CA2090858A1 - Kininogenase inhibitors - Google Patents

Abstract

Kininogenase inhibitors, optimally not exceeding the size of a hexapeptide, represented by (II), wherein A and B = amino acyl (including amino acyl analogue) the same or different forming a dipeptide group the amino acid of A carrying a terminal group and being any amino or imino-acid residue (but preferably of D-configuration) and of B being a lipophilic amino-acid residue of D- or L-configuration but not proline or a proline analogue, or a conformational analogue of said dipeptide group wherein the peptide link is replaced by -CH2-NH- ('reduced'), -CH(OH)-CH2- ('hydroxy'), -CO-CH2 ('keto'), -CH2-CH2- ('hydrocarbon') or other conformational mimic of the peptide bond and in (III) the side chain R1 is that of a basic amino acid or amino acid analogue (preferably of L-configuration), and R is H or lower alkyl(C1-C4) or C.alpha. or the peptide link comprising -N(R)- is replaced leading to a conformational mimic as above; Y = a binding enhancing or carbonyl activating group preferably selected from H (when A or B
must be cyclohexylalanine, preferably D if at A or L if at B) or alkyl (C1-C20) or fluoroalkyl (C2-C12); substituted oxymethylene; thiomethylene; sulphoxymethylene; sulphonylmethylene;
aminomethylene; hydrazino-methylene; -CH2-Het (where Het = a substituted or unsubstituted heterocycle); substituted amino (but when the resulting compound is a secondary alkylamide B must not be phenylalanine); an amino-acid group or its ester or amide; a carboxylic secondary amide or primary amide, when B must be cyclohexylalanine or adamantylalanine or other bulky lipophilic, non-aromatic amino-acid (not Ala Leu Ile Val Nva Met Nle Phe Tyr Trp Nal (1)); tertiary-carboxamide; carboxy-alkyl group or its ester or amide or amino acyl derivative.

Description

WO 92/0437~ 2 ~ 9 ~ 3 ~ 8 PCJ/GB91/0147~

KI~iIliOGENASE INHIBITORS
FIELD OF INVENTION

The invention relates to enzyme in~i~it c-. ?n~ _^
treatment of disease.

BACKG~OUND - KININS

Kinins are natural vasoac~ive pep.ides !iberated n ~:~e body from high molecular weigh_ precursors (kinincgens) -y ~he act.ton of selective proteases known as kininoaenases.

There is evidence for the involve~en- of kinir.s ir. _he following pathological states:

(a) Conditions associated with vasodilatat on and hypotension, e.g. septic, anaphylact.c and hypovolaemic shock; carcinoid syndrome and dumpinq syndrome (b! Condition~ involving inflammation, e.g. acute ar.hritis, pancreatitis, local thermal injury, c-ush in -y and brain oedema (C) Conditions involving bronchoconst-i^~ on, especially for example the initial, acute aiie_sic reaction in asthma (d) Allergic inflammation, particularly allergic rhinitis and conjunctivitis, together generally known as hay fever, and the bronchial inflammation and consequent occlusion found in the non-acute but serious and even fatal inflammatory phase of asthma.

The kinins (bradykinin, kallidin and Met-Lys-bradyk -.in) are potent mealators of inflammation. Their main actions a-e as follows:

(a) They increase capillary permeability which leads to exudate formation and oedema (b) They are potent vasodilators in arterioles and therefore reduce blood pressure and increase blood flow SIJ'E3~T~,'JT~ SHEET

W092/04371 2 ~ 9 ~ 8 ~ 8 PCT/GB91/0147 ' 2 (c) They induce pain (d) They contract bronchial smooth muscle (e) They activate pAospholipase A2 and ~hus s~imuiate the biosynthesis of prostaglandins (PG'C: wni^h mediate some of their ac~ions.

In reaard to prostaglandins, i. mav ~e noted th2t ~e_~ain ac~ ons of ~inlns, particularly pain and vascular ~er~eaDil .y above, are potentiated by PG's, although PG's Ihemselves do not cause pain nor do thev induce vascular ?erDeabil _y Zt -h2 concen~~ationS found in inflamed tissue. PG's .herefo-e 2C=
as either mediators or potentiators of kinins.

In spite of the above knowledae of ~inins and .he~~
actions, relatively little attention has been paid eo reduction of their action. In asthma treatment for exampie clinical attention is primarily directed to the acute bronchoconstrictiVe reaction, for which there are effective drugs. Deaths continue to occur f~om the gradually developing bronchial occlusion, and at present not only are there no clinically effective inhibitors of kinin release available but the concept of kinin release inhibition, at least in treating allergic inflammation, appears to be new. The only substance that is in fact a kinin release inhibitor and has at_ained clinical significance is aprotinin ('T-asylol', Bayer, trade mark), a proteinase inhibitor isolated from bovine tissues (lungs, lymph nodes and pancreas). It is a strongly basic protein (pI = 10.5) of MW = 6,500 comprising a single peptide chain of 58 residues. However, aprotinin is primarily a trypsin inhibitor (Xi = lO l3M) and is some l06-times less active against kinin release. It has been found marcinallv beneflcial in acute pancreatitis, a serious condition, where it inhibits the activation by trypsin of zymogens or pançreatic serine proteinases, and in traumatic - haemorrhagic shock. Aprotinin has to be administered parenterally, and it freauently produces a painful reaction at the injection site.

CUESTiT'~"_ SnEET

2 ~ PCT/GB91/~147g BACKGROUND - KININOGENASES

The kininogenases are serine proteinases, that is to say proteinases in which the hydroxy group of a serine residue is the nucleophile involved -in forminq the substrate transition state. They liberate the ~inins (bradykinin, kallidin) from the kininogens by limited proteolysis. There are severa kinds of ~inino~enase:-(a) Tissue kallikrein (TX, also called glandular kallikrein GT or urinary kallikrein UX) which is found in the pancreas, salivary glands, intestines, kidney and urine. It has MW = 30,000 and acts preferentially on low molecular weight kinino~en (LMWX) to release the kinin kallidin (KD). Tissue kallikrein has no potent and fast acting endogenous inhibitor present in plasma.

(b) Plasma kallikrein (PK) occurs in plasma as an inac_ive zymogen which is activated by Factor XIIa, and is part of the intrinsic coagulation cascade. It has MW
= l00,000 and its preferred substrate is high molecular weight kininogen (HMWK) from which it releases bradykinin (BK). Plasma kallikrein is rapidly and effectively inhibited in plasma, by endogenous inhibitors known as cl-inactivator and ~2-macroglobulin.

(c) Mast cell tryptase which, while not as ac~ive as the kallikreins in kinin release, we have found to occur in large amounts in the mast cells of the lung tissue of asthmatics.

BACXG~OUND - XININOGENS

The kininogens which are the natural substrates for the kininogenases (they act also as potent inhibi~ors, Xi approx.
ll, of cysteine proteinases such as cathepsins B, H and L, calpain ~nd papain) occur in two types:

~;UBSTi . 'J I - Sl :,E~

W092/04371 PCT/GB91/0147~

(a) Low molecular weight kininogen (L.~WK~ ~ith moiecula~
weight in the ranae _O,000 - 7G,~00 dependir.g on species of o-igin and degree of glycosyi~~ion.

(b) High molecular weight .~ininogen (~MWK) Wl-~.
~olecular weight in the ranqe 88,000 ~ ,OOO whic:-, in addi_ion to serving as an alternative precurso- of ~ .s and a cysteine proteinase inhibito~, aisc plays ~n obligatory -ole with plasma ~al~ rein in _he initia~ 0?.
o- ~he ~nt-inisic coaaulation cascade.

The two kininogens, whose mRNA's are transc-i~ed f-om the same gene, have identicai ?ri~arV secuences throughout -he ~-ter~inal or heavy chain (H-chain) region, the kinin region ana the fi_st twelve amino acids of the C-terminal or light chair.
(L-chain). At this point thei- s~ cl~lres dive ge, HMWK
having a lonaer L-chain (MW approximately 45K) than LMWK
(~.8K).

The cleavage of human HMWK by plasma kallikrein is for example shown schematically in Fig. i, with details of the sequence at the cleavage sites in Fig. 2 and a ~ore detailed sequence in Fig. ~ where _he conven~ional nu~Dering _--esidues adajcent to a cleavage site is shown for cleavaae cite I. After excision of one or other kinin sequence, the H- and L-Chains are held together by a single disulphide bridge:-SU5STIIU T ~ SHEET

Wo 92/04371 5 2 ~3 ~ v 8 PCl/GB91/01479 S - S , ~ S -S
H-chain 3K ~ L-erlaIc ~MWK ~1 OOK

P~ i00~;

S - S ----~ - S
H-chain L-cna s 3~, -lK
~- isure ' . Clea~ ce ~~ ~ ir'~ _ . ?~ O~!e__l _^ne.~ne PK PK
TK ~--Cleavage site II TK
-ile-Ser-Leu-l~et, Lys, Arg-Pro-Pro-Gly-2he-Ser-Pro-Phe-Arg,Ser-Ser-.~rg-Iie-Gly-~' 380 ~" Bradykinin ~' 3~
Cleavage I , C;eavage site I
site III ~ ~ Kallidin .' Figure 2. Cleavage of human kininogens by PK and TK: De~ails of sequence PK,TK

Pj P4 P3 P2 P~?2 P3 P~ P~
-Phe-Ser-Pro-Phe-Arg Ser-Ser-.~rg-Ile--Gly-38 j 389 390 394 r igure 3 . Sequences flanking ciea~, age site I i human H.~IWK

~iU~TlTUTE SHEET

W092/04371 2 ~ S ~ ~ 5 8 P~r/GBgl/0l47~J

As shown, plasma kallikrein and tissue kallikrein act at a single site to free the kinin C-terminal site, cleaving between residues 389 and 390, but at sites one residue apar~, either side of residue 380, to free the N-terminal of bradykinin (by PK) or kallidin (by TK).

The role of PK and HMWK as clotting factors in _he intrinsic cascade does not involve the enzymatic release of Xinins. However many of the ef fects of PX and all those o' TK
do involve such release, being mediated by the kinins released from the respective substrates HMWK and LMWX through selective proteolysis.

INDICATIONS

The main clinical indications for kininogenase inhibitors are inflammatory conditions, particularly allergic inflammation (e.g. asthma and hay fever). A fuller list of indications is given below:

(l) Allergic inflammation (e.g. asthma, rhino-conjunctivitis [hay fever~, rhinorrhoea, ur icaria (2) Inflammation (e.g. arthritis, pancreatitis, gas~ritis, inflammatory bowel disease, thermal injury, crush injury, conjunctivitis) (3) Smooth muscle spasm (e.g. asthma, angina) (4) Hypotension (e.g. shock due to haemorrhage, septicaemia or anaphylaxis, carcinoid syndrome, dumping syndrome) (5) Oedema (e.g. burns, brain trauma, angioneurotic oedemc whether or not as a result of treatment with inhibitors of angiotensin converting enzyme) SyBCTl~u,_ SHe:ET

W092/04371 2 ~ 8 PCT/GB91/0147~

(6) Pain and irritation (e.g. burns, wounds, cuts, rashes, stings, insect bites) STATEMENT OF INVENTION

In one aspect the invention provides a method Gf treatment (including prophylactic treatment) of an inflammatory or other condition se~ out 'n the indicat ons above, particularly an allergic inflammatory condition, wherein an effective amoun~ of a pep~ide or peptide-anaiogue kininogenase inhibitor is administered topically or systemically to a patient suffering from or at risK of the condition. It is believed that for optimum activity, administrability and stability in the body the compounas should not exceed the size of a hexa~eptide, that is to say should not comprise more than six amino acid or amino acid analogue residues; the presence of further residues, particularly in a pro-drug from which residues are cleaved in the body to give the compound primarily exerting the desired effect, is however not excluded.

