AU735214B2

AU735214B2 - Enzyme inhibitors

Info

Publication number: AU735214B2
Application number: AU84219/98A
Authority: AU
Inventors: Paul D Jenkins; Michael D. Jones; Michael Szelke
Original assignee: Ferring BV
Current assignee: Ferring BV
Priority date: 1993-12-03
Filing date: 1998-09-11
Publication date: 2001-07-05
Anticipated expiration: 2014-11-30
Also published as: AU8421998A

Description

Our Ref: 699579 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): Ferring B V Maarstraat 9 PO Box 3129 2130 KC Hoofdorp THE NETHERLANDS DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Address for Service: Invention Title: Enzyme inhibitors The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 ENZYME

INHIBITORS

Background DP-IY (EC 3.4.14.5) is a membrane-bound serine protease first identified in, rat kidney by its ability to cleave dipeptides from the IN-terminus of certain peptidies (Hopsu-Havu,

V.K.

and Glenner, Histochemie, 1966, 7, 197). The dipeptides must be of the type X-Pro or K-Ada Where X any amino acid. X-Proline is more efficiently cleaved than K-Ala.

DP-J.V is widely distributed in mammalian tissues and is found in great abundance in the kidney, intestinal epithel~ium and placenta (Yaron, A. and Naider, Critical Reviews in Biochemn. MoI. Biol. 1993, 28 31). In the humtan 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, eg.

The immune response: DP-IV expression is increased in T-cells upon rn-itogenic or antigemic stimulation (Matten, T. et al., Scand. Immunol. 1991, 3 3 737). It has been reported that inhibitors of DP-IV and antibodies to DP-1V suppres3 the proliferation of mitogen- and anti gen-stinulated T-cells in a dose-dependlant manner (Schon, E. et al., Biol. Chem. Hoppe-Seyler, 1991, 372 305 and refs. within).

.Various other functions of T-lyrnphocytes such as cytokine production, E1-2 mediated cell proliferation and B-cell helper activity have been shown to be dependant on DP-IV activity (Schon, E. et al., Scand. J. lnhmunol. 1989, 29 127).

Recently, DP-IV inhibitors based on boroproline where reported (Flentke, G.R. et al., Proc. Nat!. Acad. Sci. USA, 1991, 88 1556) which, although unstable, were effective in inhibiting anti gen-induced lymphocyte proliferation and IL-2 production in mw-inc 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. Irnmunol. 1992, 8, 192). Other recent papers also provide evidence for the involvement of DP-rV in the immune response (eg. Tanaka, T. et al., Proc. Nat!. Acad. Sci. NY, 1993, 90, 4586; Hegen,

M.

et al., Cell Immun. 1993, 146, 249; Subramanyan, M. et al., J. Jmmnunol. 1993, 150, 2544).

-29- The importance of DP-rV is artibured by some nvestigators EQ its cell-Sur-face association with the transmembrane phosphatase CD45 (Torimoro, Y. et al., Immunol. 1991, J.7214)7 The CD45 DP-IV association is possibly disrupted by DP-1V inhibitors or non-active site ligands. CD45 is known to be an integral component Of T-cell signalling.

Recently, a press release from the Pasteur Institute in Paris (and subsequently a presentation by A.G. Hovanessian at the 8th CenL Gardes Meeting, Paris, 25-27th October 1993) reporTed that DP-IV was essential for the penetration and infectivity of HIV- 1 and HIV-2 viruses in O)41 T-cells. The French group claimed that DP-IV interacted with and may have cleaved the V3 loop of the gpl20 envelope glyco-protein of the virus. They also reported that inhibitors or antibodies to DP-I'V successfully prevented entry of the virus into cells. It was known previously that there is a selective decrease of CD26 expression in T-cdlls from IV-1 infected ***individuals (Valle-Blazquez, M.v et al., I. Immwwli. 1992, 149, 3073), and that EHY- I Tat protein binds to DP-IV (S ubramanyam, M- et ad., ImmwzoL 1993, 150, 2544).

It has been shown recently that lung endothelial DP-IV is an adhesion molecule for lung-metastaric rat breast and prostate carcinoma cells (Johnson, R.C. et al-, J. Cell.

Biol. 1993, 121, 1423). DP-IY is known to bind to fibronccrin and some mtueastatic tumour cells are known to carry large amounts of fibronectin on their surface.

DP-WV has been shown to associate with the enzyme adenosine deaminase

(ADA)

on the surface of T-cells (Kameoka, 1. et al., Science, 1-993, 261, 466). ADA deficiency causes severe combined imnmunodeficiency diseas (SCIID) in humans.

This A.DA-CD26 interaction may provide clues to the pathophysiology of SCID.

High levels of DP -IY expression have been found in human skin fibrobl[ast cells from patients with psoriasis, rheumatoid arthritis (RA) and lichen planus (Raynaud, F. et al., J. Cell. Physiol. 1-992, 151, 378).

W(f) igh DP-rV activity has been found in tissue homogenates from patients with benign prostate laypertrophy and in prostarosomes. These arre prostate derived organelles important for the enhancement of sperm forward modiliy (Vanhoof, G. et al., Eur.!. Clin. Chem. Clin. Biochem. 1-992, 30, 333).

-3 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.

substance P, growth hormone releasing factor and members of the glucagon/vasoactive intestinal peptide family (Menthein, R. et al., Eur. J. Biochem.

1993, 214 829).

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).

There are also a number of other reports of raised (or sometimes lowered) levels of DP-IV in various pathological conditions.

*o 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: Immunosuppressants, eg. in organ transplantation; cytokine release suppressants eg.

in various autoimmune diseases such as inflammatory bowel disease, multiple sclerosis, RA.

Drugs for the prevention of HIV entry into T-cells and therefore useful in the prophylaxis and treatment of AIDS.

Drugs for the prevention of metastases, particularly of breast and prostate tumours to the lungs.

Agents to treat dermatological diseases, eg. psoriasis, lichen planus.

S Drugs to suppress sperm motility and therefore act as male contraceptive agents.

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 30 90 min at pH 7) mentioned above. (Bachovchin et al., WO 91/16339, October 1991) having K i 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 i 0.1 jpM). Amino-acyl proline aldehydes claimed in the same German patent cannot be synthesised due to a facile intramolecular condensation of the N-terminal amino group with the aldehyde function.

-4- We now disclose highly potent competitive inhibitors of DP-IV (with K i values in the 10-6 10-10 range) which are also chemically stable (ti 24 They fall into three broad groups of compounds (Groups I, II and I).

GROUP I 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 not at its active site). Such Group I compounds could be useful as immunosuppressants; 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.

S. 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 resulting 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 CD45 (ii) the gp 120 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.

SGROUP m 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

E-A-B

(Group hI) where B is

(CH

2

I

v(C"H 2

M

CH

n =l1or2; m=0, lor2; X C2' 0, 3, S-QSO 2 NiH or NRI where R, lower allkyl (CI to C;;

V

V. V V V 0*Ve

V

V.

U.

V

.c.

V

Vt V V V V. C

V

R

A is attached to Y; -CH or.=C (when the -CO group of A is replaced with CH= or R C.N, CHO, B (OH) 2

C=-C-R

7 or CH=.N-Rg;

R-

7 H, lower alkyl (CI to C6), CN, NO, 2 O04 C0 2 R9 or COR9;

R

8

OR

9 OCOR9, or OBn;

R

9 =lower alkyl (C 1 C6); and either co or both E'S may be absent.

The structure of A is dependent on the natur of R in moiety B and on the nature of the group to which the resulting compound belongs.

