HUT64084A - Process for producing pharmaceutical compositions comprising quininogenase 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 R<1> 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

The present invention relates to enzyme inhibiting compounds and to the treatment of diseases using these compounds. The enzyme inhibitors of the invention are primarily useful for inhibiting the production of quinine in the living body.

Quinines are known to be released by natural vasoactive peptides, which are selective proteases in the body from high molecular weight precursors (quininogens) by the action of quininogens.

Kinines are known to play a role in the following pathological conditions:

(a) conditions associated with vasodilation and hypotension such as septic, anaphylactic and hypovolaemic shock; carcinoid syndrome and dumping syndrome,

b) inflammatory processes such as acute arthritis, pancreatitis, local burns, bruising and cerebral edema,

(c) conditions associated with bronchoconstriction, particularly initial asthma allergic reactions,

d) allergic inflammations, in particular allergic rhinitis and conjunctivitis, known hay fever, and bronchial inflammation and consequent obstruction that occur in the non-acute but often fatal inflammatory phase of asthma.

Quinines (bradykinin, kallidin and Met-Lys bradykinin) are potent mediators of inflammatory processes. For example, their main effects are:

a) Increase capillary permeability leading to mucus formation and edema, ···········································································································•

b) effective vasodilators in small arteries, thereby lowering blood pressure and increasing blood flow,

(c) pain inducers,

(d) tightening of the bronchial smooth muscle,

e) activate the phospholipase A ₂ enzyme and thus stimulate the biosynthesis of prostaglandins (PG), which mediate many of their effects.

With regard to prostaglandins, it should be noted that certain effects of quinines, in particular their pain mediation and vascular permeability effects, are enhanced, although the PGs themselves do not induce pain or induce vascular permeability at defined concentrations in inflammatory tissues. PGs are therefore either mediators or enhancers of the kinin.

Despite the above-described knowledge about quinines, relatively little attention has been paid so far to reducing their effects. For example, in the treatment of asthma, attention is primarily given to acute bronchoconstriction reactions, for which effective drugs are known. Cases of death from the progressive development of bronchial obstruction, and currently not only the absence of clinically effective quinine release inhibitors, but the concentration of quinine release itself, at least in the treatment of allergic inflammatory processes, appear new. The only agent that is actually known and clinically relevant as a quinine release inhibitor is aprotinin (Trasylol, Bayer), which has been isolated from a proteinase inhibitor and bovine tissue (lung, lymphatic and pancreatic). This ······································································································································ ··· a single peptide chain of 58 members. However, primarily as a known trypsin inhibitor aprotinin (Ki = 10 ^"13 M) and 10 ⁶ times less active against kinin release. It has been shown to have marginal benefits in acute pancreatitis, a very serious condition in which this substance inhibits trypsin activation of the zymogens of the papase serine proteinases and has also been used in traumatic-hemorrhagic shocks. Aprotinin should be administered parenterally and often painful reactions occur at the injection site.

Quininogenases are serine proteinases, i.e., proteinases in which the hydroxy group of the serine group is the nucleophile, which is involved in the formation of the substrate transition state. It releases quinines (bradykinin, kallidin) from quininogens with limited proteolysis. Several types of kininogenases are known:

(a) Tissue kallikrein (also called TK, glandular kallikrein, GT or urinary kallikrein, UK) is found in pancreas in salivary glands, intestines, kidneys and urine. It has a molecular weight of 30,000 and acts primarily on low molecular weight quininogen (LMWK) and promotes the release of quinine kallidin (KD). Tissue kallikrein does not have a potent and fast-acting endogenous inhibitor in plasma.

b) Plasma kallikrein (PK) is produced in plasma as an inactive zymogen which is activated by factor XIIa and is part of the internal coagulation process. Molecular weight 100000 and

Its preferred substrate is the high molecular weight quininogen (HMWK) from which bradykinin (BK) is released. Plasma kallikrein is rapidly and potently inhibited in plasma by endogenous inhibitors, also known as cl-inactivators and α2-macroglobulins.

c) Hizo-cell tryptase, which is not as active as kallikrein in quinine release, has been found to be present in large amounts in mast cells in lung tissue of asthmatic patients.

The kininogens which are the natural substrates for the kininogenases (effective an inhibition of cysteine proteinases such as cathepsins Β for example, H and L, calpain and papain and, Ki = about 10 ^-11.) Occur in two types:

(a) low molecular weight quininogen (LMWK), with a molecular weight of between 50000 and 70000, depending on the species of origin and the degree of glycosylation;

(b) high molecular weight quininogen (HMWK), molecular weight

88,000 to 114,000, and serve as an alternative precursor for quinines, a cysteine proteinase inhibitor, and, together with plasma kallikrein, play a role in initiating internal coagulation processes.

The two quininogens, whose mRNAs are transcribed from the same gene, share the same primary sequence throughout the N-terminal or heavy chain (H-chain) region, the quinine region and the first twelve of the C-terminal or light chain (L-chain) of the amino acid. At this point their structure is different, HMWK has a longer L-chain (MW = about 45 K) than LMWK

-6 (4.8 Κ).

For example, the cleavage of human HMWK by plasma kallikrein is schematically illustrated in Bar 1, Figure 2 illustrates in more detail the sequence of the cleavage site, and more fully illustrates the sequence of Figure 3, where conventional numbering of groups adjacent to the cleavage site is shown. cleavage site. After excision of one or another quinine sequence, the H and L chains are linked by a single disulfide bridge, as shown in Figure 1.

The figures show that plasma kallikrein and tissue kallikrein act at one site to release the C-terminal site of quinine, cleaved between groups 389 and 390, but act on bradykinin (by PK) and kallidin (by TK) on both sides of group 380. To release its N-terminal.

The role of PK and HMWK as coagulation facilitators in the internal cascade processes is not mentioned in the enzymatic release of kinin. However, many of the effects of PK and all of the effects of TK include such release, which is mediated by kinins, which are released from their respective substrates of HMWK and LMWK by selective proteolysis.

·· · * ·· * · ··········································································································•

-7s- s s-s i

I

I

H · chain

L chain

HMWK -100K

PK <OOK s-s

H · chain

Figure 1. Effect of HMWK on PK (Figure 1)

PK

4 ·

TK

III cleavage site

Figure 2

II cleavage site

3rad cinema

Kailidin ·,

Effects of Human Quininogens on PK and TK Sequence Details

PK.TK ^P 2 ^P 3 ^P 4 ^P 5

-Phe-Ser-Pro-Phe-Arg ----- Ser-Ser-Arg-Ile — Gly385 389 390 394 ^P 5 ^P 4 ^P 3 ^P 2 ^P 1

pi

Figure 3. Cleavage sequences at the I site of human HMWK

-8As shown, plasma kallikrein and tissue kallikrein act as a single site to release the C-terminal site of quinine, between the 389 and 390 groups, and on one side of the 380 group, bradykinin (with PK) or kallidin (with TK). To release the N-terminal end.

The role of PK and HMWK as coagulation facilitators in the internal cascade process does not involve the enzymatic release of kinins. However, many of the effects of PK and all of the effects of TK include such release, which is mediated by selective proteolysis of the corresponding kinin released from the HMWK and LMWK substrates.

indications

The main clinical indications for quininogenase inhibitors include inflammatory conditions, particularly allergic inflammations such as asthma and hay fever. A more complete list of these indications is given below:

1) allergic inflammations such as asthma, purulent conjunctivitis, (hay fever), runny nose, urticaria;

2) inflammations such as arthritis, pancreatitis, gastritis, inflammatory bowel disease, burns, bruising, conjunctivitis;

3) smooth muscle spasms such as asthma, angina;

4) hypotension such as haemorrhagic shock, bacterial bleeding or anaphylaxis, carcinoid syndrome, dumping syndrome;

5) edema such as burns, brain trauma, anginoneuretic

• * ··

edema as a result of treatment with angiotensin converting enzyme inhibitors, independently of these;

6) pain and irritation such as burns, wounds, cuts, skin rashes, stings, insect bites.

The present invention relates to a method of treatment including prophylactic treatment of inflammatory or other conditions based on the above indications, in particular allergic, inflammatory conditions comprising administering an effective amount of a peptide or peptide analogue kininogenase inhibitor topically or systemically to patients suffering from or suffering from they are at risk. For optimum activity, dosing ability and stability in the body, the size of the compounds should not exceed the size of a hexapeptide, i.e. no more than six amino acids or amino acid analogs, but the presence of additional groups is particularly pro-drug groups from which groups may be cleaved. and compounds which produce the desired effect in a primary manner are not excluded.

The method of treatment of the present invention is particularly useful for treating an allergic inflammatory phase of asthma, wherein an effective amount of a quininogenase inhibitor, such as an effective amount of a mast cell tryptase inhibitor, is administered topically or systemically to a patient suffering from or suffering from said disease.

The present invention also relates to the manufacture of pharmaceutical compositions suitable for use as described above.

........ · •••• · * ♦ • ··· · · · · · · · · · · ···

For the topical or systemic treatment of conditions, particularly allergic, inflammatory, and more particularly asthma, wherein a quininogenase inhibitor is mixed with a pharmaceutically acceptable diluent or carrier.

Above, the kininogenase inhibitor is preferably, but not necessarily, the novel type disclosed herein, which, without limiting it to synthetic low molecular weight compounds, and selectively inhibits the action of quininogenases and thereby blocks the release of quinine from the kininogens. Inhibitors are peptide analogs of a size not exceeding the size of the hexapeptide, expressed in amino acid or amino acid analogue group, having the amino acid sequence corresponding to the cleavage site of the kininogens, analogous enough to bind to the active site of the kininogenase, they can be hydrolyzed, remain bound and inactivate the enzyme.

The inhibitors of the present invention are described in formula (I)

ABCY (I) wherein A is a P3 group, B is the P ₂ group, C represents the Ρχ group and Y is a carbonyl-activating or binding group, and A, B and C are each amino acyl or amino acyl an analog group that binds to peptide bonds or similar structures and thus mimics a peptide chain. Outside these important groups

• · · ·

Of course, other groups may also be present, such as aminoacyl or aminoacyl analog groups.

More specifically, the compounds of the present invention are represented by Formula II.

A - B

R

II which formula

A and B are the same or different aminoacyl groups (including aminoacyl analogs) which form a dipeptide group and A may also carry a terminal group (other than hydrogen) and may be any amino or imino acid group. (but preferably in the D configuration) and B may be a lipophilic amino acid residue having the D or L configuration but different from proline or proline analogue or a structurally analogous group to said dipeptide group wherein the peptide bond is -CH2-NH-. (reduced), -CH (OH) -CH 2 - (hydroxy) -CO-CH ₂ (keto), -CH ₂ CH ₂ (hydrocarbon) or other peptide bond structural mimics, and groups of formula

-12R '{X • · · <· · «· · · * · λ · · ··· · · · · ·

ι R

III ^R1 side chain is a basic amino acid or amino acid analogue side chain (preferably in L configuration) and R is hydrogen or C 1-4 alkyl or C ^or -N (R) -peptidkötés substituted with, when structurally mimic as above we win groups. For example, C is ^a nitrogen atom.

