WORLD INTELLECTUAL_ PROPERTY ORGANIZATION
`-
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) Iniemamflal Patent Classification 6 i
`
`(11) International Publication Number:
`
`WO 95/29691
`
`A61K 38/00, 31/675, A01N 57/00, C07F
`9/02, 9/547, 9/28, 9/06, 9/22, C07D
`223/00, 225/00, 295/00, 279/04, 279/06,
`279/10, 279/12, 265/04, 265/30
`
`_
`_
`_
`(43) International Publication Date:
`
`9 November 1995 (09.11.95)
`
`(21) International Application Number:
`
`PCT/US95/05345
`
`(22) International Filing Date:
`
`28 April 1995 (28.04.95)
`
`(81) Designated States: CA, JP, MX, European patent (AT, BE,
`CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT,
`SE).
`
`I
`
`(30) Priority Data:
`08/234,181
`
`28 April 1994 (28.04.94)
`
`Published
`With international search report.
`
`US
`
`(71) Applicant: GEORGIA TECH RESEARCH CORPORATION
`[US/US]; Office of Technology Licensing, Centennial Re-
`search Building, Georgia Institute of Technology, 400 Tenth
`Street, N.W., Atlanta, GA 30332-0415 (US).
`
`(72) Inventors: POWERS, James, C.; 698 Upton Road, N.W., At-
`lanta, GA 30318-2524 (US). BODUSZEK, Bogdan; Pil-
`czycka 107/6, PL-54-150 Wroclaw (PL). OLEKSYSZYN,
`Jozef; 69 Thomdike Street, Arlington, MA 02174 (US).
`
`(74) Agent: COLTON, Laurence, P.; Deveau, Colton & Marquis,
`Suite 1400, Two Midtown Plaza, 1360 Peachtree Street,
`NE., Atlanta, GA 30309-3214 (US).
`
`(54) Title: PROLINE PHOSPHONATE DERIVATIVES
`
`(57) Abstract
`
`Peptidyl derivatives of diesters of a-aminoalkylphosphonic acids, particularly those with proline or related structures, their use in
`inhibiting serine proteases with chymotiypsin-like, trypsin-like, elastase-like, and dipeptidyl peptidase IV specificity and their roles as anti-
`inflammatory agents, anticoagulants, anti-tumor agents, and anti-AIDS agents.
`
`.<¢~‘
`
`Page 1 Of 41
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`Astraleneca Exhibit 2099
`
`Mylan V_. Astraleneca
`IPR2015-013-10
`
`AstraZeneca Exhibit 2099
`Mylan v. AstraZeneca
`IPR2015-01340
`
`Page 1 of 41
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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international
`applications under the PCT.
`
`AT
`AU
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`CS
`CZ
`DE
`
`Austria
`Australia
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cote d'Ivoire
`Cameroon
`China
`Czechoslovakia
`Czech Republic
`Germany
`Denmark
`Spain
`Finland
`France
`Gabon
`
`United Kingdom
`Georgia
`Guinea
`Greece
`Hungary
`Ireland
`Italy
`Japan
`Kenya
`Kyrgystan
`Democratic People's Republic
`of Korea
`Republic of Korea
`Kazakhstan
`Liechtenstein
`Sri Lanka
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`Mali
`Mongolia
`
`Mauritania
`Malawi
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Slovenia
`Slovakia
`Senegal
`Chad
`Togo
`Tajikistan
`Trinidad and Tobago
`Ukraine
`United States of America
`Uzbekistan
`Viet Nam
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`W0 95/2969]
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`PCT/US95l05345
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`l
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`PROLINE PHOSPHONATE DERIVATIVES
`
`STATEMENT OF GOVERNMENT INTEREST
`
`This invention was made with government support under Grants No. HL34035
`
`and HL29307 awarded by the National Heart, Lung and Blood Institute of the National
`
`Institutes of Health. The United States government may have certain rights in this
`
`invention.
`
`BACKGROUND OF THE INVENTION
`
`1 .
`
`Field of the Invention
`
`This invention relates to a novel class of peptidyl derivatives of aromatic diesters of
`
`ot-arninoalkylphosphonic acids useful for selectively inhibiting elastase, selectively
`
`inhibiting chymotrypsin-like enzymes, selectively inhibiting trypsin-like enzymes and
`
`selectively inhibiting dipeptidyl peptidase IV (DPP-IV). The diesters of ot-
`
`aminoalkylphosphonic acids are analogues of natural ot-amino acids. This invention also
`
`relates to a method for controlling tumor invasion, treating inflammation and controlling
`
`blood coagulation in patients using the novel compounds of the present invention. We
`
`have found that peptidyl derivatives of aromatic diesters of ot-arninoalkylphosphonic acids .