Group I Compounds

R=H

A is an ct-aumino-acyl group derived from an C-amino-acid bearing a cycloaliphatiid side-chain C 4 to CIO, mono or bicyclic) whose ring may contain one or more heteroatorns e.g. L-cyclohexylglycine, L-cyclopentylglycine, L-decahydronaphthylglycine, L-piperidylglycine; or A is a P-amino-acyl group of general formula

CH-NH-

2 CCA (IH

CO-

where p 1 6 and the ring may also contain one or more heteroatornS replacing CH- 2 unit(s).

-6 Both ot and r3-amino acyl groups in above may contain unsaturation in their rings e. g.

CO-

H

2 N COand also may contain one or more heteroatoms.

R =CN; or CH=N-Rs A is as defined in above but in addition may be derived from any L-a-amrino acid bearing a. lipophilic side-chain, eg. fle.

R CH{Oor B(OH) 2 A is a 1-amino-acyl group as defined in above. The resulting A-B compounds are stable, unlike a-amninoacyl derivatives of the same type which undergo a facile intramolecular cyclisation. In compounds B(OH) 2 may be present as a boronate ester eg.

0 Me0 -B Me Mee Me Me these being labile in water giving the free boronic: acids.

-7- Group 11 COrnDOunds Where R ,CN, C-=C-R 7 or CH=N-RS, A is an a-amino acid derivative whose side-chain carries a functional group which is derivatised to Produce a long, chain terminating in various groups R 3 A may be of the following, three types of structure:

H

2 N

H

2

N

-(CH

2 o tH)S 2

D

Co

CO

where a 1 G-(CH2)b-(R4)q-R 3 G 0 H rNe 0-12; q o DI -D with G *0; R4 Z-NH-(CH,2- or NI{-Z-(CHA2)- where c 12 and Z C O, GB 2 or SO 2 and

R

3 C0 2 H or ester any lower alkyl, fluoroallcyl or cycloalkyl (CI to Cg), *or aromatic or heteroarornatic (5 or 6-membered ringsmono- or bicylic) ester] thereof;, CONH 2

CONHNH

2

CONR

5

R

6

CONHNR

5

SR

6

POH

*P03 (or ester thereof e.g. as defined under CO 2 S0 3 K- SO7NH: 2

SO

2

NRSR

6 OH; OR 5 aryl or heteroaryl 5 or 6-meinbered rings, monocyclic or bicyclic) (including substituted aryl or heteroaryl with substituents preferably chosen from F, C3, I, Br, OH, OR 5 N02, S0 3 H, SOZNH 2 S02NRSR 6 N2' NRSR 6 C02RS. CE 3

GN,

CONH

2

CONR

5

R,

6 NHC02R5. CH(:NRS)NR 5

R

6

NH-CR(:NR

5

)NRSIR

6 and R 5

NH

2

NR

5

R

6

NHCO

2

R

5

NHSO

2

NR

5

R

6

NHCOR

5 Nil-S%0R 5

NH-CH(:NR

5

)NRS

5

R

6

NHCONR

5

R,

6 sugar (which may be attached via an ether or a glycosidic bond); CO-aminosugar (attached via the -NH 2 eg. glucosamine or galactosamine; NHCO-aminosugar, or N:HCS-aminosugar.

In the above definition of R 3 "sugar" refers to any carbohydrate or oligosaccharide, andRS and R, 6 are independently selected from H and ailkyl, fluoroalcyl and cycloallcyl groups (of up to 8 atoms), aryl, heteroaryl and alkyiheteroaryl groups (of up to 11 atorns) or R 5 and R 6 together comprise a chain and (C 3 to C 8 8-

H

2 N

H

2

N

CO or COD where R 1 Me; the ring may also contain more heteroatoms; E J-(CH)b-(R 4 )q-R 3 J =CO, CH 2 or S0 2 and a, b, q, R 3 and R 4 as defined under (i)

H

2 N R 2

H

2

N

or OL %co OL CO **where R7 H or Me; the ring may also contain one or more heteroatoins; L =(CH 2 )d-(COIr-(CH 2 )b-(R 4 )q-R 3 or (CH 2 )e-NRL-(CH 2 )b-(R 4 )q-R 3 r =O0or1; d 4; e 2 4; and b, q,R 3 and R 4 as defined under Grout) Ml Group M compounds are defined by the general formula:

A-B

where (o CH 2 0, NIH, CO, S, SO 2 Ph or NMe and, independently,

E=CR-

2 0, NH, CO, S, S0 2 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 Qii or but in this case the terminal group R 3 in each A residue is deleted and replaced with a shared symmetrical group (-xg]which connects the two halves of the dime-, w may be absent, in which case both E's are joined together to constitute the chain linkcing the two A-B moieties; alternatively both E'S may be absent in -which case (o solely joins the two A-B moieties.

-9 The structure of co-C must of course be chemically feasible eg. NH-CO-NH, CO-NH-CO-, S0 2 -NMe-SO 2 it will be obvious to those skilled in the art which structures are not feasible, eg. -NH-NH-NH-. A specific possible example is shown in Table 7.

In such compounds as described under Groups II and III certain -CH 2 groups present in the long chains could be replaced with known bioisosteres eg. without affecting inhibitory or binding activity towards DP-IV. Also such groupings as

-CONHCH

2

CH

2 NHCO if they occur could be replaced by eg.

-CO-N N-CO- S Further, for compounds in Groups I, II and I any amide bond connecting A and B or any amide in the side-chains of A (in Groups II and II) may be replaced by known bioisosteres of amides eg.

-CO-N replaced by -CO-C- CF=C -CH-N

K'

CH=C -CS-N See Table 8 for examples of such replacements.

B iochemistry 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 (Km 0.8 lM) 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 mg ml- pH 7.8, DP-IV 25 .U ml- 1 inhibitor (in 10 mM acetate pH The reaction was started by the addition of substrate and readings taken every 30 s for 7.5 min, excitation at 395 nm, emission 450 nm. K values were determined using Dixon plots.

10 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 character-ised by FAB mass spectrometry and purity assessed by reverse phase hplc; all intermediates were characterised by 1H NMR.

Table 9 shows selected IC, values against DP-IV determined for inhibitors of different structural types.

.9 0% 11 Table 1 Examples of Group I (a) A R Calculated FAB Mass x R m Formula MoI. WL spec. [M+H1+

I~.

CH

2 H 1 Cj 1 H2ON 2

O

CH

2 H 1 C 12 H22N 2

O

OH

2 H 1 C 10 H2DN 2 0 196.2 210.2 184.2 208.2 197.2 211.2 185-2 209.2

CH

2 H 1 C 12 H2DN 2

O

CH

2 H I C 11 H2ON 2 O 9. 9.

196.1 197-2 12 Calculated FAB Mass X R m Formula 6 trans

CH

2 H 1 C, IH 2

N

2 0 196.1 1 97.2 7 trans

CH

2 H I C 11 1-1 1

N

2 0 o 0 0* 0 0 0 0 a trans 9 trans

CH

2 H 1 C 1

H

18

N

2 0

CH

2 H 1 C 11

H

14

N

2 0

CH

2 H 1 C 1 3

H

24

N

2 0 194.1 182-1 190-T 224.2 195-2 183.2 191.2 225.2 13 Table 2 Examples of Group I (b)

RI

A R

I..