Particularly, the invention provides a method of _reatment of the allergic inflammatory phase of asthma, wherein an effective amount of a kininogenase inhi'oitor sucn as a mast cell tryptase inhibitor is administered topically or systemically to a patient suffering -rom or at risk of the condition.

The invention extends further to a method of preparation of a medicament for the topical or systemic treat~ent (including prophylactic treatment) of conditions as above particularly for allergic inflammatory conditions and especially for asthma as above, wherein a kininogenase inhibitor is associated with a pharmaceutically acceptable diluent or carrier to constitute said medicament.

In the a~ove, the kininogenase inhibitor is conveniently SU~STITUTE SHEET

W092/04371 2 0 ~ O ~ ~ 8 PCT/GB91/0147g but not essentially of the novel kind now described in which in another aspect, without limitation to any particular clinical indication, the invention provides synthetic, low molecular weight compounds that selectively in~ibit kininogenases and thus block the release of kinins from kininogens. The inhibitors are peptide analoaues, desirably (as above) not exceeding the size of a hexapeptide in terms of amino acid or analogue residues, based on the known amino acid sequence of the kininogens at cleavage site I, wnich analogues have sufflcient similarity to the cleavage site sequence tO
bind to the active site of the kininogenase but are no hydrolysable and therefore remain bound, inactivating the enzyme.

The inhibitors are essentially of the structure below, in which A represents the P3 residue, B the P2 residue, C the Pl residue and '' a carbonyl-activating or binding group the structure being:-A - B - C - Y

where A, B and C are amino acyl or amino acyl analogue groups linked by peptide bonds or conformational analogues thereof giving a peptide mimic. Other residues in addition to these essential ones may of course be present, including amino acyl or amino acyl analogue residues.
In more definitive terms the compounds are represented by SUBS~E,l,;lE SHEET

WO92/04371 2 ~ 5 ~ PCT/GBg1/0147~

~ `~ Y
A - B - ~ , /
F~ I

C

wherein A and B = amino acyl (including amino acyl analogue) the same or different forming a dipeptide group the amino acid of A optionally carrying a terminal group (other than hydroqen) and being any amino or imino-acid residue (but preferably of D-configuration) and of B being a lipophilic amino-acid residue of ~- or L-configuration but not proline or a proline analogue, or a conformationai analogue of said dipeptide group wherein the peptide link is replaced by -CH2-NH- ('reduced'), -CH(OH)-CH2-('hydroxy'), -CO-CH2- ('keto'), -CH2-CH2-('hydrocarbon') or other conformational mimic of the peptide link and in:-SUBS T ITUTE Sl IEET

WO92/04371 2 3 ~ O 8 ~ 8 PCT/GB91/0147~ -~

the side chain Rl is that of a basic amino acid or amino acid analogue (preferably o L-configuration) and R is H or lower alkyl (cl -C4) or C~ or the peptide link comprising -N(R)- is replaced leading to a conformational mimic as above. For example C~ may be replaced by nitrogen.

Y = a binding enhancing or carbonyl activating group for example selected from H (but only if A or B is cyclohexylalanine, preferably D if a~
A or L if at B) or alkyl (Cl - C20) or fluoroalkyl (C2 - Cl2); substituted oxymethylene; thiomethylene; sulphoxy-methylene; sulphonylmethylene; aminomethylene;
hydrazino-m~thylene; -CH2-Het (where Het = a substituted or unsubstituted heterocycle);
substituted amino (but when the resulting compound is a secondary alkylamide B must not be phenyl-alanine); an amino-acid group or its ester or amide; a car~oxylic secondary amide or primary amide, when B must be a bulky lipophilic, non-aromatic amino-acid e.g.
cyclohexylalanine, adamantylalanine (not Ala Leu Ile Val Nva Met Nle Phe Tyr Trp Nal (l));

CUBSTITUTE S:~EET

WO92/04371 ? ~ PC~/CB91/014~3 tertiary-carboxamide: carboxy-alkyl group or its ester or amide.

_n the above context substituents are suitably c~mon functional groups that increase binding af4inity t3 the enzyme and/o~ improve pharmacological pro~ertles. ~urth~r in considering conformational analogues or mimics a dipeptide mimic ~s a structure containing non-natural amino acid ~amino acid analogue) residues or which is non-peptidic and which in I holds the side-chains of A and B o- B and C or all o4 them in a c~nformation simila- to Ihat ~resent ln the parent peptide when bound to ~he ac~ive site cf he enzyme. T~ may also contain features favourable for other interactlons ~ith the enzyme, e.g. hydrogen bonding. A mimic may be chosen f~om he published work on such analogues.

Fo- example, the following are mimics of the dipeptide DPro-Phe (Ph may be rerlaced by -CH2Ph)~-ZH ~ ~ CO H ~ ~ COzn (i~ '?~e^~cea' mimi^ 'i ) 'Hydrcxv' or 'ii , 'Ket^' ^-hyc.cxyetr.y!^r.= ri~ ersmethylene Ph Ph Ph H ~ .~" CO2H / ~ .~" CO,H H (CH2)nC02n O o n=1-4 (iv) ~v) (vi) ('~ydrccar~cn' mimic Figure ~, DPro-Phe m~ cs SUBSTITU T r Si-icET

w092/04371 2 ~ ~ O ~ ~ 8 PCT/GB~1/0147~ _ The preferred compounds represented by the above general formula are now considered.

~ referred residues for A are imino-acids, (e,g. 3-proline or an analogue of proline e.g. pipecoiinic acid, azetidine carboxylic acid etc.); lipophilic amino acids (e.g. DPhe, DCha, DChg); strongly basic amino acids (e.g. D-Arg cr a guanicinopAenylalanine) and for B they are L-Phe, -Cha, L-~Nal, L-Tal, L-(4F)Phe L-(NMe)Phe or other substituted phenylalanines. A and B may also be the N-alkyl (cl-c~J or C~-alkyl (Cl-C7 e.g. methyl, benzyl) anaiogues of these amino acids. Suitable terminal groups for A include lower aikyl (preferred) or acyl (not excluding amino acyl), alkyl sulphonyl (straight chain or branched or cyclic), amino-alkyl, carboxy alkyl, hydroxy alkyl or any other common protecting group encountered in peptide chemistry.

Groups suitable as group Y are specific to the present invention in that they are part of the structure giving the required binding to the active site and are not merely non-interfering end groups. They form a binding group which increases affinity to the enzyme and/or a group which activates the adjacent carbonyl by rendering it more electrophilic. Specific groups are included in the following formula:

- /
X 11 , SUBSTITUTE Sl~,EET

WO92/04371 2 ~ ~ a ~.J 8 PCT/GB91/~147g where in a peptide link t~ residue B the ~-nit~ogen may ~e free or substituted for example by ~ethyl or other C1-C4 alkyl and thus Rl and R are as before but particularly Rl =
3-guanidinopropyl or other guanidinoalkyl group or an ami~ino-alkyl or aminoalkyl group, also ~ara- or me~a substit~ted guanidino or amidino-benzyl or pro~ected forms of the aDove (the basic nitrogens may zlso be alXyl~ted e.g. with Me, Et~, and where Y = groups as aiven below, rirst in more aenerzl terms and then in terms of ~ore det~iied preferences, subjec~ in both cases tO ~he provisos expressed in defining the compounds of the invention earlier. The de~ailed preferences are given in groups under roman numerals, which are also indicated, in brackets, with the first listing wAich is:-(I) Y = H (representing aldehydes) or alkyl including fluoroalkyl (representing ketones) (II-III-IV) Y = -CH2Q

where Q = -oR2 or -SR~ or -SOR2 or -S0222 or -NHR2 or - ~: or -D1 \ R' (note that -~ represents a ring in which D is an ato~ O r that ring) wherein R2, R~ and R~ are as below SUBSTITUTE SHEET

WO 92/043~1 2 ~ 8 1~ Pcr/GB91/0147g ( V-VI ) Y = -rH,~ '`ON O--~ .~ _ -C.~-~ C~6 C0~~

wnereln R~, R' and R~ are as be!ow (~'l--VIII) Y = amino acyl or group 'orming a substituted amide or hydrazide (IX-X-XI) Y = a group forming an ~-keto amide e.g.
- COR 9 or -CO-D or --~o.~

anc ln ~hich further:-R2 = alkyl or substituted alkyl includingaryl or aryl alkyl and -CH~R3 where R3 =
fluoroalkyl R4 and R5 the same or different but not both hydrogen = H or C,-C20 alkyl (which may be further substituted), acyl or alkyl sulphonyl -~ is a heterocyclic ring (D = nitrogen or carbon in Group IV and N in Groups VI and X) optionally unsaturated and optionally with further hetero atoms and substituents SUBSTITUTE SHEET

W092/04371 ~ ~,3 ~ PCT/GBgl/0l479 R6 = hydrogen, alkyl, hydroxy~lXyl, aminoalkyl, alkylaminocarbonyl R3 = -NH2 as sucn o- alkylated, or amino acvl ~ he listing of more detailed preferences, again ~ithin the provisos expressed earlier, is -Group TY = H; alkyl including brancned alkyl (C.-C 0,; zryi alkyl: or cycloalkyl (Cl~c20); perfluoroalkyl or partially fluorinated alkyl (C2-Cl~); [e.g. Y = Me;
-CH(CH2CH2CH2CH3)2; -CH(CH~CH2C~C~3)CH2 -cyclohexyl;
-CH2CF2CF2CF~; -CF~CH~CH CH3;.

Group ~I

Y = -CH2QR2 where Q = O, S, SO, SO2, NH and where R' = Alkyl, branched alkyl or acyl (Cl-Cl2); or cycloalkyl (Cl-C20); or aryl or aryl alkyl; or -CH2-R3 where R3 = perfluoroalkyl or partially ,luorinated alkyl, branched or not (Cl-Cl2).

Group __ Y = -CH2N
R' R~, R5 the same or different = alkyl, branched alkyl, cycloalkyl, acyl, alkylsulphonyl, carboxyalkyl (the carboxyl group may be further derivatized to form an ester or amide with an amino-acid or dipeptide), carbamoyl, sulphamoyl, N-dialkylamino-, arylalkyl, haloalkyl including fluoroalkyl, cyanoalkyl, alkoxyalkyl, SUBSTITUTE SHEET

W092/04371 2 ~ 9 0 ~ ~ 8 PCT/CB91/0147g 1~
hydroxyalkyl, mercaptoalkyl, aminoalkyl and derivatives thereof e.g. esters, amides and thioeslers; or one of R~
or R~ = hydrogen Group T~' Y = -CH~ - ~

where D = nit~ogen or carbon and -~ ~s a saturated or unsa~urated heterocyclic -_ng or a bicycli_ -ing system, each is ~ - 8 membered, where there may be other hetero-atoms (N, S, O) and carbons or nitrogens may op~ionally be substituted by alkyl, branched alkyl, cycloalkyl, carboxyalkyl, carboxy (attached lo carbon), amino, alkoxy, alkoxymethyl or (carbon) as carbonyl or other groups beneficial for interaction with the enzyme.

Grou~ V

Y = -CH~CH(R6)CoNR'R5 ~.~, ?.5 as defined -. Gr_u? ~.
?~6 = hydroaen lower alkyl, branched alkyl, cycloalky , hydroxyalkyl, amino-alkyl, alkyla~inoczr~onyl.

Grou? VI

Y = -CH~CH~R6)CO~

p,6 as defined in Group V and - ~
as defined in Group I-i (but with D = N) SUBSTITUTE SHEET

WO92/04371 2 3 3 ~ 8 PCT/CB91/01479 Group VII

Y = an amino-acid residue cr any amide ~seccnGa-r c-ter_iary) or ester of _hat ~esidue, ~ 5-c^nfiguration. Pref2r-ed residues a_e C4 lipoph~
a~ino-acids e.~. no~leu^~e, cyo'onex~Jlal2-.i-.e, ho~oc~clohexylaianine,cyclohexylclycine,~~r~~utviclv- -.e.