`
`are potent inhibitors of chymotrypsin-like enzymes, elastases, blood coagulation enzymes,
`
`tryptases, kallikreins, and therefore they are useful as anti-tumor, anti—inflammatory and
`
`anticoagulant agents. We have also found that dipeptide proline phosphonates are
`
`inhibitors of dipeptidyl peptidase IV (DPP-IV, enzyme number EC 3.4.14.5, also known
`
`as CD26) and are thus useful in treatment of immune system disorders and acute
`
`respiratory distress syndrome (AIDS).
`
`2.
`
`Description of the Related Art
`
`Serine proteases play critical roles in several physiological processes such as
`
`digestion, blood coagulation, complement activation, fibrinolysis, and reproduction.
`
`Serine proteases are not only a physiological necessity, but also a potential hazard if they
`
`are not controlled. Blood coagulation serine proteases are responsible for vascular clotting,
`
`cerebral infarction and coronary infarction. Chymotrypsin-like enzymes and plasrnin are
`
`involved in tumor invasion, tissue remodeling, and clot dissociation. Uncontrolled
`
`proteolysis by other serine proteases such as elastase may cause pancreatitis, emphysema,
`
`'
`
`rheumatoid arthritis, inflammation and adult respiratory distress syndrome. Accordingly,
`
`specific and selective inhibitors of these proteases should be potent anticoagulants, anti-
`
`inflamrnatory agents and anti-tumor agents useful in the treatment of protease-related
`
`diseases. In vitro proteolysis by trypsin, chymotrypsin or the elastase family is a serious
`
`problem in the production, purification, isolation, transport or storage of peptides and
`
`proteins.
`
`Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5, CD26) is a post-proline cleaving
`
`enzyme which will remove the dipeptides AA-Pro (AA = amino acid residue) from the N-
`
`10
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`15
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`30
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`PCT/US95/05345
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`2
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`terminus of proteins or polypeptides. DPP-IV has been found in a variety of mammalian
`
`cells and tissues including kidney, placenta, blood plasma and on the surface of certain T-
`
`lymphocyte subsets. Despite extensive studies, the biological role of DPP-IV in
`
`mammalian systems has not been completely established, although a number of functions
`
`have been postulated. DPP-IV may participate in the metabolism and uptake of proline-
`
`containing peptides in the intestine and kidney and may be involved in fibronectin-mediated
`
`cell movement and adhesion. DPP-IV may also play a role in the metabolism or catabolism
`
`of collagen which has a high frequency of Gly—Pro sequences. DPP-IV in human plasma
`
`has been shown to cleave N-terminal Tyr-Ala from growth hormone-releasing factor and
`
`cause inactivation of this hormone. DPP-IV is also involved in T-cell activation and
`
`regulation of T-cell proliferation. Thus, inhibitors of DPP-IV may have therapeutic utility
`
`.
`
`in the modulation of the rejection of transplanted tissue by the host organism. Recently
`
`DPP-IV or CD26 has been postulated to act as a cofactor for entry into HIV in CD4+ cells
`(Callebaut, C., Krust, B., Jacotot, E., Hovanessian, A. G. T cell activation antigen,
`CD26, as a cofactor for entry of HIV in CD4+ cells. Science. 1993, 262, 2045-2050).
`
`Thus inhibitors of DPP-IV should have therapeutic utility in the treatment of AIDS.
`
`BRIEF SUMMARY OF THE INVENTION
`
`The proline phosphonates derivatives of this invention have the following general
`structure:
`
`X
`
`(
`’N
`AA
`
`IP\’
`0’ oz‘
`
`and
`
`I
`
`X
`’N"( '02
`’oP\
`0
`
`02‘
`
`or a pharmaceutically acceptable salt thereof, wherein Z and Z1 are the same or different
`
`and are selected from the group consisting of C1_6 perfluoroalkyl, phenyl, phenyl
`
`substituted with J, phenyl disubstituted with J, phenyl trisubstituted with J, and
`
`pentafluorophenyl; J is selected from the group consisting of halogen, C 1-5 alkyl, C 1-6
`
`perfluoroalkyl, C1_6 alkoxy, N02, CN, OH, CO2H, amino, C1_6 alkylarnino, C242
`
`dialkylarnino, C1-5 acyl, and C1_6 alkoxy-CO-, and C1_6 alkyl-S-; X is selected from the
`
`group consisting of (a) a single bond, (b)-CH2-, (c) -CH2CH2-, (d) —CH2CH2CH2-, (e)
`-CH2CH2CH2CH2—, (f) -Y-, (g) -CH2-Y-‘, (h) -Y-CH,g-, and (i) -H, H-, wherein Y is O
`
`or S; and AA is selected from the group consisting of (a) the structure NH2-CHR-CO-
`
`where R is selected from the group consisting of C1_6 alkyl and C1_6 fluorinated alkyl,
`
`(b) a side chain blocked or unblocked alpha amino acid residue with the L, D or DL
`
`configuration at the ot-carbon atom selected from the group consisting of alanine, valine,-
`
`leucine, isoleucine, proline, methionine, methionine sulfoxide, phenylalanine, tryptophan,
`
`serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid,
`
`lysine, arginine, histidine, phenylglycine, norleucine, norvaline, alpha-arninobutyric acid,
`
`10
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`15
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`20
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`25
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`30
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`35
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`Page 4 of 41
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`WO 95/29691
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`_
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`PCTfUS95/05345
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`citrulline, hydroxyproline, ornithine, homoarginine, O-methylsedne, O—ethylserine, S-
`
`methylcysteine, S-ethylcysteine, S-benzylcysteine, NH2-CH(CH3CHEt2)-COOH, alpha-
`
`aminoheptanoic acid, NH2—CH(CH2-1-napthyl)-COOH, NH2-CH(CH2-2-napthyl)—
`
`COOH, NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH2-cyclopentyl)—COOH, NH2-
`
`5
`
`CH(CH2-cyclobutyl)—COOH, NH2-CH(CH2-cyclopropyl)—COOH, 5,5,5-trifluoroleucine,
`
`and hexafluoroleucine, (c) an amino acid residue selected from the group consisting of
`
`beta-alanine, glycine, epsilon-arninocaproic acid, and sarcosine, (d) H, and (e)
`
`C6H5CH2OCO-.