H-lie H-Lys(Z) H-Pro

C

Calculated X m R R Formula MaL Wt-

H

2 1 H CN C 11 HjgN 3 0 209.3

;H

2 1 H CN C 19 H~sN 4

O

3 358.2

~H

2 I H CN C 10

H,

5

N

3 0 1 93.1 FAB Mass spec-. IM+H+ 210.2 359.2 194.1 212.2 212-2 236.3

OH

2 I H CN C 9

H,

3

N

3 0S

S

a

CH

2 1 H ON C 9

H

13

N

3 0S

OH

2 1 H ON C 13

H-

2

IN

3 0 211.1 211.1 2352 H 2

N'

CH

2 1 H ON C 12 Hj9N 3 0 2212 2221

H

2

N'

14 m RI R Formula Calculated mol. Wt

CH

2 1 H CN CIIH 1 9

N

3 0 0 H-lie H-lie 209.2.' 227.1 227.1 FAB Mass spec. IM+H1 4 210.2 228.1 228.1

C

10 H 1 17 N4 3 0S ClOH17N3OS

S

21 HN II..I 0 22 H-Lys(Z) S 1 H CN C 12

HISN

3 0S S i H CN C, 1

-H

24

N

4 0 3

S

S I H CN CIIH 1 7

N

3 0S 253.1 376.2 239.1 211.1 223.2 241.1 259.1 243.1 243.1 254.1 377.2 240.2 212-2 224.2 242.1 260.1 244.1 244.2 H-le H-He H-lie H-lie H-lie H-lie 0

CH

2

S

s0 2 S .,,110 C 0 H1 7 N30 2

C,

2

H-

2 1

N

3 0

C

11

H

19

N

3 0S C 0 H 1 7

N

3 0 3

S

ClaH1 7

N

3 0 2

S

C 10

H

1 7

N

3 0 2

S

15 X m R' R Formula Calculated FAB Mass M6I.-WI. s rec. M+HI- Forrrula

CH

2 1 H CN C 12 H,qN 3 0 221.2 222.2 31yY NH 0 0 32

NH

2 0 33

NH

2 34 O0 NH2 0a

CH

2 1 H CN C, 2

H

19

N

3 0

C

CH

2 1 H *CN

C

11

H

17

N

3 0

CH

2 1 H ON

C

11 1 17

N

3 0

CH

2 1 H CN C 12

H

1

N

3 0 221.2 207.2 207.2 219.1 2222 208.2 208.2 220.1 r CH2 I H CN

C

12

H

1

N

3 0 219.1 220.1 16 x m RI R Formula Calculated FAB Mass Mol. WI. spec. IM+H1+

CH

2

CH

2 1 H CN C1 2

H-

19

N

3 0 221.2 222.2 220.1 1 H CN C 12

HI

7

N

3 0 219-1 NH2 0 17 rable 3 Examples of Group I (c) A R Calculated FAB Mass X R Formula Mol. Wt. spec. [M+H1+

CH

2

CHO

C

1 2 H20N 2

O

2

C

11

H

18

N

2 0 2

CH

2 CHO 1 H 2N 0

OH

2 CHO 1 CjjHjqN 2 0 2

CH

2 6* 1 C2DH33BN 2 0 3

CH

2 13 1 C 2 1 HZBNZ0 3

CH

2 B* 1 C 21 H358NZ0 3

CH

2 B3 1 C 2 d4MBN 2 0 3 224.2 210.2 210.2 360.3 374.3 374.3 372-3 225.2 211.2 211.2 361.3 375.1 375.1 373.3

NH

2 0 43 N~

NH

2 0 18 x R m Formula Calculated FAB Mass Mol. Wt spec. rM+H1+ NH 2 0

CH

2 1 C 21 1 H3BN 2 0 3 372-3 373.3 B' O~a 19 Table 4 Examples of Group 11 (i)

Q

H 2 N N-k, 0 Calculated FAB Mass No. n X mn R Formula McI. Wt. spec. [M+Hj+ 46 47 48 49 50 51 52 53 54 56 57

-OONHGH

2 C0 2 Bn OH 2 1

.CONHCH

2

CO

2 H OH 2 1

*OONH(CH

2 3 00 2 H OH 2

I

-CONH(CH

2 2 C0 2 8n CH 2 1

-CONH(CH

2 2 00 2 H OH 2 1

-CONH(CH

2 5 CO.Bn OH 2

I

-CONH(CH

2 5 C0 2 H OH 2 1

-OONH(CH

2 3 C0 2 8n OH 2 1

-CONHGR

2 00 2 Bn OH 2 1

-CONHCH

2

CO

2 H CH 2 1

-CONH(CH

2

),CO

2 Bn OH 2 1

-CONH(C"

2 3 00 2 83n OH 2 1 O 1 7 H23N 3

O

4

C

1 0 H0 17 3 0 4

O

1 2

H

21

N

3 0 4 CISH25N 3

O

4

C

11 HjqN 3 0 4 021 H3 1

N

3 0 4

C

14 H25N 3

O

4 019 H 27

N

3 0 4 ClSH25N 3

O

4 CIIH~gN 3 0 4 O 19

H

27

N

3 0 4 C2DH2N 3 0 4 333.2 243.1 271.2 347.2 257.1 389.3 299.2 361.2 347-2 257.1 361.2 375.2 334.2 244.2 272.2 348.2 258.2 390.3 300.2 362.2 348.2 258.1 362.3 376.3 58 2 .CONH(CH 2 ):3O 2 H OH 2 1 H CO 13 H23N3O 4 285.2 286.2 No. n X m R

S.

S S S S S. S S. @5.555

S

59 2 -GONH(CH 2 ),00 2 8n 2 -OONH(0H 2 )SC0 2

H

61 2 -CONH(CH 2 2 C0 2

H

62 2 -CONH(CH 2 7

CO

2 Bn 63 2 -CONH(CH 2 7 G0 2

H

64 2 -CONH(0H 2 7 C0NH-

(CH

2 3

NHZ

2 -OONH(0H 2 6 C0NH-

(CH

2 ),C0 2 B~n 66 2 .CONH(CH 2 6 00NH-

(CH

2 5 00 2

H

67 2 -OONH(CH 2 7 C0NH- (CH2).JNH 2 68 2 -CONH(CH 2 1 00 2 83n 69 2 -CONH(CH 2 11 C0 2

H

2 -CONH(CH,) 6 C0 2 Bn 71 2 .CONH(CHA) 6 0 2

H

72 2 -OONH(CH 2 5 C0NH-

CH

2

CF

3

CH

2

CH

2

CH

2

OH

2

CH

2

OH

2 Formula.- C22HMN 3 0 4

C

15

H

27

N

3 0 4

C

12

H

2 j N 3 0 4

C

24 H3 7

N

3 0 4

C

17 H3 1

N

3 0 4 C28H45NS 5 -Calculated mol. Wt.

403.3 313.2 271.2 431.3 341.3 531.3 530.4 440.3 397.3 487.3 397.3 417.3 327.2 FAB Mass spec. [M+iHJ+ 404.3 314.2 272.2 4.32.4 342.5 532.3 531.2 441.3 398.3 488.4 398.3 418.3 328-2

OH

2 1 H C29H4N 4

O

5 H C22H4cN 4

OS

H C 2 OH39NS0 3 C29H4N 3 0 4 021 H 3 4 C23H3N 3 0 4

C

16 H29N 3 0 4

OH

2 1 H C 17 H29F 3

N

4

O

3 394.2 395.3 21 No. n X M R Frmla Calculated FAB Mass Mol. Wt. Spec. H O 19 H29F7N 4

O

3 494.2 495.2 6.S *9 0*S ,60.6 9599 .fee 73 2 -OONH(CH 2 5 C0NH- OH 2 1

CH

2 (C F 2 2

CF

3 74 2 -GONH(CH 2 )SC0NH- OH 2 1

(CH

2 6 0H 2 -CONH(CH 2 5 C0NH- CH 2 1 (0H 2 3 ph 76 2 -OONH(0H 2 5 00NH- OH 2 1 (0H 2 4 Ph 77 2 -OONH(0H 2 )SCON- OH 2 1 ('80) 2 78 2 -CONH(CH 2 5 00N- OH 2 1 (r'Hx) 2 79 2 -CO.NH(CH 2 )SCONH- OH 2 1

CH

2 Ph 2 -OONH(CH 2 4 C0 2 Bn OH 2 1 81 2 -CONH(CH 2 4 C0 2 H- OH 2

I

82 2 -CONH(0H 2 5 CONH- OH 2 1

CH

2

CH

3 83 2 -CONH(CH 2 6 0H OH 2 1 84 2 -CONH(CH 2 5 C0-1 -P OH 2 1 2 -CONH(CH 2 5 00NH 2

CH

2 1 H C 21 H114N 4

Q

4 H C 24 H38N 4

O

3 H C25H40N 4

O

3 H C23H4N 4

O

3 H C 27

H

52

N

4 0 3 H CU2H3N 4

O

3 412.3 430.3 444.3 424.3 480.4 402.3 389.2 299.2 340.3 299.2 380.3 312.2 413.2 431.2 445.2 425.3 4.81.4 403.4 390.3 300.3 341.3 300.3 381.4 313.3

C

21 1-H 3 1

N

3 0 4 C 14 H25N 3

O

4 C 7 H3N 4

O

3

C

15 H29N 3

O

3 C201-13N 4

O

3

CISHZSN

4 0 3 22 No. n X m R Calculatet Formula Mol. Wt.