G--cu~ '.---_ R' Y = - N-R~

R7 = H (when however B is not phenylalanine unless 28 is carboxylalkyl or derivatized carboxylalkylj; or aikyl, branched alXyl (Cl - Cl2), cycloalkyl (C1-C20) car~oxyalkyl or bis'carboxyl)alkyl, which may be derivatized at the car~oxyl c-oup to form an amide e.g.
with an amino-acid (preferred is z~ainine) ^- a subs~i~uted a~ine; N'-dialkylamino; N'-~lkylamino-, 2~ = R7 the same or differen_ but excluding H.

Group IX

Y = -CO-R9 but only if 3 is a bulky non-aromat~c lipophilic amino acid o- its N~ alkyl (c. - _A;
derivative (e.g. cyclohexylal~nine but excludi.. Ala, - Leu, Ile, Val, Nva, Met, Nle, Phe, Tyr, ~=p, Nal(l) and their N-methyl derivatives) where R9 = NH2, N'-alkylamino (where the alXyl groups include brznched and/or cycloalkyl); an amino-acid residue.

SUBSTITUTE SHEET

W092/04371 PCr/GB91/0147g --2~ 8 1~

Grou~ X

Y = -CO-~

-3J as defined in Group IV (bu- ~ = N) Grou~ XI

= -CO-NR~R~

as defined in Grou~ ~~_, bu- no_ ~:

The following examples illustrate the invention. They are given in the form of:-- Nine tables of compounds with reference number, structure, molecular on as deter~ined by FA3 ( fas~
atom bomDardment) spect-omet-y and class of compound (the same as the 'groups' referred to ea_lier herein) - Elght detailed exampies of svnthesis - Twelve synthesis schemes, as ;efer~ed to ~n ~he detailed examples - Table of abbreviations - Description of in vit_o tests of inhioi'ion of kininogenases and in vivo tests of ef ficacy aaair.st asthma A11 s~ructures of inte-mediates we~e ve~i~iec ~ NMR.

SUBSTI T UTE SHEET

wo 92/04371 ? ~ S ~ PCr/GB91/0147 Aldehvdes [M+Hl~ Class S Me-DPhe Cha Arg-H 473 1 hiome~hvlene Analo~ues and SulDhomethvlene Analogues [~I+Hl- Class 17 H-DPro Phe Arg CH,S(CH,)1Me 519 II
H-DPro Phe Arg CH2SnBu 505 II
61 H-DPro Phe DLArg CH,SO,nBu 537 II

E~hers [M+Hlt Class 23 H-DPro Phe Arg CH20CH,(CF2)3CHF2 647 II
62 H-Pro Phe Arg CH,OCH,CF3 515 II
63 Boc-DPro Phe DLArgCH20CH,CF3 615 II
64 H-DPro Phe DLArgCH20CH,CF3 515 II
H-DPro Cha DLArg CH20CH,CF3 521 II
66 H-DPr~ Phe Arg CH20CH(Me)CF,CF2CF3629 II
67 H-DPro Phe Arg CH20CH2CF3 515 T~
68 H-DCha Phe Arg CH20CH2CF3 571 II
69 H-DArg Phe ArgCH,OCH,CF3 574 II
H-DChg Phe ArgCH20CH,CF3~ 557 II
54 H-DPro Phe Arg CH20(CH2)5CH3 517 II
H-DPro Phe Arg CH,OPh 509 II

SljJ~ I j, 'J I E SHEE~

wo 92/04371 2 3 ~ 8 3 PCr/GB91/0147~J

Aminomethvlene and related analo~ues l~+Hl- aass 3i H-DPro Phe ArgCH2N[(CH.)sMel2 600 III
71 H-DPro Phe DArg CEi2(RSjTha 556 IV
72 H-DPro Phe ArgCH2(RS)Tha 556 IV
73 H-DPro Phe Arg CH2 1-Pip-3-(RS)CO,Et 572 IV
7J, H-DPro Phe DLArg CH, l-Pip-3-(RS)CH,O(CH2)3.~11e 586 IV
H-DPro Phe DLArg CH, 1-Pip-3-(RS)CH~NEt, 585 ~
76 H-DPro Phe DLArgCH, 1-Pip-3-CONEt, 599 IV
77 H-DPro Phe DLArg CH, 1-Pip-3-(RS)CO~Bu 599 78 H-DPro Phe DLArg CH, 1-Pip-3-(RS)CON(nBu)~ 655 l~
79 H-DPro Phe DLArg CH2 1-Pip-4-(1'-Pip) 583 I~
H-DPro Phe Arg CH2(NMe)AhaNHnBu 615 I'v 81 H-DPro Phe Arg CH,-Abn 540 IV
82 H-DPro Phe Arg CH,-Hvp(OnBu)NHEt 629 IV
83 H-DPro Phe Arg CH2-Sar Pro NHEt 628 m 84 H-DPro Phe Arg CH2-tHyp(aBu)ROnBu 644 IV
H-DPro Phe DLArg CH2Pic NEt2 641 IV
86 H-DPro Phe DLArg CH2'DHyp(aBu)BOnBu 644 IV
87 H-DPro Phe DLArg CH2pro~coNEt2 5999 IV
88 H-DPro Phe DLArg CH21-Pip-3(RS)CO2H 544 IV
89 H-DPro Phe Arg CH21-Pip-3(RS)CH,CONEt, 613 IV
H-DPro Phe Arg CH2N(CH,Ch)(CH2)5Me 612 m 91 H-DPro Phc DLArg CH2N(OC)2 657 m 92 H-DPro Phe Arg CH2N(Et)Ch 542 m 93 H-DPro Phc Arg CH2N(Me)(CH2)4N H~ 517 m 94 H-DPro Phe Arg CH2N(Me)nBu 502 m H-DPro Phe DLArg CH,NnBu2 544 m 96 H-DPro Phe DLArg CH2N[nBu]SO,nBu 594 m 97 H-DPro Phe DLArg CH2NlnBu](CH2)3C ONH, 573 m 98 H-DPro Phc DLArgCH2N[I'Hex~(CH2)~NH2 629.5 m 99 H-DPro Phc DLArg CH2N[llHex](cH2)5NH2 601 m 100 H-DPro Phc DLArgCH2N[nHex](CH2)3NH2 573 m 101 H-DPro Phe DLArg CH2N[nBu~(CH2)4Ph 620 m SUe~TlTUTE SHEET

WO 92/04371 ~ l 8 PCr/G891/0147 TABLE 4 (cont.) Aminomcthvlene Ketones and reiated analoc~ues lM+Hl~ Cl~ss 102 H-DPro Phe DLArgCH1~[nHex](cH~)6cO~H~ 643 ~I
103 H-DPr~ Phe DLArg CH,N[~He%i(CH,j6.~H~ 615 iII
104 H-DPro Phe DLArg CH~NrnHexl(CH2)7OH630 m 105 H-DPro Phe DLArg CH~NrnHex](CH2)~NHAc 671 m 106 H-DPro Phe DLArg CH,~lrnHex](CH,)6CONHE~ 671 III
107 H-DPro Phe DLArg CH~N[nHex](CH2)6CO,.\~le 658 III
108 H-DPro Phe DLArg CH,NrnHex](CH,)6CO,H 644 III
109 H-DPro Phe DLArg CH,~'rnBu](CH,)3CO~Et. 6'9 lII
110 H-DPro Phe DLArg CH,i~:'Bu](CH,)3CO.~HE~ 601 lII

Keto Isostere Ccetainin~ Analo~ues [M+H]+ Class H-DPro Phe DLArg~GlyPro-NHEt 599 VI
111 H-DPro Phe Arg~Gly P~NHE~ 599 VI
112 H-DPro Phe Arg~Gly Arg-NH2 530 V
113 H--~ Phe DLArg~JlyAla-NH2 545 V
114 H-DPTo Phe i .~rg~Gly Aha-NH, 587 V
115 H-DPro Phe DLArg~lyAha-NH~Bu 643 V

Subs~ate Analo~ues [M+H~t Class 45 H-DPro Phe ArgChg-NH2 557 VII
116CPr-CO Phe ArgChg-NH, * VII
117~IeSO2 DPro Phe Arg Chg-NH2 635 VII
118MeCO DPro Phe Arg Chg-NH2 599 VII
119H-DArg Phc ArgSer-NH, * VII
120H-DCha Phe Arg Ser- NH2 * VII
121H-DPhe Phe ArgSer- NHi ~ V~
122H-DPic Phc Arg Ser- NH2 * VII

SUBSTITUTE SHEET

WO 92/0~371 2 0 ~ 8 - pcr/GB91/o1479 TABLE 6 (cont ~
Substra~e Analo ues [MfHl Class 1~3 H-DPic Phe Arg Chg-NH~ ~ VII
1~1 H-DPro Phe ArgSer-NH~ ~ VII
1~5 H-DPro Cha Arg Ser-NH~ * VII
126 H-DPro Cha ArgGly-NH, * VII
127 H-DPro Phe Arg Ser-Arg-lNH. ~ VII
1~8 H-DPro Phe Arg Lys-N~ VII
1~9 H-DPro Phe Arg Aha-NH. ~ VII
130 H-DPro Phe Arg Phe-~H, 565 VII
131 H-DPro Phe Arg Leu-NH, 531 VII
13~ H-DPro Phe ArgIle-NH, 531 VII
133 H-DPro Nal Arg Ser-NH, ~ VII
131 H-DPro Phe Arg DAha-NH, 531 VII
135 H-DPro Phe Arg Aha-NH¢CH,)3.Ue 587 VII
136 H-DPro Phe Arg Nleucinol 518 VII
137 H-DPro Phe ArgSesOnBu NH2 561 VII
138 H-DPro Phe Arg Cha-NH, 571 VII
139 H-DPro Phe ArgAda-NH2 623 VII
140 H-DPro Phe ArgHch-NH, 585 VII
141 H-DPro Nal ArgCha-NH, * VII
142 H-DPro Cha Argcha-NH2 VII
143 H-DPro PhepNO,ArgSer-NH, ~ VII
144 H-DPro Nal Arg Ile-NH2 * VII
145 H-DPro Phe ArgIlePr~NH, * VII
146 H-I)Pro Phe ArgAhaPro-NH2 VII
147 H-DPro Nal ArgAha-NH, * VII
148 H-DPro Cha ArgNpg-NH, * VII
149 H-DPro Cha ArgHch-NH, * VII
150 H-DPro Nal ArgHch-NH, * VII
151 H-DPro Phe ArgNpg-NH2 545 VII
152 H-DPro Phe ArgChg-1-Pip 625 VII
153 H-DPIo Phe ArgChg-NH(CH2)5Me 641 VII
154 H-DPro 4-Fph Arg Chg-NH2 575 V~
Sa~sfactory am~no acid analysis obtained SUBC T ITU I E SHEET