`
`A therapeutically effective amount of these compounds can be used to inhibit
`
`10
`
`dipeptidyl peptidase IV in mammals.
`
`A therapeutically effective amount of these compounds can be used to treati AIDS in
`
`mammals.
`
`'
`
`A therapeutically effective amount of these compounds can be used to prevent tissue
`
`transplant rejection in mammals.
`
`15
`
`It is an object of this invention to define a novel group of specific inhibitors for
`
`tI'§”QSin, elastase, chymotrypsin and other serine proteases. Inhibitors are compounds that
`
`~»-.:‘~':luce or eliminate the catalytic activity of the enzyme. Trypsin and trypsin-like
`
`=5.-.r=:'ies normally cleave peptide bonds in proteins and peptides where the amino acid
`
`er:
`
`resniue on the carbonyl side of the split bond (P1 residue) is Lys or Arg. Elastase and
`
`20
`
`elastase-like enzymes cleave peptide bonds where the P1 amino acid is Ala, Val, Ser, Leu
`
`and r..=-’-ilter similar amino acids. Chymotrypsin and chymotrypsin-like enzymes hydrolyze
`
`peptide bonds where the P1 amino acid is Trp, Tyr, Phe, Met, Leu or other amino acid
`
`residue which contain an aromatic or large alkyl side chain. All of the above enzymes have
`
`extensive secondary specificity and recognize amino acid residues removed from the P1
`
`25
`
`residue.
`
`It is a further object of this invention to define new protease inhibitors, especially
`
`inhibitors for chymotrypsin and chymotrypsin-like enzymes, elastase inhibitors, blood
`
`coagulation enzyme inhibitors and tryptase inhibitors. These inhibitors are useful for
`
`controlling tumor invasion, blood coagulation and various inflammatory conditions
`
`30
`
`mediated by serine proteases. The inhibitors of this invention are useful for treating
`
`diseases such as vascular clotting, inflammations, tumor invasion, pancreatitis, emphysema
`
`or infantile and adult respiratory distress syndrome. The inhibitors of this invention are
`
`also useful for controlling hormone processing by serine proteases and for treating diseases
`
`related to tryptases such as inflammation and skin blistering.
`
`35
`
`It is yet another object of this invention to define a novel group of specific inhibitors
`
`useful in vitro for inhibiting trypsin, elastase, chymotrypsin and other serine proteases of
`
`similar specificity. Such inhibitors could be used to identify new proteolytic enzymes
`
`encountered in research. They can be used in research and industrially to prevent undesired
`
`proteolysis that occurs during the production, isolation, purification, transport and storage
`
`Page 5 of 41
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`WO 95/29691
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`PCT/US95/05345
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`4
`
`of valuable peptides and proteins. Such proteolysis often destroys or alters the activity
`
`and/or function of the peptides and proteins. Uses can include the addition of the inhibitors
`
`to antibodies, enzymes, plasma proteins, tissue extracts or other proteins and peptides
`
`which are widely sold for use in clinical analyses, biomedical research, and for many other
`reasons.
`
`It is yet another objective of this invention to define a novel group of specific
`
`inhibitors for DPP-IV. The inhibitors are useful for controlling the immune system,
`
`inhibiting the process of organ transplant rejection, for treatment of AIDS, and related
`disorders.
