CA2141785A1 - Diamino acid derivatives as antihypertensives - Google Patents

Abstract

Novel diamino acid derivative dual inhibitors of neutral endopeptidase and angiotensin converting enzyme of formula (I), wherein:
Z is amino, lower alkylamino, di-(lower alkyl)amino R9C(O)NH-or an optionally substituted guanidino group; R1 is hydrogen or R7R8N-; R2 is hydrogen, lower alkyl, cyclolower alkyl, aryllower alkyl or heteroaryl lower alkyl; and R3 is hydrogen, lower alkyl or cyclo lower alkyl; or R2 and R3, together with the carbon to which they are attached, comprise a 3-7 membered carbocyclic ring; R4 is hydrogen, lower alkyl, aryl lower alkyl or heteroaryl lower alkyl; R5 and R6 are independently hydroxy, lower alkoxy, amino, aryl lower alkoxy, lower alkylamino and di-(lower alkyl)amino; R7 is R9C(O)- or R10SO2-; and R8 is hydrogen, lower alkyl, aryl lower alkyl or aryl; or R7 and R8, together with the nitrogen to which they are attached, comprise a 5-7 membered ring; R9 is lower alkyl, aryllower alkyl, aryl, heteroaryl lower alkyl, heteroaryl, lower alkoxy, aryl lower alkoxy, amino, alkylamino or dialkylamino; R10 is lower alkyl, aryl lower alkyl, aryl, heteroaryl lower alkyl, amino, lower alkylamino, di-(lower alkyl)amino or heteroaryl; n is 1, 2, 3, 4 or 5;
and m is 1, 2, 3, 4 or 5; or a pharmaceutically acceptable addition salt thereof, useful in the treatment of cardiovascular disorders, are disclosed.

Description

WO 94/03481 . PCl/lJS93/07137 ~`

DlAMlNO ACID DERlVATl\lES AS ANTltlYPERTENSIVES

BACKGROUND OF THE INVENTlON

The present invention relates to a,~d-diamino acid derivatives which are dual inhibitors of neutrai endopeptidase and angiotensin converting enzyme, useful in the treatment of cardiovascular disorders.
Cardiovascular disorders which may be treated with t 0 compounds of the present invention include hypertension, congestive ~
heart failure and renal insufficiency. ~-The renin-angiotensin system is a complex hormonal system comprised of a large molecular weight precursor~
angiotensinogen, two processing enzymes, renin and angiotensin 5 ~converting~ enzyme (ACE), and a vasoactive ~rnediator, ~ngiotensin II (A
Fhe~ enzyme~ renin catalyzes the cleavage of angiotensinogen into ~-the-~ decapeptide~angiotensin I (A I), ~which has minimal biological ~activity on~its ~own and is oonverted into the active octapeptide A II by - ~ ~ ACE. A II has ~multiple biological actions on the cardiovascular system, ~ ;~

2 0 including vasoconstriction, activation of the sympathetic nervous system, sbmulation of aldosterone production, antinatriuresis. stimulation of v ascular grou th and stimulation of cardiac ~rowth. A II functions as a ; -pressor~hormone and ~is ~involved in the pathophysiology of several forms~ of ~hypertension.
25 ~ Angiotensin converting enzyme (ACE) is a zinc~
metalloprotease which converts A I to A II. Inhibitors of this enzyme, which have been widely studied, include the drugs captopril, enalapril, lisin~pril anld sp'irapril.~ Aîthough a major mode of ac~ion of ACE
inhibitors involves prevention of formation of the vasoconstrictor peptide A II, it has been reported in Hy~ertension, 1~ 4 (1990) p. 363-370 that .`
ACE cleaves a variety of peptide substrates. including the vasoactive peptides bradykinin and substance P. Prevention of the degradation of bradykinin by ACE inhibitors has been demonstrated, and the activity of the ACE inhibitors in some conditions has been reported in Circ. Res., SUBSTITUTE SHEET :~

WO 94J03481 PCl/US93/07137 .
.
7~

66, 1 (1990) p. 242-248 to be ~ediated by elevation of bradykinin levels rather than inhibition of A II formation.
Neutral endopeptidase ~EC 3.4.24.11; enkephalinase;
atriopeptidase; NE~) is a zinc-containing metalloprotease which cleaves 5 a variety of peptide substrates on the amino terminal side of aromatic amino acids. Substrates for this enzyme include, but are not limited to, atriat natriuretic ~actors (ANF), brain natriuretic peptide, met and leu enkephalin, bradykinin, neurokinin A, and substance P.
Inhibitors of NEP lower blood pressure and exert ANF-like 10 effects such as diuresis and increased cyclic guanosine 3',5'-monophosphate (cGMP) excretion in some forms of experimental hypertension. The antihypertensive action o~ NEP inhibitors is mediated through ANF because antibodies to ANF will neutralize the reduction in blood pressure.
U.S. 4,749,688 also established the antihypertensive action of NEP inhibitors and that co-administration of an AC~ inhibitor and a~NEP inhibitor results in a greater reduction of blood pressure than observed with eRher agent alone. The antihypertensive effect is best man~iiested under conditions in which the renin angiotensin system is 2 0 ` suppressed,~ as reported by Sybertz et al in J. Parmacol. EXD. Ther., 250,-~ 2 ~1989) pp. 624-631 and in Hvoertension. 15, 2 (1990) pp. 152-161.
For examplel NEP inhibitors reduce blood pressure effectively in the Desoxy-corticosterone ~salt (DOCA) hypertensive rat, a volume-dependent, renin-suppressed model of hypertension, but are less 25 effective under conditions in which the renin-angiotensin system is activated, such as in the spontaneously hypertensive rat (SHR) and in the two kidney Goldblatt hypertension model. Studies in the SHR and in `-the two-kidney Goldblatt hypertension model using a prodrug of the NEP
inhibitor N-l2(Sj-mercaptomethyl-3-(2-mèthylphenyl)propionyl]-30 methionine in combination with the ACE inhib}tor spirapril demonstrated the greater efficacy of the combination compared to either drug alone.
However, this interaction was inhibited in SHR which had been ~- nephrectomized, a manipulation which markedly suppresses renin ~- levels.
:: ' SUBSTITUTE SHEET