WO 92/0437l 2 ~ rj g PCI/C~9l/01479 Arnides ~I+Hlt Class 47 H-DPro Phe Arg N[(CH.)5~1e](C~ Ch ~ 5 YrrI
15i H-DPro Phe Arg N(Me)nBu 488 VIII
1i6 H-DPro Phe Arg NH(CH2)3CO Arg-NH. * V~I
157 H-DPro Phe Arg NH(CH2)3NH. 475 vm 1i8 H-DPro Phe Arg NH(CH.)4CO Arg-NH, ~ VIII
li9 H-DPro Phe Arg NH(CH2)4NH~ ~ Y~I
160 H-PTO Phe Arg NH(CH.)sCO Arg-NH~ 687 VIII
161 H-DPro Phe Arg NH(CH.)5NH. 503 ~v'lII
162 H-DPro Phe Arg NH(CH2)6cO Arg-NH2 701 VIII
163 H-DPro Phe Arg ~H(CH,)7CO Arg-NH. 715 vm 164 H-DPro Phe Arg NH(CH2)7CONH(CH2)3l\/le615 VIII
165 H-DPro Phe Arg NH(CH.)7l~AC 573 VIII
166 H-DPro Phe Arg NH(CH,)7NH, 53l VIII
167 H-DPro Phe Arg NH(CH2)7CONH2 559 vm 168 H-DPro Phe Arg NH(CH2)7CO-GlY Gly ATg-~H2 829 vm 169 H-DPro Phe ArgNH(CH2)7CO-GlyArg-NH 772 VIII
170 H-DPro Phc ArgNH(CH2)7CO-GlyGly-GlyArg-NH2 886 vm 171 H-DPro Phe Arg N[nHex]2 586 VIII
172 H-DPro Cha Arg NHCH2Ch 520 VIII
173 H-DPro c~.~al Arg NHCH2Ch 564 VIII
174 H-DPro ,BNal Arg NHCH2Ch 564 vm 175 H-DPro His ArgNHCH2Ch 504 vm 176 H-DPro(4Me)Phe Arg NHCH2Ch 528 vm 177 H-DPro Phe NarNHCH2Ch 500 vm 178 H-DPic Phe Arg NHCH2Ch 528 V~I
179 H-Drlc Phe Arg NHCH.Ch 576 vm 180 H-DThi Phe Arg NHCH.Ch 570 vm 181 nBu DPTo Phc Arg NHCH2Ch 570 vm * Satisfacto~y amino acid analysis obtaincd SUBSTlTUrE SHEET

wo 92/04371 2 () ~ O ~ ~ 8 2 PCI/GB91/01479 Ketones [M+H~- Class ~9 H-DPro Phe ArgCH3 117 48 H-DPro Phe Arg(CF2)2CF3 **
53 H-DPro Phe ArgCH~CH~nHex~. ***
** Molecular ion not de ^:ed a-Ketoarnides [M+H]T Class 11 H-DPro Phe DLLys CON[nBu]2 530 XI
H-DPro ChaDLArgCON[T~Bu]~ *** XI
*** [M+Hl~notavailable SUBSTITUTE SHEET

wo 92/0437I 2 ~ PCr/GB91/0147~

, EXAMPLE I

~le-DPhe-Cha-Arg-H
The synthesis of 5 was carried OUt according to Scheme I. Arabic nume~als underlined e.g. 1 refer to structures in these schemes. Roman numerals in parentheses e.g. (i) reter to reaction steps.
(i) Isobutvl chloroforrnate (10.'~ mmol) was added to a solution of Boc^Arg(Z.)OH (9.23 mmol) and N^methvlmorpholine (11.08 mmol) in dry l~F (25 cm3) at -20C. After ^0 mins the solid was filtered off and the filtrate added to a solution of sodium ~orohyd~ ;e (10.3 mmol) in water (10 cm3) at 0C. After 3 hours 0.3 M K}.S04 was added. the crude product extracted wi~n EtOAc and purified by flash chromatography on silica with EtOAc - petrol (4:6~. The alcohol 1 was isolated as a white solid (97%).
(ii) The Boc ~oup of I (4.75 mmol) was removed with sa~. HCl/Dioxan and the product acylated with Boc-Cha-ONSu (9.5 mmol) in CH~CI2 (20 cm3) at 0C in the presence of N-methylmorpholinc.
After two hours the reaction was worlced up using standard procedures and the crude product purified by flash chromatography on silica with EtOAc - petrol (4:6). The pure alcohol was isolated as a colourless oil (90%).
(iui) The Boc group of 2 (4.27 mmol) was removcd with sat. HCI~lDioxan and she producs reacted with Z(NMe)DPhe-OH (5.12 mmol) in she presence of HOBt (10.2 rnmol), water soluble carbodiirnide (6.1 mmol) and N-methylmorpholine in DMF (20 cm3) at 0C. Afser 18 hours the reaction was wor~ed up using ssandard procedures and the product purified by flash chrornatography on silica with EtOAc -pesrol (1:1). The pure alcohol 3 was isolased as a colourless oil - (52%).

SUBSTi T UTE SHEET

wo 92/0~371 pcr/Gs91/o:47~
2f~9~8 (ivj The alcohol 3 (2.22 mmol) was dissolved in CH,C12/AcOH ~30:1) and Dess-Mamn Per;odinane (4.5 mrnol) added. After 2~ nours a~
room temperature the reaction muxture was diluted with EtOAc and poured into a solution of sodium thiosulphate (32 mmol) and sat.
NaHC03. The crude product was purified by flash chrornatography on silica with EtOAc-petrol (3:7). The pure aldehyde _ was isoia~ed as a colourless oil (75%).
(v) The aldehyde _ (1.6~ mmol) was dissolved in MeOH/H,O/AcOH
(90:9:1, 50 cm3) and hyarogenated over 59O Pd/C. The crude material was purified by mplc on *Vydac Cl8 (15-25 Il) using ~eCN/H,O/TFA to give pure ~ (CH-851) as a white solid (780 mgj. Tlc EtOAc-Pv-AcOH-H,O (30:20:6:11), RF 0.66 on silica.
After hydrolysis at 110C/2' hrs with 6N HC1 peptide content based on Cha was 40%. FAB mass spec [M+H]+ = 473 (Calc. mJz = 472).

EXAMPLE II
11 H-DPro-Phe-Lys-CONnBu2 (see Scheme 11) (i) TcbocONSu (14.8 mmol) was added to a solution of H-Lys(Z)-OMe. HCl (12.2 mmol) and triethylamine (14.8 mmol) in CH,CI2 (50 cm3). After 3 hours at room temperature the reaction was worked up using standard procedurcs and the product purified by flash chroma~ography on silica using EtOAc - petrol (7:13). The purc estcr 6 was isolatcd as a colourless oil (100%).

(ii) Diisobu~ylaluminium hydride (1.5 M solution in toluene, 50 rnmol) was added to a solution of 6 (12.2 rnmol) in dry toluene (100 crn3) at -78C over a period of 20 minutes. After a further 15 minutcs methanol (10 cm3) was added followed by a saturated solution of Rochellc's salt (100 cm3). After 2~ hours the reaction was wor~ed up using standard procedurcs and thc product purificd by flash chrornatography on silica using EtOAc - petrol (3:7). The pure ald~hyde Z was isolated as a colourless oil (49%).

Tradc narnc ~U~3~ H 7''LJ'i E SHEET

wo 92/04371 2 ~ J ~ PCI'/GB91/01479 (iii) Potassium cyanide (18 mrnol~ and 1 M h~drochlorie aeid ~30 em;) were added to 2 solution of Z (5.98 mmol) in ethyl acetate (30 em3) After 18 hours at room temperature the reaetion was worked up using standard procedures and the produet purified by flash chromatography on silica using EtOAc - petrol (4:6). The pure cyanohydrin 8 was isoiated as a colourless oil (88%).

(iv) A 4 M solution of HCl in dioxan (50 cm3) was added to a solution of 8 (5.28 mmol) in dry me~hanol (15 cm3) at OC. After 18 hours at room temperature an ice/water mixture (15 cm3) was added.
After 3 days at 4C soiid KHC03 wæ added. The reaetion was worked up using standard proeedures and the product purifîed bv flash chromatography on silica using EtOAc - petrol (11:9). The pure ester 8b was isolated as a yellow oil (59%).

(v) Aetivated zine dust was added in srnall portions to a solution of 8b (3.1 mrnol) in AeOHJH2O (9:1, 25 em3). After 1~ hours at room temperature the zine was filtered off, the filtrate evaporated in vaeuo and the residue wæ taken up in EtOAe. This solution was washed with sat. ~JaHCO3, water, brine, dried (Na,SO4) and evaporated in vaeuo. The amine 2 was isolated as a colourless oil (85%).

(vi) The amine 2 (2.63 mmol) was aeylated with Boe-Phe-ONSu (3.04 mrnol) in CH2Cl2 (30 em3) at OC in the presenee of N-methyl morpholine. After 3 hours the reaction was worked using standard proeedures and the erude produet purified by flash ehromato~aphy on siliea with EtOAelPet Ether (6:4). The pure ester 10a was isolated æ a eolourless oil (92%).

SUBS, ITU T _ SHEET

WO 92/04371 2 0 ~ 0 8 5 8 pcr/Gs91/ol47~ ", (vii) The Boc group of 10a (2.41 mmol) was removed using sat.
HClJDioxan and the product acylated with Boc-DPro-ONSu ~2.g'~
mmol) in CH2C12 (30 cm3) at OC in the prescnce of N-methyl-morpholine. After 3 hours the rcaction was worked up using standard procedures and the product purified by flash chromatography on silica using EtOAc/Petrol (3:1). The pure ester 10b was isolated as a colourless oil (64%).

(viii) Li~hium h,vdroxide (1.6 mmol) and water (3 cm3) were added to a solution of 10b (I.S4 mmol) in THF (30 cm3). After 4 hours a~
room temperature the THE was removed in vacuo, the pH of the residue adjusted to pH 4 with 1 M citric acid and extracted with CHCI3. The organic extracts were washed with brine, dried (Na,SO4) and evaporaled in vacuo. The pure acid 10c was isolated as a colourless oil (70%).

(ix) Pentafluorophenol (1.3 mmol) and water soluble carbodiimide (1.3 mmol) were added to a solution of 10c (1.07 mmol) in CH2CI2 (20 cm3) at OC. After 2~ hours dibutylatnine (2.1 mrnol) was added to this solution,at OC and the pH adjusted to pH 9 with DEA. After 18 hours at room temperature the reaction was worked up using standard procedures and the product purified by flash chromatography on silica using EtOAc/Petrol (7:3). The pure arnide 10d was isolated as a colourless oil (48%`).

(x) Dess-Martin Pcriodinanc (0.97 mmol) was added to a solution of 10d (0.52 mmol) in CH2C12 (100:1, 40 cm3). After 2 hours at room temperature further Dess-Mamn Periodinane (0.52 mmol) was added. After a further 3 hours the reaction mLsture was diluted with EtOAc and poured into a solution of sodium thiosulphate (7.3 mmol) in water and sat. NaHCO3 were added. The crude product was purified by flash chroma~tography on silica with EtOAc/pcsrol (9:11). The pure keto amide 10e was isolated as a colourless oil (57%).

SUBSII,~)E S~lEi~T

WO 92/04371 2 ~ 9 ~ 8 Pcr/cB91/01479 2c~

(xij The Boc group of 10e (0.24 mmol) was removed using sat.
HCUDioxan. The resultar.; produc; was dissolved in A.,OH~I-,O
(9:1) and hydrogenated over 5% Pd/C. The crude material was purified by mplc on *Vydac Cl8 (15 - 25 ~1) using MeCNlH20~IFA
to give 11 (CH-1463 89.7 mg). Hplc, *Novapak C~8, 4 11 (8 x 100 mrn), linear gradient 20 -- 80% 0.1% TFA/MeC~' into 0.1 S~
TFAIH,O over 25 min at 1.5 ml min-l indicates the presence of two epimers D-Arg (40%) ~t 11.2 min and L-Arg (60%) at 12.6 min.
After hvdrolysis at 110C/'2 h with 6N HCI. amino acid analvsis Phe 0.93, Pro 1.07.