`
`These and other objects are accomplished by the present invention which defines
`_
`novel peptidyl derivative of aryl diesters of ot-arninoalkylphosphonic acids. These
`
`phosphonate derivatives are potent inhibitors of serine proteases including chymotrypsin-
`
`like enzymes, trypsin-like enzymes, elastase-like enzymes, DPP-IV and other enzymes
`
`with other substrate specificities. The phosophonates are stable in buffer or plamsa, and
`
`inhibit the serine proteases to give stable inhibited enzyme derivatives. The phosphonates
`
`can be used both in vitro and in vivo.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Peptidyl derivatives of aryl diesters of ot-aminoalkylphosphonic acids have be
`
`found to be excellent inhibitors of several serine proteases including bovine thrombin,
`
`human factor XIIa, human plasma kallikrein, bovine trypsin, rat skin tryptase, human
`
`leukocyte elastase, porcine pancreatic elastase, bovine chymotrypsin, human leukocyte
`
`cathepsin G, DPP-IV, and rat mast cell protease II. The diesters of ot-
`
`aminoalkylphosphonic acids are analogues of natural ot-amino acids and are designated by
`
`the generally accepted three letter abbreviations for the amino acid followed by the
`
`superscript P. For example diphenyl ot-(N-benzyloxycarbonylarnino)ethylphosphonate
`which is related to alanine is abbreviated as Cbz-AlaP(OPh)2.
`
`10
`
`15
`
`20
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`25
`
`CH3
`,OPh
`|
`Bzl-O-CO-NH CH fixoph
`0
`
`Complete Structure of Cbz-AlaP(OPh)2
`
`30
`
`Peptidyl derivatives of aryl diesters of ot-aminoalkylphosphonic acids inhibit serine
`
`proteases by reaction with the active site serine to form "phosphonylated" enzymes, which
`
`due to the similarity of phosphorus atom to the tetrahedral intermediate formed during
`
`peptide hydrolysis, show remarkable stability. The enzyme catalytic apparatus is required
`
`to activate the phosphorus atom for nucleophilic substitution and reaction with enzyme.
`The activation is mainly due to precise interaction with the S1 pocket of various serine
`
`35
`
`proteases. The following figure shows the reaction course of a phosophonate with a serine
`protease. The phosphonate first binds to the enzyme (below left) andvthen reacts to form a
`
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`PCTfUS95l05345
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`covalent bond with the active site serine residue (below right). Slow aging can take place
`
`with loss of the phenoxy group (below center).
`
`Substrate
`
`Binding Site
`
`31
`
`Active Site
`
`S1
`
`_
`_
`Active Site
`
`$3195
`
`/Series
`R I
`__l
`—|—:| O-H-.--His57
`_
`I _
`___m—§ R_CO_NH_ I _PaO '
`R CO NH CH fisoph
`CH l$\OPh
`'2'.
`N
`
`O
`
`I
`
`\
`
`oxyanion
`hole
`
`N
`
`S1
`
`Active Site
`
`_
`
`Ser
`
`______> R_CO_NH_CiH_I?,g- H-HIS57
`
`IO\
`
`*3
`N
`
`*2‘
`N
`
`oxyanion
`hole
`
`Peptides with a C-terminal phosphonate residue which is an analog of valine (e.g.
`ValP(OPh)2) are potent and specific irreversible inhibitors of elastase and elastase-like
`
`enzymes. The peptides with C-terminal phosphonate residues related to phenylalanine,
`
`other aromatic amino acids or amino acids with long aliphatic side chains are potent and
`
`specific inhibitors of chymotrypsin and chymotrypsin—like enzymes. The peptides with C-
`
`terminal phosphonate residues related to ornithine, arginine or containing a C-terminal
`
`diphenyl ester of oL-amino-0L-(4-amidinophenyl)methanephosphonate are specific and
`
`potent inhibitors of trypsin and tiypsin-like enzymes. Dipeptides with the C-terrninal
`
`phosphonate residues related to proline or homoproline are specific and potent inhibitors of
`
`DPP-IV. The structures of two inhibitors are shown below. The dipeptide phosphonate
`on the left has a proline phosphonate derivative (abbreviated -Prop-) at the C-terminal end
`
`10
`
`15
`
`of the dipeptide, while the derivative on the right has a homoproline phosphonate (or
`piperidyl phosphonate, abbreviated -PipP-) as the C-terminal residue.