WO 94/03481 PCI`/US93iO713 f r~

-3~

An explanation of this interactive effect of ACE inhibitors and NEP inhibitors on blood pressure is that suppression ~of the renin angiotensin system allows for full expression of the ANF-like antihypertensive effect of the iNi_P inhibitor. A Il and ANF exert opposite effects on the cardiovascular system and it has been proposed by Johnston et al in Am. J. M~ 87, (Suppl 6) (1990) p. 24S-28S that these two hormonal systems act to counterbalance one another.
Compounds possessing dual activity as NEP-ACE
inhibitors have been reported. European Patent Publication EP
0358398 discioses cycloalkyl-substituted glutaramides of the formula R2~ (A3~ R

1, c~ N~ R3 . ~ ~
O Co2R4 wh~rein: ~ A completes a 5 or 6 membered carbocyclic ring; R1 is H or 15~ allcyl; ~R~ and~ R4 are H, alkyl, cycloalkyl, benzyl or an alternative biolabile -ester-forming group, Y is a bond or an aikylene group; R2 is H, aryl, heterocyclyl, or a carboxamido, carbamoyl, sulfamoyl or sulfonamido ;~ ~ group; and R3 is a group of the torrnula ``

~ N~

2 0 : ~ H or H , -~.

; ~ ~wherein the il~enzenè'~ring of said group is opti~nally substituted, European Patent Publication EP OA74553 discloses actinoin derivatives of the formula , ~
SlJBSTlTUTE S~EET

WO 94/03481 PCI/US~3/07137 ~ 5 R~,O

CH,~

wherein R1 is sulfoxymethyl, carboxyl, carboxarnido. hydroxyamino-carbonyl or alkoxycarbonyl; and R2 is hydroxy, alkoxy, hydroxyamino or sulfoxyamino;
European Patent Publication EP 0481522 discloses compounds of the ~ormula A2 or 10 wherein:
: ~ Al and A2 are independently H or -CC)OR4; provided that where A1 is H, A2 is -COOR4, and where A1 is COOR4, A2 is H;
Bl and B2 are independently H, OH, Cl-C4 alkoxy, aryl or !`aryl(C1-C4 all~1); or, where B1 and B2 are attached to adjacent carbon atoms, B1 and B2 together with the carbons to which they are attache~ comprise a , , benzene or methylenedioxy ring;
R2 is H, C1-Cg alkyl, -CH2-O-(CH2)2-O-CH3, aryl or aryl(Cl-C4 alkyl);
R3 is H, acetyl, -CH2-O-C(O)-CCH3, or benzoyl;

SUBSTITUTE SHEET

W O 94/034Xl ~CT/US93/0713, ' ~L4'.~ ~5 R4 is H, -CH2-~-C(O)-CCH3, C1-C4 alkyl, diphenylmethyl, aryl or aryl(C1-C4 alkyl); and nisOor1;
having activity as inhibitors of both neutral endopeptidase and 5 angiotensin converting enzyme; -and Gros, et al., Proc. Natl. Acad. Sci. USA, 88, (1991) pp 4210 - 4214, discloses dual inhibitors of ACE and NEP of the formula ~ N ~COOR~

:;
wherein R is H or CH3C(O)-, R1 is H or benzyl, and R2 is H or methyl.
SUMMARY OF THE INYENTION
, ~ ~
15 ~ Novel compounds of ~he present invention are represented by ~he~fDrmula I ~ -C~ ~R6 20 wherein 1 Z iis am~no, lower alkyl'amino, di-(lower alkyi)amino R9C(O)NH-, or a guanidino group of the formula R1 1 R12NC(=NR13)N(R14j- or R1 1 R12NC(NR13R14)=N-, wherein R1 1, 2, R13 and R14 are independently hydrogen or lower alkyl, or wherein 25 R1 1~ and R12, or R13 and R14, taken together with the nitrogen atom to which they are attached, comprise a 5 or 6-membered ring;
R1 is hydrogen or a group of the formula R7R8N~
, ~, SU BSTITUTE SHEET ;`
,~ ~ ` ,, ~VO 9~/03481 PCI`/US93/07137 t 7~

R2 is hydrogen, lower alkyl, cyclolower alkyl, aryllower alkyl or heteroaryllower alkyl; and R3 is hydrogen, lower al~yl or cyc!olower alkyl; or R2 and R3, together with the carbon to which they are attached, comprise a 3-7 membered carbocyclic ring;
R4 is hydrogen, lower alkyl, aryl lower alkyl or heteroaryllower alkyl;
Pt5 and R6 are independently selected from the group consisting of hydroxy, lower alkoxy, amino, aryllower alkoxyl lower alkylamino and di(lower alkyl)amino;
-~ 10 ~ R7 is R9C(Q)- or R1S02-; and R8 is hydrogen, lower alkyl, aryllower alkyl or aryl; or R7 and R8, together with the nitrogen to which they are attached, comprise a 5-7 membered ring;
R9 is lower alkyl, aryllower alkyl, aryl, he~eroaryllower alkyl, heteroaryl, lower alkoxy, aryllower aikoxy, amino, lower alkylarnino or 15~ di-(lower alkyl)amino;
R10 is~ ower alkyl, aryl 10wer alkyl, aryl, heteroaryl lower ,.
alkyl, ~amino, lower alkylamino, di-(lower alkyl)amino or he~eroaryl;
n is l, 2, 3, 4 0r 5;
mis~1 2 3 40rS-20~ or a ph-rrnaceutically acceptable addition salt thereof.

Preferred are compounds of the formula I wherein n is 4 , ~ , i and m~ is 1. Also preferred are compounds of the formula I wherein R4 is4-hy~roxyphenylmethyl. Anothergroup of preferred compounds is 25~ ~ ~ tha~ wherein. Z is~amino,~benzyloxy-C(O)NH- or C2HsNHC(=NC2Hs)NH-. Yet another ~roup of preferred compounds is that wherein~ R1 is H or CH3S02NH- and R6 is hydroxy, lower alkoxy or aryllower alkoxy.
More preferred ;are compounds of' the formula Ia, Ib or Ic:

"~
~"~
, ~ ~
"~
", ~

-:

,~,, SU~STITUTE SHE~T

WO 94/03481 PCr/ljS93/07137 ;~ 5 Z~ ~X Fl2 O

OH Ia N~ NJ~ ~6 wherein r is 0, 1, 2 or 3 ~OH Ib N~X N~ N~ R6 ~C~NHSO2-(bw6ralkyl) Especially preferred are compounds of the formula Ia, Ib or Ic, wherein Z is amino, R9C(ojNH- or R1 1R12NC(=NR13)N(R14)-; R~ ;
is H or (~1~3SO2NH-; R2 is iso-propyl, propyl or benzyl; R5 and R6 are indepsndently hydroxy, lower alkoxy or aryllower alkoxy; R9 is aryllower alkoxy; R1l and R13 are alkyl; and R12 and R14 are hydrogen.
; ~ The invention also relates to pharmaceutical compositions comprjsing a c~rnpound qf the formula I, and to methods of treatment of cardiovascular diseases comprising administering a compound of the fom~ula I to a mammal in need of such treatment.

