, glycine, tyrosine,
Glutamic acid, pyroglutamic acid, tryptophan, glutamine, valine, nor
Valine, isoleucine, lysine, leucine, norleucine, thioproline, homo
Proline, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), 2,3-dihydroindole-2-carboxic acid, α-naphthylglycine, O-phenylglycine,
4-amidinophenylglycine, 4-phenylproline, 4-amidinophenylalani
, O-benzyl tyrosine, ω-acetyl lysine, α-aminobutyric acid, citrulline, e
Moscitrulline, ornithine, O-methylserine, O-ethylserine, S-methylcysteine
In, S-ethylcysteine, S-benzylcysteine, homoserine, 4-dehydrop
Lorin, penicillamine, β- (2-thienyl) alanine, NH_Two-CH (CH_TwoCHEt_Two) -COOH, α
-Aminoheptanoic acid, NH_Two-CH (CH_Two1-naphthyl) -COOH, NH_Two-CH (CH_Two(-2-naphthyl) -C
OOH, NH_Two-CH (CH_Two-Cyclohexyl) -COOH, NH_Two-CH [CH- (cyclohexyl)_Two] -COOH, NH_Two-CH (CH_Two-Cyclopentyl) -COOH, NH_Two-CH [CH- (cyclopentyl)_Two] -COOH, NH _Two -CH (CH_Two-Cyclobutyl) -COOH, NH_Two-CH [CH- (cyclobutyl)_Two] -COOH, NH_Two-CH (CH_Two -Cyclopropyl) -COOH, NH_Two-CH [CH- (cyclopropyl)_Two] -COOH, 5,5,5-triflu
Ololeucine, hexafluoroleucine, (S) -azetidine-2-carboxylic acid, (S) -pipecoric acid, (S) -oxazolidine-4-carboxylic acid, (R) -thiazolidine-4-carboxylic acid
Acid (L-thioproline), sarcosine; f) a residue selected from the group consisting of 3-aminobenzoic acid, ε-aminocaproic acid, and β-alanine; g) Y-NH-CO-NH-; h) Y'O_TwoCCH (NHCo-Y) -CH_Two-; i) Y'NHCO-; j) CH_Three-O-CO-Y '-NH-CO-; k) CH_Three-CH_Two-O-CO-Y '-NH-CO-; where Y is C₁-C₆Alkyl, aryl or H, and Y ′ is C₁-C₆Alkyl,
And a pharmaceutically acceptable salt thereof. 2. X is a compound of the general formula (AA) p-aa-, wherein p is 0, 1, 2, 3, 4 or 5 residues AA, which are identical or identical in one molecule. AA and aa are α-aminocarboxylic acids having an optionally substituted C ₁ -C ₆ alkyl, aryl, aralkyl group at the alpha position, and pharmaceutically acceptable salts thereof. The compound of claim 1, which is a moiety of 3. X is a compound of the general formula (AA) p-aa- wherein p is 0, 1, 2, 3, 4 or 5 residues AA, which are identical or identical in one molecule. AA and aa may be selected from the group consisting of: alanine, methionine, methionine sulfoxide, arginine, homoarginine, phenylalanine, aspartic acid, proline, hydroxyproline, asparagine, serine, cysteine, threonine. , Histidine, glycine, tyrosine, glutamic acid, pyroglutamic acid, tryptophan, glutamine, valine, norvaline, isoleucine, lysine, leucine, norleucine, thioproline, homoproline, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) , 2,3-dihydroindole-2-carboxylic acid, α-naphthylglycine, α-phenylglycine, 4-amidi Nophenylglycine, 4-phenylproline, 4-amidinophenylalanine, O-benzyltyrosine, ω-acetyllysine, α-baminobutyric acid, citrulline, homocitrulline, ornithine, o-methylserine, o-ethylserine, S-methylcysteine, S- ethyl cysteine, S- benzyl cysteine, homoserine, 4-dehydroproline, penicillamine, 9- (2-thienyl) _{alanine, NH 2 -CH (CH 2 CHEt} 2) -COOH, α- amino heptanoic acid, NH ₂ - CH (CH _{2-l-naphthyl) -COOH, NH 2 -CH (CH} 2 -2- naphthyl _{Le) -COOH, NH 2 -CH (CH} 2 - _{cyclohexyl) -COOH, NH 2 -CH [CH-} ( cyclohexyl ) ₂ ] C
_{OOH, NH 2 -CH (CH 2} - _{cyclopentyl) -COOH, NH 2 -CH [CH-} ( _{cyclopentyl) 2] -COOH, NH 2 -CH} (CH 2 - cyclobutyl) -COOH, NH ₂ -CH [CH- (Cyclobutyl) ₂ ] -COOH, NH ₂ -CH