EXAMjPLE III
17 H-DPro-Phe-Arg-CH,S(CH,)4CH3 (see Scheme m) (i) Boc-Arg(Z2)OH (46.1 mmol) was dissolved in dry THF (200 cm3).
N-methylmorpholine (50.85 mmol) and isobutyl chloroformate (50.73 mrnol) were added at -20C. After 20 min. this mixture was added to a solution of diazomcthane (0.1 mole) in Et2O at -5C.
After 2 hours the diazoketone 12 was isolated as a yellow solid.
(ii) The diazoketone 12 (46.1 mrnol) in dry THF was treated with HBr (69.15 mmol) in EtOAc at -20C followed by addition of sat.
NaHCO3 after 45 mins. The crude product was extracted with EtOAc and crystallised fTom EtOH to give pure Boc-Arg(Z2)CH2Br, 13, (85%).
(iii) l-Pentanethiol (1.27 mmol) in dry DMF (5 cm3) was treated with sodium hydride (1.4 mmol). After 30 rnins Boc-Arg(Z2)CH2Br 13 (1.27 mmol) was added-40C for 20 mins and -5C for 2~ hours.
After addition of 0.3 M KHS04 and extraction of the crude product with EtOAc, flash chrornatograpy or silica with EtOAc - pet~1 (15:85) yielded tlA_ pure thiomethylene compound 14 as a colourless oil (74%).
Trade name SUBS, ITUTE ~HEET

WO 92/0437l 2 U 3 ~ 8 ~ 8 PCr/GB9~ 479 ., (iv) The Boc pro~ecting group of 1~ (0.94 rnmol) was removed using sat HCl/~ioxan and the resulting prociuc~ was acyiated with Boc-Phe-OPfp (1.13 mmolj in CH2CI2 at OC in the presence of DEA. The crude product was purified by flash chromatography on silica with EtOAc - petrol (3:7) yielding the pure thiomethylene analogue 15 as ~ colouriess oil (S5%).
(v) The Boc protecting group of 15 (0.5~ mmol) was removed using sat.
HCI/Dioxan. The resulting product was dissolved in D~IF and treated with Boc-DPro-OH (0.63 mmol) in the presence OI- HOBt ( 1.05 mmol), water soluble carbodiirnide (0.76 mmol) and N-methylmorpholine. After a standard work-up the crude material was purified by flash chromatography on silica with EtOAc - petrol (4:6) yielding the pure thiomethylene compound 16 as a colourless oil (74%).
(vi) The Boc protecting group of 16 (0.38 mmol) was removcd using sat.
HClfDioxan. The resultant product was dissolved in AcOH/H2O
(9:1) and hydrogenated over 5% Pd/C. The crude material was purified by mplc on ~Vydac C18 (15-25 ~), using MeCN/H20~ A
to give pure 17 tCH-574, 41 mg). Hplc, ~Novapak Cl8, 4~L (8 x 100 mm~, linear gradient 20 ~ 80% 0.1% TFA/MeCN into 0.1%
TFA/H20 over 25 min at 1.5 ml min-l indicates the presence of two epimers D-Arg (<5%) at 9.8 min and L-Arg (>95%) at 11.1 min.
After hydrolysis at 150C/1.5 h with 6N HCl, amino acid analysis Phe, 0.80; Pro, 1.00.
All analogucs in Table '' were synthesised by the descTibed method.
61 was synthesised by the oxidation of 60 with meta-chloroperoxybenzoic acid.
Trade name SUBSTITU T t- SHEET

wo 92/04371 21~ ~ O ~ ~ 8 PCr/GB91/0l479 EXAMPLE IV
'3 H-DPro-Phe-Arg-CH~OCH~(CF~)3CHCF~ (see Scheme IV) (i) IH, IH, 5H-Octafluoro-l-pentanol (2.45 mmol) in dry DMF (8 cm3) was treated with sodium hydride (1.83 mmol). After 30 mins the bromoketone 13 (1.65 mmol) was added at -40C and left at this temperature for 30 mins and -5C for 2~ hours. Addition of 0.3 M
KHSO4 and extraction with EtOAc gave the crude product which was purified by flash chromatographv on silica using EtOAc - pe~rol (15:85). The pure fluoroether 18 was isolated as a colouriess oil (69%).

(ii) The fluoroether 18 (1.13 mmol) was dissolved in MeOH (40 cm3), sodium borohydride (1.18 mmol) was added to this solution at 0C.
After 15 min 0.3 M KHSO4 was added and the m.ixture extracted with EtOAc giving the pure compound 19 as a colourless oil (88%).
(iii) The Boc pro~ecting group of 19 (1.0 mmol) was removed with sat.
HCUDioxan. The resulting pr~duct was dissolved in CH2Cl~ and acylated with Boc-Phe-OPfp (1.2 mmol) in the presence of DE~A at 0C. After a standard work up the crude product was purif1ed by flash chroma~ography on silica with EtOAc - petrol (3:7) yielding the pure product ~0 as a colourless oil (55%).
'iv) 20 (0.55 mrnol) was deprotected ~ith sat. HCI/Dioxan and acylated with Boc-DPn~OPfp (1.63 mmol) in CH2C12 at 0C in the prcsence of DEA. After a standa~d work up the crude product was purified by flash chroma~ography on silica with EtOAc - petrol (4:6) yielding the pure product 21 as a colourless oil (48%).

SUBSTI' UTc SIJ,EET

wo 92/04371 2 0 9 ~ 8 ~ 8 pcr/GBg1/o1479 (v) ' 1 (0.2~ mmol) was dissolved in CH,Cl,/AcOH (30:1 ) and Dess-Martin Perio~linane (0.48 rnmoij was aaaed. Afier 2 hours a~
room temperature the reaction mixture was diluted with EtOAc ana poured into a solution of sodium thiosulphate (3.5 mmol) in water and sat. NaHCO3. The crude product was purified by flash chromato~raphy on silica with EtoAc - petrol (7:13) yielding the pure fluoroether '~ as a colourless oil (64%).
(vi! The fluoroether ~ (0.16 mmol) was deprotected and purified as described in Example III (vi). Pure ~3 (CH-619) was isolated as a white solid (50.9 mg). Hplc, *Novapak Cl8 411 (8 x 100 mrn), linear gradient 20 ~ 80% 0.1% ~ .VMeCN into 0.1ao TFA/H,O over '5 mins at 1.5 ml min l indicated a single product (TR = 11.5 min).
After hydrolysis at 150C/1.5 hr with 6N HCl, ~mino acid analysis Phe, 1.00; Pro, 1.2.
All analogues in Table 3 were synthesised by the described medhod except 54 and 55 which were synthesised by methods oudined in Schemes xm and XIV respectively.

EXAMPLE V
31 H-DPro-Phe-Arg-CH,N[(CH2)5CH3~2 (see Scheme V) DL
(i) Boc-Arg(Z2)OH (18.5 mmol) was dissolved in CH2C12 (50 cm3).
To this solution at 0C was addcd trichloroethanol (20.35 mmol)~
water soluble carbodiimide (22.2 mmol) and dimethylaminopyridine (0.93 mmol). After 3 hours the reaction was worked up using standard procedures giving the pure ~ichloroethyl derivative 24 as a colourless oil (100%).
Trade name SUeSTl i l,' I c SnEET

WO 92/04371 2 0 S ~ 8 pcr/GB9l/ol479 (iij ~ (18.1 mmol) was depro~ec~ed with sa~. HCVDioxan and acylated with Boc-Phe-ONSu (27 2 mmol) in CH2CI~ a~ 0C in ~he presence of N-methylmorpholine. After 3 hours the reaction mixture was worked up using stanaard procedures and the crude producl was purified by flash chromatography on silica with E~OAc - petrol (2:8) vielding rhe pure product 25 as a white solid (97%).
(iii) '5 (17.1 mmol) was deprotecsed with sat. HCVDIoxan and acylated wi~h Boc-DPro-ONSu (26.2 mmol) in CH,CI. a~ 0C in ~he presence of N-methylmorpnoline. Af~er 3 hours the reaction was worked up using standard procedures and the crude product purified by flash chromatography on silica with EIOAc - petrol (35:65) giving the pure protected tripeptide 26 as a colourless oil (94%).
(iv) Activated zinc powder was added to a solution of 26 (16.47 mmol) in glacial acetic acid. After 3 hours at room temperature the zinc was filtered off, the filtrate evaporated and the crude product purified by flash chromatography on silica with EIOAc - petrol -AcOH (74:25:1) giving the pure tripeptide 27 as a white solid (91%).
(v) The prolected tripeptide 27 (15 mrnol) was dissolved in dry THF
(40 cm;), N-methylmorpholine (18 mmol) and isobutvl-chloroformale (16.6 mmol) were added at -20C. After 20 mins the mixture was added to a solution of diazomethane (35 mmol) in Et10 at -5C. After 2~ hours the diazoketone 28 was isolated as a yellow oil.
(vi) The diazoketone 28 (15 mmol) in dry TE~ was treated with HBr (22.5 mmol) in EtOAc at -20C followed by addition of sat.
NaHCO3 after 45 mins. The cmde product was extracted with EtOAc and purified by flash chmmatography on silica with EtOAc -petrol (1:1). The pure bromoketone 29 was isolated as a white solid (72%).

SUESTi, '~ H c SHEET

WO 92/04371 2 ~ ~1) 8 5 8 PCr/GB91/0147~

(vii) I)ihexylarnine (1.25 mmol) and NaHCO3 (0.8 mmol) were added to a solution of bromoke~one 29 (0.23 mmol) in dr,v l~lF (5 cm3).
After 18 hrs at room temperature 0.3 M KHS04 was added to the reaction mixture and the crude product was pu~ified by flash chromatography on silica with EtOAc - petrol (35:6~). The protected aminomethylene ketone 30 was isolated as a yellow oil (54%).
(viii) The aminomethvlene ketone 30 (0.1' mmol) was depro~ec~ed and purified as described in Example III (vi). Pure 31 (CH-694) was isola~ed as a white solid (41 mg). Hplc, linear gradient 20 ~ 80%
0.1~7o TFA/MeCN into 0.1ao TFA/H,O over 25 mins at 1.5 ml min~;
indicated the presence of ~wo epirners D-Arg (50%) at 11.2 min and L-Arg (50%) at 12.5 mins. After hydrolysis at 110Cl2' hrs with 6N HCI, arnino acid analysis Phe, 0.91; PTO, 1.09.
A11 analogues in Table 4 were synthesised by the describcd metho~
The required arnines were synthesised by standard synthetic methods sucn as reductive amination and ~he Curtius rearrangemcnt.

EXAM.DLE ~tl 40 H-DPro-Phe-Arg~Gly-Pro-NHEt (see SchemeVI) (i) H2C(CO2Tce)2 (6.81 mmol) was treated with sodiurn hydride (5.67 mmol) in dry THF (30 cm3). After 45 mins the bromoketone 13 (4.52 mmol) was added at -5C. After 2t hours 0.3 M KHS04 was added. the crude product extracted with EtOAc and purified by flash chromatography on silica with EtOAc - petrol (2:8). The pure Boc-Arg(Z2)CH,CH(CO2Tce)~ 32 was isolated as a colourless oil (83%).
(ii) Achvated zinc was adided to a solution of 32 (3.65 mmol) in glacial acetic acid. After 2t hours at room temperature the zinc was filDd off, the filtrate evaporated and the diacid 33 isolated.