`: ~ :

.

SUBSTIl~VTE SHEET

WO 94/03481 PCr/US93/07137 DETAILED DESCRIPTION

As used herein, the term "lower alkyl" means straight or branched alkyl chains of 1 to 6 carbon atoms, and ~lower alkoxy"
similarly refers to alkoxy groups having 1 to 6 carbon atoms. Cyclolower alkyl means cyclic alkyl groups of 3 to 6 carbon atoms.
The term ~aryl~ means a phenyl or naphthyl group substituted by 0-5 substituents independently selected from the group consisting of lower alkyl, hydroxy, lower alkoxy, lower alkenoxy, lower alkoxycarbonyloxy, lower cycloalkoxycarbonyloxy, lower alkylsulfonamido, arylsulfonamido, ha!o, trifluoromethyl, phenyl, phenoxy or phenylthio, or wherein two substituents attached to adjacent carbons together comprise a fused 5- or 6-membered ring containing 0 or 1 heteroatoms selected from N, O and S, e.g. dihydrobenzofuranyl, dihydrobenzothienyl, or indanyl.
Theterm 'heteroaryl" meansfuranyl, thienyl, pyrrolyl, benzoturanyl, benzothienyl, indolyl, indazolyl or pyridyl, substituted by ~4 substltuents independently selected from the group consisting of lower~alkyll hydroxy, lower alkoxy, iower alkenoxy, lower aikoxycarbonyloxy, lower cycloalkoxycarbonyloxy, lower alkylsulfonamido, arylsulfonamido, halo, trifluoromethyl, phenyl, phenoxy orphenylthio.
The term ~halo~ means fluorine, chlorine, bromine or iodine radicals.
~ Certain compounds of the invention are acidic, e.g., those cornpounds which possess a carboxyl group. These compounds form pharmaceutically acceptable salts with inorganic and organic bases.
Examples ot such salts are the sodium, potassium, calcium, aluminum, gold and silver salts. Also included are salts formed with pharmaceutically acceptable amines such as ammonia, alkyl amines, - h3/droxyalkylamines, N~methylglucamine and the like.
- ~ The salts may be formed by conventional means, as by reacting the free acid form of the product with one or more equivalents ^~ h of the appropriate base in a solvent or medium in which the salt is ~ 35 insoluble, or in a solvent such as water which is then removed in vacuo ,~:

~ ~ SU BSTITUTE SHEET

WO ~4/034~ i ~'~1 / l ;S93/()7 1 37 or by freeze-drying or by exchanging the cations of an existing salt for another cation on a suitable ion exchange resin.
Certain compounds of the invention, e.g., those with a basic Z group, also form pharmaceutically acceptable salts with organic ;~
5 and inorganic aclds. Examples of suitable acids for such salt formation are hydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic, salicylic, malic, fumaric, ~uccinic, ascorbic, maleic, methanesulfonic and other mineral and carboxylic acids well known to those skilled in the art.
The salts are prepared by contacting the free base form with a sufficient 10 amount of the desired acid to produce a salt in the conventional manner.
The free base forms may be regenerated by treating the salt with a suitable dilute aqueous base solution such as dilute aqueous sodium hydroxide, potassium carbonate, ammonia and sodium bicarbonate.
The free base forms differ from their respective sait forms somewhat in 15 certain physical properties, such as solubility in polar solvents, but the salts are otherwise equivalent to their respective free base forms for purposes of this invention. ~;
Compounds of the formula I may have asymmetrical carban~ atoms in addition to those designated and therefore include various stereoisomers. The invention includes all such isomers both in pure form and in admixture, including racemic mixtures.
The following solvents and reagents emptoyed in preparing compounds of the present invention are identified by the abbreviations indicated: diethyl ether (Et2O); ethyl actetate (EtOAc);
methanol (MeOH); ethanol (EtOH); dimethylf~rrnamide (DMF);
tetrahydrofuran~(THF); acetic acid (AcOH); 1-(3-dirnethylaminopropyi)-3-ethylcarbodiimide hydrochloride (EDCI); dicyclohexylcarbodiimide (DCC); 1-hydroxybenzotriazole hydrate (HO8T); p-toluenesulfonic acid (p~TSAj; trifluoroacetic!acidi(ltFA);
Compounds of the present invention can be prepared via ~`
methods known to those skilled in the art.
One method of preparing c~mpounds of the formula I is outlined in Scheme A. A N-protected amino acid of the formula II, wherein PG is a protecting group, e.g. benzyloxycarbonyl, is coupled ` ~ 35 with an amine of the formula III using a dehydrative coupling agent, .
~ SUBSTITUTE SHEET `
:
:::

WO ~4~034~1 PCr/lJS93/07137 ..... ..

e.g. EDCI or DCC, and HOBT, in a suitable soivent, such as DMF. The N-protecting group of resulting peptide IV is removed, e.g. in the case of a benzyloxycarbonyl protecting group, by hydrogenation over PdlC, to fonn an amine of the fomnula V. The amine of formula V is then coupled, using known methods, to a carboxylic acid of the formula VI to -foml a compound of the formula I.

. .
, ,. .

,,:' ,~' ~ ' ' ' . , ~,: - : .

SU~STITUTE SHEET
`-: ~

vo 94/03481 Pcr/~ 3/ul13/
: . .
X~ 35 Scheme A

O ~.

X ~ H2N`JJ~
(CH2)m N COOH + 5~6 H -II
PG = N-protecting group III
.~
1 ) EDCI, H(3BT
triethylamine, DMF ~
2) deprotection :

N~ N protecting _ O R4 V PG = H

Rl COOH

R~N~
o O R4 SUBSTI~UTE SHEET

WO 9~1/034~1 PCT/US93/07137 r 7~5 :,"1.