(CH ₂ - _{cyclopropyl) -COOH, NH 2 -CH [CH-} ( cyclopropyl) _2] -COOH, 5,5,5-trifluoro-leucine, hexafluoro leucine, (S) - azetidine-2-carboxylic acid , (
(S) -Pipecoric acid, (S) -oxazolidine-4-carboxylic acid, (R) -thiazolidine-4-carboxylic acid (L-thioproline), 3-aminobenzoic acid, sarcosine, ε-aminocaproic acid, β-alanine Wherein the α-amino residue is a blocked or unblocked side chain, and the α carbon element is L,
And a pharmaceutically acceptable salt thereof]. 4. The compound according to claim 2, wherein AA is lysine or ornithine, and the amino side chain thereof is involved in intramolecular covalent bonding. 5. X is M- (AA) p-aa- wherein p, AA and aa are as defined in claim 2-4; M is selected from: a) optional -CONH replaced by_Two, -CSNH_Two, -SO_TwoNH_Two, Phenyl-SO_Two-, Phenyl-CH _Two SO_Two-, 2-furylacryloyl; and b) acetyl, adamantyloxycarbonyl, benzyloxycarbonyl, benzoyl, benzyl, t-butoxycarbonyl, t-butyl, 2,4-dinitrophenyl
, Formyl, fluorenylmethoxycarbonyl, 4-methoxybenzyl, tosyl,
Trifluoroacetyl, trityl, phthaloyl, phenylalkylcarbonyl,
2-indanylacetyl, 2- (1,2,3,4-tetrahydronaphthyl) acetyl, 4- (4-benzylphenoxy) alkyl, and pharmaceutically acceptable salts thereof]. Compound. 6. X is (M-) AA-aa- (wherein (M-) means the presence or absence of M, aa is proline, and AA is as defined in claim 2-4. )
A compound according to claim 1-5. 7. The method of claim 1, wherein X is (M-) AA-aa-, wherein (M-) represents the presence or absence of M, and AA and aa are both proline. Compound of 5. 8. aa is alanine and R1 and R2 can be further selected from the following, in addition to the definition of claim 1: 1) H, halogen, NO ₂ , CN, OH, COOH m) amino, C ₁ -C ₆ alkylamino, C ₂ -C _{1 2} dialkylamino n) C ₁ -C ₆ acyl o) C ₁ -C ₆ alkoxy -CO- p) C ₁ -C ₆ alkyl -S-, and pharmaceutically 6. The compound of claim 2-5, which is a chemically acceptable salt. 9. The compound of claim 2-8, wherein AA bound to at least aa is proline or phenylalanine, and a pharmaceutically acceptable salt thereof. 10. The compound of claim 9, which is the compound Phe-Ala-diphenylphosphonate or Pro-Ala-diphenylphosphonate and pharmaceutically acceptable salts thereof. 11. The compound of the following general formula: 2. The compound of claim 1 having the formula (wherein R1, R2, X are as defined in claims 1-9, and pharmaceutically acceptable salts thereof) (also referred to as Group 1 compounds). 12. The following: di (3-acetamidophenyl) 1- (benzyloxycarbonyl- (S) -prolyl) pyrrolidine-2 (R, S) -phosphonate (10d); Cetamidophenyl) 1- (benzyloxycarbonyl- (S) -prolyl) pyrrolidine-2 (R, S) -phosphonate (10e); 5-di (4-methylsulfonylaminophenyl) 1- (benzyloxy- Carbonyl- (S)-
(Prolyl) pyrrolidine-2 (R, S) -phosphonate (10f); di (3-ureylphenyl) 1- (benzyloxycarbonyl- (S) -prolyl) pyrrolidine-2 (R, S) -phosphonic acid Salt (10 g); di [4- (N-benzoylglycylamino) phenyl] -1- (benzyl-oxycarbonyl-
(S) -Prolyl) pyrrolidine-2 (R, S) -phosphonate (10h); di [4- (N-benzyloxycarbonylglycylamino) phenyl] -1- (benzyloxycarbonyl- (S)- Prolyl) pyrrolidine-2 (R, S) -phosphonate (10i): di [4- (N-benzyloxycarbonyl- (S) -alanylamino) phenyl] -1- (benzyloxycarbonyl- (S)- Prolyl) pyrrolidine-2 (R, S) -phosphonate (10j); di [4-((S) -pyroglutamylamino) phenyl] -1- (benzyloxycarbonyl- (S)