Y is a bond-enhancing carbonyl activating group such as hydrogen (only when A or B is cyclohexylalanine, preferably in configuration D for A and in configuration B for L) or C 1-20 alkyl or C 2-12 fluoroalkyl. group; substituted oxymethylene; thiomethyl group; sulfoxides methylene group; sulfonyl-methylene group; aminomethylene group; hydrazino-methylene;

-CH 2 - Het where Sev is a substituted or unsubstituted heterocyclic group; a substituted amino group, but when the resulting compound is a secondary alkylamide group, B cannot be a phenylalanine group; an amino acid residue or an ester or amide thereof; a carboxylic acid secondary amide or primary amide group if B is to be a bulky lipophilic, non-aromatic amino acid group such as cyclohexylalanine, adamantylalanine, but * · * «· 9 ·» ·. . · .. · .... .. · '. ·

-13Ala, Leu, Ile, Val, Nva, Met, Nle, Phe, Tyr, Trp, Nal (1); tertiary carboxamide group; a carboxyalkyl group or an ester or amide thereof.

The substituents mentioned above are preferably known functional groups which increase the binding affinity to the enzyme and / or improve the therapeutic effects. As to structural analogs or groups mimicking dipeptide groups, those structures include non-natural amino acid (or amino acid analog) groups, or which are non-peptide-like groups and which in the compound of formula I are A and B or B and C or both. which is structurally similar to the group present in the parent peptide when bound to the active side of the enzyme. It may also contain groups that are more favorable for interaction with other enzymes, such as hydrogen bonding. Mimicking groups can be any of the groups published for such analogs.

For example, dipeptide mimicking groups of DPro-Phe (Ph may be replaced by CH ₂ Ph) include:

CO ₂ H

Ph

i) Reduced mimicking ii) Hydroxy or iii) Keto or group hydroxyethylene ketomethylene mimicking group

arc)

v) vi) hydrocarbon mimicking group

Figure 4 DPro-Phe imitation group

Preferred compounds of the above formula are described below.

Preferred groups in this context are imino acids such as D-proline or a proline analog such as pecolic acid, azetidine carboxylic acid, etc .; lipophilic amino acids such as DPhe, DCha, DChg; highly basic amino acids such as D-Arg or guanidino phenylalanine, and B are preferred: L-Phe, L-Cha, L-aNal, L-Tal, L- (4F) Phe, L- (NMe) Phe or other substituted phenylalanines. A and B may also be N-C 1-4 alkyl or C 1 -C 7 alkyl, such as methyl, benzyl analogs of these amino acids. Preferred terminal groups in this case include, for example, lower alkyl or acyl, but not limited to aminoacyl, alkylsulfonyl, which may be straight or branched or cyclic, aminoalkyl, carboxy, alkyl, hydroxyalkyl, or other protecting groups commonly known in peptide chemistry.

Suitable Y groups are those that are part of the structure that provides the desired bond to the active site and are not merely non-interfering end groups. They form binding groups that increase the affinity for the enzyme and / or groups that activate the adjacent carbonyl group by making it more electrophilic. Such groups are described, for example, by the formula (IV):

ARC

Wherein the α-nitrogen in the peptide bond to the B group may be free or substituted, for example with methyl or other C 1 -C 4 alkyl, so that R ¹ and R are as defined above, but particularly preferably R ¹ is 3-guanidino-propyl; or another guanidinoalkyl group or an amidinoalkyl or aminoalkyl group, and may also be a para or meta-substituted guanidino or amidinobenzyl group or a protected form of these groups, the basic nitrogen atom may also be alkylated such as methyl or ethyl, and in which group

Y is as defined above, primarily and in the most general sense, followed by particularly preferred groups, in both cases the above conditions apply. The preferred groups are described in more detail in the following groups, indicated by Roman numerals:

denotes H (denotes aldehyde) or alkyl, including fluoroalkyl (denotes ketones), denotes CH ₂ Q, denotes -OR ² or -SR ² or -SOR ² or Q

you are a group

-NHR ² or N

R ⁴ or -D, wherein -D_J is a ring in which D is a member of the ring, and • «

-17V VI

R ^2, R ⁴ and R ⁵ are as defined below, is

-CH ₂ CHR ⁶ CON

R ⁴

Or R ⁵

-ch ₂ chr ⁶ co-d 2) wherein R ⁴ , R ⁵ and R ⁶ are as defined below,

VII VIII

ΙΧ-Χ-ΙΧ which is above

Y is an aminoacyl group or a group which forms a substituted amide or hydrazide,

Y is an α-keto-amide-forming group such as COR ⁹ or -CO-D, or / ^{R 4}

A group represented by the formula -CON ^ RS

R ² is alkyl or substituted alkyl, including aryl or arylalkyl, and CH ₂ R ³ where R ³ is fluoroalkyl,

R ⁴ and R ⁵ are the same or different, but both cannot be hydrogen or C 1-20 alkyl optionally substituted or acyl or alkylsulfonyl, -D 4 is a heterocyclic ring wherein D is nitrogen - or carbon atom above (IV) ·· ····

-18 or N in the above groups (VI) and (X), which ring may be unsaturated and may optionally contain further heteroatoms and substituents,

R ⁶ is hydrogen, alkyl, hydroxyalkyl, aminoalkyl, or alkylaminocarbonyl,

R ⁹ is -NH 2, which may also be alkylated, or amino-acyl.

The above groups are described in more detail below, along with the conditions already given above:

Group I

Y is H; C 1 -C 20 straight or branched chain alkyl; arylalkyl; or C 1-20 cycloalkyl; C2-C12 perfluoroalkyl or partially fluorinated C2-C12 alkyl; for example, Y may be Me; -CH (CH ₂ CH ₂ CH ₂ CH ₃ ) ₂ ; -CH (CH ₂ CH ₂ CH ₂ CH ₃ ) CH ₂ ~ -cyclohexyl; -CH2CF2CF2CF3.

Group II

CH2QR Y ^2, wherein Q is 0, S,

SO, SO ² , NH and R ² are straight or branched alkyl or acyl groups having 1 to 12 carbon atoms; or C 1-20 cycloalkyl; or aryl or arylalkyl; or -CH 2 R ³ group wherein R ³ represents a perfluorinated alkyl or partially fluorinated linear or branched alkyl group, wherein the alkyl from 1 to 12

-19szénatomos.

Group III

Y is

Y = -CH ₂ N

wherein R ⁴ and R ⁵ are the same or different and may be alkyl, branched alkyl, cycloalkyl, acyl, alkylsulfonyl, carboxyalkyl (the carboxyl group may also be derivatized and thus be an ester or amide formed with an amino acid or a dipeptide), carbamoyl, sulfamoyl, Ν-dialkylamino, arylalkyl, haloalkyl such as fluoroalkyl, cyanoalkyl, alkoxyalkyl, , hydroxyalkyl, mercaptoalkyl, aminoalkyl and derivatives thereof, ester, thioester or amide derivatives thereof, or -R ⁴ and R ⁵ is hydrogen.

Group IV

Y is Y = -CH ₂ -D 4 where D is nitrogen or carbon and represents a saturated or unsaturated heterocyclic or bicyclic ring having 5 to 8 members and which further heteroatoms (N, S, O) and carbon or nitrogen are optionally they may be substituted with alkyl, branched alkyl, cycloalkyl,

carboxyalkyl, carboxy (attached to carbon), amino, alkoxy, alkoxymethyl or carbonyl groups or other groups which are capable of interacting with the enzyme.

Group Y

Y is -CH ₂ CH (R ⁶ ) CONR ⁴ R ⁵ wherein R ⁴ and R ⁵ are as defined above for group (III) and R ⁶ is hydrogen, lower alkyl, branched alkyl, cycloalkyl; , hydroxyalkyl, aminoalkyl, alkylaminocarbonyl.

Group VI

Y is -CH ₂ CH (R ⁶ ) COD 1), wherein R ⁶ is as defined above for (V) and -iP) same as for (IV), except that D = N.

Group VII

Y is an amino acid group or any amide group (secondary or tertiary) or an ester of these groups in the L or D configuration. Preferred groups are lipophilic amino acid residues such as norleucine, cyclohexylalanine, homocyclohexylalanine, cyclohexylglycine or tert-butylglycine.

Group VIII

Y is

R7

Y = -NR ⁸ • · · ·

-21 wherein R ⁷ is H (when B is not phenylalanine if only R ⁸ is not carboxyalkyl or derivatized carboxyalkyl) or alkyl, C 1 -C 12 branched alkyl, C 20 -cycloalkyl, carboxyalkyl or bis (carboxyl) alkyl groups in which the carboxyl group may be even functionalized, such as an amide formed with, for example, an amino acid, preferably arginine, or a substituted amine, N'- dialkylamino, N'alkylamino,

R ⁸ is as defined for R ⁷ except hydrogen.

Group IX

Y is -CO-R ⁹ , but only when B is a bulky non-aromatic lipophilic amino acid or its N ^a -C 1-4 alkyl-

a derivative such as cyclohexylalanine but excluding Al, Leu, Ile, Val, Nva, Met, Nle, Phe, Tyr, Trp, Nal (1) and N-methyl derivatives thereof, and R ⁹ is NH ₂ , N ¹ -alkylamino, (alkyl may be branched and / or cycloalkyl) or amino acid.

Group X

Y is -CO-D ^), wherein ^ is as defined above for group (IV) (but D = N).

Group XI

Y is a C0-NR ⁴ R ⁵ group wherein • • · · · · ·

-22R ⁴ and R ⁵ are as described in Group (III) as defined but is not hydrogen.

The following examples further illustrate the invention. The examples are given in the following forms:

- nine tables of compounds with reference to their structure, molecular structure determined by FAB, and Roman numerals to which of the preceding groups, eight detailed preparation examples, twelve reaction schemes referenced in the detailed examples, a description of the in vitro assay for inhibition of kinases and an in vivo assay for efficacy against asthma.

The structure of the intermediates was confirmed by NMR.