Al~ernatively, an amine of the formula V can be prepared by coupling an azido carboxylic acid of the formula XXI with~an amine of the formula m using a dehydrative coupling agent, e.g. EDCI or DCC, .
and HOBT, in a suitable solvent, such as DMF. The resultin~ azido amide is hydrogenated over a suitable catalyst, e.g. Pd on carbon, to give the desired amine.
o N3~(CH3~n N~COOH + ~R6 111 H _ ~:

- ~ 1) EDCl, HOBT .
triethylamine, ; DMF
2) H2, 1 0% Pd/C

C~N~ ~R6 V
" ~
o R
,, ;. . '' Compounds of the formula II can be prepared by reacting an amine of the formula VII, wherein PG is a protecting group, e.g.
15 benzs/loxycar~onyl,~l with ia trif~ate of the formula VIII, wherein LG is lower alkoxy or ary!lower alkoxy. The reaction is carried out in a suitable solvent, e.g., in the presence of a non~nucleophilic strong base, e.g. Pro~on Sponge~, giving rise to a compound of the formula IX, wheréin LG and ~PG are as defined above. The C(O)LG ester group of ZO IX is hydrolyzed to give a compound of the forrnula II, by treating with base, e.g. sodium hydroxide, in a suitable solvent, e.g. methanol, or SUBSTITUTE SHEET -1/03~1 PCr/l,'S93/07137 . . ' .

altematively treating with acid, e.g. TFA, in a suitable solvent, e.g.

PG--N~ X ~ LG
(CH 2) m NH2 o ::
VII VIII

proton sponge R5~0 2 :
PG (CH2)l N~C~
H
5: ~ 0 ~:

hydrolysis ~: ~ r :
.

,, . ~ . - ~ .
Amines of the formula III are commercially available or ~-0 can be ~prepared via known procedures. Where chiral amines of the formula III are not available, they can be separated from racemic amines via known methods. For example, a racemic amine of the -formula III, wherein R6a js lower alkoxy, is protected with a suitable N
protecting group PGa, e.g. where PGa is benzyloxycarbonyl, by ~;
15 ~ treatment with b~nzylchloroformate and a base, such as triethylamine, in i a solvent, such as dioxane. The N-protected compound af formula ',``
XVIII is hydrolyzed with an aqueous base, such as sodium hydroxide solution, in a suitable solvent, e.g. THF to form a carboxylic acid of the forrnula XIX. The racemic acid XIX is enantioselectively esterified with Su 8STl-rlJTE SI~EET

~J ~/ U~ PCI /US93~07137 , 21'~ ~ 7 ~ ~5 an alcohol of the formula F~6bOH, wherein R6b is lower alkoxy or aryllower alkoxy, e.g. by treating with the alcohol, papain, L-cysteine and 1 M citrate buffer, to give a chiral ester of the formula XX. The N-protecting group is rerrioved via known methods, e.g. in the case of N- -benzyloxycarbonyl protection, by hydrogenation in a suitable solvent, ~
such as ethanol, in the presence of a palladium catalyst, to produce the - `
corresponding amine of formula III.
o ,~:

H2Nyl~R~ -- '-~ PG'~N~ 6, racemic - III XVIII
-~ : O
~: hydrolysis ~ H ,¦¦~
-: ~ XVIII~ ~ pGa ~ OH
f ~4 enantioselective H ll ~:
esterification N ~
~ PGa~ ~ R~b :
:. , : _ .
R4 ' :

deprotection J~
~K ~ ~~ ~ R4 I I l Amines of the fonnula VII are commercially available or can be prepared by~procedures well known in the art. For example, -~
amines of the formula VIIa, wherein pGb is benzyloxycarbonyl and Rsa `"~
~; ~ 20 is hydroxy, lower alkoxy, or aryllower alkoxy can be prepared via the SU BSTITUTE SI~EET

34~ 1 rj Pc~ JS93/07137 ., .

- 1 5- ~
":
following procedure. A diamino acid X is converted to the N,N
bis(benzyloxycarbonyl) derivative XI, by treating with benzylchloroformate and a base, e.g. sodium hydroxide, in a suitabie solvent, e.g. toluene/water. Compound XI is cyclized to the oxazolidinedione XII, e.g. by heating with thionyl chloride in a suitable ;
solvent, such as CH2C12. The oxazolidinedione XII is then selectively hydrolyzed to the amino acid VIIai wherein R5b is hydroxy, by treating with acid, e.g. HCI, in an appropriate solventi e.g. acetone.

~ O ~
COOH C aJI~ COOH

(CH2)m NH2 ~ (CH2~N--PGb X ~oluene/water XI H .~

XI~ b H ~ ~GO ~-reflux PG - N~ N

- ~ XII

Xll H
acetone p6b N~
(CH2)m NH2 Amines o~ the forrnula VIIa, whereln R5b is lower alkoxy, are prepared by reacting an oxazolidinedione of formula XII with an alcohol of for T ula RsCH, wherein R5C is lower alkoxy, and thionyl chloride, in an appropriate solvent, e.g. CH2Cl2.
20~ ~ ~ Amines~ of the formuta VIIa, wherein Rsb is t-butyloxy, are prepared by reacting a so!ution of an amine of formula VIIa in a suitable solvent, e.g. dioxane, with isobutylene gas in a sealed vessel.

SU BSTITUTE SHEET

: ~

9~/034~1 ~ PCr/US93/0713 Amines of the formula VIIa, wherein R5b is aryllower alkoxy, are prepared by heating an amine of the formula ~IIa ~th an alcohol of the ~ormula R5dH, wherein ~5d iS aryllower alkoxy, and an acid, e.g. pTSA, in a suitable solvent, e.g. toluene.
Triflates of the formula VIII are prepared by reacting an alcohol of the formula XIII with trifluoromethanesulfonic anhydride and a base, such as pyridine, in a solvent, such as CH2C12.

R3 R2 (CF3S02)20 R3 HO~ C~ LG _ ~ ~ ~ .
CH2C12 CF3s~20 Ic XIII VIII

Alcohols of the formul? XIII are commPrcially available or `can be prepared via~known: methods. For example, alcohols of the ~-formllla ;~ wherein LGa is t-butoxy can be prepared from an acetoxypropionic acid XIV. Treating the acid XIV with isobutylene and 15 an~acid, such concentrated H2S04, in a solvent, such as CH2Cl2, in a seaied vessel provides~the t-butyl ester XV, which is selectively hydrolyzed with diaminoethane in a suitable solvent, such as DMF, to give ~the~ alcohol XIII.

isobutylene CH3~ ~; O COOH - ~ CH3 ~ ~
XIV ~ 2 2 o XV ~r ~';
diaminoethane ~- ~ DMF
, ~20: LGa= t-butoxy To prepare an alcohol of the formula XIII1 wherein LGb is wer alkoxy or aryllower a!koxy, treat the corresponding hydroxy acid XVI with a lower alkyl halide, e.g. methyl iodide, or a lower arylalkyl :,- : : , SUBSTITUTE SHEET

WO 94/03481 P~/US93/07137 2~ t~ ~35 -17~

halide, e.g. benzyl chloride, in the presence of a suitable base, e.g.
cesium carbonate, in a solvent such as DMF.