SU''ST,TUT-- SHEET

wo 92/04371 2 ~ pcr/GB~ 47~

(iii) A solution of the diacid 33 in toluene was heated at reflux for 45 mins. The solvent was evaporated and the crude produc~ purified by flash chromatography on silica with EtOAc - petrol - AcOH
(60:39:1). The Boc-Arg(Z2)~Gly-OH 34 was isolated as a colourless oil (70% from ~).
(iv) Trichloroethanol (2.82 mmol), wa~er soluble carbodiimide ( '.~1 mmol) and dimethylaminopyridine (0.117 mmol) were added to a solution of 31 (2.34 mmoi) in CH~CI, (50 cm;) at 0CC. After 2.
hours the reaction was worked up using s~andard procedures and the crude product purified by flash chromatography on silica with E~OAc - petrol (85:15). The trichloroe~hyl derivative 35 was isolated as a colourless oil (83~o).
(v) The Boc pro~ecting group of 35 (1.85 mmol) was removed using sat.
HClJDioxan and the resulting product acylat~ ' with Boc-Phe-OPfp (6.04 mmol) in CH~CI, in the presence of DEA. After 2 hours the reaction was worked up using standard procedures and the crude product purified by flash chromatography on siiica with E~OAc -petrol (2:8). The pure product 36 was isolated as a colourless oil (85%).
(vi) The Boc protecting g~up of 36 (1.58 mrnol) was removed using sat.
HCl/Dioxan and the resulting product acylated with Boc-DPro-OPfp (5.12 mmol) in C~2Cl2 in the presence of DEA.
After 2 hours the reaction was wor~ed up using standard procedures and thc crude product punfied by flash chromatography on silica with EtOAc - petrol (35:6~i). The pure produc~ 32 was isolaud as a colourless oil (79%).
(vii) Activated zinc dust was added to a solution of 37 (1.13 rnmol) in glacial acetic acid. After 2~ hours at room temperature the zinc was filtered off, the filtrate cvaporated and thc crude product purificd by flash chrornatography on silica with EtOAc - petrol - AcOH
(70:29:1). The product 38 was isolatcd as a colourless oil (76%).

SUB~ T ITUTE SHEET

wo 92/04371 2 ~ 9 ~ 8 ~ 8 pcr/GBg1/~1479 (viii) The protected keto isostere 38 (0.26 mrnol) was converted to its Pfp ester by treatment with Pfp-OH (0.29 mmol) and water soluble carbodiimide (0.31 mmol) in CH,C12 (8 cm3) at 0C for 2~ hours.
This Pfp ester was coupled at 0C to H-Pro NHEt . HCl salt (0.78 mrnol) in the presence of DEA. After 18 hours the reaction was worked up using standard procedures and the product purined bJ
~lash chromatography on silica with CHCl3-~leOH-AcOH (97:2:1).
The product 39 was isolated as a colourless oil (91 C~o).
(ix) The protected keto isostere containing anaiogue 39 (0.23 m.mol) was deprotected as described in Example m (vi). Pure 40 (CH-595) was isolated as a white solid (40 mg). Hplc, linear gradient 10 ~ 50%
0.1% TFA/MeCN into 0.1% TFA/H,O over 25 mins at 1.5 ml min l indicated the presence of two epimers D-Arg (46%) at 10.1 min and L-Arg (54%) at 11.7 min. After hyarolysis at 150C/1.5 hrs with 6N HCl amino acid analysis Phe, 0.91; Pro, 1.09.
All analogucs in Table 5 were synthesised by the described method.

EXAMPLE VII
45 H-DPro^Phe-Arg-Chg-NH, (see Scheme VII) (i) Boc-Phg-OH (19.9 mmol) was dissolved in AcOH/H~O (9:1, 100 cm3) and hydrogenated over Rh/C at 60 p.s.i. for 3 days. The catalyst was filtered off and the solvent removed to give Boc-Chg-OH 41 (100%).
(ii) Water soluble carbodiimide (4.1 mmol) and HOBt (4.3 mmol) were added to a solution of 41 (3.9 mmol) in CH~Cl,IDMF (2:1, 60 cm3) at room temperature. After 30 mins 35% arnmonia solution (0.8 cm3) was added. After a further 3 hours at room temperature the reaction was wor~ed up using standard procedures and the product recrystallised from EtOH to give the pure arnide 42 as a whitc solid (60%).

SUE~STiTl,' T E SHEET

WO 92/04371 ~ .3 8 Pcr/cB91/0l47~

(iii) The Boc group of 42 (0.77 mmol) was removed with sa~.
HCVDioxan to give the amide 43 as a white solid (100%).
(iv) The protected tripeptide 27 (0.38 mmol) was dissolved in DMF (5 cm3). 43 (0.76 mmol), HOBt (0.76 mmol) water soluble carbodiimide (0.46 mmol), and N-methylmorpholine were added at 0C. After 18 hours at room temperature the reaction was worked up using stand~rd procedures and the product purified by flash chromatography on silica with CHCI3/MeOH (99:l). rne pure pro~ected tetrapeptide 44 was isolated as a white solid (69%).
(v) The protected tetrapeptide 41 (0.27 mmol) was deprotected and purified as described in Example III (vi). Pure 45 (CH-640) was isolated as a white solid (58 mg). Hplc, linear gradient 10 1 50%
0.1% TFA/MeCN into 0.1% TFAJH20 over 25 rnin at 1.5 ml min-l, single peak detected at 14.2 tnin. After hydrolysis at 110C/22 hrs with 6N HCl, amino acid analysis, Arg, 0.96; Phe, 1.00; Pro, 0.95.
All analogues in Tablc 6 were synthesised by the described method or by other standard peptide coupling methodology. (M. Bodansky & A. Bodansky, The Practic~ of Pep~ide Synthesis, Springer-Verlag, 1984) EXAMPLE vm 47 H-DPro-Phe-Arg-N[(CH2)sCH3](CH2)3Ch (see Scheme Vm) (i) Thc protccted tripcptidc 27 (0.32 mmol) was dissolved in DMF (5 cm3), HN[(CH2)5CH3](CH2~3Ch (0.96 rnrnol), HOBt (0.64 col), water soluble carbodumide (0.38 rnmol) and N-methylmorpholine were added al OC. After 18 hours at room temperature the reaction was worked up using standard proccdures and thc product purified by flash chrornatography on silica using EtOAclHexanc (4:6). The amide 46 was isolated as a colourless oil (32%).

SU5~,TH 1_:-, c SHEET

W O 92/04371 2 ~ 9 ~ ~ 3 8 PC~r/GB~1/01479 (iij The pro~ec~ed tripeptide arnide 46 (0.1 mmol) was deprotected and purified as described in Example I~ (vi). Pure 47 (CH-~85) was isola~ed as a white solid (17 mg). Hplc, linear gradient 40 - 90%
0.1% TFAtMeCN into 0.1% TFAJH70 over ~5 min at 1.5 rnl min-l, single peak detected at 10.7 min. After hydroiysis at 110C for 22 hrs with 6N HCI, ~nino acid analysis Phe, 1.01: P~, 0.99.
All analogues in Table 7 were syn~hesised by the described method.
The required tripeptide presursors were iynthesised in d similar manner to 27 or by standard peptide coupling methodology. (M.
Bodansky & A. Bodansky, The Prac~ice of Peptide Synthesis, Springer-Verlag, 1984.) Required arnines were either cornmercially available or synthesised via the Curtius rearrangement.
nBu-DPro-OH for 181 was synthesised by reductive arnination.

SU~STI I U-, E SHEET

wo 92/04371 ~ T~ ~ ~ 8 PCr/GBg1/ot47g Scheme I
(Syn~hesis of comDound O

æ (i) isuococl/~TMMrrHF z, Boc-Arg-OH Boc-Arg 2 O~
N;IBH~,H~O

(ii) HC!/Dio,Y~n Boc-Ch2 ON'Su/~i~l.M
CH,CI~

Z~ (iiij l-lCI/Din,Y;~n ~ Z.
Z(~TMe)DPhe-Ch~-Arg ROH ~ Boc-Cha-Ar~ 2OH
Z(NMe)DPhe-OH
3 HOBt/wscd NMMtDMF

(iv) Dess Martin Periodinane CH.CI~tAcOH

Z, (v) H" Pd/C
Z(~Me)DPhe-Ch~-Arg-H ~, Me-DPhe-Cha-Arg-H
MeOHlH~O/AcOH 5 SlJ~S T i T ~, . E Si~EET

WO 92/04371 2 ~ ~ a ~ ~ 8 pcr/GBg~ 47s~

Scheme I I
(Syn~nesls or comwuna ~) Z ('~ cbocOXSu Z
H-L~,s-O~Vle . ~iCI ~ Tcboc-Lys-~)~le E:,.',/CH,C;, 5 ~ii) DIBALToluenc !

,z iiii., ~c~ ,z .. boc-LysQ~r~ ~ T~boc-Lys-H
HCI/H,O
Q~ EtOAc (iVJ ~CIJDioxan !
~leOH/0C
H,0/4C ~

7 (Vj Z.~/AcOH Z
Tcooc Lys~CO,~Ie ~H-Ljs~CO~lc 8b 2 (vi) Boc-Phe ONSu CHCl2/N~I ~

Z (vii-~ HCllDioxan Z
Boc-DPro-Phe L~s2~CC~Me 4 Boc-Phe-Lys~HCO,.?~e Boc-DPro-ONSu VC~.Cl~ 10a (viiij LiOH/H,O ¦ .
1~ 1 - Z (ix) PWH/wscdJCH,CI, Z
Boc-DP;o-Phe-Lys~rO2H . ~ Boc-DPro-Phe-Lys~CON"Bu~
!Cc nBu,NHlD~ 10d (x) Dess Maran Pcnodinane CHCl,,/AcOH r (xi) HC~JDioxan ,Z
H-DPro-Phc-Lys-CONnBu, ~ , Boc-DPro-Phe-Lys CONnBu, H2, PdJC
Ll . AcOH/H20 lOc SUBSTIIUTE SHEET

WO 92/04371 j . PCI/GB91/0147 Scheme I~T
(Svnthesis of eompound ~) Z.(i) iBuOCOCI/~MrrHF Z~
Boc-Ar~-OH ~Boe-Ar~-Cr~.
(ii) CH.N~ . ~
-(ii) HBr/E:OAe THF I

Z~ (iii) CH~(CH~)S Na æ' Boe-Arg-S(CH,)4CHt Boe-Ar-CH.Br D~LF ~.

(iv) HCl~Dioxan Boe-Phe-OPfp DIEA/CH2CI, æ (v) HC.JDiox~n æ
Boe-Phe-Arg-CH~S(CH~)4CH~ ~ Boc-DPro-Phe-Arg-C~I S(Cr.. ).C-~
Boc-DPro-OPfp 16 -- DEA/CH~CI~ --(vi) HCI/Diox~n H, Pd/C
AeOH/H.O r H-DPro-Phe-Ars-CH S(CH~)4CI-I, CU~S-, I I U ~ SI IEET.

WO 92/04371 2 a 9 ~ ~ a ~ PCI/CB91/014 Sc;~leme ~V
(Synrhes2s of compound ~) z.(i) CHF.(CF.)3CH,O ~'~; æ
Boc-Arg-CH ,Br ~ Boc-Arg-CH~OCH~CF~)3C~F~
DMF

(ii) NaBHJ~eOH i æ (iii) HCl,'Diox2n æ
Boc-Phe-Ar ,HCH~OCH~tCF.)3CHF, --- Boc-.'~rt~HrH.OCH.(CF.)~Cr.r~
~n ~oc-Phe-OPf? 1O
DIEA/CH,CI.