• · · · • · · · · · · · · · · · · · · · · · ·

-231. spreadsheet

Aldehydes [M + H] ⁺ Class

Me-DPhe Cha Arg-H

473 I

Table 2

Thiomethylene analogs and sulfomethylene analogs

[M + H] ⁺ class 17 H-DPro Phe Arg CH ₂ S (CH 2) ₄ Me 519 Π 60 H-DPro Phe Arg CH ₂ S ⁿ Bu 505 Π 61 H-DPro Phe DLArg CH ₂ SO ₂ ⁿ Bu 537 Π Table 3 Ethers [M + H] ⁺ class 23 H-DPro Phe Arg CH ₂ OCH ₂ (CF ₂ ) ₃ CHF ₂ 647 Π 62 H-Pro Phe Arg CH ₂ OCH ₂ CF ₃ 515 Π 63 Boc-DPro Phe DLArg CH ₂ OCH ₂ CF ₃ 615 Π 64 H-DPro Phe DLArg CH ₂ OCH ₂ CF ₃ 515 Π 65 H-DPro Cha DLArg CH ₂ OCH ₂ CF ₃ 521 II 66 H-DPro Phe Arg CH ₂ OCH (Me) CF ₂ CF ₂ CF ₃ 629 Π 67 H-DPro Phe Arg CH ₂ OCH ₂ CF ₃ 515 Π 68 H-DCha Phe Arg CH ₂ OCH ₂ CF ₃ 571 Π 69 H-DArg Phe Arg CH ₂ OCH ₂ CF ₃ 574 Π 70 H-DChg Phe Arg CH ₂ OCH ₂ CF ₃ 557 Π 54 H-DPro Phe Arg CH ₂ O (CH 2) ₅ CH ₃ 517 Π 55 H-DPro Phe Arg CH ₂ OPh 509 Π

Table 4

Aminomethylene and its Analogs ······························································· ·

[M + H] ⁺ class H-DPro Phe Arg CH ₂ N [(CH ₂ ) ₅ Me] ₂ 600 m H-DPro Phe DArg CH ₂ (RS) Tha 556 ARC H-DPro Phe Arg CH ₂ (RS) Tha 556 ARC H-DPro Phe Arg CH ₂ l -Pip-3- (RS) CO ₂ Et 572 ARC H-DPro Phe DLArg CH ₂ 1 -Pip-3- (RS) CH ₂ O (CH ₂ ) 3Me 586 ARC H-DPro Phe DLArg CH ₂ l-Pip-3- (RS) CH ₂ NEt ₂ 585 ARC H-DPro Phe DLArg CH ₂ l-Pip-3-CONEt ₂ 599 ARC H-DPro Phe DLArg CH ₂ l-Pip-3- (RS) CONH ⁿ Bu 599 ARC H-DPro Phe DLArg CH ₂ l-Pip-3- (RS) CON ( ⁿ Bu) ₂ 655 ARC H-DPro Phe DLArg CH ₂ l-Pip-4- (l'-Pip) 583 ARC H-DPro Phe Arg CH ₂ (NMe) AhaNH ⁿ Bu 615 ARC H-DPro Phe Arg CH ₂ -Abn 540 ARC H-DPro Phe Arg CH ₂ -Hyp (.O ⁿ Bu) NHEt 629 ARC H-DPro Phe Arg CH ₂ -Sar Pro NHEt 628 m H-DPro Phe Arg ΟΗ ₂ -Ήνρ (Ο ^η Βιι) ^δ Ό ^η Βιι 644 ARC H-DPro Phe DLArg CH ₂ Pic NEt ₂ 641 ARC H-DPro Phe DLArg CH ^ HypíCPBuj ^ Bu 644 ARC H-DPro Phe DLArg CH ₂ Pro ^B CONEt ₂ 599.9 ARC H-DPro Phe DLArg CH ₂ 1-Pip-3 (RS) CO ₂ H 544 ARC H-DPro Phe Arg CH ₂ l-Pip-3 (RS) CH ₂ CONEt ₂ 613 ARC H-DPro Phe Arg CH ₂ N (CH ₂ Ch) (CH ₂ ) ₅ Me 612 m H-DPro Phe DLArg CH ₂ N (Oc) ₂ 657 ΠΙ H-DPro Phe Arg CH ₂ N (Et) Ch 542 tendon H-DPro Phe Arg CH ₂ N (Me) (CH ₂ ) ₄ NH ₂ 517 ΠΙ H-DPro Phe Arg CH ₂ N (Me) ⁿ Bu 502 m H-DPro Phe DLArg CH ₂ N ⁿ Bu ₂ 544 m H-DPro Phe DLArg CH ₂ N ["Bu] SO ₂ ⁿ Bu 594 m H-DPro Phe DLArg CH ₂ NrBu] (CH ₂ ) ₃ CONH ₂ 573 m H-DPro Phe DLArg CH ₂ NPHex] (CH ₂ ) ₇ NH ₂ 629.5 m H-DPro Phe DLArg CH ₂ NrHex] (CH ₂ ) ₅ NH ₂ 601 ni H-DPro Phe DLArg CH ^ PHexKCH ^ Nfy 573 m H-DPro Phe DLArg CH ^ FBuKCH ^ Ph 620 m

• · · · · · · · · · · · · · · · · · · · · · · · · · · ·

-254. Table Cont.

Aminomethylene Ketones and their Analogs · «·· ♦ · ···· ··

[M + H] ⁺ Qassim 102 H-DPro Phe DLArg CH ₂ N [ ^T1 Hex] (CH ₂ ) ₆ CONH ₂ 643 ΠΙ 103 H-DPro Phe DLArg CH ₂ N [ ⁿ Hex] (CH ₂ ) ₆ NH ₂ 615 ΠΙ 104 H-DPro Phe DLArg CH ₂ N [ ⁿ Hex] (CH ₂ ) ₇ OH 630 hl 105 H-DPro Phe DLArg CH ^ fHexKCH ^ NHAc 671 ΠΙ 106 H-DPro Phe DLArg CH ₂ N [ ^Tl Hex] (CH ₂ ) ₆ CONHEt 671 ΙΠ 107 H-DPro Phe DLArg CH ₂ N [ ⁿ Hex] (CH ₂ ) ₆ CO ₂ Me 658 m 108 H-DPro Phe DLArg CH ₂ N [ ^7l Hex] (CH ₂ ) ₆ CO ₂ H 644 m 109 H-DPro Phe DLArg CH ₂ N [ ^T 'Bu] (CH ₂ ) ₃ CONEt ₂ 629 ni 110 H-DPro Phe DLArg CH ₂ Nf ⁿ Bu] (CH ₂ ) ₃ CONHEt 601 hl

Table 5

Keto isostere containing analogs

[M + H] ⁺ class 40 H-DPro Phe DLArg ^ Gly Pro-NHEt 599 VI 111 H-DPro Phe Arg ^ ly Pro-NHEt 599 VI 112 H-DPro Phe Arg ^ Gly Arg-NH ₂ 530 V 113 H-DPro Phe DLArg ^ Gly Ala-NH ₂ 545 V 114 H-DPro Phe DLArg ^ Gly Aha-NH ₂ 587 V 115 H-DPro Phe DLArg ^ Gly Aha-NIT'Bu 643 V

Table 6

Substrate Analogs [M + H] ⁺ Class

45 H-DPro Phe Arg Chg-NH ₂ 557 vn 116 CPr-CO Phe ArgChg-NH ₂ * vn 117 MeSO ₂ DPro Phe Arg Chg-NH ₂ 635 vn 118 MeCO DPro Phe Arg Chg-NH ₂ 599 vn 119 H-DArg Phe Arg Ser-NH ₂ * vn 120 H-DCha Phe Arg Ser-NH ₂ * vn 121 H-DPhe Phe Arg Ser-NH ₂ * vn 122 H-DPic Phe Arg Ser-NH ₂ * vn

• · · ♦

Table 6 continued

Substrate Analogs [M + H] ⁺ Qass

123 H-DPic Phe Arg Chg-NH ₂ * vn 124 H-DPro Phe Arg Ser-NH ₂ * vn 125 H-DPro Cha Arg Ser-NH ₂ * vn 126 H-DPro Cha Arg Gly-NH ₂ * vn 127 H-DPro Phe Arg Ser-Arg-NH ₂ * vn 128 H-DPro Phe Arg Lys-NH ₂ * vn 129 H-DPro Phe Arg Aha-NH ₂ * vn 130 H-DPro Phe Arg Phe-NH ₂ 565 vn 131 H-DPro Phe Arg Leu-NH ₂ 531 vn 132 H-DPro Phe Arg De-NH ₂ 531 vn 133 H-DPro Nal Arg Ser-NH ₂ * vn 134 H-DPro Phe Arg DAha-NH ₂ 531 vn 135 H-DPro Phe Arg Aha-NH (CH ₂ ) ₃ Me 587 vn 136 H-DPro Phe Arg Nleucinol 518 vn 137 H-DPro Phe Arg SerOBu NH ₂ 561 vn 138 H-DPro Phe Arg Cha-NH ₂ 571 vn 139 H-DPro Phe Arg Ada-NH ₂ 623 vn 140 H-DPro Phe Arg Hch-NH ₂ 585 vn 141 H-DPro Nal Arg Cha-NH ₂ * vn 142 H-DPro Cha Arg Cha-NH ₂ * vn 143 H-DPro PhepNO ₂ Arg Ser-NH ₂ * vn 144 H-DPro Nal Arg Ile-NH ₂ * vn 145 H-DPro Phe Arg Ile Pro-NH ₂ * vn 146 H-DPro Phe Arg Aha Pro-NH ₂ * vn 147 H-DPro Nal Arg Aha-NH ₂ * vn 148 H-DPro Cha Arg Npg-NH ₂ * vn 149 H-DPro Cha Arg Hch-NH ₂ * vn 150 H-DPro Nal Arg Hch-NH ₂ * VII 151 H-DPro Phe Arg Npg-NH ₂ 545 vn 152 H-DPro Phe Arg Chg-l-Pip 625 vn 153 H-DPro Phe Arg Chg-NH (CH ₂ ) ₅ Me 641 vn 154 H-DPro 4-Fph Arg Chg-NH ₂ 575 vn

♦ Satisfactory amino acid analysis obtained

Table 7

Amidok [M + H] ⁺ Class

47 H-DPro Phe ArgNKCH ^ iMeKCHihCh 626 vm 155 H-DPro Phe Arg N (Me) ⁿ Bu 488 vm 156 H-DPro Phe Arg NH (CH ₂ ) ₃ CO Arg-NH ₂ * vm 157 H-DPro Phe Arg NH (CH ₂ ) ₃ NH ₂ 475 vm 158 H-DPro Phe Arg NH (CH ₂ ) ₄ CO Arg-NH ₂ * VNI 159 H-DPro Phe Arg NH (CH ₂ ) ₄ NH ₂ * vm 160 H-Pro Phe Arg NH (CH ₂ ) ₅ CO Arg-NH ₂ 687 vm 161 H-DPro Phe Arg NH (CH ₂ ) ₅ NH ₂ 503 vm 162 H-DPro Phe Arg NH (CH 2) ₆ CO Arg-NH ₂ 701 vm 163 H-DPro Phe Arg NH (CH ₂ ) ₇ CO Arg-NH ₂ 715 vm 164 H-DPro Phe Arg NH (CH ₂ ) ₇ CONH (CH ₂ ) ₃ x 4e 615 vm 165 H-DPro Phe Arg NH (CH ₂ ) ₇ NHAc 573 vm 166 H-DPro Phe Arg NH (CH ₂ ) ₇ x NH ₂ 531 vm 167 H-DPro Phe Arg NH (CH ₂ ) ₇ CONH ₂ 559 vm 168 H-DPro Phe Arg NH (CH ₂ ) ₇ CO-Gly Gly Arg-NH ₂ 829 vm 169 H-DPro Phe Arg NH (CH ₂ ) ₇ CO-Gly Arg-x NH ₂ 772 vm 170 H-DPro Phe Arg NH (CH ₂ ) ₇ CO-Gly Gly-Gly Arg-NH ₂ 886 vm 171 H-DPro Phe Arg N [Hex] ₂ 586 vm 172 H-DPro Cha Arg NHCH ₂ Ch 520 vm 173 H-DPro anal Arg NHCH ₂ Ch 564 vm 174 H-DPro βΝπΙ Arg NHCH ₂ Ch 564 vm 175 H-DPro His Arg NHCH ₂ Ch 504 vm 176 H-DPro (4Me) Phe Arg NHCH ₂ Ch 528 vm 177 H-DPro Phe Nar NHCH ₂ Ch 500 vm 178 H DPIC Phe Arg NHCH ₂ Ch 528 vm 179 H DTIC Phe Arg NHCH ₂ Ch 576 vm 180 H DThi Phe Arg NHCH ₂ Ch 570 vm 181 ⁿ Bu DPro Phe Arg NHCH ₂ Ch 570 vm

* Satisfactory amino acid analysis obtained

Table 8

Ketones [M + H] ⁺ Class

49 H-DPro Phe ArgCH ₃ 417 I 48 H-DPro Phe Arg (CF ₂ ) ₂ CF ₃ * ♦ I 53 H-DPro Phe ArgCH ₂ CHPHex] ₂ *** I

♦♦ Molecular ion not decoded

Table 9 a-Ketoamides [M + H] ⁺

class

H-DPro Phe DL Lys CON ^ Bu ^ 530

H-DPro Cha DL .Arg CON [ ⁿ Bu] ₂ *** ♦♦♦ [M + H] ⁺ Not Available «··« · * «♦ · 4 · • ·· · ♦ ·· ♦ · ··· «·· · • 9 9 · 9 · · · · · · · · · · · · · · · · ·

-29I. example

Me-DPhe-Cha-Arg-H (Compound 5)

The title compound 5 was prepared according to Scheme I. In the following, Arabic numerals refer to the compounds shown in this scheme. Small Roman numerals in parentheses refer to reaction steps.