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`6
`
`R
`
`X. +H3NJ\'r
`
`N: >
`OAr
`o «.P’
`O \OAr
`
`R
`
`X. +H3N/Kir
`
`N
`
`OAr
`o .P’
`O \OAr
`
`P
`
`Additional specificity as well as increased activation toward reaction with the
`
`enzyme can be introduced into the inhibitor molecule by variation of the amino acid
`
`sequence in the peptide portion of the structure. In fact there is a good agreement between
`
`5
`
`the sequence of enzyme substrates such as a peptidyl p-nitroanilides and the sequence of an
`
`effective peptidyl phosphonate inhibitor. The best inhibitors have the sequence of the best
`
`peptidyl p—nitroanilide substrate for a particular enzyme. For example, the best inhibitor for
`chymotrypsin and chymotrypsin-like enzymes is Suc—Va1-Pro-PheP(OPh)2 which has an
`
`amino acid sequence that is analogous to Suc-Val-Pro-Phe—NA, an excellent substrate for
`
`10
`
`these enzymes. With human leukocyte elastase, the two best inhibitors (MeO-Suc-Ala-Ala-
`Pro-ValP(OPh)2 and Boc-Val-Pro-ValP(OPh)2) have an amino acid sequence similar to
`
`MeO-Suc-Ala-Ala-Pro-Val—NA and Boc-Val-Pro—Val-NA , two excellent substrates for this
`
`enzyme. For bovine thrombin, the best phosphonate inhibitor is dipheny1Boc-D-Phe-Pro-
`
`arnino(4-amidinophenyl)methanephosphonate hydrochloride, which corresponds to Boc-D-
`
`l5
`
`Phe-Pro-Arg-NA, which is excellent substrate for thrombin, and D—Phe-Pro-Arg—H which
`is an excellent peptide aldehyde inhibitor of thrombin and an anticoagulant. Since good
`
`substrate sequences are known in the literature for other serine proteases, it is possible to
`
`predict the structure of additional excellent phosphonate inhibitors for these enzymes. It is
`
`also possible to design good phosophonate inhibitors for serine proteases based on on the
`
`20
`
`peptide sequences found in other potent reversible and irreversible inhibitors for those same
`
`serine proteases reported in the literature
`
`25
`
`30
`
`Examples of phosphonate inhibitors for various enzymes are given below:
`Cbz-Gly—Leu-PheP(OZ)2
`for cathepsin G and RMCP H
`MeO-Suc-Ala-Ala-Pro-MetP(OZ)2
`for Cathepsin G
`Suc-Pro-Leu-PheP(OZ)2 and Boc-Ala-Ala-PheP(OZ)2
`for RMCP I
`Boc-Gly-Leu-PheP(OZ)2, Suc—Phe-Leu-PheP(OZ)2
`for human and dog skin chymase
`Boc-Ala-Ala-GluP(OZ)2
`for S. aureus V-8 protease
`Cbz-Gly-Gly-ProP(OZ)2
`for human prolyl endopeptidase
`Ala-ProP(OZ)2
`for DPP IV
`i
`Suc-Ala-Ala-Pro-ValP(OZ)2
`for PPE
`Suc-Lys(Cbz)-Val-Pro-ValP(OZ)2, adarnantyl-SO2—Lys(COCH2CH2CO2H)—Ala-
`Va1P(OZ)2, adamantyl-CH3CH2OCO-Glu(O-t-Bu)-Pro-ValP(OZ)2, adamantyl-SO2-
`L.V5(CO'C6H4CO2H)'A1a'V31P(OZ)2
`for human leukocyte (neutrophil) elastase
`Suc-Ala-Ala-Pro-LeuP(OZ)2
`for elastolytic proteinase from "Schistosoma mansoni"
`
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`Glu-Phe-X and Dns-Ala-Phe-X
`
`D—Val-Gly-X and Dns-Glu-Gly-X
`
`for plasmin
`
`for factor Xa
`
`Cbz-Phe—X and Cbz-Trp-X
`
`for porcine pancreatic and human
`
`Cbz-Lys-X
`
`Cbz-Gly-X
`
`plasma kallikreins
`
`for human skin tryptase
`
`for human lung tryptase
`
`Cbz-Ile-Ala-Gly-X
`
`for factors IXa, Xa, XIa, XIIa and
`
`10
`
`Glu-Gly-X
`
`Dn_s_—Phe~Pro-X
`‘Dns—Ile-Pro-X
`
`Cbz-Trp-X
`
`Cbz-Gly-X
`
`Cbz-Phe—X
`
`15
`
`Cbz-Phe-Gly-X
`
`bovine plasma kallikrein
`
`for urokinase
`
`for plasminogen activator
`
`for activated protein C
`
`for bovine factor IXa
`
`for bovine factor Xa and XIa
`
`for bovine factor Xlla
`
`for trypsin
`
`20
`
`25
`
`30
`
`35
`
`where Z represents an aryl group, a substituted aryl group or a highly fluorinated alkyl
`group and X represents ArgP(OZ)2 or an aryl diester of ot-amino-ot—(4-
`
`amidinophenyl)methanephosphonate [NH2-CH(AmPh)PO(OZ)2].
`
`The inhibitory potency of peptidyl derivatives of aryl diesters of ot-
`
`aminoalkylphosphonic acids is also determined by the electronic property of the Z group.
`
`More electron withdrawing groups such as nitro, cyano, halogen, etc. on the aryl ring can
`
`make the phosphorus atom in the inhibitor more electrophilic and accelerate reaction with
`
`the active site serine of the serine protease. Reactivity toward serine proteases can also be
`
`obtained by using derivatives where the Z groups are highly fluorinated alkyl groups.
`
`However increased reactivity can also result in low chemical stability and in extreme cases,
`
`compounds may be too hydrolytically unstable for practical purposes if the Z group is too
`
`electronegative. Phosphonates where the Z group is not sufficiently electron withdrawing
`
`will be chemically very stable and will react very slowly with serine proteases or not at all.