(iv) HCI/Dioxan Boc-DPro-OPfp DIEA/CH,Cl, æ
Boc-DPro-Phe-ArgHCH,OCH2(CF2)3CHF2 (v) Dess Martin Pcriodinane CH2Cl~!AcOH I

Boc-DPro Phc-Arg-CH20CH2(CFi)3CHF2 (vi) HCVDiox~n H2 PdlC
AcOHIH20 ~

H-DP~Phc-Arg-CH~OCH2(CF2)3C~F2 Sl ;Li~ i i I ' J i-_ S~EET

WO 92/04371 ~ ) 8 PCT/CB~1/01474 Scheme ~' (Svn~nesis ot compound l `, Z~ (i) Tce-OH/wscd Z.
Boc-Arg~-OH ~Bor-Arg-OT-^
D ~IAP~CH.C;. , .
(iij HCUDiox:~n 30c-?he-O~iSu I
CH.C!./~
Z~ (iii`) HClJDiox~n Z.
Boc-DP.o-Phe-Arg-OT.o Boc-?ho-Arg-O 1 C' ~$ Boc-DP o-ONSu ^c -- CH~CI~
(iv) Zn/AcOH

Z, iv) BuOCOC,/~ I/T.. - ~, ~oc-DP o-Ph~-Arg-OTc ~ Boc-DPro-Phe-Arg-Cr~' tEt.O ' 8 (vi) HBr/E~OAc ~,F
z (vii) HN[(CH.?sCH312 Z' Boc-DPro-Ph^-Arg-CH~N[(cH~)scH3]2 ~ -- Boc-DPro-Phe-Arg-CH.Br ~HCO~/l'HF ,,9 HC~lDiox~n I H" Pd,'C.AcOH H.O

H-DPro-Phe-Ar~-CH~N~(CH2)5cH3l2 3l ~;~JB~T,T~ SH~ET

WO 92/04371 2 ~ 9 ~ ~ ~ 8 Pcr/GB91/~)147~

Scheme VT
(Svn~hesis of comr)o~nd 4()) Z, ;i`~ N~lr CH(CO~TceJ~ Z
Bc~-~rg-C~ r ~~ec- ~rg-ch~cH(cc)~
3 _ (ii') Zn/AcOH

Z. ~ iii) Toiuene / ~ Z~
Boc-Arv'Gl,v-OH ~ -- 3oc~Arg-cH~cH( (iv) ¦ Tee-OI llw~c~
l D~AP/CH~CI~
z ~ ;v) HCllDiox~n Z~
Boc-Arv!~Gi,v-OTce Boc-Phe-Arg~Giv-OTce ~c Boc-Phe-OPf?
~~ DIEA/CH.C!.
(vi) HCI/Diox~n Boc-DPro-OPfp CH~CI,~DIEA ~ r Z2 (vii) ZntAcOH æ
Boc-DPro-Phe-Arg~Gly-OH - ----Boc-DPro-Phe-Arg~'~Gl,v-OT.e (viii) ¦ PfpOHtwscdtCH.CI.
HPro NHEt/DEA
~ z, (ix) HCltDiox~n Boc-DPro-Phe-ArgXGly-Pro-NHE~ ~H-DPro-Phe-Arg~Gly-Pro-NHE~
H2, PdtC
39 AcOHlH,O 40 SUBSTITU ~ - SHEET

WO 92/04371 2 3 ~ 8 PCr/GB91/01479 Seheme vn (Synthesis of compound ~) (i) H2, Rh/C
Boe-Phg-OH ~ Boe-Chg-OH
AcOH/H~O/60 psi (ii) HOB~Jwsed D~IF/CH.Cl, ~H3 (iii) HCVDioxan ~r H-Chg-NH. HCI ~I Boe-Chg-l~iH.

(iv) Boe-DPro-Phe-Arg-OH 27 HOBt/wsedJDMF

Z~ (v) HCl/Dioxan Boe-DP`To-Phe-Arg-Chg-.~H, -- H-DPro-Phe-Arg-Chg-NH~
44 H,, PdlC 45 SUE3;~-ri, U I E SHEET

WO 92/04371 2 ~ 3 a 8 ~ 8 6 PCl'/GB91/1)147 Scheme Vl~
(Synthesis of compound ~ ~

z" (i) HN~(CH,)5CH~j (C~ Ch z, Boc-DPro-?he-~rg-OH ~ ~ Boc-DPro-Phe-Ar~-.`.[(C'.i.~5CH3j(C','.;3C:.
HOBt/wscd~lF

(ii`) HCI/Diox~n H. PdJC
AcOHlH.O

H-DPro-Phe-Arg-N[(CH.)5CH3](CH,)3Ch , SUE,STi,UTE SHErT

WO 92/0~371 2 ~ 9 i~ ~ ~ 8 PCI/GB91tO1479 Scheme IIY
;Svn~r,esis o~ com~ou-d 18 Boc-Arg30H

¦ Dcss-Ma,~n I Penodinane CH.Cl,;AcOH

~ CF3CF,CF,I Z~
30c-Arg-H ---~Boc-ArgHCF1CF.C~:, Z. ~ HF,~ souna HClJDioxan Boc-Phe-ONSu ~TMMJC~cl2 ~

~ HC'vDioxan Z~
8c~c-DP~o-?h.:-ArgHCF2CF,CF; ~ Boc-Phe Arg~CF.CF,CF, Boc-DPro-O~Su ~IC~.Cl~
Dess Ma~n Penoainane CH2CI,JACOH

æ HCVDioxan 30c-DP o-Phe-Arg-Cr.CF1G3 ~-H DPro-Phe-Arg-CF1CF2CF3 H ,. Pd/C
AcOH/~I.O 1 8 Sl i3S T iTU I E SHEET

wo 92/04371 2 ~ 9 ~ ~ ~ 8 Pcr/GB91/0147g Scheme X
(Symhesis of compound 49) ~- ZnJAcOH . Z~
Boc-DPro-Phe-Arg-CH,Br ~ Boc-DPro-Phe-Arg-CH~
'9 HCVDiox~n H,, PdJC
AcOHlH~O i H-DPro-Phe-Arg-CH3 WO 92/04371 ~ ~) 'j 8 PCr/CBgl/n147~

Scheme Xl (SynLncs~s o~ compound S~) Z, Na~ O~)-OMe Z, 30c-A~g CH2Br DMF Boc-Arg-C}~.0 ~ OMc (i) .~'aBHJMcOH
(ii) TBDMS TriflaLe Lu~denelcH2cl2 Z' ~ CcsTic 3mmonium ni~aLc æ
30c Arg ~ C~i~O}~. ~i C~/H O 30c Arg~D~sc~o~ oM~
I ~1) 3~ss Mamn Peno~nane CH,Cl~/AcOH
(.i) B. 2/McOH/H20/~aHCO3 Z.~
Boc-ArQ~'sCCi,.~,lc ~C~)io~an Z' Boc Cha4NSu CH2CI,~NMM
~Cl'Dioxan Boc DPro4NSu ~MhU~l2Cl2 ~ ~

,- ~'aO~ OH æ
Boc DPro Cha ArglBDMsco~.H Boc DPro-Cha Arg~CO.Me OC
(i) ~OH~wscdJCH,C;, (ii) ~u,,~r~

Boc-DP ro Cha-Arg~CON"BU2 ~ Bx DP~-Cha-Arg-CON~Bu2 (u) Dess-Ma~ssn P~iodinane (i) HC~ioxan (ii) H2PdtC
MeOH/H20 HCl ~ ~
H-DPr~Cha An CO2N'Bu, SO

SUBSTlTUTc ~ -tT

WO 92/04371 2 a 9 a 3 ~ 8 ~ pcr/GB9l/ol47~
~cheme XII
;Svnur:esis or compour.c ~j iBuOCOC!,~IJl~ Z
On~-OH - ~ ~oc-Om3O~
~aBHJH,O
H~ PdJC
.~cOH/H~O

Bzl Boc-Om~OH hCHO/CH~C~ /~csO~ 3Oc~ OH
~aBH~eOH
Z ONS u E~ ~/CH Cl I

BzL ZDess-:.~arn;l Pe:ioain~ne Bzl.Z
Boc-C;~ ~OH Boc-O~n-H
CH,Cl1,AcOH

H-C(C0713zl)~ N~mD an~exCHfC~ 2 nHexV60C

CO-H)~ H, Pd/C ~Hex.C(CO,Bz!), MeOH
~,rroluene n~ RedAl~Toluene .. cx~CHCO,H ~ nHcx~CHCH,OH

hlsCVEt,.~/C~a, ~
, ~

nHcx~C~CH2Br LiBrlAcctone nHcx ,CHCH~OMs 5'' ( cont . ) SUBSTITUTE SHEET

2 ~ 5 ~ PCr/GB91/0147~
, Scheme XII (corlt.) ~ex.~ .C~i,BI -~ Boc OrnC ~.C.~.'.eY.

HCl/Dioxan Boc-Phe ONSu CH2Cl~,~MM

30C~ r~e.~ r~ r.C'v'Diox~n Bz!. Z
.. . .... ~.... x. ~ Boc-Phe C~:l~CH.C'.~'-^x Boc-DP~o-O~'Su/N~
CH,CI, H. PdlC
.~,cOH/~G
CH,S-C ~ ~ z Boc-DPro-?hc- irn~HCX~C.~n.~-x. EtOHJ~ ~ Boc2-DP~o Phe-ArgQ~CY.~CY.nHc%, Dess Manin Penodinane CH2a2ACOH

H-DP o-?he-~g-CH,C~nH:x, ~ HCllDioxan Boc DPro Phe-Arg-C~l.C~Hex~
H, Pd/C
c, AcOEVH.O

2 ~ 9 0 3 5 8 PCI/GB91/0147 Scheme xm (Synthesis of compound ~) ~ PhCOCO~HtICF æ
Boc-DPro-Phe-Arg-CHqBr ~ ~Boc-DPro-Phe-Arg-CH,OCOCOPh DMF
'9 ~HCOJH,Orl~F

æ CH,(CH,)5VAg,O Z~
Boc-DPro-Phe-Arg-CH,O(CH,)cCHl ~ Boc-DPro-Phe-Arg-CH~OH
CH,CI, HCU.Dioxan ~ H2, Pd/C AcOH/H2O

H-DPro-Phe-Arg-CH20(CH2)sCH3 SUBSTITUTE SHEET

WO 92/04371 2 ~ 9 ~ PC~/GB91/01479 Scheme XlV
(Synthesis of compound ~) Z^ PhOH/KF Z' Boc-DPro-Phe-Arg-CH,Br D~IF ~ Boc-DPr~Phc-Arg-CH~OPh ,9 HCVDioxan H, Pd/C AcOH/H.O ~

H-DPro-Phe-Arg-CH,OPh SUBSTITUTE SHEET

WO 92/04371 ~ ~ ~ a ~ ~ 8 pcr/GB9l/o147g ABBREVIATIO\IS l,SED

Abn 3-Az~bicvcio~3 ~ non3ne Ac ~ce:~ i ' cOH Ace:ic ~cid Ad~ Ad:lm n ~ i;mine Ah I ~-Aminone < lnoic ~cid ( ~i'orie~c no Boc ~ert-Butviox-c~rbonvl Bu Butvl Ch Cvciohexvl Ch~ Cvclohe~,vi3i~nine Chg C,vclohexvlglvcine Cpr Cvclopropvl DIEA Diisopropvlethvl~mine D~1AP '-Dimerhvl:lmino-pvridine 3~F Dimemvlform~mide E;OAc E:h,vi 3cet~te FAB Flst A~om Bomb3rdment ~-Fph 4-Fluorophenvl~l~nine Hch Homoc,vcloheYvl~l,qnine HOBt l-Hvdroxvbenzorriqzole hplc high perrorrn3nce liquid chrom3rogr3pilv Hvp 4-Hydroxyproiine 'Hvp trlns-4-~lvdroxyprniille SUBSTI I UTE S~EET

wo 92/04371 2 ~ 8 PCT/GB91/0147 ke:o isoat_r. -COC~ -.~ Ie Methyi .\ leCN Ace:oni~rile .~leOH Meth;lnoi ;npic medium pressure ( ~ree~tive I iicuid .: .om~to~ ?n~
pn~hyi;~ nine IM i~-~vlethylmorphoiine .~c, ~leopentyh~iycine 0~: 0~
OH H-droxy isoste.e -CY.O~i-ONSu hydroxysuccinimide Petrol Petroieum e~her 60 - ~0C
P.fp Pen~ iuorophenyl Phe-4NO~ '-Nitrophenyi~l~nine Ph~ Phenyi~lycine P.c Pipecolinic~cid Pip Piperidyl Py Pyridine R Reduced isostere -CH~-S~r S~rcosine (N-methyl ,iycine) T~3AF Tetr~butyl~mmonium fluoride TBDMS ~ert-Bu~yldimeshyisilvi Tcboc ( I -Dime~hyl- I -trichloromeshyl)eshoxy c~rbonyl Tcc 2.~ ~-Trichloroethyl SUBSTI-rUTE SHEET

wo 92/04371 pcr/GB9l/ol479 2 1~ 5 8 n~!a i . 3 . ~ -T, .~.c~n ~ lhe x~n- droa~epy l Tesahyarofuran I hi Thicnyialanine .ic ~ 3~'-Te~~h-aroiso~inol:ne-3-car~ox-ii. ac:d ~i. L~in ia-e. cl;:oma~og~pny wscd water soluble carbodiimide Z BenzyloxycarDonvl !; G ; ~