(i) 10.2 mmol of isobutyl chloroformate ester are mixed with 9.23 mmol of Boc-Arg (Z2) OH in 25 ml of dry THF at -20 ° C and 11.08 mmol of N-methyl- morpholine. After 20 minutes, the solid was filtered and the filtrate was added at 0 ° C to 10.3 mmol of sodium borohydride in 10 mL of water. After 3 hours, 0.3 M KHSO4 solution was added and the crude product was extracted with EtOAc and purified by flash chromatography on silica gel (EtOAc / petroleum ether = 4/6). The alcohol 1 was isolated as a white solid (97%).

(ii) a compound of formula 1, 4.75 mmol of the Boc group was removed by treatment with a mixture of saturated HCl / dioxane, followed by 9.5 mmol of Boc-Cha-ONSu acylated with 20 mL CH2C12 at 0 ° C ^_ N-methyl in the presence of morpholine. After 2 hours, the reaction mixture was worked up in a known manner and the crude product was purified by flash chromatography (silica gel, EtOAc / petroleum ether = 4/6). The alcohol 2 is thus obtained in the form of a colorless oil (90%).

(Iii) 4.27 mmol of the compound of formula 2 above are removed. the Boc group was treated with saturated HCl / dioxane and the resulting product was reacted with 5.12 mmol of Z (NMe) Dphe-Oh in the presence of 10.2 mmol of HOBt, 6.1 mmol of water-soluble carbodiimide and N-methylmorpholine. ml in DMF at 0 °. After 18 hours, the reaction mixture was worked up in a known manner and the product was purified by flash chromatography (silica gel, EtOAc / petroleum ether = 1/1). The alcohol 3 is thus obtained in the form of a colorless oil (52%).

(iv) 2.22 mmol of the alcohol of formula 3 above is dissolved in CH2Cl2 / ACOH (30/1) and 4.5 mmol of DessMartin Periodinane is added after 2.5 hours at room temperature and the reaction mixture is diluted with EtOAc and 32 ml of sodium are added. -thiosulphate containing saturated NaHCO3 solution. The crude product was purified by flash chromatography (silica gel, EtOAc / petroleum ether-3/7). This gave the aldehyde 4 as a colorless oil (75%).

(v) Aldehyde 4 (1.65 mmol) was dissolved in 50 mL

MeOH / _H2 O / AcOH = 90/9 / l of palladium on carbon hydrogenation catalyst and a mixture of 5%. The crude product was purified by MPLC (* Vydac C ^ g (1,525 gm) MeCN / H ₂ O / TFA) to give pure compound (CH-851) of 5 as a white solid in the amount of 780 mg. (Tlc EtOAc-Py-AcOHH ₂ O = 30/20/6/11, on silica gel). After hydrolysis with 6N HCl solution at 110 ° C for 2 hours, the peptide content is 40% relative to Chara (FAB mass spec [M + H] ⁺ = 474 (Calc.

-31m / z = 472).

II. example

H-DPro-Phe-Lys-CO 2 ⁿ BU ² (Compound 11, Scheme II) (i) 14.8 mmol TcbocONSu was mixed with 12.2 mmol H-Lys (Z) -OMe.HCl in 50 mL CH 2 Cl 2. and 14.8 mmol of triethylamine. After 3 hours at room temperature, the reaction mixture was worked up in a known manner and the product was purified by flash chromatography (silica gel, EtOAc / pertol = 7/13). This gave the ester 6 as a colorless oil (100%).

(ii) Add 50 mmol of diisobutylaluminum hydride as a 1.5 molar solution in toluene to 12.2 mmol of the above compound 6 in 100 ml of anhydrous toluene at 78 ° C for 20 minutes. After an additional 10 minutes, methanol (10 mL) was added followed by a saturated solution of Rochelle's salt (100 mL). After 2.5 hours, the reaction mixture was worked up in a known manner and the product was purified by flash chromatography (silica gel, EtOAc / petroleum ether = 3/7). This gave 7 as a colorless oil (49%).

(iii) 18 mmol of potassium cyanide and 30 mL of 1 M hydrochloric acid were mixed with 5.9 mmol of compound 7 in 30 mL of ethyl acetate. After 18 hours at room temperature, the reaction mixture was worked up in a known manner and the resulting product was purified by flash chromatography (silica gel, EtOAc / petroleum ether = 4/6). This gave cyanohydrin 8 as a colorless oil (88%).

· · · · · · · · · · · · · · · · · · · · · · ···

-32 (iv) (V) (Vi) ml of 4M hydrochloric acid in dioxane was mixed with 5.28 mmol of 8 in 15 ml of anhydrous methanol at 0 ° C and after 15 hours at room temperature 15 ml of ice water was added. After 3 days solid KHCO3 was added at 4 ° C, the reaction mixture was worked up in a known manner and the product was purified by flash chromatography (silica gel, EtOAc / pertol ether = 11/9). The ester 8b was obtained as a yellow oil (59%).

Activated zinc powder was added in small portions to a solution of 8b (3.1 mmol) in AcOH / H2O = 9/1 (25 mL), and after 1.5 hours at room temperature the zinc was filtered off, the filtrate was concentrated in vacuo and the residue dissolved in EtOAc. The resulting solution was washed with saturated NaHCO 3 solution, then dried over Na ₂ SO 4 and concentrated in vacuo. The amine 9 was obtained as a colorless amine (85%).

The amine of Formula 9 (2.63 mmol) was acylated with Boc-Phe-ONSu (3.04 mmol) in CH ₂ Cl ₂ (30 mL) at 0 ° in the presence of N-methylmorpholine. After 3 hours, the mixture was worked up in a known manner and the crude product was purified by flash chromatography (silica gel, EtOAc / petroleum ether = 6/4). The ester 10a was obtained as a colorless oil (92%). The Boc group from 2.41 mmol of 10a was removed by treatment with saturated HCl / dioxane and the product was acylated with 2.92 mmol of Boc-DPro-ONSu in 30 mL of CH ₂ Cl ₂ at 0 ° in the presence of morpholine. After 3 hours, the reaction mixture (vii) • ·

Work up in a known manner and purify by flash chromatography (silica gel, Et 2 O / petroleum ether = 3/1). This gave the ester 10b as a colorless oil (64%).

(viii) 1.6 mmol of lithium hydroxide and 3 mL of water were mixed with 1.54 mmol of 10b in 30 mL of THF. After 4 hours at room temperature under vacuum, the THF was removed, adjusted to pH 4 with 1 M citric acid, and extracted with CHCl3. The organic extracts were washed with brine, dried over Na ₂ SO 4 and concentrated in vacuo. This gives the acid 10c as a colorless oil (70%).

(ix) 1.3 mmol of pentafluorophenol and 1.3 mmol of water-soluble carbodiimide were mixed with 1.07 mmol of 10c in 20 mL of CH ₂ Cl ₂ at 0 °. After 2.5 hours, 2.1 mmol dibutylamine was added at 0 ° and the pH was adjusted to 9 with DIEA. After 18 hours at room temperature, the reaction mixture was worked up in a known manner and the product was purified by flash chromatography (silica gel, EtOAc / petroleum ether = 7/3). The resulting amide 10d is obtained as a colorless oil (48%).

(x) 0.97 mmol of Dess-Martin periodinane was added to 0.52 mmol of 10d compound dissolved in 40 mL of CH ₂ Cl ₂ . After 2 hours at room temperature, an additional 0.52 mmol of Dess-Martin periodinane was added, and after a further 3 hours the reaction mixture was diluted with EtOAc and 7.3 M sodium thiosulfate was added with water and saturated NaHCO 3. The product obtained by flash chromatography • ·

-34 (silica gel, EtOAc / petroleum ether = 9 / l). The ketoamide 10e is obtained as a colorless oil (57%).

(xi) 0.24 mmol of the compound of formula 10e is removed by treatment with saturated HCl / dioxane.

The product was dissolved in 9/1 ACOH / H2O and hydrogenated over 5% palladium on carbon. The crude product was purified by mplc (Vydac C ^ g, 15-25 μτα, MeCN / H ₂ O / TFA). 89.7 mg of compound 11 (CH-1463) are obtained. HPLC analysis (Novopak C g, 4 μ, 8 x 100 mm, linear gradient from 20 to 80%, 0.1% TFA / MaCN, 0.1% TFA / H ₂ O for 25 minutes at 1.5 mL / min), showing the presence of 2 epimer D-Arg (40%) at 11.2 min and L-Arg (60%) at 12.6 min. After hydrolysis with 6N hydrochloric acid at 110 ° C for 22 hours, Phe = 0.93, Pro = 1.07 according to amino acid analysis.

III. example

_{H-DPro-Phe-Arg-CH2} -S _(CH2) 4CH3 (17 Compound Scheme III) 46.1 mmoles of Boc-Arg _(Z2) OH (i) was dissolved in 200 ml of dry THF was added 50.85 mmol of N-methylmorpholine and 50.73 mmol of isobutyl chloroformate at -20 °. After 20 minutes, 0.1 molar diazomethane in Et ₂ O was added at -5 °. After 2 hours, diazoketone 12 is obtained as a yellow solid.

(ii) Dissolve 46.1 mmol of diazoketone 12 in anhydrous THF, add 69.15 mmol of HBr in EtOAc at -20 °. · · ···· ·· ····

After -35 minutes, saturated NaHCO3 solution was added. The crude product was extracted with EtOAC and crystallized from EtOH to give BocArg (Z2) CH2Br 13 (85%).

(iii) 1.27 mmol of 1-pentanethiol in 5 ml of anhydrous DMF are mixed with 1.4 mmol of sodium hydride. After 30 minutes, 1.27 mmol of 13 were added at 40 ° C for 20 minutes and -5 ° C for 2.5 hours. 0.3 M KHSO4 was then added and the product was extracted with EtOAc and flash chromatographed on silica gel (EtOAc / petroleum ether = 15/85) to give 14 as a colorless oil (74%).