2SH4N 4

O

3 452-4 .1.

86 2 -CONH(0H 2 5

CQNH-

(OH 2 )gCH 3 87 2 -CONH(CH 2 5 C0NH-

(CH

2 6

CH

3 88 2 -GONH(CH 2 5

CONH-

CH

2 Ch 89 2 -CQNH(CH 2 5 C0NH-

(CH

2 3

NHZ

90 2 -CONH(CH 2 5

CONH-

(CH

2 3

NH

2 91 2 -CONH(CH 2 5

CONH-

(CH-

2 3 -Gua 92 2 -CONH(CH 2 5

CONH-

Ph(4-SO 3

H)

93 2 -CONH(CH 2 )S00NH-4- SPip(1 -Bn) 94 2 CONH(CH 2 5 C0NH- 4-Pip 2 -CONH(CH 2 4

N(Z)-

(CH

2 3

NHZ

96 2 -OONH(0H 2 4

NH-

(CH

2 3

NH

2

OH

2 1 H C22H 42

N

4

O

3 I H C22H4N 4

O

3

OH

2 1 H G26H 4

N

5

O

5 1 H C

OH'

2 H C 18 IH45N 5

O

3

CH

2 1 H C, 9

H

37

N

7 0 3

OH

2 1 H CZ 21 H2N 4

O

6

S

410.3 408.3 503.3 369.3 411.3 468.2 485.3 395.3 595.3 J Ag Mass spec [M+HI+ 453.5 411.4 409.4 504.4 370.3 412.4 469.2 486.3 396.3 596.3

OH

2

OH

2 CH2 1 H C27H43N 5 0 3 H C2OH 37

N

5

O

3 H C32H4N 5

O

5

OH

2 H C 1 6HMN 5 0 2 3272 328.2 23 No. n X m R Formula 97 2 -CONH(0H 2 5

CO

2 Bn 98 3 -CONH(CH 2 6

CONH-

(0H 2 ),00 2 8n 99 3 -OONH(CH 2 6 C0NH-

(CHA)

5 0 2

H

100 3 -CONH(CH 2 5

CO

2 8n 101 .3 -CONH(CH 2 )S00 2

H

102 2 -SO 2

NH(CH

2 5 C0 2

H

103 2 -CONH(CH 2 )sNH-G-

OH

2 1

OH

2 1

OH

2 1

OH

2

I

OH

2 1

OH

2 1

CH

2 1 C23H3N 4

O

4 C3OH4N 4

O

5 H C23H4N 4

O

5 Calculated MoI. Wt.

428.3 544.4 454.3 417.3 327.2 349.2 547.4 FAB Mass sPec. fM+l-11 429.3 545.2 455.3 418.2 328.2 350.2 548.5 0* 0 ft..

0* 0* 0 v 000*** C3H35N3O 4

C

16 H29N 3

O

4 C0 14

H

27

N

3 0 5

S

C

24 H45N 5

O

7

'S

24 Table Examples of Group 11 (ii)

NHQ

(CH

2

X

H 2 N*Y NI, R 0 Calculated FAB Mass No. n X m R Formula 104 1 -OO(CHA) 6 0 2

H

105 1 -OO(CH 2 )rC0 2 Bn 106 3 -CO(H 2 4 00 2

H

107 3 *CO(CH 2 4 C0 2 Me 108 4 -CO(CH 2 5

NH

2 109 4 -CO(OH 2 3

NH

2 110 4 -CO(CH- 2 3

NHSO

2 fPp 111 4 -CO(CH 2 3 NHCOPfp 112 4 -C0(CH 2 3

NH.SO

2

CH

2

CF

3 113 4 CO(C0 2 11 NHC0-

(CH

2 6

NHZ

114 4 -CQ(CH 2 11

NH-

CO(CH

2 )rNH 2 CH~2'

OH

2

OH

2

OH

2

OH

2

CH

2

OH

2

CH

2

OH

2

CH

2 C 15

H

27 N30 4 C22H3N 3 0 4

C

1 5HV.N 3 0 4

C

16 H29N 3 0 4

C

16 H32N 4 0 2

O

14 H28N 4

O

2 C2OH 27

F

5

N

4 0 4

S

C

2 1 H2F 5

N

4

O

3

C

1 6 H29F3N 4

O

4

S

Mol. Wt. spec. [M+H1+ 313.2 314.3 403.3 404.3 313.2 314.3 327.2 328.3 312.3 313.3 2B4.2 285.2 514.2 515.2 478.2 479.2 430.2 431.3 H C 3 7 H63N 5 0 5 657.5 658.6

OH

2 H C29H5N 5 0 3 523.4 524.4 25 No. n X m R Formula 115 4 -CO(CH 2 5

NHCO

(CH

2 5

NHCO(CH

2

NHZ

116 4 -CO(CH 2 )5NHCO- (0H 2 5

NHOO(CH

2 5

NH-

2 117 4 -CO(CH 2 3 00 2

H

118 4 -CO(CH 2 3 00 2 Bn 119 4 .CO(CH 2 6

NH

2 120 4 -CO(CH 2 7

NH

2 121 4 -OO(0H 2 16 .Me 122 4 -CO(CH 2 )r-Gua 123 4 -S0 2

(CH

2 7

CH

3 124 4 CO(COI) 1

NH

2 125 4 -COCH 2

NHZ

126 4 -CO(CH 2 2

NHZ

127 4 CO(CH 2 )3"NHZ 128 4 -CO(0H 2 2

NH

2

CH

2

OH

2 1 H C2BHSN 6

O

4 H C36H-1 6

O

6

OH

2

OH

2

OH

2

OH

2

OH

2

CH

2

OH

2

OH

2

CH

2

CH

2

OH

2

OH

2 O 15

H

27

N

3 0 4 C0pH3N 3

O

4 o 1 7

H

34

N

4 0 2 ClSH36N 4

O

2 C29H5N 3

O

2

O

18 -13N 6 0 2

C

18

H

37

N

3 0 3

S

C22H4N 4

O

2 COH3ON 4

O

4 0211-H3N 4

O

4 O22H3N 4

O

4

C

12

H-

24

N

4 0 2 Calculated MCI. wt.

