United States Patent (19)
`Jenkins et al.
`
`USOO5939560A
`Patent Number:
`11
`(45) Date of Patent:
`
`5,939,560
`Aug. 17, 1999
`
`54 INHIBITORS OF DP-MEDIATED
`PROCESSES, COMPOSITIONS AND
`THERAPEUTIC METHODS THEREOF
`
`Lotti et al., European Journal of Pharmacology, 162:
`273–280 (1989).
`
`75 Inventors: Paul D. Jenkins, Romsey; D. Michael
`Jones, Nr. Romsey; Michael Szelke,
`Romsey, all of United Kingdom
`73 Assignee: Ferring B.V., KC Hoofdorp,
`Netherlands
`21 Appl. No.:
`08/647,887
`22 PCT Filed:
`Nov.30, 1994
`86 PCT No.:
`PCT/GB94/02615
`S371 Date:
`Aug. 27, 1996
`S 102(e) Date: Aug. 27, 1996
`87 PCT Pub. No.: WO95/15309
`PCT Pub. Date:Jun. 8, 1995
`Foreign Application Priority Data
`30
`Dec. 3, 1993 GB United Kingdom ................... 93248O3
`Dec. 6, 1993 GB United Kingdom ................... 9324981
`(51) Int. Cl. ............................................... A61K 38/05
`52 U.S. Cl. ............................ 548/535; 514/19; 548/400;
`548/405
`58 Field of Search ............................... 514/19, 548/535,
`548/400, 405
`
`56)
`
`References Cited
`
`Primary Examiner-Cecilia J. Tsang
`ASSistant Examiner David Lukton
`Attorney, Agent, or Firm-Foley & Lardner
`
`57
`
`ABSTRACT
`
`A-B (Groups I and II)
`
`e-A-B
`
`e-A-B
`
`e-A-B
`
`e-A-B
`(Group III)
`
`(CH2) Ils. X
`
`-Y N CH Y (CH2)n
`
`(1)
`
`(2)
`
`(3)
`
`(4)
`
`U.S. PATENT DOCUMENTS
`
`R
`
`5,200,412 4/1993 Whittaker ................................ 514/293
`FOREIGN PATENT DOCUMENTS
`1221238 2/1971 United Kingdom.
`WO91/16339 10/1991 WIPO.
`WO93/08259 4/1993 WIPO.
`
`OTHER PUBLICATIONS
`Demuth et al., Federation of European Biochemical Societ
`ies, 320(1): 23–27 (Mar. 1993).
`Patents Abstracts of Japan, 1(120): 2929 C 77 (Oct. 12,
`1977).
`
`Compounds Selected from those of general formula A-B
`(Groups I and II) and (group III), (1, 2 and 3) where B is
`(4) and A is selected from specified aminoacyl compounds
`are inhibitors of DP-IV mediated processes.
`
`8 Claims, No Drawings
`
`Merck Exhibit 2183, Page 1
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`1
`INHIBITORS OF DP-MEDIATED
`PROCESSES, COMPOSITIONS AND
`THERAPEUTIC METHODS THEREOF
`
`BACKGROUND
`DP-IV (EC 3.4.14.5) is a membrane-bound serine pro
`tease first identified in rat kidney by its ability to cleave
`dipeptides from the N-terminus of certain peptides (HopSu
`Havu, V. K. and Glenner, G. G., Histochemie, 1966, 7, 197).
`The dipeptides must be of the type X-Pro or X-Ala where
`X=any amino acid. X-Proline is more efficiently cleaved
`than X-Ala.
`DP-IV is widely distributed in mammalian tissues and is
`found in great abundance in the kidney, intestinal epithelium
`and placenta (Yaron, A. and Naider, F, Critical Reviews in
`Biochem. Mol. Biol. 1993, 28 (1),31). In the human immune
`System the enzyme is expressed almost exclusively by
`activated T-lymphocytes of the CD4" type where the
`enzyme has been shown to be Synonymous with the cell
`Surface antigen CD26.
`The exact role of DP-IV in human physiology is not
`completely understood but recent research has shown that
`the enzyme clearly has a major role in human physiology
`and pathophysiology, e.g.
`(a) The immune response: DP-IV expression is increased
`in T-cells upon mitogenic or antigenic stimulation (Mattern,
`T. et al., Scand. J. Immunol. 1991, 33, 737). It has been
`reported that inhibitors of DP-IV and antibodies to DP-IV
`Suppress the proliferation of mitogen- and antigen
`stimulated T-cells in a dose-dependant manner (Schön, E. et
`al., Biol. Chem. Hoppe-Seyler, 1991, 372, 305 and refs.
`within).
`Various other functions of T-lymphocytes Such as cytok
`ine production, IL-2 mediated cell proliferation and B-cell
`helper activity have been shown to be dependant on DP-IV
`activity (Schon, E. et al., Scand. J. Immunol. 1989,29, 127).
`Recently, DP-IV inhibitors based on boroproline where
`reported (Flentke, G. R. et al., Proc. Natl. Acad. Sci. USA,
`1991, 88, 1556) which, although unstable, were effective in
`inhibiting antigen-induced lymphocyte proliferation and
`IL-2 production in murine CD4 T-helper cells. Such
`boronic acid inhibitors have been shown to have an effect in
`Vivo in mice causing Suppression of antibody production
`induced by immune challenge (Kubota, T. et al., Clin. Exp.
`Immunol. 1992, 89 192). Other recent papers also provide
`evidence for the involvement of DP-IV in the immune
`response (eg. Tanaka, T. et al., Proc. Natl. Acad. Sci. NY,
`1993, 90, 4586; Hegen, M. et al., Cell Immun. 1993, 146
`249; Subramanyan, M. et al., J. Immunol. 1993, 150, 2544).