(iv) 0.94 mmol of compound 14 is deprotected with saturated HCl / dioxane solution and then acylated with 1.13 mmol of Boc-Phe-OPfp in CH ₂ Cl ₂ at 0 ° in the presence of DIEA. The crude product was purified by flash chromatography (silica gel, EtOAc / petroleum ether = 3/7) to give thiomethylene analog 15 as a colorless oil (55%).

(v) 0.52 mmol of the compound of formula 15 was deprotected by treatment with saturated HCl / dioxane solution and the resulting product was dissolved in DMF and treated with 0.63 mmol of Boc-DPro-OH in 1.05 mmol of HOB and 0.76 mmol of water soluble carbodiimide and N-methylmorpholine. The product is then worked up in a known manner and purified by flash chromatography on silica gel (EtOAc / petroleum ether = 4/6). This gives 16 thiomethylene compounds of the formula: Colorless ····

In the form of -36 oil (75%) · (vi) 0.38 mmol (16), the Boc protecting group is removed by treatment with saturated HCl / dioxane solution. The product was dissolved in AcOH / H2O = 9/1 and hydrogenated over 5% palladium on carbon. The crude product was purified by MPLC (Vydac C ₁₈ , 15-25 μ, MeCN / H ₂ O / TFA) to give 41 mg of 17 (CH-574). The product was assayed by HPLC (Novapak C13, 4 μ, 8x100 mm, linear gradient of 20 -> 80% 0.1% PFA / MeCN in 0.1% TFA / H ₂ O into, for 25 minutes in 1.5 ml of / min), the assay shows the presence of two epimers, D-Arg (<5%) at 9.8 minutes and Arg (> 95%) at 11.1 minutes. After hydrolysis with 6N hydrochloric acid at 150 ° for 1.5 hours, Phe = 0.80, Pro = 1, according to amino acid analysis.

All analogs summarized in Table 2 were prepared according to the method described above. Compound 61 was prepared by oxidation of compound 60 with meta-chloroperoxybenzoic acid.

ARC. example

H-DPro-Phe-Arg-CH ₂ OCH ₂ (CF ₂ ) ₃ CHCF ₂ (Compound 23, Scheme IV) (i) 2.45 mmol of 1H, 1H, 5H-octafluoro-1-pentanol in 8 mL of anhydrous

After stirring for 30 minutes at -40 ° C, 1.63 mmol of bromoketone 13 is added and the mixture is kept at this temperature for 30 minutes and then at -5 ° C. 2.5

4

4

44 ··· · ·· ···· ·· ··

-37 (ii) (iii) (iv) hours. Then 0.3 M KHSO4 was added and the product was extracted with EtOAc and purified by flash chromatography on silica gel (EtOAc / petroleum ether = 15/85). The fluoro ester of the formula was obtained as a colorless oil (69%).

Dissolve 1.13 mmol of the fluoroether 18 in 40 mL of MeOH, add 1.18 mmol of sodium borohydride at 0 °, and after 15 minutes add 0.3 M KHSO4 and extract with EtOAc. This gave 19 as a colorless oil (88%).

19 mmol of the compound of formula (19) are deprotected with saturated HCl / dioxane and the resulting product is dissolved in CH ₂ Cl ₂ and acylated with 1.2 mmol of Boc-Phe-OPfr at 0 ° in the presence of DIEA. The product was isolated in a known manner and purified by flash chromatography on silica gel (EtOAc / petroleum ether = 3/7) to give 20 as a colorless oil (55%).

The compound (0.55 mmol) is deprotected with saturated HCl / dioxane and then acylated with Boc-DPro-OPfp (1.63 mmol) in CH ₂ Cl ₂ at 0 ° in the presence of DIEA. The product was recovered in a known manner and flash chromatographed on silica gel (EtOAc / petroleum ether = 4/6) to give 21 as a colorless oil (48%).

0.24 mmol compound 21 was dissolved in CH2 Cl ₂ / AcOH = 30 / l mixture was added Dess-Martin periodinane, 0.48 mmol. After 2 hours at room temperature (V)

Μ 4 ······· · · · · · · · · · · · · · · · · · · · · · · · · · · ·

The -38a mixture was diluted with EtOAc and then added with water and saturated NaHCO3 to 3.5 mol of sodium thiosulfate. The crude product was purified by silica gel flash chromatography (EtOAc / petroleum ether = 7/13) to give the fluoroether 22 as a colorless oil (64%).

(vi) deprotecting 0.16 mmol of the fluoroether compound 22 and purifying according to Example III (vi). Purification afforded 23 (CH-619) as a white solid (50.9 mg). According to the HPLC assay performed (Novapak Οχθ 4μ, 8x100 mm, linear gradient from 20 to 80%, 0.1% TFA / MeCN, 0.1% TFA / H ₂ O over 25 min, 1.5 mL / min ) a single product is present (TR = 11.5 minutes). After hydrolysis with 6N hydrochloric acid at 150 ° C for 1.5 hours, Phe = 1.0, Pro = 1.2, according to amino acid analysis.

The analogs summarized in Table 3 are prepared as described above, except for compounds 54 and 55 which are prepared according to Schemes XIII and XIV, respectively.

Example V

H-DPro-Phe-Arg-CH ₂ N [(ch ₂ ) ₅ ch ₃ ] ₂

DL (Compound 31, Scheme V) (i) Dissolve 18.5 mmol of Boc-Arg (Z ₂ ) OH in 50 mL of 4L. · ·

Trichloroethanol (20.35 mmol), water soluble carbodiimide (20.35 mmol) and dimethylaminopyridine (0.93 mmol) were added at -0 ° C to -39CH2Cl2. After 3 hours, the mixture was worked up in a known manner to afford the trichloroethyl derivative 24 as a colorless oil (100%).

(ii) Deprotection of 18.1 mmol 24 with saturated HCl / dioxane and 27.2 mmol Boc-Phe-ONSu in CH 2 Cl 2 at 0 ° in the presence of N-methylmorpholine. After 3 hours, the reaction mixture was worked up in a known manner and the crude product was purified by flash chromatography on silica gel (EtOAc / petroleum ether = 2/8) to give 25 as a white solid (97%).

(iii) 17.4 mmol of 25 are deprotected with saturated HCl / dioxane and acylated with 26.2 mmol of Boc-DPro-ONSu in CH 2 Cl 2 at 0 ° in the presence of N-methylmorpholine. After 3 hours, the mixture was worked up in a known manner and the product was purified by flash chromatography on silica gel (EtOAc / petroleum ether = 35/65) to afford the protected tripeptide 26 as a colorless oil (94%).

(iv) To a solution of 16.47 mmol (26) in glacial acetic acid was added activated zinc powder, after 3 hours the zinc was filtered off, the filtrate was concentrated and the resulting crude product was flash chromatographed on silica gel (EtOAc / petroleum ether / AcOH = 74/25 / l). yielding pure tripeptide 27 as a white solid (91% 0).

9 · »· ·« · 9 ** ·· • · · · * * * • ve · ·· • «« · · · · · · · · · ···

-40 (v) 15 mmol of the protected tripeptide 27 are dissolved in 40 ml of anhydrous THF, 18 mmol of N-methylmorpholine and 16.6 mmol of isobutylchloroformate are added at -20 °, and after 20 minutes the mixture is taken up in Et2O. 35 mmol of diazomethane in solution at -5 °. After 2.5 hours, diazoketane 28 is obtained as a yellow oil.

(vl) Diazoketane 28 (15 mmol) was treated with HBr in anhydrous THF (22.2 mmol) in EtOAc at -20 ° and saturated NaHCO 3 was added after 45 min. The crude product was extracted with EtOAc and flash chromatographed on silica gel (EtOAc / petroleum ether = 1/1) to give bromoketone 29 as a white solid (72%).

(vii) 1.25 mmol of dihexylamine and 0.8 mmol of NaHCO3 are mixed with 0.23 mmol of bromo ketone 29 in 5 mL of dry THF, and after 18 hours, 0.3 mol of KHSO4 are added and the crude product was purified by flash chromatography on silica gel (EtOAc / petroleum ether = 35/65). This afforded the protected aminomethylene ketone 30 as a yellow oil (54%).

(viii) The aminomethylene ketone 30 (0.12 mmol) is deprotected as in Example III (vi) and the compound is purified. 41 mg (CH-694) of 31 is obtained in the form of a white solid. According to the HPLC analysis performed (linear gradient from 20 to 80%, 0.1% TFA / MeCN, 0.1% TFA / H ₂ O in 25 min, 1.5 mL / min), two epimers are present, D-Arg (50%) at 11.2 min and L-Arg (50%) at 12.5

-41percnél. After hydrolysis with 6N hydrochloric acid at 110 ° C for 22 hours, Phe = 0.91, Pro = 1.09, based on the amino acid analysis.

The analogs summarized in Table 4 are prepared as described above. The required amines are prepared by known procedures such as reductive amination and Curtius rearrangement.

VI. Example I H-DPro-Phe-Arh ^R Gly-Pro-NHEt (Compound 40, Scheme VI) (i) 6.81 mmol H2C (CO2Tce) 2 was mixed with 30 mL dry THF with 5.67 mmol sodium hydride and After 45 minutes, bromo ketone 13 (4.52 mmol) was added at -5 °. After 2.5 hours, 0.3 M KHSO4 was added and the crude product was extracted with EtOAc and purified by silica gel flash chromatography (EtOAc / petroleum ether = 2/8). This gave 32 as a colorless oil (83%).

(ii) To a solution of 32 (3.65 mmol) in glacial acetic acid is added activated zinc. After 2.5 hours at room temperature, the zinc is filtered off and the filtrate is evaporated to give the diacid 33.

(iii) The diacid (33) is dissolved in toluene and heated at reflux for 45 minutes. The solvent was then evaporated and the crude product was purified by flash chromatography on silica gel (EtOAc / petroleum ether).

-42ter = 60/39 / l). This gave 34 as a colorless oil (70% from 32).

(iv) Trichloroethanol (2.82 mmol), water-soluble carbodiimide (2.81 mmol) and dimethylaminopyridine (0.117 mmol) were added to compound 34 (2.34 mmol) in CH 2 Cl 2 at 0 °. After 2.5 hours, the mixture was worked up in a known manner and the crude product was purified by flash chromatography on silica gel (EtOAc / petroleum ether = 85/15) to give trichloroethyl 35 as a colorless oil (83%).

(v) 1.85 mmol of 35 are deprotected using saturated HCl / dioxane and the resulting product is acylated with 6.04 mmol of Boc-Phe-OPfp in CH ₂ Cl ₂ in the presence of DIEA. After 2 hours, the reaction mixture was worked up in a known manner and the crude product was purified by flash chromatography on silica gel (EtOAc / petroleum ether = 2/8) to give 36 as a colorless oil (85%) · (vi) 1.58 mmol from 36 deprotecting the Boc group using saturated HCl / dioxane and acylating the resulting product with 5.12 mmol Boc-DPro-OPfp in CH ₂ Cl ₂ in the presence of DIEA. After 2 hours, the reaction mixture was worked up in a known manner and the crude product was purified by silica gel flash chromatography (EtOAc / petroleum ether = 35/65) to give 32 as a colorless oil (79%).