-Prolyl) pyrrolidine-2 (R, S) -phosphonate (10k); di {4-[-(S)-(2-methoxycarbonyl-2-acetamido) ethyl] -phenyl} l- (benzyloxy Carbonyl- (S) -prolyl) pyrrolidine-2 (R, S) -phosphonate (101); di (4-methoxycarbonylphenyl) l- (tert-butyloxycarbonyl- (S) -prolyl) pyrrolidine- 2 (R, S) -phosphonate (10m); di {4-[(ethoxycarbonyl) methylaminocarbonyl] phenyl} 1- (benzyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (R, S ) -Phosphonate (lOn); di {4- [2- (methoxycarbonyl) ethylaminocarbonyl] phenyl) 1- (benzyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (R, S) -phosphonate Acid salt (10o); di [4- (n-propylaminocarbonyl) phenyl] 1- (benzyloxycarbonyl- (
(S) -Prolyl) -pyrrolidine-2 (R, S) -phosphonate (10p); di (3-acetamidophenyl) 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonate Acid hydrochloride (11d); di (4-acetamidophenyl) 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (11e); di (4-methylsulfonyl (Aminophenyl) 1-((S) -prolyl) -pyrrolidine-2 (R, s)-
Phosphonic acid / hydrochloride (11f); di (3-ureylphenyl) 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonic acid / hydrochloride (11 g); di [4- ( N-benzoylglycylamino) phenyl] -1-((S) -prolyl) -pyrrolidine-2
(R, S) -phosphonic acid hydrochloride (11h); di [4- (N-glycylamino) phenyl] -1-((S) -prolyl) -pyrrolidine-2 (R, S) -phosphonic acid / 3 Hydrochloride (11i); di (4- (S) -alanylaminophenyl) -1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonic acid trihydrochloride (11j); (4- (S) -pyroglutamylaminophenyl) -1-((S) -prolyl) -pyrrolidine-2 (R, S
) -Phosphonic acid / hydrochloride (11k); di {4-[-(S)-(2-methoxycarbonyl-2-acetamido) ethyl] -phenyl} -1- (S) -prolyl) pyrrolidine-2-phosphone Acid hydrochloride (11l); di {4-[(ethoxycarbonyl) methylaminocarbonyl] phenyl} 1-((S) -prolyl) pyrrolidine-2 (R, S) -phosphonate (11n); di { 4- [2- (methoxycarbonyl) ethylaminocarbonyl] phenyl} 1-((S) -prolyl) -pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (11o); di [4- (n- Propylaminocarbonyl) phenyl] 1-((S) -prolyl) -pyrrolidine-2
12. The compound of claim 11, wherein the compound is selected from the group consisting of: (R, S) -phosphonic acid hydrochloride (11p); and pharmaceutically acceptable salts thereof. 13. The compound represented by the following general formula: Wherein R 1, R 2, and X are as defined in claims 1-11, and pharmaceutically acceptable salts thereof, are 2,2 ′ biphenyl diesters of α-aminoalkylphosphonic acids. Compound of 1. 14. The following: 2,2′-biphenyl 1- (benzyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (R
, S) -phosphonate (17a); 2,2′-biphenyl 1- (t-butyloxycarbonyl- (S) -prolyl) -pyrrolidine-2 (R
, S) -phosphonate (17b); 2,2′-biphenyl 1-((s) -prolyl) -pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (19
The compound of claim 13, which is selected from the group consisting of: 15. The compound having an inhibitory activity on DPP IV of 2,2′-biphenyl 1-((
15. The compound of claim 14, which is s) -prolyl) -pyrrolidine-2 (R, S) -phosphonic acid hydrochloride or a pharmaceutically acceptable salt. 16. A compound of the general formula: Wherein R 1 is as defined in claim 1, X is as defined in claim 2-5, A is H or C ₁ -C ₆ alkyl or haloalkyl (perfluoroalkyl ) Or a pharmaceutically acceptable salt thereof (Group 3 compound). (17) the compound is 2- (2'-hydroxyphenyl) phenylmethyl 1- (S
17. The compound of claim 16, which is) -prolyl) -pyrrolidine-2 (R, S) -phosphonic acid hydrochloride (18). 18. The method according to claim 18, wherein the compound is DPP IV, DPP II, PO, FAPα, lysosomal Pro-X.