`The importance of DP-IV is attributed by some investi
`gators to its cell-Surface association with the transmembrane
`phosphatase CD45 (Torimoto, Y. et al., J. Immunol. 1991,
`147, 2514). The CD45-DP-IV association is possibly dis
`rupted by DP-IV inhibitors or non-active site ligands. CD45
`is known to be an integral component of T-cell Signalling.
`(b) Recently, a press release from the Pasteur Institute in
`Paris (and Subsequently a presentation by A. G. Hovanessian
`at the 8th Cent. Gardes Meeting, Paris, Oct. 25–27th 1993)
`reported that DP-IV was essential for the penetration and
`infectivity of HIV-1 and HIV-2 viruses in CD4" T-cells. The
`
`15
`
`25
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`5,939,560
`
`2
`French group claimed that DP-IV interacted with and may
`have cleaved the V3 loop of the gp120 envelope glyco
`protein of the virus. They also reported that inhibitors or
`antibodies to DP-IV Successfully prevented entry of the
`Virus into cells. It was known previously that there is a
`selective decrease of CD26 expression in T-cells from HIV-1
`infected individuals (Valle-Blazquez, M. et al., J. Immunol.
`1992, 149,3073), and that HIV-1 Tat protein binds to DP-IV
`(Subramanyam, M. et al., J. Immunol. 1993, 150, 2544).
`(c) It has been shown recently that lung endothelial DP-IV
`is an adhesion molecule for lung-metastatic rat breast and
`prostate carcinoma cells (Johnson, R. C. et al., J. Cell. Biol.
`1993, 121, 1423). DP-IV is known to bind to fibronectin and
`Some metastatic tumour cells are known to carry large
`amounts of fibronectin on their Surface.
`(d) DP-IV has been shown to associate with the enzyme
`adenosine deaminase (ADA) on the Surface of T-cells
`(Kameoka, J. et al., Science, 1993, 261, 466). ADA defi
`ciency causes Severe combined immunodeficiency disease
`(SCID) in humans. This ADA-CD26 interaction may pro
`vide clues to the pathophysiology of SCID.
`(e) High levels of DP-IV expression have been found in
`human skin fibroblast cells from patients with psoriasis,
`rheumatoid arthritis (RA) and lichen planus (Raynaud, F. et
`al., J. Cell. Physiol. 1992, 151, 378).
`(f) High DP-IV activity has been found in tissue homo
`genates from patients with benign prostate hypertrophy and
`in prostatoSomes. These are prostate derived organelles
`important for the enhancement of Sperm forward motility
`(Vanhoof, G. et al., Eur: J. Clin. Chem. Clin. Biochem. 1992,
`30, 333).
`(g) DP-IV has been shown to be responsible for the
`degradation and inactivation of circulating peptides with
`penultimate proline or alanine at the N-terminus, eg. Sub
`stance P, growth hormone releasing factor and members of
`the glucagon/vasoactive intestinal peptide family
`(Menthein, R. et al., Eur: J. Biochem. 1993, 214,829).
`(h) Raised levels of DP-IV have been observed in the
`gingiva of patients with periodontitis (Cox, S. W. et al.,
`Arch. Oral. Biol. 1992, 37, 167).
`(i) There are also a number of other reports of raised (or
`sometimes lowered) levels of DP-IV in various pathological
`conditions.
`It follows from the above that potent inhibitors of DP-IV
`may be useful as drugs for the treatment of human disease.
`Such inhibitors could be useful as:
`(a) Immunosuppressants, eg. in organ transplantation;
`cytokine release Suppressants eg. in various autoim
`mune diseaseS Such as inflammatory bowel disease,
`multiple Sclerosis, RA.
`(b) Drugs for the prevention of HIV entry into T-cells and
`therefore useful in the prophylaxis and treatment of
`AIDS.
`(c) Drugs for the prevention of metastases, particularly of
`breast and prostate tumours to the lungs.
`(d) Agents to treat dermatological diseases, eg. pSoriasis,
`lichen planus.
`(e) Drugs to Suppress Sperm motility and therefore act as
`male contraceptive agents.
`
`Merck Exhibit 2183, Page 2
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`5,939,560
`
`3
`(f) Agents beneficial in benign prostate hypertrophy.
`Inhibitors of DP-IV
`The only competitive inhibitors of DP-IV enzyme activity
`reported so far are the unstable boronic acids (t/230-90 min
`at pH 7) mentioned above. (Bachovchin et al., WO
`91/16339, October 1991) having K values in the nanomolar
`range for DP-IV, and simple amino-acid pyrrolidides or
`thiazolides (Neubert et al., DD 296 075 A5, November
`1991) which have only modest potency (K-0.1 uM).
`Amino-acyl praline aldehydes claimed in the same German
`patent cannot be Synthesised due to a facile intramolecular
`condensation of the N-terminal amino group with the alde
`hyde function.
`We now disclose highly potent competitive inhibitors of
`DP-IV (with K. values in the 10-10' range) which are
`also chemically stable (t/2>24 h). They fall into three broad
`groups of compounds (Groups I, II and III).
`GROUPI
`These are molecules designed to bind tightly in the active
`site of DP-IV and to inhibit its proteolytic activity without
`interfering with attachment of any accessory ligands which
`may bind to the surface of DP-IV (i.e. not at its active site).
`Such Group I compounds could be useful as immunoSup
`preSSants, anti-HIV infectivity agents, agents to SuppreSS
`release of certain cytokines (eg. IL-2, IL-6, Y-INF) from
`activated T-cells. The boronic acids and pyrrolidides
`referred to earlier also fall into this category.