`
`Such non—reactive inhibitors would -include derivatives where Z is simple alkyl groups (e.g.
`
`peptidyl derivatives of alkyl diesters of ot-aminoalkylphosphonic acids). Thus the
`
`phosphonate ester groups (Z) should represent a balance between these two competing
`
`factors and we find that diphenyl esters (Z = Ph) are one way to obtain a balance between
`
`increased reactivity and stability in solution.
`
`Diphenyl esters of ot-aminoalkylphosphonate can be synthesized by a previously
`
`described method (Oleksyszyn et al., 1979, Synthesis, 985, incorporated by reference).
`
`Di(substituted phenyl)esters of ot-aminoalkylphosphonate can also be prepared by the same
`
`procedure using tris(substituted phenyl) phosphite instead of triphenyl phosphite.
`
`Perfluoroalkyl diesters can be synthesized by a method involving transesterification
`
`(Szewczyk _et al., Synthesis, 1982, 409-414, incorporated by reference). Alternatively, the
`
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`Page 9 of 41
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`synthesis of diesters of ot-aminoalkylphosphonic acids and their peptides can be performed
`by esterification of the phosphonic acid moiety as described previously (Bartlett et al.,
`Bioorg. Chem. , 1986, 14, 356-377, incorporated by reference).
`The dipeptides which contain 2-pyrrolidylphosphonate (proline phosphonate) or 2-
`piperidylphosphonate (homoproline phosphonate) were synthesized using the reactions
`outlined in the following scheme. The proline phosphonate HCl-ProP(OR)2 was
`
`synthesized by reaction of diphenyl phosphite or di-(4-chlorophenyl) phosphite with 1-
`pyrroline trimer (Petrillo, E. W., Spitzrniller, E. R. Synthesis of 2-Phosphopyrrolidine
`and Its Substitution for Proline in an Inhibitor of Angiotensin-Converting Enzyme, Tet.
`Lett. 1979, 51, 4929). Subsequent coupling of HCl-ProP(OR)2 (HC1-ProP(OPh)2, 1 or
`HCl-ProP(OPh-4-C1)2, 3) with the N-blocked amino acid Cbz-AA—OH using the DCC
`method gave the dipeptide phosphonate Cbz—AA-ProP(OR)2. Deblocking of the dipeptides
`was accomplished by hydrogenolysis in the presence of acid or by the use of 30% HBr in
`AcOH to give compounds 6, 9, 10, and 12. Similarly, the homoproline derivative
`PipP(OR)2-HCl (2, 4, or 5) was synthesized by reaction of diphenyl phosphite or di—(4-
`halophenyl) phosphite with 2,3,4,5-tetrahydropyridine trimer (Solodenko, V. A., Kukhar,
`V. P. Synthesis of DL—(2-Piperidyl) Phosphonic Acid, Zh. Obsh. Khim. 1987, 57,
`2392). The intermediate PipP(OR)2-HCl was then coupled with Cbz-AA—OH using the
`DCC method to give the dipeptides Cbz-AA-PipP(OR)2. Subsequent deblocking of Cbz-
`AA-PipP(OR)2 with hydrogenolysis in the presence of acid or HBr in AcOH gave
`
`compounds 7, 12, 13, and 14.
`HCl-ProP(OPh)2
`(HCI-PipP(OPh)2
`HC1-ProP(OPh-4-Cl)2
`HC1-PipP(OPh-4-Cl)2,
`HC1-PipP(OPh—4-F);
`
`\D®\l¢\Ul-Bull»)!-I
`
`I-| ¢
`
`hi hi
`
`F-I N
`
`I-| DJ
`
`r-A A
`
`HCI-Ala-ProP(OPh)2
`
`AcOH-Ala-PipP(OPh)2
`
`AcOH-Ala-PipP(OH)(OPh)
`HBr-Phe-ProP(OPh)2
`
`2HBr-Lys—ProP(OPh)2
`2HCl-Lys—PipP(OPh)2
`HCl-Ala-ProP(OPh-4Cl)2
`
`HC1-Ala-PipP(OPh-4Cl)2
`HC1-Ala-PipP(OPh-4-F)2
`
`20
`
`25
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`30
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`W0 95/29691
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`.
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`_
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`PCTfUS95l05345
`
`a’b
` N
`Q +HPO(OZ)2 _:_»
`
`< :N
`
`Cl" +H2N
`
`l oz
`
`,P’
`0’ ‘oz
`
`X
`
`_ +
`
`H3N
`
`Fl’
`
`)\n/N
`0 O’! \
`02
`
`I02 <—-————cbz-NH
`dore
`
`C
`
`R‘
`
`£02
`)\[(N _
`O ’»P\
`0 oz
`
`(a) heat under argon; (b) HCI gas in ether; (c) Cbz-NHCHR'COOH, DCC;
`aReagents:
`(d) Pd/C, H2, H* (e) 30% HBr/AcOH
`
`The diphenyl phosphonate moiety is very resistent to chemical hydrolysis and at pH
`7.5 we did not observe any hydrolysis after several days (monitored by 31P NMR).