R3 F 2 LGb-X . .

HO~COOH cesium carbonate XIII `;~
XVI DMF LGb = lower alkoxy or lower arylalkoxy . ` X = halogen ~Hydroxy acids of the formula XVI are commerciaily ^
available or can be prepared via known procedures. For example, -~
compounds of the formula XVI can be prepared from amino acids of the formula XVII by treating with H2SO4, sodium nitrite and water.

H2SO4 x,' H2N~ COOH - ~ XVl XVII

Compounds :of the formula XXI (above) can be prepared by reacting an:~amino ester of the formula XXII with a triflate of the 5 . :formula~XXIII, in the presence:of an acid scavenger, such as Proton Sponge~, :followed by hydrolysis o1 the resulting azido ester XXIV.

.,, , . I

' ' ' ~ ' ~ ` .

:, ' 0 ~4/034X~ PCl/US93/07137 ~
;21A1785 R5`~
~R3 N3- (CH2)", OSO2CF3 + H2N C--OC(CH3)3 XXIII ~ XXII :
acid scavenger ~ r R~ //
~R3 XXIV
.
N3- (CH2)m H C--C)C(CH3)3 hydrolysis Triflates of the formula XXIII can be prepared from a D
hydroxy acid XXV by esterification of the carboxylic acid to give the diol 5 ~VI. The primary alcohol is seiectively converted to the analogous azide ~VII, followed by reaction with triflic anhydride to give the ; ~ ~ triflateXXlII.

; : ~ HO~O ! i:

=
. - HO--~CH ~m OH HO--(CH~)~OH

XXVI

~: SU8STITUTE S~EET

wo s4to34sl Pcr/usg3/o7l37 ~.
.. , . ii~
2~17~35 P(C6H5)3~ R5 /YO
CBr4~ LiN3 ~

DMF N3 ` (CH2)m OH
XXVII ```
:`
~cF~s02)2o pyridine XXVII - --------~ XXIIl ~.
Altematively! cornpounds of the formula XXVXI, wherein m=1, c~n be prepared via esterification of (R)-2-hydroxy~3-azido-propionic acid XXVIII. The hydroxy acid XXVIII can be prepared - from commercially availabl 3,4-isopropylidene-D-mannitol via known methods.
~- 10 ~ _ COOH
esterification OH ~ XXVII
XXVIII
,~
Carboxylic acids of the formula VI, diamino acids of the forrnula X, acetoxypropiortic aoids XIV, amino acids Qf the formula 15 ~ ~ XVII and amino esters of formula XXII are commercially available or can be prepared by methods well known in the art.
The ACE inhibitory activity of the compounds of the present ir~lvention is d~termined Yia in Yitro tests using standard test procedures for AGE inhibition well known to thos& skilled in the art. Similarly, NEP
irlhibitory acti-lity is ldetennined via in vitro tests using standard test procedures for NEP inhibition well known to thoss skilled in the art.
~- ~ The antihypertensive effects of dual inhibitors of the present invention are determined according to the following procedures.
DOCA Sal~ Mod~l For the DOCA salt hypertension model, male Sprague Dawley rats weighing 100 1~0 g are anesthetized with ether and the ~ ~ .
.
SUBSTITUTE S~EET
:.

WO 9~/03481 PCI/US93/07137 2~4~785 right kidney is removed. Three pellets containing Doc acetate ;
(desoxycorticosterone acetate, DOCA, 25 mg/pellet) are implanted subcutaneously. Animals recover from surgery, are maintained on -normal rat chow and are allowed free access to a fluid of 1/~, NaCl and 5 0.2% KCI instead of tap water for a period of 25-30 days. This procedure results in a sustained elevation in blood pressure and is a slight modification of published procedures (e.g. Brock et al, 1982~ that have been used to produce DOCA salt hypertension in the rat.
On the day of the study, animals are again anesthetized 10 with ether and the caudal artery is cannulated for blood pressure measurement. Patency of the caudal artery cannula is maintained with :;~
a continuous infusion of dextrose in water at a rate of 0.2 mUhr. Animals are placed into restraining cages where they recover consciousness.
- ~ Blood pressure is measured from the caudal artery catheter using a `-5 ~ Statham~pressure transducer attached to a Beckman oscillographic recorder. In~ addition,~a cardiovascular monitoring device (Buxco ~Eletronics, Inc.) and a digital computer are used to calculate average blood pressures.
After an equilibration period of at least 1.5 hr., animals are 0 20 ~ dose`d subcutaneously (1 mLlkg) with vehicle ~methylcellulose, hereinafter MC) and dual NEP-ACE inhibitor, and blood pressure is monitored for the next 4 hours.

ANF Potentiation Model ~ ~ Male spontaneously hypertensive rats (SHR), 16-18weeks old, 270-350 9, are an~sthetized with ether and the abdominal aorta is cannulated through the tail artery. The animals are then placed into - ~ restrainers to recover fromj anelsthesia (ir;l less than 10 min.? and remain inside throughout the experiments. Through a pressure transducer (Gould P23 series) analog blood pressure signals aro registered on a ~ ~ Beck~nan 612 recorder. A Buxco digital computer is used to obtain - - ~ mean arterial pressures. Patency of the arterial cannula is maintained with a continuous infusion of 5% dextrose at 0.2 mL/hr. Animals are ;~ allowed a 90-min equilibration period. The animals first undergo a challenge with an ANF such as atriopeptin II (AP II) or AP28 at 30 SUBSTlTUTE SHEET

WO 94/03481 . PCI`/US93/0713, i ~41785 `~

-2 1 - :~

Ilg/kg iv and at the end of 60 min. are treated with drug vehicle or a diamino acid dual inhibitor subcutaneously. A second ANF challenge is administered 15 min. Iater and blood pressure is monitored for the next -90 min.