`GROUP II
`These are evolved from Group I compounds; however
`they contain long-chain extensions to the Side-chains of the
`amino-acid defined as A in the general Structure. The result
`ing compounds bind tightly to the active-site of DP-IV but
`the long-chain extensions protrude from the enzyme active
`Site and Serve to prevent the attachment of any other ligand
`which may bind to the surface of DP-IV. Such compounds
`could have the same uses as Group I compounds but in
`addition could block the interaction of DP-IV with (i) CD45
`(ii) the gp120 V3 loop of HIV-1 (iii) tumour cell surface
`fibronectin (iv) any other ligand important for T-cell
`activation, Virus entry into T-cells or tumour cell adhesion.
`GROUP III
`This group comprises novel dimers in which two active
`site directed inhibitors of DP-IV are linked via the side
`chains of their amino-acid residues designated A in the
`general Structure by a long chain. Such dimers can inhibit
`two molecules of DP-IV concurrently and also prevent
`accessory ligands binding to the Surface of DP-IV. These
`dimers would have the same uses as Group II compounds
`but may be more effective.
`The invention provides inhibitors of DP-IV mediated
`processes, the inhibitors being of general formula:
`A-B (Groups I and II) or
`
`15
`
`25
`
`35
`
`40
`
`where B is
`
`(CH2)
`—y
`N CH1 (CH2)n:
`
`R
`
`n=1 or 2,
`m=0, 1 or 2;
`X=CH, O, S, SO, SO,
`NH or NR where R =lower alkyl (C. to C);
`A is attached to Y,
`-Y=-N, -CH or =C (when the -CO group of A is
`replaced with CH=or CF=);
`R=H, CN, CHO, B(OH), CEC-R, or CH=N-Rs;
`R=H, F, lower alkyl (C. to C), CN, NO, OR, COR
`or COR;
`Rs=Ph, OH, OR, OCOR, or OBn;
`Ro=lower alkyl (C-C); and either () or both e's may be
`absent.
`The structure of A is dependent on the nature of R in
`moiety B and on the nature of the group to which the
`resulting compound belongs.
`Group I Compounds
`(a) R=H
`A is an a-amino-acyl group derived from an O-amino-acid
`bearing a cycloaliphatic side-chain (e.g. C to Co., mono or
`bicyclic) whose ring may contain one or more heteroatoms
`e.g. L-cyclohe Xylglycine, L-cyclope ntylglycine,
`L-decahydronaphthylglycine, L-piperidylglycine;
`O
`A is a B-amino-acyl group of general formula
`
`CH-NH
`/
`2
`(CH2)
`
`CH-CO
`
`where p=1-6 and the ring may also contain one or more
`heteroatoms replacing CH2 unit(s).
`Both C. and f-amino acyl groups in (a) above may contain
`unsaturation in their rings e.g.
`
`NH2
`
`s
`
`o
`
`HN CO
`
`also may contain one or more heteroatoms.
`(b) R=CN: C=C-R, or CH=N-Rs
`A is as defined in (a) above but in addition may be derived
`from any L-O-amino acid bearing a lipophilic Side-chain, eg.
`Ile.
`(c) R=CHO or B(OH)
`A is a f-amino-acyl group as defined in (a) above. The
`resulting A-B compounds are stable, unlike C-aminoacyl
`
`45
`
`50
`
`55
`
`60
`
`e-A-B
`
`()
`
`e-A-B
`
`(Group II)
`
`65
`
`Merck Exhibit 2183, Page 3
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`S
`derivatives of the Same type which undergo a facile intramo
`lecular cyclisation. In compounds (c) B(OH) may be
`present as a boronate ester eg.
`
`5,939,560
`
`/ O
`o R
`
`O
`
`Me
`
`Me
`
`Me
`Me
`
`O
`1.
`- B
`
`O
`
`\,
`
`s
`
`Me
`
`these being labile in water giving the free boronic acids.
`In a preferred embodiment, A is Selected from the group
`consisting of T1, T2 and T3, wherein
`T1 is a 3-amino-acyl group of the formula
`
`15
`
`1. CH-NH
`(CH2)
`
`SéH-CO
`
`25
`
`35
`
`wherein p is an integer of 1-6, and wherein the ring present
`in T1 optionally contains one or more heteroatoms, and
`wherein the ring present in T1 optionally has one or two Sites
`of unsaturation and wherein the carbonyl group of T1 is
`optionally replaced with CH= or CF=;
`T2 is an O-amino acyl group bearing a cycloaliphatic side
`chain, wherein the ring present in T2 optionally con
`tains one or more heteroatoms, and wherein the ring
`present in T2 optionally has one or two Sites of
`unsaturation, and wherein the carbonyl group of T2 is
`optionally replaced with CH= or CF=;
`T3 is an L-C.-amino acid bearing a lipophilic Side chain,
`wherein the carbonyl group of T3 is optionally replaced
`with CH= or CF=;
`with the provisos that,
`(a) A is T1 only if R is H, -CHO or -B(OH)2;
`(b) A is T2 only if R is H; and
`40
`(c) A is T3 only if R is CN, C=C-R, or CH=N-Rs.