(vii) 1.13 mmol for a solution of 37 in glacial acetic acid. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

-43activated zinc powder was added and after 2.5 hours at room temperature the zinc was filtered off, the filtrate was concentrated and flash chromatographed on silica gel (EtOAc / petroleum ether / AcOH = 70/29 / L). This gave 38 as a colorless oil (76%).

(viii) 0.26 mmol 38 of the protected keto isostere is converted to its Pfp ester by treatment with 0.29 mmol Pfp-OH and 0.31 mmol water soluble carbodiimide in 8 mL of CH ₂ Cl ₂ at 0 ° for 2.5 hours. under. The resulting Pfp ester was coupled at 0 ° to 0.78 mmol H-Pro-NHEt.HCl salt in the presence of DIEA. After 18 hours, the mixture was worked up in a known manner and the product was purified by flash chromatography on silica gel (CHCl3 / MeOH / AcOH = 92/1) to give 39 as a colorless oil (91%).

(ix) 0.23 mmol of the protected keto-isosterone compound 39 was deprotected as described in Example III (vi) to afford 40 mg of purified compound 40 (CH-595) as a white solid. According to the HPLC analysis performed (linear gradient from 10 to 50%, 0.1% TFA / MeCN, 0.1% TFA / H ₂ O over 25 min, 1.5 mL / min), two epimers are present, D-Arg (46%) at 10.1 min and L-Arg (54%) at 11.7 min. After hydrolysis with 6N hydrochloric acid at 150 ° for 1.5 hours, Phe = 0.91, Pro = 1.09 according to amino acid analysis.

The analogs of Table 5 were prepared as described above.

• · ·· ·

VII. example

H-DPro-Phe-Arg-Chg-NH ₂ (Formula 45, Scheme VII) (i) Dissolve 19.2 mmol of Boc-Phg-OH in 100 mL of AcOH / H 2 O = 9/1 and in the presence of Rh / C catalyst. Hydrogenate at 60 psi for 3 days. The catalyst was filtered off and the solution was evaporated to give Boc-Chg-OH 41 (100%).

(ii) 4.1 mL of water soluble carbodiimide, 4.3 mmol of HOBt were triturated with 3.9 mmol of 41 in 60 mL of CH ₂ Cl ₂ / DMF = 2/1 at room temperature and after 30 min 0.8 mL of 35% ammonia solution. After a further 3 hours at room temperature, the mixture was worked up in a known manner and the product was recrystallized from EtOH to give amide 42 as a white solid (60%).

(iii) 0.42 mmol of Boc is removed from the compound 42 using saturated HCl / dioxane to give amide 43 as a white solid (100%).

(iv) 0.38 mmol of the protected tripeptide 27 are dissolved in 5 mL of DMF, 0.76 mmol of 43, 0.76 mmol of HOBt and 0.46 mmol of carbodiimide and N-methylmorpholine are added at 0 ° C. temperature. After 18 hours at room temperature, the reaction mixture was worked up in a known manner and the product was purified by flash chromatography on silica gel (CHCl3 / MeOH / = 99/1). This afforded the protected tetrapeptide 44 as a white solid (69%).

-45 (v) 0.27 mmol of the protected tetrapeptide 44 is deprotected and the product is purified according to Example III (vi). 58 mg (CH-640) of compound 45 are obtained in the form of a white solid. According to the HPLC analysis performed (linear gradient from 10 to 50%, 0.1% TFA / MeCN, 0.1% TFA / H ₂ O over 25 min, 1.5 mL / min), a single peak was found. At 2 minutes. After hydrolysis with 6N hydrochloric acid at 110 ° C for 22 hours, Arg = 0.96, Phe = 1 and Pro = 0.95 according to amino acid analysis.

The analogs summarized in Table 6 are prepared as described above or by other known peptide coupling procedures (M. Bodansky & A. Bodansky, The Practice of Peptide Synthesis, Springer-Verlag, 1984).

VIII. example

H-DPro-Phe-Arg-N [(CH ₂ ) 5] (CH ₂ ) ₃ Ch, Scheme VIII (i) 0.32 mmol of the protected tripeptide 27 are dissolved in 5 mL DMF and added at 0 ° C. 96 mmol HN [(CH ₂ ) 5] (CH ₂ ) ₂ Ch, 0.64 mmol HOBt, 0.38 mmol water soluble carbodiimide and N-methylmorpholine. After 18 hours at room temperature, the reaction mixture was worked up and the product was purified by flash chromatography on silica gel (EtOAc / hexane = 4/6) to give amide 46 as a colorless oil (32%).

(ii) The protected tripeptide amide 46 (0.1 mmol) is deprotected and the product is purified by purification.

-46III (vi). 17 mg (CH-985) of 17 is obtained in the form of a white solid. According to HPLC analysis showed (linear gradient of 40 -> 90% 0.1% TFA / MeCN into 0.1% TFA / H ₂ O over 25 minutes, 1.5 ml / min), single peak can be detected At 10.7 minutes. After hydrolysis with 6N hydrochloric acid at 110 DEG for 22 hours, Phe = 1.1, Pro = 0.99 according to amino acid analysis.

The analogs summarized in Table 7 are prepared as described above. The desired tripeptide precursors are prepared in a similar manner to 27 or using known peptide coupling procedures (M. Bodansky & A. Bodansky, The Practice of Peptide Synthesis, Springer-Verlag, 1984). The required amines are either commercially available or can be obtained by Curtius rearrangement. For BU 181, ⁿ BU-DPro-0H is prepared by reductive amination.

• · • · ······

Preparation of -47I

Z, (i) 'BuOCOCl / NMM / THF i * ·

Boc-Arg-OH

NaBFVHoO

Z,

Boc-Arg-OH (ii) HCl / Dioxan Boc-Cha-ONSu / NMM

CH ₂ Cl ₂

Z.

Z (NMe) DPhe-Cha-Arg 6 OH (iii) HCl / Dioxane

Z,

I

Boc-Cha-Arg - OH

Z (NMe) DPhe-OH

HOBt / WSCD

NMM / DMF (iv) Dess Martin Periodinane

CH ₂ Cl ₂ / AcOH

(v) H ₂ , Pd / C

Z (NMe) DPhe-Cha-Arg-H> · Me-DPhe-Cha-Arg-H

MeOH / H ₂ O / AcOH • · · ·

-48 • · · · ······· · · · ζ

H-Lys-OMe. HCI

Ζ

Tcboc-Lys HCN

8a ✓ I (arc HCVDioxan

MeOH / O ° C

H, 0/4 ° C v

Tcboc-Lys ^ CO ₂ Me

8b

Z

Boc-Phe-Lys-OPROM COnJv'e

10b arc) LiOH / H-10

THF '

Z

Boc-DPro-Phe-Lys®HC0 ₂ H

10c

H-DPro-Phe-Lys-CON ⁿ Bu ₂

Preparation of Compound Scheme II (i) TcbocONSu

--------------------------->.

Et ₃ N / CH ₂ Cl ₂ (iii ^- ) KCN

HCVH ₂ O

ErOAc

Z

Tcboc-Lys-OMe (ii) DIBAL / ToIucL ·

Z

Tcboc-Lys-H arc) Zn / AcOH Z

-----------------> H-Lys2HCO ₂ xMe (vi) Boc-Phe-ONSu CHCWNMM ▼

(vii) HCVDioxan

Boc-DPro-ONSu

NMM / CH ₂ Cl ₂

Z

Boc-Phe-Lys CCKM

10a

[ix) PfpOH / wscd / CH ₂ Cl ₂ Z ^a Bu ₂ NH / DEEA (x) (xi) HCVDioxan -4 ---------------------------- U Dess Martin Periodinane CHCiyAcOH Boc-DPro- r z ³ he-Lys-CON ⁿ Bun

H ₂ , Pd / C AcOH / H ₂ O

10c · * «« · · 4 «4 4 ♦ ♦ 4 4 4 4 * * * 44 * 44

Preparation of compound -49III

Z, Boc-Arg-OH (i) 'BuOCOCl / NMM / THF

------------------>

(ii) CH ₂ N ₂

Z ₂

Boc-Arg-CHNi (ii) HBr / EtOAc

THF

Zn ι "

Boc-Arg-S (CH ₂ ) ₄ CH ₃ (iii) CH ₃ (CH ₂ ) S'Na ⁺

-4 DMF -----------------------

Boc-Arg-CH ₂ Br (iv) HCl / Dioxan

Boc-Phe-OPfp

DIEA / CH ₂ C1 ₂ y

z, ι ~

Boc-Phe-Arg-CH ₂ S (CH ₂ ) ₄ CH ₃ (v) HCl / Dioxan

Boc-DPro-OPfp

DEA / CH ₂ Cl ₂

Z,

I -> - Boc-DPro-Phe-Arg-CH ₂ S (CH ₂ ) ₄ Cl (vi) HCl / Dioxane Hn Pd / C AcOH / H ₂ O

H-DPro-Phe-Arg-CH ₂ S (CH ₂ ) ₄ CH ₃ · «·« · «· ···· ·······································• ··· «·« «· 4 · ·» · ·· 9 ·

Preparation of Compound -50IV (i) CHF ₂ (CF ₂ ) ₃ CH ₂ O'Na ⁺

Boc-Arg CHnBr

► ----------

DMF

Z, i

Boc-Arg-CH ₂ OCH ₂ (CF ₂ ) ₃ CHF ₂ (ii) NaBH 1 / MeOH

Zn

BoC'Phc-Arg2HcH ₂ OCH ₂ (CF ₂ ) ₃ CHF ₂ (iii) HCl / Dioxan «<----------------------- Boc-Phe- OPfp

DIEA / CH ₂ C1 ₂ v

Zo

Boc-Arg5ÜCH ₂ OCH ₂ (CF ₂ ) ₃ CHF ₂ (iv) HCl / Dioxan Boc-DPro-OPfp DIEA / CH ₂ Cl ₂ v

Boc-DPro-Phe-Arg-CH ₂ OCH ₂ (CF ₂ ) ₃ CHF ₂ (v) De ss Martin Periodinane CH ₂ Cl ₂ / AcOH

Z, i "

Boc-DPro-Phe-Arg-CH ₂ OCH ₂ (CF ₂ ) ₃ CHF ₂ (vi) HCl / Dioxan H ₂ Pd / C AcOH / H ₂ O

H-DPro-Phc-Arg-CH ₂ OCH ₂ (CF ₂ ) 3 CHF ₂

0 * 5 • · · • ·

Preparation of Scheme V Compound

Z, i

Boc-Arg-OH (i) Tce-OH / wscd

---------------->

DMAP / CH ₂ Cl ₂ z,

I ~

Boc-Arg-OTce

Z ₂

Boc-DPro-Phe-Arg-OTce (iv) Zn / AcOH v

Z-, i ~

Boc-DPro-Phe-Arg-OTce (ii) HCl / Dioxane Boc-Phe-ONSu CH ₂ Cl ₂ / NMM v (iii) HCl / Dioxan Z ₂

Boc-DPro-ONSu

CH ₂ Cl ₂ / NMM

Boc-Phe-Arg-OTce (v) 'BuOCOCl / NMM / THF

CH ₂ N ₂ / Et-> 0

Zn

I *

Boc-DPro-Phe-Arg-CHL '_; (vi) HBr / EtOAc in THF

Zn

Boc-DPro-Phe-Arg-CH ₂ N [(CH ₂ ) ₅ CH ₂ ] ₂ (vii) HN [(CH ₂ ) ₅ CH ₃ ] ₂ ◄ ----------- tz, i *

Boc-DPro-Phe-Arg-CH ₂ Br (fruits)

NaHCO ₂ / THF

HCl / dioxane

H ₂ , Pd / C, AcOH. H ₂ O v

H-DPro-Phe-Arg-CH ₂ N [(CH ₂ ) ₅ CH ₃ ] ₂ • · ·

Preparation of compound -52VI

Z>

I "

Boc-Arg-CH ₂ Br (i) Na ⁺ 'CH (CO ₂ Tce) ₂

Zo

Boc-Arg-CH ₂ CH (CO ₂ Tce _;; (ii) Zn / AcOH

25, ι ~

Boc-Arg-GIy-OH (iii) Tolud / Δ

-4 --------------- J

Boc-Arg-CH ₂ CH (CO ₂ H) ₂

(arc)

Tce OH / WSCD

DMAP / CH ₂ Cl ₂

Z,

I ·

Boc-Arg-Gly-OTce (v) HCl / Dioxan Z,

I

--------------------- ► Boc-Phe-Ars ^ Jlv-OTce

Z2

Boc-DPro-Phe-Arg-Gly-OH

Boc-Phe-OPfp 36 DIEA / CH ₂ C1 ₂ (Vi) HCl / dioxane Boc-DPro-OPfp CHoCk / DIEA 1 (vii) Zn / AcOH 21

ι • 4 ------------------------ B oc - D Pro- Phe - A rg ^ G 1 v -OTce

PfpOH / wscd / CHoCl ₂

HPro-NHEI / DIEA (ix) HCl / Dioxan

--------------------------------->.