672.5 538.4 313.2 40:3.3 326.3 340.3 465.4 368.3 375.3 39,6.4 39%.2 404.2 418.3 256.2 FAE3 Mass S~ec. fM+Hj+ 673.6 539.4 314.3 404.3 327.3 341.3 466.4 369.3 376.3 397.4 391.3 405.3 419.3 257.2 26 X m R Formula 129 130 131 132 133 134 135 136

S

-CQ(CH

2 5

NHZ

-OOCH

2 -Gua

-OO(CH

2 2

NH

2

-CO(CH

2 2 -Gua

-CO(C.H

2 3 -Gua

-CO(CH

2 5 -Gua

-OO(CH

2 6

NH

2

-CO(CH

2 7

NH

2

CH

2

OH

2

OH

2

OH

2

OH

2

OH

2

OH

2

CH

2

O

24 H38N 4

O

4

C

13 H26N 6

O

2

C

13 H26N 4

O

2

O

14 H29NSO 2 O 1 5 H3()N 6

O

2

C

18 H33N 5 0 2 O 1 -1H3N 5

O

2 Calculated FAB Mass 446.3 447.4 298.2 299.3 270.2 271.3 312.2 313.3 326.3 327.3 35.3 355.3 351.3 352.4 365.3 366.3 27 Table 6 Examples of Group 11 (Iii) H 2N No. R M Y Calculated FAB Mass Forula Mol. Wt. spec. IM+H1-' 137 H -OCH 2 C0NH(CH 2 5 C0 2

H

138 H -OCH 2 00NH(CH 2 5 C0 2 8n 139 H -OCH 2 C0NH(CH 2 4 C0 2 8n 140 H -OCH 2 C0NH(CH 2 4 C0 2

H

141 CH 3 -00 H 3 142 CH 3 -0C 2

H

5 143 CH 3 -0(CH 2 5

CH

3 144 CH 3

-OCH

2 C0NH(CH 2 5

S-

C0 2 8n 145 CH 3

-OCH

2 C0NH(CH 2 5 SC0 2

H

CH

2

OH

2

CH

2 1 H C 15 H27N 3 0 5 1 H C22H 3

N

3 0 5 i H C21 H 31

N

3 0 5

OH

2 I H C 14 H25N 3 0 5 329.2 419.3 405.2 315.2 186.1 200.1 256-2 433.3 343.2 330.3 420.3 406.3 316.3 187.2 201.2 257.3 434.3 344.3

OH

2 CH 2

OH

2

OH

2

OH

2 CgH, 1 8

N

2 0 2 Cj 0 H2DN 2 0 2

C

14 H29N 2 0 2 CZ3H3N 3 0 5 I H C 16 H29N 3 0 5 28 Calculated FAB Mass NO. R X m Y Formula Ill.

146 CH 3

-OCH

2

CONH(CH

2 4

CO

2 8n 147 CH 3

-OCH

2

CONH(CH

2 4 C0 2

H

CH

2 1 H C22H3N 3

O

5

CH

2 1 H C 15 H27N 3

O

5 419.2 329.2 420.3 330.3 a 29 Table .7.

Example of Group III No. Structure Formula Cluae A is 0 NH(C.H 2 12 NH 0 O-WSe.[+j 148

H

2 N C32H5N 8

Q

4 614.4 615.4 00 30 Table 8 Specific examples of compounds A-B, containing amnide bond bicisosteres.

Calculated FAB Mass

A-B.

Formula Mol. Wt. spec. [M+HI+

S

SS 0

S

S. S 09 t: 0* 0**S

S.

S

S.

S S

S.

*5.e S S

CIIH

21

N

C 1 2 Ht N1 2 C 2

H,N

2

C,

1

H

2 ONiS 167.2 192.2 192.2 200.1 168.2 193.2 193.2 201.2 5

S

S S

H

2

N'

31 Table 9 Selected Ki values against OP-IV.

No. Ki (M) 2 6.4 x1 O- 7 7.6 x10-6 11 2-2 1.7 x10-9 23 5.0 x10 10 3.7 x108 38 9.8'x10-1 44 2.0 x10- 9 59 1.5 X10- 7 66 1.8xi10- 7 :97 5.0 x 10-10 *110 2.5X 1- 136 1.7 x10 4 143 9.4 x 10- 7 150 1.7 x10- 4 32 Schematic Representations for General Preparation of all Classes of Compounds Table 1 Compounds can be made by an adaption of the general route described by E. Schdn et al., Biol. Chem. Hoppe-Seyler, 1.991, 372, 305-311.

Table 2 R: CN x r Boc-A-OH. HN -1YNH 2 0 x PyBop K'' CH'2 Boc-A-N Nil 2 E t 3 N 0 x H--N CN POCd 3 pyridine, irnidazole x K Qn Boc-A-N -J1.

CN

X=S mCBA x Boc-A-N -KI

CN

(O)y 'On H-A-N

-JC

y 1, 2 33 R: -CEI=NPh Boc-A-ONSu HN OH x Boc-A-N y0 x K Boc-A-N

OH

DMPT

CH

2 C1 2 hNH- 2 Toluene, A R: CH=N OR'

()R'ONH

2 HC1 pyridine, DMIF Boc-A-N H-A-N Boc-A-N N Ho.- H-A-N For R Ac Py, AC 2 0 K (H C 2 C1 2 b-Bc-- N OAc (RI =H) x N N- OAc R -C-=CR Ph 3 P, CBr4 Zn, CH 2

CI

2 x Boc-A-N __Br Br x nBuLi KO n (ii) C R (iii) H+ Table 3 j0 R= -B R CHO

H

Prepared by method of: W.W. Bachovchin et al., J. Biol. Chem., 990, 265, 3738-3743.

H-A-N

CHO~

34 Table .4 P Protecting groups; P 1 p 2 Groups as described in corresponding tables) R =CN_

OP

OH

Boc-N ft

,NH

2 Boc-N' ft PyBop, CH 2 Cl 2 Et 3

N

remove P (ii) HONSu, WSCD f a 0*0* /JNH(CH 2 p2 (1 X

H-

2

N(CH

2 )mP' (ii) modify PI P 2 if required Boc-N H1 0 ONSu 0. x 00 0 A

NH(CH

2 {pyridine, irnidazole H(CH- 2 )mp 2 On x Boc-N

J'FN

S 0 2 C1 01 x Boc-N N A 0

H+

On x

H

2 N "rN

CN

0

SO

2

NH(CH

2 )mP 2 On Boc-N KrN- 1

R

H

(ii) Modify PI p 2 if required (IV) complete synthesis as above (IV) was prepared via method of G. Luisi et al., Ter. Lett., 1993, 34, 239 1-2392.

For R H, modify above procedure as described for Table 1 examples.

35 Table R =CN ()n

OH

Boc-N--

H

HN -,-yNH 2 0 PyBop, CH- 2

C

2 Et 3

N

NlHW )n floc-N

H,

NH

2 a a.

ab a ~e.

a.

a. a Remove W 0 (ii) P(GH 2 M)I. ONSu P(CH7.mSO 2 C1 (for sulphonamide /NHCO(CH2)P Boc-N N NH- 2 00 modify P P' if. required GOi POC1 3 pyridine, irnidazole x N

CN

NHCO(CHTI4'

H'

2 N )"rNA 0 R H, modify above Procedure as described for Table 1 examples.

36 Table 6 Use method described for Table 5 examples for preparation of (VI) from (V) R OW

OH

Boc-N R' OH Boc-NN

MV)

*6

S

*4 Nail (ii) RI Br (iii) H+

R

H

2

N

Y CN, -C=N~h, -C=NOR,

_-CR

2

(VI)

Nail (ii)

BOH

0 r OCH3 R 0 Boc-N)r H4 0 0

NH(CH)

7

,P

LiOH, H 2 0, dioxafl (ii) H.