`Group II Compounds
`Where R=H, CN, C=C-R, or CH=N-Rs, A is an
`C.-amino acid derivative whose side-chain carries a func
`tional group which is derivatised to produce a long chain
`terminating in various groupS R. A may be of the following
`three types of Structure:
`
`45
`
`(i)
`HN
`HN
`)— (CH2)-CO-D or )— (CH2), -SO2-D
`
`CO
`
`CO
`
`50
`
`55
`
`where a=1-5; D=G-(CH-)-(R)-R; G=O, NH, or
`NMe;
`b=0-12; q=0-5;
`D'=D with G zO;
`R=Z-NH-(CH-)- or NH-Z-(CH),
`c=1-12 and Z=CO, CH or SO; and
`R=COH or ester e.g. any lower alkyl, fluoroalkyl or
`cycloalkyl (C. to Cs), or aromatic or heteroaromatic (5
`or 6-membered rings, mono- or bicylic) ester thereof;
`CONH; CONHNH; CONRR, CONNRR; POH
`
`60
`
`65
`
`where
`
`6
`(or ester thereof e.g. as defined under COH); SOH;
`SONH; SONRR, OH, ORs, aryl or heteroaryl (e.g.
`5 or 6-membered rings, monocyclic or bicyclic)
`including substituted aryl or heteroaryl with substitu
`ents preferably chosen from F, Cl, I, Br, OH, OR, NO,
`SOH, SONH, SONRR, NH, NRR, CORs,
`CF, CN, CONH, CONRR, NHCORs, CH(NR)
`NRR, NH-CH(NR)NRR and Rs); NH; NRR;
`NHCOR; NHSONRR: NHCORs; NH-SOR;
`NH-CH(NR)NRR; NHCONRR; sugar (which
`may be attached via an ether or a glycosidic bond);
`CO-aminoSugar (attached via the -NH) eg. glu
`cosamine or galactosamine; NHCO-aminoSugar, or
`NHCS-aminosugar. In the above definition of Rs.
`"Sugar refers to any carbohydrate or oligosaccharide,
`and Rs and R are independently selected from H and
`alkyl, fluoroalkyl and cycloalkyl groups (of up to 8
`atoms), aryl, heteroaryl and alkylheteroaryl groups (of
`up to 11 atoms) or Rs and R together comprise a chain
`and (C. to Cs).
`
`HN
`
`HN
`
`(ii)
`)— (CH2)NRE or )-O- E
`f
`f
`where R'-H, Me; the ring may also contain more heteroa
`toms,
`E=J-(CH2)-(R)-Rs: J=CO, CH or SO; and a, b,
`q, R and R as defined under (i)
`
`HN
`
`R2
`
`HN
`
`)-( O )-()– OL
`f
`OL
`f
`
`(iii)
`
`where R=H or Me; the ring may also contain one or more
`heteroatoms,
`L=(CH2)-CO)-(CH2)-(R)-Rs or (CH2)—
`NR'-(CH-)-(R)-Ra: r=0 or 1; d=0–4; e=2–4;
`and b, q, R and R as defined under (i).
`Group III
`Group m compounds are defined by the general formula:
`
`e-A-B
`
`e-A-B
`
`where ()=CH, O, NH, CO, S, SO, Ph or NMe and,
`independently, e=CH, O, NH, CO, S, SO, Ph or NMe.
`These compounds are Symmetrical dimers. They may
`have any B Structure as defined previously. A may be chosen
`from any group II structure (i), (ii) or (iii)), but in this case
`the terminal group R in each A residue is deleted and
`replaced with a shared Symmetrical group e--e) which
`connects the two halves of the dimer, may be absent, in
`which case both es are joined together to constitute the
`chain linking the two A-B moieties; alternatively both es
`may be absent in which case solely joins the two A-B
`moieties.
`
`Merck Exhibit 2183, Page 4
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`5,939,560
`
`8
`-continued
`/
`/
`/
`- CH-N : CHEC : - CS-N .
`
`7
`The structure of e--e must of course be chemically
`feasible eg. NH-CO-NH, CO-NH-CO-, SO
`NMe-SO; it will be obvious to those skilled in the art
`which structures are not feasible, e.g. -NH-NH-NH-. A
`specific possible example is shown in Table 7.
`In such compounds as described under Groups II and III
`certain -CH2-groups present in the long chains could be
`replaced with known bioisosteres eg. -O- without affect
`ing inhibitory or binding activity towards DP-IV. Also such
`groupings as -CONHCHCH-NHCO if they occur could
`be replaced by eg.
`
`/ \
`- CO-N
`N - CO
`
`15
`
`Further, for compounds in Groups I, II and III any amide 20
`bond connecting A and B or any amide in the Side-chains of
`A (in Groups II and III) may be replaced by known bioi
`Sosteres of amides eg.
`
`/
`/
`/
`- CO-N replaced by - CO-C-; CF=C. :
`\
`
`25
`
`See Table 8 for examples of Such replacements.
`Biochemistry
`All compounds were tested in Vitro against pure human
`DP-IV (purchased from M & E, Copenhagen, Denmark).
`Inhibition of DP-IV was determined using the fluorescent
`substrate Ala-Pro-AFC (Ki 0.8 uM) at three concentrations
`for each inhibitor. A typical assay (total volume 0.4 ml)
`comprised sodium Hepes 83.3 mM, EDTA 1.67 mM, BSA
`1.5 mg ml pH 7.8, DP-IV 25 uU ml, inhibitor (in 10 mM
`acetate pH 4.0). The reaction was started by the addition of
`Substrate and readings taken every 30 S for 7.5 min, exci
`tation at 395 nm, emission 450 nm. K. values were deter
`mined using Dixon plots.
`Chemistry
`152 Examples of compounds Synthesised are shown in
`Tables 1-8 followed by schemes and experimental details
`for the preparation of different Structural types. All final
`products were characterised by FAB maSS Spectrometry and
`purity assessed by reverse phase hplc, all intermediates were
`characterised by "H NMR.