`Furthermore, they show excellent stability in human plasma. For example Suc-Val-Pro-
`PheP(OPh)2 has a hydrolysis half-time in human plasma of about 20 hrs. These
`
`5
`
`experiments demonstrate that the phosphonate inhibitors are remarkably stable in buffer
`
`and plasma. Thus they can be used under a variety of conditions. Phosphonates have the
`
`additional advantage of being very stable in plasma and will have a high effectiveness in
`
`10
`
`vivo due to their long lifetimes. Additionally, the inhibitor-enzyme complex is very stable
`
`and the enzyme did not regain any activity after several hours in the case of chymotrypsin
`
`and after several days no recovery of activity was observed in the the cases of elastases and
`
`trypsin. These experiments show that it is possible to decrease or eliminate the enzyme
`
`activity and biological function of serine proteases for extended time periods.
`Either racemic mixtures of the diphenyl 0L-arninoalkylphosphonate residue or pure
`
`15
`
`diastereomers can be used in the inhibitor structures. The racemic compounds are more
`
`easily synthesized and are obtained from less expensive starting materials. The pure
`optically active ot-aminoalkylphosphonate derivatives required in the synthesis are more
`
`difficult to synthesize and require more expensive starting materials. In the case of the
`
`20
`
`peptidyl phosphonate inhibitors which are mixtures of two diastereomers, only one will
`
`usually react with the enzymes. The pure diastereomers will possess higher inhibition rates
`and could be used at lower concentrations.
`“
`
`Peptidyl derivatives of aryl diesters of ot-arninoalkylphosphonates may be used in
`
`vivo to treat diseases resulting from abnormal or uncontrolled blood coagulation or diseases
`
`25
`
`caused by uncontrolled proteolysis by elastase, chymotrypsin, trypsin and related serine
`
`proteases. These inhibitors may be used in vitro to prevent proteolysis which occurs in the
`
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`10
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`process of the production, isolation, purification, storage or transport of peptides and
`proteins.
`
`The novel peptidyl derivatives of aryl diesters oL—arninoalkylphosphonates of the
`
`present invention have the following structural formula:
`
`(
`’N
`A A
`
`X
`
`, p(
`0’ oz‘
`
`and
`
`.
`(\ X
`’N'—< ’OZ
`A A
`,,P\
`0 02‘
`
`or a pharmaceutically acceptable salt, wherein
`Z and Z1 are the same or different and are selected from the group consisting of C1-
`6 perfluoroalkyl, phenyl, phenyl substituted with J, phenyl disubstituted with J, phenyl
`trisubstituted with J, and pentafluorophenyl;
`J is selected from the group consisting of halogen, C1_6 alkyl, C 1-5 perfluoroalkyl,
`C1_6 alkoxy, N02, CN, OH, COZH, amino, C1_5 alkylamino, C2_ 12 dialkylamino, C1—5
`acyl, and C 1-6 alkoxy—CO-, and C 1_5 alkyl-S-;
`
`X is selected from the group consisting of
`
`(a)
`
`(13)
`
`(c)
`
`(d)
`
`(e)
`
`(0
`
`(g)
`
`(h)
`
`(i)
`
`a single bond,
`
`-CH2-.
`
`-CH2CH2-,
`
`—CH2CH2CH2-,
`
`—CH2CH2CH2CH2-,
`
`—Y—,
`
`—CH2—Y-,
`
`-Y-CH2—, and
`
`—H. H-.
`
`wherein Y is O or S; and
`
`AA is selected from the group consisting of
`(a)
`the structure NH2—CHR-CO-
`
`where R is selected from the group consisting of C1_5 alkyl and C1_6
`fluorinated alkyl,
`
`a side chain blocked or unblocked alpha amino acid residue with the
`(b)
`L, D or DL configuration at the oc-carbon atom selected from the group
`consisting of alanine, valine, leucine, isoleucine, proline, methionine,
`methionine sulfoxide, phenylalanine, tryptophan, serine, threonine,
`cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid,
`lysine, arginine, histidine, phenylglycine, norleucine, norvaline, alpha-
`aminobutyric acid, citrulline, hydroxyproline, omithine, homoarginine, O-
`methylserine, O-ethylserine, S-methylcysteine, S-ethylcysteine, S-
`
`10
`
`p 15
`
`20
`
`25
`
`30
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`1 l
`
`benzylcysteine, NH2-CH(CH2CHEt'_;_)-COOH, alpha-aminoheptanoic acid,
`
`NH2-CH(CH2-1-napthyl)-COOH, NH2-CH(CH2—2-napthyl)-COOH,
`
`NH2-CH(CH2-cyclohexyl)-COOH, NH2-CH(CH3-cyclopentyl)-COOH,
`
`NH2-CH(CH2-cyclobutyl)—COOH, NH2-CH(CH2-cyclopropyl)—COOH,
`
`5
`
`5,5,5-trifluoroleucine, and hexafluoroleucine,
`
`(c)
`
`an amino acid residue selected from the group consisting of beta-
`
`alanine, glycine, epsilon-arninocaproic acid, and sarcosine,
`
`(d)
`
`(e)
`
`H, and
`
`C5H5CH2OCO-.