A1 Challen~e Assav Male Sprague-Dawley rats weighing 270-345 g are anesthetized with ether and the abdominal aorta cannulated via the caudal -~
(ventral tail) artery with polyethylene tubing (PE10 fused to PE~0). A jugular ~-vein is also cannulated with polyethylene tubing and both cannulae exteriorized at the back of the neck. The catheters are filled with heparinized saline (0.2%) and sealed. Animals are then returned to their cages and fasted overnight. The next day, place the animals into plastic `
restrainers. Blood pressure is recorded from the arterial catheter. Patency of the arterial cannula is maintained by a continuous infusion of 5% dextrose in water at a rate of 0.2 mUhr. .
After a 30 min stabilization period, animals are challenged with an~iotensin I (AI) and angiotensin II (AII) (0.3 ,ug/kg dissolved in physiological~ saline solution and injected iv in volumes of 100 ~LUkg followed by a 100 1l~ flush) twice at 5-10 min. intervals during a control period. The diamino acids are then administered orally (via a feeding need!e) in 0.4% aqueous methylcellulose vehicle in a volume of 2 mUkg. ;~
AI and AII chalienges are repeated at 30 min. intervals for the next 6 hr.

FSHR Assay Male spontaneously hypertensive rats (SHRj, 16-18 weeks old, are pretreated via oral gavage with 50 mg/kg of furosemide (in a volume of 4 mlJkg) the evening qefore the day of testing. The rats were fasted ovemight, but had ad libftum access to water, and were pretreated with - 30 furosemide (as before) the morning before surgical preparation on the day ;
- of testing. The pretreated rats are anesthetized with ether, the caudal (ventral tail) artery cannulated with polyethylene tubing (PEsO), and their blood pressure and heart rate recorded. The rats are placed in plastic ca~es to recover.

SUB~;TlrUTE SHEET

~VO ~4/03481 PCT/US93tO7137 '~
~4~7~5 `

After a 90 minute stabilization period, the diamino acids are -administered orally as solutions or suspensions in 0.4% aqueous methylcellulose vehicle via a feeding needle. The amino acid or vehicle is given in a volume of 4 mUkg. ;
Alternatively, the diamino acids (0.4%) dissolved or suspended in a vehicle composed of 10% (vh) ethanol, 20% 0.1N (tris[hydroxymethyl]- ;;-aminoethane) and 70% methylcellulose, are administered via a subcutaneous (sc) route. A volume of 2 mL/kg is used to deliver the drug or vehicle in these sc studies.
~ '`'`
~ ~ ~ ANF has been shown to exert beneficial hemodynamic and ~
- ~ renal actions in congestive heart~ failure (CHF) with the exception of the ~.
most severe states, in which its actions may be blunted. ANF and the r enin angiotensin system also act as physiological antagonists of one 15 ~anothèr~in~Gt~F. Therefore,it~iscontemplatedthatadualNEP-ACE
inh~or~will~be~ usèful in~the treatment of CHF. ~ ~Measurements of the de~diuresis and natriuresis, as well as hemodynamics, are used to ~ételmb~ the~effi-acy ol the~ prFsent combination in the treatment of 20~ A variety of pharmaceutical dosage forms are suitable, ~1 pr~ferably for~ oral~ or parenteral administration, although mechanical systems~such~as transdermàl dosage~forms are also contemplated.
The~ daily~dosages of the compounds of this invention for i~
2 S trèatment ~of hypertension ~or congestive heart failure are about O.3 mgJkg tQ ~abaut 100 mg/kg of mammalian weight per day administered in single or divided doses. The exact dose to be administered is deterrnined by the attending clinician and is dependent on the potency of the cornpound àdrninistered, the age, weight, condition and response of the patient.
Generally, in treating humans having hypertension or congestive heart failure, the compaunds of this invention can be administered in dosage~ ranges of about 1 .O to 50 mg/lcg.

, ~ ..

SUBSTITUTE SI~EET ~
~ - ' wo ~4/0348~ '3 : $~

4~7~5 , Typical oral formulations include tablets, capsules, syrups, elixirs and suspensions. Typical injectable formulations include -~
solutions and suspensions.
The typical pharmaceutically acceptable carriers for use in 5 the fomnulations described above are exemplified by: sugars such as lactose, sucrose, mannitol and sorbitol; starches such as cornstarch, tapioca starch and potato starch; cellulose and derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose;
calcium phosphates such as dicalcium phosphate and tricalcium ~-10 phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone;
polyvinyl alcohol; stearic acid; alkaline earth metal stearates such as magnesium stearate and calcium stearate; stearic acid; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil and corn oil;
non-ionic, cationic and anionic surfactants; ethylene glycol polymers;
15 betacyclodextrin; fatty alcohols; and hydrolyzed cereal solids, as well as othernon~toxic compatiblefillers, binders, disintegrants, buffers, preseNatives, antioxidants, lubricants, flavoring agents, and the like commonly used in pharmaceutical formulations.
Following are examples of procedures for preparing ~ -~ 20 compounds of formula 1.

; : PREPARATION 1 .

; 6 s--Ob'N~`NH2 O
: .
Steo A: In an ice-water bath, to 2(S),3-diamino-propionic acid (32 g) in water (1001 mL) add, with stirring, concurren~ly solutions of b!enzyll`
- chioroformate (76 mL) in toluene (20 mL) and 4N NaOH (133 mL) over 30 min. Stir at 20-25C for 45 min and then add additional benzyl 30 chloroformate (34 mL) in toluene (10 mL) and 4N NaOH (61 mL) concurrently over 10 min. Stir the mixture at ambient temperature for 2 hr. Add diethyl ether (Et2O) (200 mL) and stir for 10 min. Filter and .

:: SUE~STITUTE SHEET

W() ~ )3~X~ PCi/US93/07i37 '~4~785 .~:

wash the solid with Et2O (200 mL). Extract the aqueous solution with Et2O. Combine the aqueous solution with th~ solid and add concentrated HCI to pH 1-2. Extract the aqueous solution with EtOAc . ~-(250 mL twic~). Dry the EtOAc ~xtract (MgS04) then.concentrate in vacuo to give 2(S),3-bis-(benzyloxycarbonylamino)propionic acid, [a]23-6D =-13.7 (MeOH).

- Step B: Suspend the product of Step A (2 9) in CH2CI2 (25 mL) and treat with thisnyl chloride (1.6 mL). Stir the resulting mixture at - 10 room temperature for 1 hr and then heat under reflux for 1.5 hr.
Concentrate the mixture in vacuo and triturate with hexane (3 times).
Dissolve the residue in CH2CI2 (10 mL), treat with hexane (30 mL), and stir overnight. Filter to give 4(S)-benzyloxycarbonylaminomethyl-2,5-oxazolidinedione.