2

N(CH

2 )mP, PyBop (iii) H+ R- -0

H-

2

N

37 Table 7 Standard coupling, dehydration and deprotection sequence siiliar to abovc~schernes.

/1-ONSu On x Boc-Nj"rN-

NH

H n

(III)

S

0.5 molar equivalent

H-

2

N(CH

2 )m1{H 2

NH(CH

2 )mNH o )n BocNH N Boc-N 2 H 00 0 0 IG() POC1 3 pyridine (ii) H 4 0

A-M(CH

2 ).Nm )n

H-

2 N H2 CN

CN

38 Table 8

R

Boc-N j 14 0 PPh 3 Toluene, R H+ 0 reflux NH-Boc Nil 2

R

H-

Boc-N H 0 (EtO), 2

POCN

LiCN, DMF (ii) SrnI 2

THF,

t BuOH 11 0 PPh 3 Toluene, reflux NII-Boc NH-Boc

NH

2 Thioarnides were prepared by the method described by K. Clausen et al. Tetrahedron, 1981, 37, 3635-3639. Other axnide bioisosteres can be prepared from literature precedent.

Spatola in "Chemistry and Biochemistry of Amino Acids, Peptides and Proteins", Vol. II, B. Weinstein Ed., Marcel Dekker, New York, 1983, p. 267).

39 Experimental Details for Specific Examples EXAMLE 1 2-(S)-Cyano-1-isoleucylpy-rolidine (U1) H-fle-

CN

Di-isopropylethylainine was added to a solution of H-ProNH 2 HC1 (225 mng, 1.50 mmol) in dry CH- 2 C1 2 (15 cm 3 until the pH was adjusted to 9. *BocfleONSu 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 3 and 0.3 N KHS0 4 solution (10 cm 3 The organic layer! was further washed with saturated NaCHO 3 solution (10 cm 3 water (10 cm 3 and brine cm 3 The solution was dried (Na 2

SO

4 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 mng of BocfleProNH 2 as a colourless foam.

'IH NNM (CDCl 3 8 (ppm); 6.90 (1H, br.s); 5.51 (1H1, br.s); 5.18 (1H, di, J 9.6 Hz); 4.62 (1H, dd, J 2.6, 7.0 Hz); 4.29 (111, dd, J 8.4, 9.2 Hz); 3.79 3.58 (2H, mn); 2.36 (lH, mn); 2.09 1.57 (5H, in); 1.43 (9H, 1.17 (1H, in); 0.95 (3H, d, J 6.6 Hz); 0.90 (3H, t, J 7.3 Hz).

Imidazole (84 mg, 1.24 mnol) was added to a solution of BodfleProNH 2 in dry pyridine (10 cm 3 under a nitrogen atmosphere. The solution was cooled to -35TC, before -the dropwise addition Of POC1 3 (0.25 cm 3 2.48 minol). The reaction was stirred at -30'C to for 60 min. The solution was then evaporated and the crude residue subjected to column chromatography (silica gel) to yi eld 180 mg of 2-(S)-cyano-1-[N-(t-butoxycarbonyl) isoleucyl]pyrrolidine as a colourless oil.

IH NMR (CDC1 3 8 (ppm); 5.14 (1H, d, I 9.2 Hz); 4.80 (1H, dd, I 2.6, 7.1 1h); 4.22 (1H, dci, J 7.9, 9.1 Hz); 3.81 (IH, mn), 3.71 (IH, mn), 2.30 2.12 (4H, mn); 1.75 (111, mn); 1.60 (li, mn); 1.42 (9H, 1.19 (1H, in); 0.97 (3H, d, J 6.9 Hz); 0.91 (3H, t, J =7.3 Hz).

1 3 C NMR (CDCI 3 6 (PPM); 171.7, 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.

40 Deprotection was carried out by stirring with triflujoioacetic acid for'60 min. Evaporation and lyophilisation from water afforded 60 m .g of -2-(S)-cyano- 1-isoleucylpyrrolidine (11) as a white, fluffy solid.

FAB Mass Spec: Calculated 209.3, Found 210.2.

IH NMR (D 2 5 (ppm); 4.3. (1H1, in); 3.64 (1H, d, J 5.6 Hz); 3.16 (2H, mn); 1.86 1.48 (5H1, mn); 0.98 (1H, mn); 0.68 (1H, in); 0.51 (3H, d, J 6.9 Hz); 0.38 (3H1, t, J 7.3 Hz).

13 NNvR (D 2 5 (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-Glu[NH(CH 2 7

CONII(CH

2 3 NHZ]pyrrolidide (64) 0 NH(CH 2 7 CONI1(CH 2 3

NIIZ

NO

H

2

N

0 Di-isopropylethylamine was added to a solution of BocGlu(OH)pyrrolidide (193 mg, 0.64 mmrol) and PyBop (500 mg, 0.96 inmol) in CH 2

CI

2 (6 cm 3 to adjust the pH of the mixture to 9. After stirring for 5 min, a solution of benzyl 8-amnino-octanoate (220 mg, 0.77 rmnol) in CH 2

CL

2 (5 cm 3 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 to 3% methanol in ethyl acetate) to obtain 344 mg of BocGlu(NH(CH 2 7

CO

2 Bnlpyrrolidide as a colourless solid.

'H NMR (CDC1 3 65}ppm); 7.35 (511, 6.63 (1H1, br. t, I 6.7 Hz); 5.65 (1H, d, J 8.3 Hz); 5. 11 (2H1, 4.36 (111, dt, J 2.6, 8.9 Hz); 3.55 3.20 (6H1, mn); 2.34 (2H1, t, J 7.3 Hz); 2.26 (2H1, dd, J 5.6, 7.3 Hz); 2.11 1.48 (1011, mi); 1.43 (9H1, 1.32 1.27 (6H, mn).

41 Hydrogen gas was bubbled through a solution of BocGlufNH(CI 2 7

CO.

2 Bnlpyrrolidide (230 mg, 0.43 inmol) in ethyl acetate (10 cm 3 containing 10% palladium on charcoal mng). 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 of BocGlu(NH(CH- 2 7

CQ

2 H]pyrrolidide as a colourless oil.

Di-isopropylethylainine was added to a solution of BocGlu(NH(CHi2)CO 2 Hilpyrrolidide (125 mg, 0.28 mmol) and PyBop (221 mg, 0.43 rnmol) in CHi 2 C1- 2 (10 CM 3 to adjust the pH of the solution to 9. After stirring for 5 min, a solution of ZNH(CH 2 3

NH

2 HCO mg, 0.37 mmol) and di-isopropylethylamine (38 mg, 0.37 mnrol) 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 to 15% methanol in ethyl acetate) to afford -151 mg of ::BocGluIN(CH) 7 CONHi(CH 2 3 NT1Z]pyrrolidide as a colourless oil.

'H NMR (CDCl 3 5 (ppm); 7.35 (5H, 6.60 (IH, br.t, J 7.2 Hz); 6.14 (1H, br.t, J 0* 7.2 Hz); 5.63 (1H, d, J 8.3 5.39 (11, br.t, J 5.6 Hz); 5.10 (2H, 4.38 (1H1, dt, J 2.3, 9.2 Hz); 3.52 3.13 (10H, in); 2.26 (2H, t, J 6.9 Hz); 2.17 (2H, t, J 7.6 Hz); 1.98 1.48 (12H, in); 1.44 (9H, 1.38 1.23 (6H, in).

A solution of BocGlu[N(CH 2 7

CONH(CH

2 3 NHZ]Pyrrolidide (14 mg, 0.022 inmol) in 4N ECI/dioxan was stirred for 45 min. The solvent was evaporated and the residue dissolved in water, filtered and lyophilised to yield 13 mg of H-Glu(NH(CH 2 7 CONH(CH-2)NHZ]pyrrolidide (64) as a colourless oil.

FAB Mass Spec: FA.B ass pec: Calculated 531.3, Found 532.3.