`Table 9 shows selected K values against DP-IV deter
`mined for inhibitors of different structural types.
`
`TABLE 1.
`
`Examples of Group I (a)
`
`No.
`
`1.
`
`A.
`
`X R n Formula
`
`FAB Mass
`Calculated
`Mol. Wt. spec. M+H"
`
`CH H 1 CHNO 196.2
`
`197.2
`
`HN
`
`O
`
`2
`
`CH H 1 C2HNO 210.2
`
`211.2
`
`HN
`
`Merck Exhibit 2183, Page 5
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`5,939,560
`
`10
`
`9
`
`TABLE 1-continued
`
`Examples of Group I (a)
`
`No.
`
`X R in Formula
`
`Calculated
`Mol. Wt.
`
`FAB Mass
`spec. M + H"
`
`CH H 1 CHNO
`
`1842
`
`185.2
`
`CH H 1 C2HNO
`
`208.2
`
`209.2
`
`CH H 1 CHNO
`
`1961
`
`1972
`
`NH2
`
`CH H 1 CHNO
`
`1961
`
`1972
`
`CH H 1 CHNO
`
`1941
`
`195.2
`
`CH, H 1 CHNO
`
`1821
`
`183.2
`
`CIS
`
`trans
`
`trans
`
`trans
`
`Merck Exhibit 2183, Page 6
`Mylan Pharmaceuticals Inc. v. Merck Sharp & Dohme Corp.
`IPR2020-00040
`
`

`

`5,939,560
`
`TABLE 1-continued
`
`Examples of Group I (a)
`
`X
`
`( ( )n v.
`
`M
`A.
`
`R
`
`No.
`
`9
`
`1O
`trans
`
`A.
`
`X R n Formula
`
`FAB Mass
`Calculated
`Mol. Wt. spec. M+H"
`
`NH2
`
`O
`
`CH H 1 CHNO 190.1
`
`1912
`
`CH H 1 CHNO 224.2
`
`225.2
`
`NH2
`
`%r O
`
`TABLE 2
`
`Examples of Group I (b)
`
`X
`R1
`s ( )n
`N
`M
`A.
`
`R
`
`X
`
`CH,
`CH,
`CH,
`
`CH,
`
`R.
`
`R.
`
`Formula
`
`FAB Mass
`Calculated
`Mol. Wt. spec. M+H"
`
`H CN CHNO 209.3
`H CN CHNO. 358.2
`H CN CHNO 193.1
`
`H CN CHNOS 211.1
`
`210.2
`359.2
`1941
`
`212.2
`
`CH,
`
`H CN CHNOS 211.1
`
`212.2
`
`No. A
`
`11 H-Ile
`12 H-Lys(Z)
`13 H-Pro
`
`14
`
`15
`
`HN
`
`O
`
`r S
`
`HN
`
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`

`13
`
`5,939,560
`
`14
`
`TABLE 2-continued
`
`Examples of Group I (b
`
`X
`R1
`N. ( )n
`N
`M
`A.
`
`R
`
`X
`
`in
`
`R.
`
`R.
`
`Formula
`
`FAB Mass
`Calculated
`Mol. Wit. spec. M+H"
`
`CH,
`
`1 H CN CHNO 235.2
`
`236.3
`
`CH,
`
`1 H CN CHNO 221.2
`
`222.2
`
`CH,
`
`1 H CN CHNO 209.2
`
`210.2
`
`S
`S
`
`S
`
`S
`
`S
`
`1 H CN CHNOS 227.1
`1 CN H CHNOS 227.1
`
`1 H CN CHNOS 253.1
`
`2281
`2281
`
`254.1
`
`1 H CN CHNOS 376.2
`
`1 H CN CHNOS 2.39.1
`
`377.2
`
`240.2
`
`O
`CH,
`S
`SO
`
`1 H CN CH NO 211.1
`2 H CN CH, NO 223.2
`2 H CN CHNOS 241.1
`1 H CN CHNOS 259.1
`
`st, to 1 H CN C10H, NOS 243.1
`
`212.2
`224.2
`242.1
`26O1
`
`244.1
`
`No. A
`
`16
`
`HN
`
`HN
`
`17
`
`18
`
`19 H-Ile
`20 H-Ile
`
`21
`
`O
`
`O
`
`O
`
`HN
`
`O
`
`22 H-Lys(Z)
`
`23
`
`HN
`
`O
`
`24 H-Ile
`25 H-Ile
`26 H-Ile
`27 H-Ile
`
`28 H-Ile
`
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`5,939,560
`
`15
`
`TABLE 2-continued
`
`Examples of Group I (b)
`
`X
`w
`( )n
`
`R
`
`R
`
`Formula
`
`Calculated
`Mol. Wt.
`
`FAB Mass
`spec. M + H'
`
`243.1
`
`244.2
`
`221.2
`
`222.2
`
`No.
`
`29
`
`H-Ile
`
`Sc O
`
`CH,
`
`31
`
`
`
`CH,
`
`221.2
`
`222.2
`
`CH,
`
`CH,
`
`CH,
`
`CH,
`
`CH,
`
`32
`
`33
`
`34
`
`35
`
`36
`
`NH2
`
`208.2
`
`208.2
`
`219.1
`
`219.1
`
`221.2
`
`222.2
`
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`5,939,560
`
`18
`
`17
`
`TABLE 2-continued
`
`Examples of Group I (b)
`
`A.
`
`No.
`
`37
`
`R.
`
`R.
`
`Formula
`
`Mol. Wt.
`
`Calculated
`
`FAB Mass
`spec. M + H"
`
`CH,
`
`1.