H-DPro-Phe-Arg-Pro-Gly NHE:

H ₂ , Pd / C

AcOH / H ₂ O

Boc-DPro-Phe-arginine ^ Gly-Pro-NHEt

Preparation of compound -54VIII

Boc-DPro-Phe-Arg-OH (i) HN [(CH ₂ ) ₅ CH ₃ ] (CH ₂ )?

----------------------- ► Boc-DPro-Phe-Arg-N [(CH ₂ ) ₅ CH ₃ ] (CH ₂ ) ₃ Ch

HOBt / wscd / DMF ,, (ii) HCIZDioxan

H ₂ Pd / C

AcOH / H ₂ O

H-DPro-Phe-Arg-N [(CH ₂ ) ₅ CH ₃ ] (CH ₂ ) ₃ Ch • · · ·

Preparation of compound -55IX

Za

Boc-Arg ^ OH

Dess-Martin Periodinane CH, ClVAcOH

Z,

I ~

Boc-Arg-H cf ₃ cf ₂ cf ₂ if ²

Boc-Arg — CF ₂ CF ₂ CF ₃

Zn / THF / U I tra h * n <a

HCl / Dioxan Boc-Phe-ONSu NMM / CH ₂ Cl ₂

Ά

3oc-DPrc-Phe-Arg-CF ₂ CF ₂ CF ₃

HCl / Dioxane ◄ ---------- Za

Boc-Phe-Arg-CF ₂ CF ₂ CF _: .

Dess Martin Periodinane CH ₂ Cl ₂ / AcOH

Boc-DPro-ONSu

NMM / CH ₂ Cl ₂

HCl / dioxane

Η », Pd / C AcOH / H-, Ο z, ι

Boc-DPro-Phe-Arg-CF ₂ CF ₂ CF ₃ >

H-DPro-Phe-Arg-CF ₂ CF ₂ CF ₃

Boc-DPro-Phe-Arg-CH ₂ Br

Preparation of compound -56X

Zn / AcOH? 2 ► Boc-DPro-Phe-Arg-CH3

HCVDioxan

H ₂ , Pd / C

AcOH / H ₂ O

H-DPro-Phe-Arg-CH ₃

Preparation of compound -57XI

Boc-Arg-CH ₂ Br

Well * Ό

OMe

DMF

B oc- Arg-CH ₂ O · I

OMe

F ²

3oc-ArgSraDMSCH ₂ OH (µ) and

Cemc ammonium nitrate ◄ -------------- MeCN / H ₂ O ° C Dess-Martin Periodinane CH ₂ Cl ₂ / AcOH

Br ₂ / MeOH / H ₂ 0 / NaHCO ₃

Boc-Arg3TSOMS _C o ₂ Me

HCVDioxán

Boc-Cha-ONSu

CH ₂ Cl ₂ / NMM

Z2

Boc-DPro-Cha-Arg <§ ^ CO ₂ H

NaOH / MeOH ◄ ---------- o ° c (i) PfpOH / wscd / CH ₂ Cl ₂ (ii) ⁿ Bu ₂ NH (i) TBAF / THF

Boc-DPro-Cha-Arg'® ^ CON ⁿ Bu ₂ (ii) Dess-Martin Pcriodinane CH ₂ Cl ₂ / AcOH (i) (ii)

NaBUf / MeOH TBDMS TriflateLutidene / CH ₂ Cl _{2 1 r}

Boc-Arg2ISSMSCH ₂ oZ ύ-OMe £

Boc-Cha-Arg ^QTBDMS CO ₂ Me

HCl / Dioxane Boc-DPro-ONSu NMM / CH ₂ Cl ₂

Z,

Boc-DPro-Cha-Arg ^Q ™ ^2Mi CO ₂ Me

Z-, ι '

Boc-DPro-Cha-Arg-CON ⁿ Bu ₂

HCl / Dioxane (i) (ii) H ₂ Pd / C MeOH / H ₂ O

MONTH "

H-DPro-Cha-Arg-CO ₂ N ⁿ Bu ₂

Preparation of Compound Scheme XII ζ

Boc-Om-OH 'BuOCOCVNMWTHF

NaBHVH.O

Z

Boc-Orn ^ OH

HoPd / C

AcOH / H ₂ O

Bzl

Boc-Orn-OH

PhCHO / CHnCb / EtsN / NigSCU ◄ ------------- NaBHa / MeOH

Boc-Om ^ OH

ZONSu y

Bzl, Z

Boc-Om ^ OH

Dess-Maran Periodinane

-------------->

Chichi / AcOH

Bzl.Z

Boc-Orn-H

Η ₂ ε (σθ ₂ Βζ1) ₂

NaH / IHF

ⁿ HexI

HexCHíCCúBzDi

NaH / THF ⁿ HexI / 60 ° C

H ₂ Pd / C

MeOH ⁿ Hex2C (CO2Bzl) ₂

Δ / Toluene y

ⁿ Hex ₂ CHCO2H

Redal / Toluen

------------>

ⁿ Hex ₂ CHCH ₂ OH

MsCl / EHN / CEWI

HeXnCHCI ^ Br

LiBr / Accton «<------------------- y ⁿ Hex ₂ CHCH ₂ OMs (cont.)

Scheme -59XII continued.

ⁿ Hex ₂ CHCH ₂ Br

Mg / THF

------------>

Bzl.Z

Boc-DPrc rhesus, Cm ^ ^ Chichek Hexi

H ₂ Pd / C

AcOH / H ₂ O v

Boc-DPrc-Phe-Cm-CH ₂ CH ⁿ Hex ₂

HCl / dioxane

Boc-DPro-ONSu / NMM CHnCl2

Bzl.Z

Boc-Orn ^GH CH2CH ⁿ Hex2

HCl / Dioxin Boc-Phe-ONSu CILC1VNMM 'T

Bzl.Z

Boc-Phe-OrnSHCHiCirHex,

H-DPro-rne-Arg-CH ₂ CH ⁿ Hex ₂

CH ₃ SC x ^ NZ ^ NHZ

EtOH / Δ

HCl / Dioxane

H ₂ Pd / C

AcOH / H ₂ O

Z?

Boc ₂ -DPro-Phe-Arg ^ CH ₂ CH ⁿ Hex ₂

Dess Marun Periodinane

CH ₂ Cl ₂ AcOH

Z,

Boc-DPro-Phe-Arg-CH rPHexj

Preparation of Compound Scheme XIII 2

Boc-DPro-Phe-Arg-CH ₂ Br

PhCOCO ₂ H / KF

------------>

DMF

Boc-DPro-Phe-Arg-CH ₂ OCOCOPh

KHCCME ^ O / THF

Boc-DPro-Phe-Arg-CH ₂ O (CH ₂ ) ₅ CH ₃

CH ₃ (CH ₂ ) ₅ VAg ₂ O ◄ ----------- CH ₂ Cl ₂ v

Boc-DPro-Phe-Arg-CH ₂ OH

HCl / dioxane

H ₂ , Pd / C AcOH / H ₂ O

H-DPro-Phe-Arg-CH ₂ O (CH ₂ ) ₅ CH ₃ ············· · · · · ·

Preparation of Compound Scheme XIV?

Boc-DPro-Phe-Arg-CH ₂ Br

PhOH / KF 2

--------------- ► Boc-DPro-Phe-Arg-CHiOPh

DMF '

HCl / dioxane

H ₂ Pd / C AcOH / H ₂ O

H-DPro-Phe-Arg-CH ₂ OPh • · · ·

-62Applied abbreviations ·· 9 9 9999

99 99 99 9 ·

9 9 9 99>

• 9 9 9 9 9 99

9999 99 9999 9999

Abn 3-azabicyclo [3.2.2] nonane Ac acetyl AcOH acetic acid Ada adamantylalanine aha 2-Amino-hexanoic acid (Norleucine) Boc tert-butoxi1 karboni1 Bu butyl ch cyclohexyl Cha cyclohexylalanine Chg cyclohexylglycine cpr cyclopropyl DIEA diisopropylethyl amine DMAP 4-dimethyl-aminopyridine DMF d imet i1-formam id EtOAc ethyl acetate FAB Spotted Atom Bombardment 4-FPh 4-fluoro-phenylalanine HCH Homociklohexil-alanine HOBt 1-hydroxybenzotriazole hplc high performance liquid chromatography Hyp 4-hydroxyproline ^t Hyp trans-4-hydroxyproline K keto isostere -COCH2- Me methyl MeCN acetonitrile MeOH methanol MPLC medium pressure prep. liquid chromato

«· · · · ······················································································ · · · · ·

At -63- chromatography naphthylalanine NMM N-Meth-1-morpholine NPG neopentyl glycine oc octyl OH hydroxy isostere -CHOH- ONSu hydroxysuccinimide petrol petroleum ether 60-80 ° pfp pentafluorophenyl Phe-4NO ₂ 4-nitrophenyl alanine Phg phenylglycine Pic pipecolic Pip piperidyl Py pyridine R reduced isostere -CH ₂ - Mud Sarcosine (N-methylglycine) TBAF Tetrabutylammonium fluoride TBDMS tert-butyldimethylsilyl Tcboc (1-dimethyl-l-trichloro-methyl) ethoxycarbonyl tce 2,2,2-trichloroethyl Tha 3,3,5-trimethyl-hexahidroazepil THF tetrahydrofuran Thi thienylalanine Tic 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid tic Thin layer chromatography WSCD water soluble carbodiimide Z -Benzyloxycarbonyl Nor norarginin

-64 ·· '···· ····· · · · · • »♦ ·· ·· ·· ·· ····

Biological tests

The compounds of the invention were tested in vitro according to known methods for the following activities:

(a) inhibition of human tissue kallikrein, plasma kallikrein, and mast cell tryptase by hydrolysis of chromogenic substrates S-2266, S-2302 and S-2266 (applied by Johansan HT et al., Int. J. Tiss. Reac., 8, 1986). 185 192). Assays were performed using different inhibitor concentrations and at least two different substrate concentrations. The Ki inhibition constant was plotted against the Dixon curve (M.