, ~
~i ~ St~ Treat the product from Step B (11 9) in acetone (75 mL) wit~ 6N HCI (90 mL) and stir at room temperature for 2 days. Remove the acetone in vacuo and wash the aqueows mixture with Et2O, twice.
Treat~the~aqueous solution with concentrated NH40H to pH 5 to give a 2 0 ~prècipitate, m.p. 218-228C. Heat a portion of the precipitate (0.6 9) with 50% MeOH/H2Q (30 mL), boil down to 20 mL, filter, and leave at room ~ . ~
~ temperature and filter to give the title compound, m.p. 231-2C.
,: , ;~ For preparation of the racemic compound, see: S. Takagi, - ~ 25 Chem. Pharm. Bull. Ja~an, 7, 616 (1959).

t-C4Hg02C ` , ; , . ~ C6HS O~N~NH
~: O
i ~ 3~
Add the product of Preparation 1 (2.5 9) to dioxane (100 mL~, then add 0.5 mL of H2SO4, stir and cool to -20C. Treat with SUBSTITUTE SHEET

~' ~4/034~1 ~Cr/l~S93/0713, ; . ~,-.
~ r~ ~ ~ iL417~
-25- ~
~, isobutylene gas (25 mL), seal the reaction vessel, and stir at room temperature for 3 days. Vent the reaction vessel, dilute with Et2O (100 mL) and 7% NaOH (50 mL). Dry the Et20 solution over MgSO4, then concentrate in vacl~o to give the title compound.

C H3CH202C . '"~
C H o H~ HCI .

O
.
Add EtOH (2.0 mL) to a solution of the oxazolidinedione from P!eparation 1, Step B ~1.0 g) in CH2CI2 (12 mL) and thionyl chloride (0.8 mL~, and heat the solution under reflux for I hr.
Concentrate the mixture in vacuo, triturate with Et2O, and filter to give a ~ white solid. Recrystallize from CH 3CN to give the title compound, m.p.
5~ 63-5C, la]~3-6D =-9.5 (MeOH).

PREPARATI~L~L

CtHs o~H~ p-TSA
o Heat, in the presence of a Dean-Stark trap, the product of - Preparation 1 (6.6 g), p~énethyl alcohol (26 mL), and p-TSA (5.5 9) in toluene (60 mL) overnight. Cool and add Et2O (100 mL), then ~ollect the sdlid and recrystallizeifrom MeOH/Et20 to give the ti~le compound, 25 m.p. 134-6C, [aI22-5D = ~9.2 (MeOH).

~ ;
.

.

~ .:

SUB~;TI~UTE S~EET

/034X I Pcr/US93/()7 1 37 ~ ~ ~ f ~1 ~17~5 .;

. -26-~9 `
~W .

C F3SO20~0CH3 O
Cool trifluoromethanesulfonic anhydride (1.5 mL) in CH ~C12 (20 mL) to -20 to^30C. Add pyridine (0.7 mL) and stir to give a precipitate. Add 3-phenyl-2(R)-hydroxypropionic acid methyl ester (1.3 g) in CH2C12 (10 mL). Adjust pH if necessary with pyridine. Warm the mixture to 0-10C, add excess lN HCI, stir 5 min., then add Et2O (60 10 mL). Extract the Et2O solution with water, 1 N NaHCO3, and aga;in with water. Dry the ~t20 solution (MgS04), then concentrate in vacuo to give the ;title compound.

Using the appropriate starting hydroxy esters, the following 15 compounds can be prepared via substantially ~he same procedure:

CF3SO2o - n~ocH2c6Hs CF3SO20~0C4H9-t o O
Prep5a PrepSb ,~ .

CF3SO20~0CH2C6H5. i .~

Prep5c CF3SO20~0CH3 -; ~ Prep5d ~SUBSTITUTE S~EET

WO 94/03481 PCI /US~3/07137 :;

PREPARAT!ON 6 Ho b~OC4Hg~t O

SteQA:Cool to -30 to ~20C! 3-phenyt-2(R)-acetoxypropionic acid (10.5 9) in CH2CI2 (200 mL), add conc. H2SO4 (0.~ mL) and isobutylene (120 mL~, seal the vessel, and stir at room temperature for 2 days. Vent the vessel, pour the reaction mixture into NaHGO3 solution, and extract with CH2C12. Dry the CH2CI2 extract (MgSO4), then 10 concentrate ~n Yacuo to give 3-phenyl-2(R)-acetoxypropionic acid t-butyl ester.

ll; Under anhydrous conditions, heat the product of Step A
p.50 g3 with diaminoethane (1.28 9) in DMF (17 mL) at 50-53C for 22 hr. Dîîute with Et2O, wash with 10% KHSO4 solution, and then water.
Con~entrate the dried (MgSO4) solution in vacuo to a residue. ; -Chromatograph the residue on silica gel (40 ~, 500 mL) using CH2Cî~
as~eîuant, to give the tîtle compound, [a~26D = -11.9 (MeOH).

20 ~ PREPARATIC)N 7 Ho~:)cH2c6H~

~; Cool D-norvaline (20.0 9) in 5% H2S04 (360 mL) to 0~5C
25 and treat with sodium nitrite (30 9) in water (140 mL) dropwise over 1 hr.
` - Warm the resulting mixture to room temperature and stir for 20 hr. I
Extract with Et2O (3x400 mL). Concentrate the dried (MgSO4) Et2O l;

, , .
~ ~ SU~STITUTE SHEE~

WO 9~/034Xl ~l ~ 3L PCr/US93/07137 t......... :

. ` :

solution in v~cvo to give 2(R)^hydroxyvaleric acid, [c~]26D = +8.4 (MeOH).
Step B: Treat 2(R)-hydroxyvaleric acid (10.22 9), from Step A, in DMF (70 mL) at 0-5C with benzyl chloride (9.6 mL) and cesium carbonate (27.3 9), warm to room temperature, and stir for 18 hr.
Concentrate the reaction mixture in vacuo and partition between water and EtOAc. Concentrate the dried (MgSO4) EtOAc s~lution in vacuo to a residue. Chromatograph the residue on a column of flash silica gel (40 ~,1600 mL) using CH2CI2 as eluant to give the title compound. ~

PREPARATI0~18 , y HO~OCH3 ' : :;