`
`H. CN
`
`C2HNO 219.1
`
`TABLE 3
`
`Examples of Group I (e
`
`A.
`
`R
`
`R
`
`Formula
`
`FAB Mass
`Calculated
`Mol. Wt. spec. M+H"
`
`CH,
`
`CHO 1 C2HNO.
`
`224.2
`
`225.2
`
`CH,
`
`CHO 1 CHNO, 210.2
`
`211.2
`
`CH,
`
`CHO 1 CHNO, 210.2
`
`211.2
`
`CH,
`
`B*
`
`1 CHBNO. 360.3
`
`361.3
`
`No.
`
`38
`
`39
`
`40
`
`41
`
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`23
`
`5,939,560
`
`24
`
`TABLE 4-continued
`
`Examples of Group II (i)
`
`(CH2) ( c),
`N
`
`R
`
`HN
`
`No.
`
`X
`
`R Formula
`
`Calculated
`Mol. Wt.
`
`FAB Mass
`spec. M+H"
`
`OH
`
`HO
`
`OH
`
`NH
`
`OH
`
`TABLE 5
`
`Examples of Group II (ii
`
`HN
`
`No.
`
`O
`
`Formula
`
`Calculated
`Mol. Wt.
`
`FAB Mass
`spec. M + H"
`
`O4
`05
`O6
`O7
`O8
`O9
`1O
`11
`12
`13
`14
`15
`16
`17
`18
`19
`2O
`21
`22
`23
`24
`25
`26
`27
`28
`29
`3O
`31
`32
`33
`34
`35
`
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`CH,
`
`313.2
`403.3
`313.2
`327.2
`312.3
`284.2
`514.2
`478.2
`430.2
`657.5
`523.4
`672.5
`538.4
`313.2
`403.3
`326.3
`340.3
`465.4
`368.3
`375.3
`396.4
`390.2
`404.2
`418.3
`256.2
`446.3
`298.2
`270.2
`312.2
`326.3
`354.3
`351.3
`
`314.3
`404.3
`314.3
`328.3
`313.3
`285.2
`515.2
`479.2
`431.3
`658.6
`524.4
`673.6
`539.4
`314.3
`404.3
`327.3
`341.3
`466.4
`369.3
`376.3
`3974
`391.3
`405.3
`4193
`257.2
`447.4
`299.3
`271.3
`313.3
`327.3
`355.3
`352.4
`
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`5,939,560
`
`TABLE 5-continued
`
`Examples of Group II (ii
`
`NHO
`?
`X n
`(CH2) ( ( )m
`N
`
`R
`
`HN
`
`O
`
`No.
`
`in Q
`
`136
`
`4 -CO(CH),NH
`
`X m R Formula
`
`FAB Mass
`Calculated
`Mol. Wit. spec. M+H"
`
`CH, 1 CN CHNO,
`
`365.3
`
`366.3
`
`TABLE 6
`
`Examples of Group II (iii
`
`R
`
`R1
`
`HN
`
`O
`
`X al ( ( )n N
`
`Y
`
`No.
`
`in Q
`
`137
`138
`139
`140
`141
`142
`143
`144
`145
`146
`147
`
`H –OCH,CONH(CH)s-COH
`H -OCH,CONHCCH)s-COBn
`H -OCHCONHCCH)-COBn
`H –OCH,CONH(CH)-COH
`CH -OCH
`CH -OCHs
`CH -O(CH2)CH
`CH -OCH2CONHCCH)s-COBn
`CH, -CCHCONHCCH)s-COH
`CH -OCH2CONHCCH)-COBn
`CH -OCHCONHCCH)-COH
`
`X m R Formula
`
`CH, 1 H CH-NOs
`CH, 1 H C2HNOs
`CH, 1 H CHNOs
`CH, 1 H CHNOs
`CH, 1 H CHNO.
`CH, 1 H CH2N2O2
`CH, 1 H CHNO.
`CH, 1 H CHNOs
`CH, 1 H CHNOs
`CH, 1 H C2HNOs
`CH, 1 H CHNOs
`
`FAB Mass
`Calculated
`Mol. Wit. spec. M+H"
`
`329.2
`419.3
`405.2
`315.2
`1861
`2001
`256.2
`433.3
`343.2
`419.2
`329.2
`
`330.3
`420.3
`4O6.3
`316.3
`187.2
`2012
`257.3
`434.3
`344.3
`42O3
`330.3
`
`TABLE 7
`
`Example of Group III
`
`No. Structure
`
`Formula
`
`FAB Mass
`Calculated
`Mol. Wt. spec. M+H"
`
`148
`
`O
`
`NH(CH2)12NH
`
`O
`
`C2H5NO 614.4
`
`615.4
`
`HN r O
`N
`
`C.
`
`CN
`
`CN HN
`
`O
`
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`5,939,560
`
`TABLE 8
`
`Specific examples of compounds A-B, containing amide bond bioisosteres.
`
`No.
`
`149
`
`A-B
`
`Formula
`
`FAB Mass
`Calculated
`Mol. Wit. spec. M+H"
`
`CHN 167.2
`
`168.2
`
`NH2
`
`150
`
`C12H2N2
`
`192.2
`
`193.2
`
`C12H2N2
`
`192.2
`
`193.2
`
`CH2N2S
`
`2001
`
`2012
`
`NH2
`
`v , //cN
`
`NH2
`
`151
`
`152
`
`HN
`
`N
`
`S
`
`TABLE 9
`
`Selected K. Values against DP-IV.
`
`TABLE 2
`
`No.