`
`10
`
`This invention also includes the use of the novel phosphonate compounds described
`
`above for inhibiting dipeptidyl peptidase IV in mammals by treatment of a mammal with a
`
`therapeutically effective amount of the novel phosphonate compound.
`
`The blocking groups which may be present on -HN-CH(R)-P(O)- or on the amino
`
`acid AA are those well known in the art of peptide synthesis. The particular choice of the
`
`15
`
`blocking group used in the compounds of the invention depends on several factors,
`
`including the blocking group's affect on enzyme specificiity, its affect on phosphonate
`
`solubility, and its utility during synthesis. Suitable blocking groups include but are not
`
`limited to carbobenzyloxy (Cbz), benzoyl, t-butyloxycarbonyl (Boc), glutaryl, p-
`
`tolylsulfonyl (Tos), methoxysuccinyl (MeO-Suc), and succinyl.
`
`20
`
`The -HN-CH(R)-P(O)- residue is derived from a blocked or unblocked alpha amino
`
`acid residue —HN-CH(R)-CO- whose alpha carbonyl group has been replaced by a P(O)
`
`group. The R group is the side chain of the alpha amino acid. The alpha amino acid
`
`residue is derived from natural alpha amino acids such as those listed in the IUPAC-IUB
`
`Joint Commision on Biochemical Nomenclature report on the Nomenclature and
`
`25
`
`Symbolism for Amino Acids and Peptides (J. Biol Chem., 260, 14-42 (1985) incorporated
`
`by reference). The choice of the particular amino acid residue used in the design of the
`
`phosphonate inhibitor will depend on the enzyme targeted for inhibition. For example,
`with chymotrypsin-like enzymes which prefer Trp, Tyr, or Phe at the P1 position of their
`
`30
`
`substrates, -TrpP-, TyrP-, and PheP- residues would be suitable phosphonate residues to
`incorporate into the P1 position of an inhibitor. With elastase-like enzymes which prefer
`Val, Ser, or Ala at the P1 position of their substrates, -ValP-, SerP-, and AlaP- residues
`
`would be suitable phosphonate residues to incorporate in the P1 position of an inhibitor.
`
`Likewise with trypsin-like enzyme -LysP- or -ArgP- residues would be suitable.
`
`Unnatural blocked or unblocked alpha amino acid residues can also be used in the
`
`35
`
`design of phosphonate inhibitors. If the target serine protease will hydrolyze a substrate
`
`containing the unnatural amino acid residue at the P1 position or if an inhibitor structure
`
`contains the unnatural amino acid residue as the P1 residue, then this residue can be used in
`
`the design of a phosphonate inhibitor. For example, chymotrypsin hydrolyzes para-
`
`fluorophenylalanine containing substrates and thus the -HN-CH(CH2C6H4F)—P(O)-
`
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`would be suitable for incoroporating into a chymotrypsin inhibitor. Likewise, trypsin will
`
`hydrolyze substrates with aminoethylcystein residues and cathespin G will hydrolyze
`aminopropylcystein residues and thus -HN-CH(CH2SCH2CH2NH2)-P(O)- and -HN-
`
`CH(CH2SCH2CH2CH2NH2)-P(O)- would respectively be suitable residues to
`
`'
`
`incorporate into inhibitors for trypsin and cathepsin G. One skilled in the art of designing
`
`inhibitors for proteolytic enzyme can list many other unnatural amino acid residues which
`
`could be used in the design of suitable inhibitors.
`
`Other aryl diesters of 0L-aminoalkylphosphonic acids have been prepared earlier for
`
`other purposes (illustrative examples: Oleksyszyn, J. et al., Synthesis, 1979, 985-986.;
`
`Vo-Quang, Y. et al., J. Med. Chem. 1986, 43, 579-581.; Kafarski, P. et al., Tetrahedron,
`
`1987, 43, 799-803.; Szewczyk, J. eta1., Synthesis, 1982, 409-414; the preceding articles
`
`are incorporated herein by reference).
`
`A few other derivatives of ot-arninoalkylphosphonic acids have been prepared
`
`recently for inhibition of serine proteases, but they are not peptidyl derivatives or are
`
`peptidyl derivatives with the phosphonic acid moiety inside the peptide chain (Bartlett et al.,
`
`Bioorg. Chem., 1986, 14, 356-377, Larnden et al., Biochem. Biophys. Res. Commun.,
`
`1983, 112, 1085-1090; the preceding articles are incorporated herein by reference).