SUB~; ~ ITUTE SHEET

WO 92t04371 _ 2 i3 9 a ~ 5 ~ pcr/GB~l/o147~

BIOLOGICAL AC~I'~'IT~

Compounds were ested n v-l-o ~~ ~he Col owi-._ ac~ivities using stanaard procedures:

(a) Inhibi.ion of human . ssue kallikrein ?lasma kall Xrei~
and mast cell tryptase hydrolysinc ~he ch~omoaeni~
suDstrates 5-2266 S-2302 and S-2266 -espec~:~ely ~e_hs~
s adaDted ~-om t~at of ~ohansen u ~ et 21., ~ n~
ss. Reac. la86 ~ 185-1a2). A series Oc 2easu.emen~s were car~ied out usina a -.umber o _ e_en.~ -.h_- =^~
concent~ations and a least .wo di feren. subs_~a=e concentrations. The inhlbitory constan~ Ki was determined a-zphically usina a Dixo~. -lc. (M. ~ixo-~iochem. ~. 195~ 5~ ~o) (b) Inhibi__~~ of kinin release from low and hich ~oiec--ia-weight kininoqens by tissue and plasma kallikrein respectively. A series of measurements were carried out using two subst.ate concent_ations. ~he act vity is calculated 2s the amount o~ kinen released per m nute this beina determ-ned by -adioimmu-.oassay usinc polyclor;al antibodies. The inhibitory ~onstan~ Ki was deter~ined graphicall-l usina a Dixon plot.

All the examples ln ~ables 1 - 9 have Ki values ln the range 10 ~ - 10 3 M against one or all of the enzymes in the chromogenic assay.

In vivo ac~ivity has been tested in well-established pharmacology models of asthma based on the sensitised guinea pig. A selection of these hibitors representing -he different chemical types pro ~ to be highly effect ve in blocking both the acute phase response and the late phase reaction their efficacy being comparable or superior to those of the topical steroids and B2-agonist currently used in asthma therapy.

SUBSTITU-I E SHEtT

WO92/04371 2 ~ 9 ~ 3 ~ ~ PCT/GB91/0147~

When the compounds of _he present invention are used as a medicine, there are no critical limitations to ~he administration methods. The present enzyme inhibitor can 3e formulated by any conventional method in pharmaceutics. ~or example, the present enzyme inhibitor may be applied in any conventicnal manner including int-avenous inject~on, intramuscular lnjectlon, instlllation, orai administration.
respiratory inhalation, instillation, -hinencnysis, ~na external sKin .rea~ment. ..lthouah ~here are no c-lt-^zl limitations to the aàminls_-atlon dosage, ~he suitable doszae is 1 to 1000 mg/day/person.

SUE~Ti';'u"l E SHEET

Claims (11)

C L A I M S

1. Kininogenase inhibitors, optimally not exceeding the size of a hexapeptide, represented by:- wherein A and B = amino acyl (including amino acyl analogue) the same or different forming a dipeptide group the amino acid of A carrying a terminal group and being any amino or imino-acid residue (but preferably of D-configuration) and of B being a lipophilic amino-acid residue of D- or L-configuration but not proline or a proline analogue, or a conformational analogue of said dipeptide group wherein the peptide link is replaced by -CH2-NH- ('reduced'), -CH(OH)-CH2-('hydroxy'), -CO-CH2- ('keto'), -CH2-CH2-('hydrocarbon') or other conformational mimic of the peptide bond and in:- the side chain R1 is that of a basic amino acid or amino acid analogue (preferably of L-configuration) and R is H or lower alkyl (C, -C4) or C.alpha. or the peptide link comprising -N(R)- is replaced leading to a conformational mimic as above Y = a binding enhancing or carbonyl activating group preferably selected from H (when A or B
must be cyclohexylalanine, preferably D if at A or L if at B) or alkyl (C1 - C20) or fluoroalkyl (C2 - C12): substituted oxymethylene; thiomethylene; sulphoxy-methylene; sulphonylmethylene; aminomethylene:
hydrazino-methylene; -CH2-Het (where Het = a substituted or unsubstituted heterocycle);
substituted amino (but when the resulting compound is a secondary alkylamide B must not be phenylalanine); an amino-acid group or is ester or amide; a carboxylic secondary amide or primary amide, when B must be cyclohexyi-alanine or adamantylalanine or other bulky lipophilic, non-aromatic amino-acid (not Ala Leu Ile Val Nva Met Nle Phe Tyr Trp Nal (1));
tertiary-carboxamide; carboxy-alkyl group or its ester or amide or amino acyl derivative.

2. Compounds according to claim 1 wherein A is selected from imino-acids, (e.g. D-proline or an analogue of proline e.g.
pipecolinic acid, azetidine carboxylic acid); lipophilic amino acids (e.g. DPhe, DCha, DChg); strongly basic amino acids (e.g. D-Arg or a homologue or analogue of Arg, e.g.
amidino- or guanidinophenylaianine); or N-alkyl or C.alpha.-alkyl (including benzyl) derivatives thereof.

3. Compounds according to claim 1 or 2 wherin 3 is selected from L-Phe, L-Cha, L-.alpha.Nal, L-Tal, L-(4F)Phe L-(NMe)Phe or other substituted phenylalanines; or N-alkyl or C.alpha.-alkyl (including benzyl) derivatives thereof.

4. Compounds according to claim 1, 2, or 3 wherein R1 is selected from 3-guanidinopropyl or other ?anidinoalkyl group, (or an amidinoalkyl or aminoalkyl group also para- or meta substituted guanidino or amindo-benzyl or preotected forms of the above; optionally basic nitrogens are alkylated (Me, Et or other).

5. Compounds according to any preceding claim wherein subject to the provisors in regard to the nature of Y expressed in claim 1, selection for Y is from:-Y = H or alkyl including fluoroalkyl Y = -CH2Q
where Q = -OR2 or -SR2 or -SOR2 or -SO2R2 or -NHR2 or or wherein R2, R4 and R5 are as below Y = -CH2 CHR6 or wherein R4, R5 and R6 are as below Y = amino acyl or a group forming a substituted amide or hydrazide Y = a group forming an .alpha.-keto amide - COR 9 or or and in which further:-R4 and R5 the same or different but not both hydrogen = H
or C1-C20 alkyl (which may be further substituted), acyl or alkyl sulphonyl is a hetercyclic ring (D = nitrogen or carbon in Group IV and N in Groups VI and X) optionally unsaturated and optionally with further hetero atoms and substituents R6 = hydrogen, alkyl, hydroxyalkyl, aminoalkyl, alkylaminocarbonyl R3 = -NH2 as such or alkylated, or amino acyl.

6. Compounds according to any claim 1 to 4 wherein subject to the provisos in regard to the nature of Y expressed in claim 1, selection for Y is from:-Group I
Y = H; alkyl including branched alkyl (C1-C20); aryl alkyl; or cycloalkyl (c1-C20); perfluoroalkyl or partially fluorinated alkyl (C2-C12); [e.g. Y = Me;
-CH(CH2CH2CH2CH3)2; -CH(CH2CH2CH2CH3)CH2-cyclohexyl;
-CH2CF2CF2CF3; -CF2CH2CH2CH3].
Group II
Y = -CH2QR2 where Q = O, S, SO, SO2, NH and where R2 = Alkyl, branched alkyl or aryl alkyl; or -CH2-R3 where R3 = perfluoroalkyl or partially fluorinated alkyl, branched or not (C1-C12).
Group III
Y = R4, R5 the same or different = alkyl, branched alkyl, cycloalkyl, acyl, alkylsulphonyl, carboxyalkyl (the carboxyl group may be further derivatized to form an ester or amide with an amino-acid or dipeptide), carbamoyl, sulphanoyl, N-dialkylamino-, arylalkyl, haloalkyl including fluoroalkyl, cyanoalkyl, alkoxyalkyl, hydroxyalkyl, mercaptoalkyl, aminoalkyl and deriJatives thereor e.g. esters, amides and thioesters, or one of R4 or R5 = hydrogen Group IV
Y = -CH2 - where D = nitrogen or carbon and is a saturated or unsaturated heterocyclic ring or a bicyclic ring system, each is 5- 8 membered, where there may be other hetero-atoms (N, S, 0) and carbons or nitrogens may optionally be substituted by alkyl, brancned alkyl, cycloalkyl, carboxyalkyl, carboxy (attached to carbon), amino, alkoxy, alkoxymethyl or (carbon) as carbonyl or other groups beneficial for interaction with the enzyme.

Gruop V

Y = -CH2CH(R6)CONR4R5 R4, R5 as defined in Group III.
R6 = hydrogen lower alkyl, branched alkyl, cycloalkyl, hydroxyalkyl, amino-alkyl, alkylaminocarbonyl.

Group VI
Y = R6 as defined in Group V and - as defined in Group IV (but with D = N) Wo 92/04371 65 PCT/GB91/01479 Group VII
Y = an amino-acid residue or any amide (secondary or tertiary) or ester of that residue, L or D
configuration. Preferred residues are of lipephilic amino-acids e.g. norleucine, cyclohexylalanine, homocyclohexylalanine, cyclohexylglycine, tertbutylglycine.
Group VIII
Y = R7 = H (when however B is not phenylalanine unless R3 is carboxylalkyl or derivatized carboxylalkyl); or alkyl, branched alkyl (C1 - C12), cycloalkyl (C1-C20) carboxyalkyl or bis(carboxyl)alkyl, which may be derivatized at the carboxyl group to form an amide e.g.
with an amino-acid (preferred is arginine) or a substituted amine; N`-dialkylamino; N`-alkylamino-;
R8 = R7 the same or different but excluding H.
Group IX
Y = -CO-R9 but only if B is a bulky non-aromatic lipophilic amino acid or its N.alpha. alkyl ( C1 - C4 ) derivative (e.g. cyclohexylalanine but excluding Ala, Leu, Ile, Val, Nva, Met, Nle, Phe, Tyr, Trp, Nal(1) and their N-methyl derivatives) where R9 = Nh2, N`-alkylamino (where the alkyl groups include branched and/or cycloalkyl); an amino-acid residue.

Group X
Y = as defined in Group IV (but D = N) Group XI
Y = -CO-NR4R5 R4, R5 as defined in Group III, but not H.

7. Any one of the compounds specifically listed in Tables 1 to 9 herein.

8. A method of treatment (including prophylactic treatment) of an inflammatory or other condition as set out in the indications (1) to (6) herein, particularly an allergic inflammatory condition, wherein an effective amount of a peptide or peptide-analogue kininogenase inhibitor is administered topically or systemically to a patient suffering from or at risk of the condition, the peptide or peptide analogue used being optionally of hexapeptide of smaller size.

9. A method of treatment of the allergic inflammatory phase of asthma, wherein an effective amount of a kininogenase inhibitor e.g. a mast cell tryptase inhibitor is administered topically or systemically to a patient suffering from or at risk of the condition.

10. A method of preparation of a medicament for the topical or systemic treatment (including prophylactic treatment) of conditions as in claim 8 particularly for allergic inflammatory conditions and especially for asthma as in claim 9, wherein a kininogenase inhibitor is associated in effective amounts with a pharmaceutically acceptable diluent or carrier to constitute said medicament.

11. A method of treatment or of preparation of a medicament as above wherein the inhibitor is a compound as claimed in any or claims 1 to 7.