`2
`7
`11
`2O
`23
`35
`38
`44
`59
`66
`97
`110
`136
`143
`150
`
`K (M)
`6.4 x 10
`7.6 x 10
`2.2 x 10
`1.7 x 10
`5.0 x 10
`3.7 x 10
`9.8 x 10
`2.0 x 10
`1.5 x 107
`1.8 x 107
`5.0 x 1010
`2.5 x 107
`1.7 x 10
`9.4 x 107
`1.7 x 10
`
`50
`
`55
`
`60
`
`Schematic Representations for General Preparation of all
`Classes of Compounds
`Table 1
`Compounds can be made by an adaption of the general 65
`route described by E. Schon et al., Biol. Chem. Hoppe
`Seyler, 1991, 372, 305-311.
`
`r ( ),
`
`Boc-A-N
`
`POCl,
`-
`NH -
`pyridine,
`imidazole
`
`O
`
`X n
`
`r ( )n
`BA--
`X E scena
`
`CN --
`
`r ( )n
`H- H-A-- N.
`
`X s
`
`CN
`
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`

`5,939,560
`
`31
`
`TABLE 4
`
`(a) R = CN
`
`O
`
`ls
`
`( )n
`
`OP
`
`X
`
`r (),
`
`HN
`
`NH2
`
`OH
`
`Boc-N
`
`H
`
`O
`
`O
`PyBop, CH2Cl2, EtN
`
`Boc-N
`
`O
`
`|- OP X
`( )n r
`N
`
`32
`
`NH2
`
`H
`
`O
`
`O
`
`(i) remove P
`(ii) HONSu, WSCD
`
`O
`
`( )n r X
`
`Boc-N
`
`H
`
`O
`
`O R
`
`pyridine, imidazole
`
`(i) HN(CH2)P
`(ii) modify P-> P2
`if required
`
`O
`
`O
`
`O
`
`- ONS
`
`( )n r - O
`1.
`
`Bs
`
`H
`
`O
`
`(III)
`
`X
`H He
`r
`Boc-N C. HN
`
`1. O
`
`r X
`
`CN
`
`CN
`
`( )n
`
`H
`
`O
`
`(b)
`
`( )n r
`Boc-N C. R
`
`SOCI
`A
`
`H
`
`O
`(IV)
`
`(i) H2N(CH2)P
`(ii) modify P->P
`
`Her
`
`SONH(CH2)P
`
`M C.
`
`Boc-N
`
`H
`
`complete synthesis as above.
`(W, P = Protecting groups; P', P’ = Groups as described in corresponding tables)
`
`(IV) was prepared via method of G. Luisi et al., Tet. Lert,
`1993, 34,2391-2392. (c) For R=H, modify above procedure
`as described for Table 1 examples.
`
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`

`35
`
`5,939,560
`
`TABLE 7
`Standard coupling, dehydration and deprotection sequence similar to above
`schemes.
`
`O
`
`|- ONS
`( )n r
`
`Boc-N
`H
`
`NH2
`
`O
`
`(III)
`
`O
`
`molar equivalent
`
`O
`
`TABLE 8-continued
`
`R
`
`H
`
`Boc-N
`
`H
`
`O
`
`Toluene,
`O -
`as
`reflux
`
`PPh3
`
`R
`
`(i) (EtO)2POCN
`LiCN, DMF
`O -
`-
`(ii) SmI2, THF,
`t
`BuOH
`
`15
`
`NH-Boc
`
`CN --
`
`CN
`
`R
`
`R
`
`NH-Boc
`
`NH2
`
`3O
`
`35
`
`Thioamides were prepared by the method described by K.
`Clausen et al. Tetrahedron, 1981, 37, 3635-3639. Other
`amide bioisosteres can be prepared from literature prece
`dent. (A. F. Spatola in “Chemistry and Biochemistry of
`Amino Acids, Peptides and Proteins”, Vol. III, B. Weinstein
`Ed., Marcel Dekker, New York, 1983, p. 267).
`EXPERIMENTAL DETAILS FOR SPECIFIC
`EXAMPLES
`Example 1
`2-(S)-Cyano-1-isoleucylpyrrolidine (11)
`
`O
`
`CN
`
`45
`
`50
`
`55
`
`60
`
`65
`
`H-Ile-N
`
`CN
`
`Di-isopropylethylamine was added to a Solution of
`H-ProNH. HCl (225 mg, 1.50 mmol) in dry CHCl (15
`cm) until the pH was adjusted to 9. BocIleONSu was added
`in one portion and the mixture Stirred for 16 h, under a
`nitrogen atmosphere. The Solvent was evaporated and the
`residue treated in the Standard way, i.e. the residue was
`partitioned between ethyl acetate (60 cm) and 0.3 N
`KHSO, solution (10 cm). The organic layer was further
`washed with saturated NaCHO Solution (10 cm), water (10
`cm) and brine (5 cm). The solution was dried (NaSO)
`and evaporated at reduced pressure. The crude product was
`passed down a short plug of Silica gel, eluting with hexane
`:ethyl acetate, (10:90 to 0:100) to yield 301 mg (92%) of
`BocIleProNH as a colourless foam.
`"H NMR (CDC1), 8 (ppm); 6.90 (1H, brs); 5.51 (1H,
`bris); 5.18 (1H, d, J=9.6 Hz); 4.62 (1H, dd, J=2.6, 7.0 Hz);
`4.29 (1H, dd, J=8.4, 9.2 Hz); 3.79–3.58 (2H, m); 2.36 (1H,
`m); 2.09–1.57 (5H, m); 1.43 (9H, s); 1.17 (1H, m); 0.95 (3H,
`d, J=6.6 Hz); 0.90 (3H, t, J=7.3 Hz).