42 EXAMPLE =HE H-Lys(CO(CH 2 3 N1{SO 2 Pfp]pyrrolidide (110) F F NHCOX NHS 2 F F F

NO

'00:0,HIN 0

ZNH(CH

2 3

CO

2 NSu (570 mg, 1.7 inmol) was added in one portion to a solution of 1-[N-(t-butoxycarbonyl)lysyl]pyrrolidine (745 mrg, 2.2 inmol) in dry CH 2 Cl 2 ThepH was adjusted to 9 with di-isopropylethylamine and the mixture stirred for 60 min. The solvent was evaporated and the residue treated in the standard procedure as described for example one. Column chromatography (100% ethyl acetate to 1596 methanol in ethyl acetate) afforded 620 mg of BocLys[CO(CH7.) 3 NIIZ]pyrrolidide.

IH NMR (CDCl 3 8 (ppmn); 7.42 (5H, 6.31 (lH, br.t, J 6.5 Hz); 5.58 (1Hi, d, J 8.9 Hz); 5.39 (1Hi, br.t, J3 6.9 Hz); 5.17 (2H, 4.44 (1H, in); 3.72 3.20 (8H, in); *2.29 (2H, t, J =7.3 Hz); 2.14 1.83 (8H, in); 1.78 1.41 (4H, mn); 1.43 (9H, s).

Hydrogen gas was bubbled through a mixture of BocLys(CO(CH 2 3 NHZ~pyrrolidide (620 mrg, 1.16 mrmol) and 10% palladium on charcoal in methanol (10 cm 3 containing one molecular equivalent of 2N HC. After 60 min, the reaction was flushed with nitrogen, and filtered. through celite. Evaporation of the solvent afforded 282 mg of BocLys(C0(CH-2)NH 2 HCI]pyrrolidide. This product was dissolved in CH 2

CI

2 (10 cm 3 and stirred, under a nitrogen atmosphere. Di-4sopropylethylainine was added to adjust the pH to 9 before the introduction of pentafluorobenzenesuLfonyl chloride (45 mrg, 0.17 mmcl). This mixture was stirred for 16 h. The solvent was evaporated and the crude material treated in the standard procedure described in example one. Column chromatography (100% ethyl acetate to 10% methanol in ethyl acetate) afforded 33 mg of BocLys(C(CH-) 3 NHS0 2 Pfp]pyrrolidide as a colourless oil.

43 'H NMR (CDC1 3 8 (ppm); 7.19 (1H, br.t, J 6.3 Hz); 6.18 (1H, br.t, J 6.6 Hz); 5.50 (1H, d, J 8.4 Hz); 4.38 (1H1, mn); 3.65 3.16 (8H, in); 2.36 (2H, t, J 6.8 Hz); 2.01 1.82 (8H, mn); 1.69 1.41 (4H, in); 1.43 (9H1, s).

This product was stirred in trifluoroacetic acid (10 cm 3 for 30 rmin. The solvent was evaporated and the residue dissolved in water, filtered and lyophilised to yield 30 mng of H-Lys(CO(CH 2 3

NHSO.

2 Pfp]Prl (110) as a colourless oil.

IFAB Mass Spec: Calculated 5 14.2; Found 515.2.

EXAMPLE FOUR H-Thr((CH- 2 5

CH

3 ]pyrrolidide (143) 0

H

2 N

N

Pyrrolidine (0.88 g, 12.4 mrnol) was added to a solution of BocThrONSu (3.0 g, rnmol) in dry CH 2

CI

2 (30 cm 3 under a nitrogen atmnosphere. The reaction was stirred for min at room temperature. The solvent was evaporated and the residue was treated in the standard procedure as described for example one. The residue was subjected to column chromatography (hexane:ethyl acetate, 30:70) to afford 2.50 g of 1-(N-(t-butoxycarbonyl)threonyl]pyrro~infe as a colourless oil.

IH NMR (CDC1 3 8 (ppm); 5.52 (1H, d, J 6.5 Hz); 4.30 (II, 7.4 Hz); 4.16 (2H, mn); 3.72 (1H, mu); 3.46 (3H, mn); 1.98 1.82 (4H, rn); 1.43 (9H1, 1.19 (3H, d, J 7.1 Hz).

44 Sodium hydride (17 mng, 0.70 mmol) was added to a solution of 1-(N-(t-butoxycarbonyl) threonyllpyrrolidine in dry THF, at 0 0 C, under a nitrogen atmosphere. The mixture was stirred at 0 0 C for 15 min before the introduction of n-hexyl iodide (200 mng, 0.94 mmcl).

The reaction was then allowed to stir at room termperature for 16 h. The solvent was evaporated and the residue treated in the standard manner as described in example one.

The crude product was subjected to column chromatography (hexane:ethyl acetate, 40:60) to afford 25 mng of BocThr[(CH 2 5 CHi 3 ]pyrroidide (143).

1 H NMR (CDC1 3 5 (ppm); 5.50 (1H, d, J 6.9 Hz); 4.48 (111, mn); 3.70 3.32 (711, in); 1.92 1.80 (6H, mn); 1.52 (211, in); 1.42 (9H, 1.30 (6H, mn); 1.22 (8H, di, J 6.9 Hz); 0.83 (3H, t, J 7.9 Hz).

BocThr[(CH 2 5

CH

3 ]pyrrolidide (20 mng, 0.06 mmiol) was stirred in 4N HCI/dioxan (5 cm 3 :for 60 min. The solvent was evaporated, the residue taken up in water, filtered and lyophilised to yield H-Thr[(CH 2 5

CH

3 ]pyrrolidide (20 mng) as an orange oil. The product was purified by reverse phase HPLC to afford 15 mng of' (143) as a colourless oil.

FAB'Mass Spec: Calculated 256.2, Found 257.3.

EXAMPLE FIVE H-fle-x(GH=Qi]JPyrrolidide (149) Nil 2 1.6 N nButyl lithium (0.50 cm: 3 0.76 inmol) was added to a stirred solution/of cyclopent3'l triphenyphosphonium bromide (287 mng, 0.69 minol) in dry THF (6 cm 3 under a nitrogen atmosphere, maintaining the temperature at -30*C. After stirring for 60 min, the solution was fur-ther cooled to -50*C subsequent to the dropwise addition of a solution of N-(t-butoxycarbony I)-L-isoleuciflal (125 mg, 0.58 mmol, prepared by the method of.

Fehrentz and Castro, Synthesis, 1983, 676), in dry TH-F (4 cm 3 After the final addition, the reaction was allowed to slowly attain room temperature, over 3.5 h.

45 The reaction was quenched. with saturated ammronium chloride solution (2 cm 3 This was diluted with water (10 cm 3 and extracted with diethyl ether (Q x 20 c 5 The combined ethereal layers were washed with water (10 cm 3 dried (Na.

2

SO

4 and evaporated to yield 187 mng of crude product. Column chromatography (90:10, hexane:Et 2

O)

afforded 53 mg of Boc-Ile-W4CH=CH~pyrrolidide as a colourless oil.

1H NMR (GDC1 3 8' (ppm); 0.84 (3H, t, J 6.9 Hz); 0.91 (3H d, J 7.3 Hz); 1.08 (1H, mn); 1.44 (9H, 1.48 (1H, in); 1.64 (5H, in); 2.24 2.45 (4H, mn); 4.08 (1H, br.s); 4.41 (1W, br.s); 5.12 (1H, dt, I 2.3, 8.9 Hz).

1 3 C NMR(CDCI 3 a (PPM); 155.8, 147.4, 119.1, 79.2, 54.8, 4.0.1, 34.2, 29.6, 28.9, 26.8, 26.6, 26.1, 15.0, 12. 1.

Treatment of this product with 4N HC1/dioxan for 35. min removed the Boc-protecting group. The reaction was evaporated, the residue dissolved in water, filtered and lyophilised to yield 24 mg of H-Ile-iVtICH=CH~pyrrolidide (149) as a foamy solid.

FAB Mass Spec: Calculated 167.2, Found 168.2.