Dixon, Biochem. J., 1953, 55, 170).

b) Inhibition of quinine release from low- and high-molecular-weight quininogens by tissue and plasma calichrin. Measurements were made using two different substrate concentrations. Activity was calculated based on the amount of quinine released per minute as determined by radioimmunoassay using polyclonal antibodies. The Ki inhibition constant was plotted against the Dixon curve.

1-9. For each of the compounds summarized in Tables 1 to 4, the Ki value was always 10 to ^{5 to} 10 to ⁹ M for one or all of the enzymes in the chromogenic assay.

·· · ·· "·· ···· ········ • 4 ♦ · · · · · · · 4 • ····» ·· »·» · · · «·« »·

Carcinogenicity assays with -65In were performed using a well-defined pharmacological asthma model in sensitized guinea pigs. Inhibitors representing a variety of chemical structures have been shown to be highly effective in both acute phase blocking and late phase reactions, comparable or superior to the topical steroids and 63-agonist compounds currently used to treat asthma.

When used as medicaments, the compounds of the invention may be administered in any form. The enzyme inhibiting compounds of the present invention may be formulated into pharmaceutical compositions in a known manner. For example, they may be used in the form of known intravenous injections, intramuscular injections, drip formulations, oral formulations, inhalation formulations, and external topical formulations. Although there is no critical limitation on the dosage to be administered, a suitable dose is 1-1000 mg / day / person.

Claims (11)

1. Quininogenase inhibitors of the formula (II) not exceeding the size of a hexapeptide Claims II which formula A and B are the same or different aminoacyl groups (including aminoacyl analogs) which form a dipeptide group and A may also carry a terminal group (other than hydrogen) and may be any amino or imino acid group. (but preferably in the D configuration) and B may be a lipophilic amino acid group having the D or L configuration but different from proline or proline analogue, or may be a structurally analogous group to said dipeptide group, wherein the peptide bond is -CH2-NH-. substituted (reduced), -CH (OH) -CH 2 - (hydroxy) -CO-CH 2 - (keto), -CH 2 -CH 2 - (hydrocarbon), or other peptide bond structural mimetics, and · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · R III ^R1 side chain is a basic amino acid or amino acid analogue side chain (preferably in L configuration) and R is hydrogen or substituted by C 1-4 alkyl or ^C or N (R) group-containing peptide is, when structurally as above we win similar imitation groups, Y is a bond-enhancing carbonyl activating group such as hydrogen (only when A or B is cyclohexylalanine, preferably in configuration D for A and in configuration B for L) or C 1-20 alkyl or C 2-12 fluoroalkyl. group; substituted oxymethylene; thiomethyl group; sulfoxides methylene group; sulfonyl-methylene group; aminomethylene group; hydrazino-methylene; -CH2-Het, where Sev is a substituted or unsubstituted heterocyclic group; a substituted amino group, but when the resulting compound is a secondary alkylamide group, B cannot be a phenylalanine group; an amino acid residue or an ester or amide thereof; a carboxylic acid secondary amide or primary amide group when B is a bulky lipophilic non-aromatic amino acid residue -68 be, for example, cyclohexylalanine, adamantylalanine, but Ala, Leu, Ile, Val, Nva, Met, Nle, Phe, Tyr, Trp, Nal (1); tertiary carboxamide group; a carboxyalkyl group or an ester or amide thereof. Compounds according to claim 1, wherein A is an imino acid such as D-proline or a proline analogue such as pipecolic acid, azetidine carboxylic acid; lipophilic amino acids such as DPhe, DCha, DChg; strongly basic amino acids such as D-Arg or a homologue or analogue thereof, such as amidino, guanidino-phenylalanine, or an N-alkyl or _C, alkyl (including also benzyl) derivatives thereof. Compounds according to any one of claims 1 or 2, wherein B is L-Phe, L-Cha, L-aNal, L-Tal, L- (4F) Phe, L- (NMe) Phe or other substituted phenyl alanine, or an N-alkyl or _C, alkyl (including also benzyl) derivatives thereof. 4. Compounds according to any one of claims ¹ to 3, wherein R ¹ is 3-guanidino-propyl or other guanidino-alkyl (or an amidino-alkyl or amino-alkyl) group, including para or meta-substituted guanidino or amidino. benzyl groups or protected forms thereof, in which the basic nitrogen is optionally alkylated (Me, Et or other). 5. Compounds according to any one of claims 1 to 3, wherein Y is as follows: - Y is H or alkyl, including fluoroalkyl Y is CH2Q, -69 where -OR ² or -SR ² or -SOR ² or SO ₂ R ² or -NHR ² or -D swept, wherein -D0 is a ring in which D is a member of the ring, and R ^2, R ⁴ and R ⁵ are as defined below, is -CH ₂ CHR ⁶ CON or a group of the formula wherein R ⁴ , R ⁵ and R ⁶ are as defined below, is an aminocyclyl or a group which forms a substituted amide or hydrazide, Y is an α-keto-amide-forming group such as COR ⁹ or -CO-D, or -CON in the formulas R ² is alkyl or substituted alkyl including aryl or aryl alkyl groups and 70-CH2R ³ group wherein R ³ represents fluoroalkyl, R ⁴ and R ⁵ are the same or different, but both cannot be hydrogen or C 1-20 alkyl optionally substituted or acyl or alkylsulfonyl, represents a heterocyclic ring, wherein D represents nitrogen or carbon in the above group (IV) or N in the above groups (VI) and (X), which ring may be optionally unsaturated and may contain further heteroatoms and substituents, R ⁶ is hydrogen, alkyl, hydroxyalkyl, aminoalkyl, or alkylaminocarbonyl, R ⁹ is -NH 2, which may also be alkylated, or amino-acyl. 6. Compounds according to any one of claims 1 to 4, wherein Y is: Group I Y is H; C 1 -C 20 straight or branched chain alkyl; arylalkyl; or C 1-20 cycloalkyl; C2-C12 perfluoroalkyl or partially fluorinated C2-C12 alkyl; for example, Y may be Me; -CH (CH ₂ CH ₂ CH ₂ CH ₃ ) ₂ ; -CH (CH ₂ CH ₂ CH ₂ CH 3) CH ₂ -cyclohexyl; -CH2CF2CF2CF3. Group II t CH2QR Y ^2, wherein Q is O, S, SO, SO ₂ , NH and R ² are straight or branched alkyl or acyl groups containing from 1 to 12 carbon atoms; or C 1-20 cycloalkyl; or aryl or arylalkyl; or CH ₂ -R ³ wherein R ³ is perfluoroalkyl or partially fluorinated straight or branched chain alkyl wherein the alkyl group is 1 to 12 carbon atoms. Ili group Y is a group wherein R ⁴ is the same or different and may be alkyl, branched alkyl, cycloalkyl, acyl, alkylsulfonyl, carboxyalkyl (the carboxyl group may also be derivatized and thus be an ester or amide formed with an amino acid or a dipeptide), carbamoyl, sulfamoyl, Ν-dialkylamino, arylalkyl, haloalkyl such as fluoroalkyl, cyanoalkyl, alkoxyalkyl, , lakil- hydroxyalkyl, mercaptoalkyl, aminoalkyl and derivatives thereof, ester, thioester or amide derivatives thereof, or -R ⁴ and R ⁵ is hydrogen. Group IV • r · » Y is Y = -Cti.2-fa / wherein D is nitrogen or carbon and represents a saturated or unsaturated heterocyclic or bicyclic ring having 5 to 8 members and which may contain further heteroatoms (N, S, O) and carbon or nitrogen atoms and which may be optionally substituted with alkyl, branched alkyl, cycloalkyl, carboxyalkyl, carboxy (attached to a carbon atom), amino, alkoxy, alkoxymethyl or carbonyl group, or other groups which are suitable for the enzyme interaction. Group Y Y is -CH 2 CH (R ⁶ ) CONR ⁴ R ⁵ wherein R ⁴ and R ⁵ are as defined above for group (III) and R ⁶ is hydrogen, lower alkyl, branched alkyl, cycloalkyl, hydroxy, alkyl, aminoalkyl, alkylaminocarbonyl. Group VI Y is -CH 2 CH (R ⁶ ) COD 1) wherein R ⁶ is as defined above for group (V) and -D 4 same as for group (IV) except D = N. Group VII Y is an amino acid group or any amide group (secondary or tertiary) or an ester of these groups in the L or D configuration. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · -73 preferred groups are lipophilic amino acid residues such as norleucine, cyclohexylalanine, homocyclohexylalanine, cyclohexylglycine or tert-butylglycine. Group VIII Y is R ⁷ Y _{8 is} Y = -NR ⁸ where R ⁷ is H (when B is not phenylalanine only R ⁸ is not carboxyalkyl or derivatized carboxyalkyl) or alkyl having 1 to 12 carbon atoms alkyl, C 1 -C 20 cycloalkyl, carboxyalkyl or bis (carboxyl) alkyl groups, the latter of which may even be functionalized, such as an amide formed with, for example, an amino acid, preferably arginine, or a substituted amine, N'-dialkylamino, N'-alkylamino, R ⁸ is as defined for R ⁷ except hydrogen. Group IX Y is -CO-R ⁹ , but only if B is a bulky non-aromatic lipophilic amino acid or its N ^a -C 1-4 alkyl derivative such as cyclohexylalanine, but excluding Ala, Leu , Ile, Val, Nva, • 4 44 »9 ν · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· 44 44 -74Met, Nle, Phe, Tyr, Trp, Nal (1) and N-methyl derivatives thereof, and R ⁹ is NH 2, N'-alkylamino, (the alkyl may be branched and / or cycloalkyl) or an amino acid residue. group. Group X Y is -CO-D (d) wherein D) is as defined above for group (IV) (but D = N). Group XI Y is CO-NR ⁴ R ⁵ wherein R ⁴ and R ⁵ are as defined above for group (III) but not hydrogen. 7. compounds specifically listed in Table II. 8. A method of treating inflammatory or other indications, including prophylactic treatment, specified in (1) to (6) herein, particularly for the treatment of allergic, inflammatory conditions, characterized in that patients suffering from or suffering from the said disorders. are administered, an effective amount of a quininogenase inhibitor peptide or peptide analogue that is optimally equivalent to or less than the size of a hexapeptide. 9. A method of treating an allergic, inflammatory phase of asthma, comprising administering to the patient or at-risk patient an effective amount of a quininogenase inhibitor, such as a mast cell tryptase inhibitor, by topical or systemic route. 10. A method as claimed in claim 8, in particular allergic, inflammatory conditions, more particularly, · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·· ·· ·· for the manufacture of a medicament for topical or systemic treatment of asthma, which comprises mixing an effective amount of a quininogenase inhibitor with a pharmaceutically acceptable carrier or diluent. 11. A method of treating a medicament or preparing a pharmaceutical composition comprising the compound of claim 17 as an active ingredient.