`
`O
`
`ls
`NH(CH2)NH
`( )n
`s' N
`
`O
`NH2
`
`Boc-N
`H
`
`N
`
`O
`
`O
`
`(III) | POCl3, pyridine
`(ii) H'
`
`O
`
`ls
`O
`|- NH(CH2)NH
`( )n
`HN P. N
`
`HN
`
`O
`
`N
`
`O
`
`N C
`
`TABLE 8
`
`(a)
`
`R
`
`Boc-N
`
`H --
`
`Toluene,
`He
`reflux
`
`H
`
`O
`
`PPh3
`
`R
`
`H
`HRH- R
`
`NH-Boc
`
`NH2
`
`(b)
`
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`

`37
`Imidazole (84 mg, 1.24 mmol) was added to a Solution of
`BocIleProNH in dry pyridine (10 cm), under a nitrogen
`atmosphere. The solution was cooled to -35 C., before the
`dropwise addition of POCl (0.25 cm, 2.48 mmol). The
`reaction was stirred at -30° C. to -20° C. for 60 min. The
`Solution was then evaporated and the crude residue Sub
`jected to column chromatography (silica gel) to yield 180
`mg (94%) of 2-(S)-cyano-1-N-(t-butoxycarbonyl)
`isoleucylpyrrolidine as a colourless oil.
`"H NMR (CDC1), 8 (ppm); 5.14 (1H, d, J=9.2 Hz); 4.80
`(1H, dd, J=2.6, 7.1 Hz); 4.22 (1H, dd, J=7.9, 9.1 Hz); 3.81
`(1H, m), 3.71 (1H, m), 2.30-2.12 (4H, m); 1.75 (1H, m);
`1.60 (1H, m); 1.42 (9H, s); 1.19 (1H, m); 0.97 (3H, d, J=6.9
`Hz); 0.91 (3H, t.J-7.3 Hz).
`'C NMR (CDC1), 8 (ppm); 1717, 155.6, 118.0, 79.6,
`56.0, 46.5, 46.0, 37.8, 29.6, 28.1, 25.0, 24.2, 15.2, 10.9.
`Deprotection was carried out by String with trifluoroacetic
`acid for 60 min. Evaporation and lyophilisation from water
`afforded 60 mg of 2-(S)-cyano-1-isoleucylpyrrolidine (11)
`as a white, fluffy solid.
`FAB Mass Spec: Calculated 209.3, Found (M+H)"=
`210.2.
`"H NMR (DO), 6 (ppm); 4.3 (1H, m); 3.64 (1H, d, J=5.6
`Hz); 3.16 (2H, m); 1.86–1.48 (5H, m); 0.98 (1H, m); 0.68
`(1H, m); 0.51 (3H, d, J=6.9 Hz); 0.38 (3H, t, J=7.3 Hz).
`'NMR (DO), 8 (ppm); 169.7, 119.7, 57.3, 48.6, 48.1,
`36.9, 30.2, 25.8, 24.5, 15.4, 11.5.
`Example Two
`
`H - GluNH(CH2)7CONH(CH2)NHZlpyrrolidide (64.
`
`O
`
`HN
`
`Di-isopropylethylamine was added to a Solution of
`BocGlu(OH)pyrrolidide (193 mg, 0.64 mmol) and PyBop
`(500 mg, 0.96 mmol) in CHCl (6 cm) to adjust the pH of
`the mixture to 9. After stirring for 5 min, a solution of benzyl
`50
`8-amino-octanoate (220 mg, 0.77 mmol) in CHCl (5 cm)
`was added. The mixture was stirred at room temp for 16 h.
`The reaction was worked up in the Standard procedure as
`described in example one. The crude residue was Subjected
`to column chromatography (1% to 3% methanol in ethyl
`acetate) to obtain 344 mg (99%) of BocGluNH(CH)
`,COBnpyrrolidide as a colourless Solid.
`"H NMR (CDC1), 8 (ppm); 7.35 (5H, s); 6.63 (1H, brt,
`J=6.7 Hz); 5.65 (1H, d, J=8.3 Hz); 5.11 (2H, s); 4.36 (1H, dt,
`J=2.6, 8.9 Hz); 3.55–3.20 (6H, m); 2.34 (2H, t, J=7.3 Hz);
`2.26 (2H, dd, J=5.6, 7.3 Hz); 2.11-1.48 (10H, m); 1.43 (9H,
`s); 1.32-1.27 (6H, m).
`Hydrogen gas was bubbled through a solution of BocGlu
`NH(CH),COBnpyrrolidide (230 mg, 0.43 mmol) in
`ethyl acetate (10 cm), containing 10% palladium on char
`
`60
`
`65
`
`5,939,560
`
`38
`coal (50 mg). After 90 min, the reaction vessel was flushed
`with nitrogen, the Solution filtered through a pad of celite
`and the solvent evaporated to yield 187 mg (98%) of
`BocGlu(NH(CH),COHpyrrolidide as a colourless oil.
`Di-isopropylethylamine was added to a Solution of
`BocGluNH(CH),COHpyrrolidide (125 mg, 0.28 mmol)
`and PyBop (221 mg, 0.43 mmol) in CHCl (10 cm) to
`adjust the pH of the solution to 9. After stirring for 5 min,
`a solution of ZNH(CH)NH2. HCl (90 mg, 0.37 mmol) and
`di-isopropylethylamine (38 mg, 0.37 mmol) was added in
`one portion. The solution was stirred for 18 h then treated in
`the Standard procedure as described for example one. The
`crude residue was Subjected to column chromatography (2%
`to 15%