EXAMPLES SIX ANT) SEVEN H*e R-yaom(HC~proiie (150) :::.H-fle((2S)-cyano-WV(CH=CH)pyrTolidideI .(151) CN

CN

NH

2 iNm 2 N-(t-Butoxycarbonyl)-L-isoleuciflaI (2.40 g, 11.2 mmol) and 2-oxy-1-tripheflylphosphoranecyclopentafle (4.61 g, 13.4 inmol, prepared by method of H.O. House and H.

B abed, 1. Org. Chem., 1963, 28, 90) were heated, at reflux, in toluene, under a nitrogen atmosphere. After 15 h, the mixture was cooled, and the solvent evaporated. Column chromatography (80:20, hexane:ethyl acetate) of the crude residue afforded 2.33 g (74%) of Bocfle-V(CH=CHIipyrrclidifl-2-ol as a colourless oil.

46 'H NMR (CDC13), 5 (ppm); 6.29 (1H, dt, J 2.6, 9.2 Hz); 4.59 (1H, br.d); 4.17 (1H, 2.82 (1H, 2.66 2.50 (2H, 2.34 (2H, t, J 7.8 Hz); 1.96 (2H, q, J 7.6 Hz); 1.44 (1H, 1.43 (9H, 1.12 (1H, 0.89 (3H, d, J 5.3 Hz); 0.88 (3H, t, J 6.9 Hz).

Diethylcyanophosponoacetate (0.30 cm 3 1.92 mmol) was added to a solution of Boclle-/[CH--CH]pyrrolidin-2-one (180 mg, 0.64 mmol) and LiCN (0.5 M in DMF, 3.84 cm 3 1.92 mmol) in dry DMF (2 cm 3 under a nitrogen atmosphere. The reaction was stirred at room temperature for 30 min. The mixture was diluted with water (20 cm 3 and then extracted with ethyl acetate (2 x 30 cm 3 The combined organic layers were washed with water (5 x 10 cm 3 dried (Na 2

SO

4 and evaporated to afford 360 mg of S crude product A portion of this crude cyano-phosphonate (284 mg, 0.64 mmol) was dissolved in dry THF, and stirred under nitrogen. tert-Butanol (47 mg, 0.64 mmol) was added, followed by the dropwise addition of a solution of samarium (II) iodide (0.1 M in THF, 19.2 cm 3 1.92 mmol). After the final addition, the reaction was stirred for a further min before the addition of 2N HC1 (20 cm 3 The mixture was extracted with diethyl ether (3 x 30 cm 3 The combined ethereal layers were washed with 10% Na 2

S

2 03 solution (10 cm 3 water (2 xlO cm 3 and brine (2 x 10 cm 3 The solution was dried

S.

(Na 2 S0 4 evaporated and the crude residue subjected to column chromatography (90:10, hexane:ethyl acetate) to yield 122 mg of a diastereomeric mixture of Boclle[2-(RS)-cyano-v(CH=CH)pyrrolidine] as a colourless oil.

1 H NMR (CDC13), 5 (ppm); 5.52 (1H, d, J 9.6 Hz); 4.5 (1H, br.s); 4.12 (1H, m); 3.35 (1H, 2.57 (1H, 2.38 (1H, 2.17 (1H, 1.91 (2H, 1.69 (2H, m); *1.53 (1K, 1.43 (9H, 1.12 (1H, 0.92 (1.5 H, d, J 7.3 Hz); 0.91 (1.5 H, d, J ,o 7.3 Hz); 0.89 (1.5 H, d, J 6.6 Hz); 0.86 (1.5 H, t, J 6.9 Hz).

Treatment of this diastereomeric mixture with 4N HCI/dioxan for 60 min removed the protecting group. Evaporation of the solvent and subsequent reverse phase HPLC of the residue afforded the two pure diastereomers.

(150), (47 mg, 60%) FAB Mass Spec: Calculated 192.2, Found 193.2 (151), (28 mg, 36%) FAB Mass Spec: Calculated 192.2, Found 193.2.

Preparative methods described herein in relation to Tables 1 8 and in examples one to seven form part of the present invention.

-47- Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising" or the term "includes" or variations thereof, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. In this regard, in construing the claim scope, an embodiment where one or more features is added to any of the claims is to be regarded as within the scope of the invention given that the essential features of the invention as claimed are included in such an embodiment.

S

S.

*0 -48- Boc Bn

BSA

nBu Ch

DMF

DMP

EDTA

FAB

Gua

IHPLC

"lix Mass Spec mCPBA Mol Wt ONSu Pfp Ph pip Pri Py PyBop ws CD z Abbreviations tert-Butyloxycarbonyl Benzyl Bovine serum albumin n-Butr'l Cyclohexyl Dime thylfonziamide Dess-Martin Periodane Ethylenediaminetetraacetic acid Fast atom bombardment Guanidinyl High performance liquid chromatography n-Hexyl Mass spectrome try rneta-Chloroperbenzoic acid Molecular weight N-O-Succinimide Pentafluorophenyl Phenyl Piperidyl.

Pyrrolid ide Pyridine Benzotriazole-l-yl-oxy-tris-pyrrolidinlo-phosphoflium hexafluorophosphate Water soluble carbodiimide Benzyloxycarbonyl

Claims

1. Inhibitors of DP-IV mediated processes selected from those of general formula A-B (Group I) where B is -Y n 1 or 2; m 0, 1 or 2; X is CH 2 S, SO, SO 2 O, or -Y -N; @0 R is H, CN, CHO or B(OH) 2 and when R is H, A is an a-amino acyl group having a C4-C1o cycloaliphatic side chain or is a P-amino acyl group of S* the general formula CH- and 0 where p is 1 to 6, the ring may also contain one or more heteroatoms replacing CH 2 unit(s); 0. when R is CN, A is as defined at and in addition may be derived from any L-a-amino acid bearing a C,-C, 0 alkyl side chain; and when R is CHO or B(OH) 2 A is a B-amino acyl group as defined under

2. Inhibitors according to claim 1, wherein B is a ring, n is 1; X is CH 2 or S; and R is CN. -0 j 50

3. Inhibitors of DP-IV mediated processes selected from those of general formula A-B (Group II). where B is Y B J1 n =l1or 2;R 0 4 m 0, 1 or 2 *X is CH 2 0, S, SO, or SO 2 Y -N; R is H or CN, A is attached to Y; and A is whr a. is wh R4 is 1-5; C2 -o H- C2 1weec s11,adZi 000 atce coprs is chai(n 2 orN (CHto whre c is 1-2,ani)i CO orCH 2 2 i OHo C oCN serteef OHCN 5 6 0 0 0 0) 00 where R1 is H or Me, 1 51 E is 4 )q-R 3 J is CO; and a, b, q, R 3 and R 4 are as defined under or A is (iii) H 2 N R, (iii) CO OL where R, is H or Me, L is (CH)d-(CO),-(CH 2 )b-(R 4 )q-R3 where r is 1, d is 1-4, and b, q, R 3 and R 4 are as defined under

4. An inhibitor of a DP-IV mediated process according to claim 1 selected from Examples 1 152 of Tables 1 to 8 herein.

5. The use of a compound according to any of claims 1 to 4 for the preparation of a medicament for inhibiting DP-IV mediated processes.

6. A method of treating or preventing disorder due..to a DP-IV mediated process in a patient, which comprises administering to the patient a DP-IV inhibiting amount of compound according to any of claims 1 to 4.

7. A pharmaceutical composition containing a DP-IV inhibiting amount of compound according to any of claims 1 to 4. 52

8. Compounds of any one of Claims I to 4 ,compositionS containing same and methods for their preparation substantially as hereinbefofe described with reference to the Examples. DATED this 10th day of May, 2001. FERIRING BV By Its Patent Attorneys DAVWES COLLISON CAVE