Carboxypeptidase or elastase, or one of these peptidases
18. The compound of claim 1-17, which has regulatory activity on a related peptidase with at least 45% amino acid sequence homology. 19. The compound of claim 1-18 for use as a therapeutic. 20. The compound may optionally be labeled with: inflammation, organ-specific or systemic autoimmune disease, non-malignant disorder of leukocytes and / or immunoglobulins, rejection of cells or tissues after transplantation, tissue Destruction and bone degenerative diseases, neuroendocrine dysfunction, glucose intolerance, obesity, functional gastrointestinal disorders, stunted or delayed growth, thrombosis and hemorrhage, vascular and cardiopulmonary disorders, neurodegenerative and affective disorders, pain, 19. The compound of claim 1-18 for use in the treatment or prevention of one of a neoplasm-related disease. 21. The compound according to claim 1, wherein the compound can be optionally labeled and used in a diagnostic or research method such as fluorescence, radioassay, imaging, in situ histochemical and cytochemical staining.
18 compounds. 22. are those wherein X is as defined in claim 1-5, R1 and / or R2, 3-AcNH, 4- AcNH, 4-MeSO 2 NH, 3-H 2 NCONH, 3-H ₂ NCONH, 4- (N-Bz-G
ly-NH), 4- (H-Gly-NH), 4 (H- (S) -Ala-NH), 4-((S) -Pyr-NH), 4-((2S) -MeO ₂ CCH (N
_{HAc) CH 2), 4-} MeO 2 C, 4- (EtO 2 CCH 2 NHCO), 4- [MeO 2 C (CH 2) 2 NHCO], 4- [CH 3 (CH 2) 2 NH
20. The compound of claims 1-18, selected from the group consisting of: CO]. 23. are those wherein X is as defined in claim 5, R1 and / or R2, 3-AcNH, 4- AcNH, 4-MeSO 2 NH, 3-H 2 NCONH, 3-H 2 NCONH, 4- (N-Bz-Gly-N
H), 4- (H-Gly -NH), 4 (H- (S) -Ala-NH), 4 - ((S) -Pyr-NH), 4 - ((2S) -MeO 2 CCH (NHAc )
CH ₂ ), 4-MeO ₂ C, 4- (EtO ₂ CCH ₂ NHCO), 4- [MeO ₂ C (CH ₂ ) ₂ NHCO], 4- [CH ₃ (CH ₂ ) ₂ NHCO] 19. The compound of claim 1-18, wherein the compound is selected and has a modulating activity, particularly an inhibitory activity, on PO. 24. Use of a compound of claims 1-18 for the manufacture of a pharmaceutical composition which inhibits serine protease activity. 25. The following: inflammation, organ-specific or systemic autoimmune diseases, non-malignant disorders of leukocytes and / or immunoglobulins, rejection of cells or tissues after transplantation, tissue destruction and bone degenerative diseases, neuroendocrine function Treatment of deficiency, glucose intolerance, obesity, functional gastrointestinal dysfunction, abnormal or delayed growth, thrombosis and bleeding, vascular and cardiopulmonary disorders, neurodegenerative and affective disorders, pain, neoplasm-related disorders Or use of a compound of claims 1-18 for the manufacture of a pharmaceutical composition for preventing. 26. One or more compounds of claim 1-18, and a suitable excipient,
A pharmaceutical formulation comprising a carrier or a diluent. 27. The pharmaceutical formulation of claim 26, further comprising one or more additional therapeutic ingredients. 28. The following: inflammation, organ-specific or systemic autoimmune diseases, non-malignant disorders of leukocytes and / or immunoglobulins, rejection of cells or tissues after transplantation, tissue destruction and bone degenerative diseases, neuroendocrine function Deficiency, glucose intolerance, obesity, functional gastrointestinal disorders, stunted or stunted growth, thrombosis and bleeding, vascular and cardiopulmonary disorders, neurodegenerative and affective disorders, pain, neoplasm-related disorders 28. The pharmaceutical formulation according to claim 27 for use in prophylaxis. 29. A method for in vitro regulating a protease with a suitable concentration of a compound of claim 1-18. 30. The method of claim 29, wherein undesired denaturation of the peptide substrate by protease activity occurs prior to measuring the peptide in a peptide assay. 31. A method for ex vivo regulating a protease by a suitable concentration of the compound of claim 1-18. 32. The method of claim 31, wherein a suitable concentration of the compound is applied to the cells or organs for transplantation. 33. A method for inhibiting protease activity in vivo by administering an appropriate concentration of the compound of claim 1-18 to a living body. 34. Inflammation, organ-specific or systemic autoimmune diseases, non-malignant disorders of leukocytes and / or immunoglobulins, rejection of cells or tissues after transplantation, tissue destruction and bone degenerative diseases, neuroendocrine function Treatment of deficiency, glucose intolerance, obesity, functional gastrointestinal dysfunction, abnormal or delayed growth, thrombosis and bleeding, vascular and cardiopulmonary disorders, neurodegenerative and affective disorders, pain, neoplasm-related disorders Or how to prevent.