~ ~ Treat 2(R)-hydroxycyclohexylacetic acid (23.00 9) in DMF
25~ mL)~at 0~5C with methyl iodide (17.0 mL) and cesium carbonate (47.4~g),~warm to room temperature, and stir for 18 hr. ~ Concentrate the r eac~tion mixture in vacvo and partition between water and Et2O.
G~oncentrate ihe dried (MgSO4) Et2O in vacuo to give an oil.
Chromatograph this oil on a column of flash silica gel (40 Il, 2000 mL) using CH2CI2 as eluant~to give the title compound. ~`

I PREPARATlON 9 ,~ NHSO

~ .
Step A: At 10-15C, add benzylchloroformate (15.5 mL) dropwise toa heterogeneous mixture of 3-methanesulfonamido-D,L-~, : ~: : ` Sl; BSTITUTE SH EET
,~

WO 94/034~l ... . PCI /VS~3/07137 .:

~ ~4i7~5 phenylalanine ethyl ester (25.00 9) and triethylamine (15 mL) in dioxane (400 mL). Warm to room temperature, stir overnight, and remo~e the dioxane in v~cuo. Partition the mixture between EtOAc (200 mL)/water (500 mL). Wash the EtOAc solution with water (500 mL), 1 N HCI (500 mL), 5% NaHCO3 (500 mL), and brine (500 mL). Concentrate the dried (MgSO4) EtOAc solution in vacuo to a residue. Chromatograph the residue on silica gel (4011, 2200 mL) using E~OAc:hexane (3:7) as eluant to give N-benzyloxycarbonyl-3-methanesulfonamido-D,L-phenylalanine ethyl ester.
1 o steD B- Add lN NaOH (107 mL) to the product of Step A (14.80 9) in THF (100 mL) and stir overnight. Concentrate the THF solution in vacuo and partition between EtOAc (700 mL)/2N HCI (200 mL~.
Concentrate the dried (MgSO4) EtOAc solution in vacuo to a residue.
Add CH2CI2 (50 mL) and concentrate in vacuo (3 times) to give N-~
- ~ 15~ benzyloxycarbonyl-3-methanesutfonamido-D,L-phenylalanine.
SteD C: Treat the~product of Step B (~2.~ 9) in EtOH (400 mL) with papain (2.56 g freshly ground), L-cysteine (0.156 9) and freshly made lM citrate buffer pW 4.5 (2.8 mL). After 2 hr, add papain (1.28 9), L-cysteine (0.078 9), and 1 M citrate buffer pH 4.5 (1.4 mL). Repeat the 20 làtter addition of papain, L-cysteine, and citrate buffer at 4 and 6hr intervals. Stir the resulting mixture for 4 days. Filter, wash with EtOH, and~ooncentrate in vacuo to a residue. Partition the residue between (1400~ mL)/ 5% NaHC03 (6xl 50 mL). Concentrate the dried (MgSO4) Et2O solution in V8CUO to give N-benzyloxycarbonyl-3-25 methanesulfonamido-L-phenylalanine ethyl ester, ~a]26D=-6.7 (MeOH).
Step~D: Hydrogenate the product of Step C (5.20 9) in EtOH (300 mL) in the presence of 10% Pd/C t2.1 9) for 5 hr at 50 psi. Filter and wash with CH2CI2. Concentrate the EtOH-CH2CI2 solution ~n V8CUO to give ttie title cornpouhd, 1C~]26D=+14.3 (MeOH).

`~ : SlJBSTl~UTE SHEET

WO 94/03~ 1 PCI / US93/07 1 37 i-''`'-'' '`
` ',2~41785 C6Hs 0~ N~`
O H O

St&p A At 0-5~C, treat the product of Preparation 3 (liberated from :~:
the respective HCI salt (9.6 9) with NH40H) in CH2CI2 (90 mL) with Proton Sponge~ (9.09) and the product Prep 5b of Preparation 5 (12.0 g) in CH2CI2 (75 mL) as described in Example 1A to give a residue.
- ChrQmatograph the residue on silica gel (40 Il, 1,300 mL) using 0.5:99.5 MeOH:CH2t:~12 as eluant to give the product : ~
CH3C~202C~ C6H5 ' :-:
C6Hs--o~D~N~I~ ~D~OC4Hg-t a]22Do+O.5~ (MeOH).

Using substantially the same procedure, the following 15~ ~ compound Gan also be prepared ~ , .
C6H CH2~H2O2C fC6H5 N~N~OC4Hg-t O H O Prepl Oa-A
a]22D:=-3.5 (MeOH) ;~
~2; Tteatlthe product of Step A (9.2 9) ir~ CH2C12 (30 n~L) with :: TFA (30 mL) at room temperature for 5 hr. C:oncentrate in vacuo. Add `~
20 Et2Oandconcentrate in vacuo. Trituratewith hexane, decantand concentrate in vacua to a residue. Chromatograph the residue on silica gel (40 ~L, 1,10û mL) using CH2CI2:MeOH:AcOH 310:10:1 to give the title compound, ta]24-5D = +0.67 (MeOH).

:~ : SUBSTITUTE S~EET

wo 94~03481 , i7?~

Using substantiaily the same procedure, the foliowing compound can also be prepared C6HsCH2CH202C ~C6i~5 ' C6H5`' 0~ N `~` N ~oH
O H O Prep1 Oa MS: mle ~17 (M+) .;:

:-PREPAR,4TlON 1 1 N3 OS(O)2CF3 `

OH ~ --OH .-Treat a cold~solution of 5 a g (R)-2-hydroxy-3-azido- ;~
15~ p~opanoic acid~in 80 mL of; methanoUbenzene (7:1) with an excess of trimethr~lsilyldiazomethane. Neutralize the excess diazomethane with ;acetic acid,~then corlcentraie the mixture to a residue. Chromatograph ~ -the residue on silica gel (30% ethyl ~acetate/hexane) to give the analogQus hydroxy ester. 1H NMR (200 MHz, CDCI3): 4.40 (dd, lH);
20 3.85~ (s, 3H), 3.66-~.50 (AB~,!JAg_12.8 Hz~ JAq-4.20 Hz~ JBq~_3~20 Hz, 2Ht~
. ~ .::

,--- , ..
.

SUBSTITUTE SHEET
.: -, :

/u~
PCrt~JS93/07 1 37 7~5 Step B

COOCH3 trifluoromethane - sulfonic anhydride COOCH3 N3 OH pyridine N3 os(o)2cF3 S The product of step A (5.0 9) is dissolved in 80 mL of CH2CI2 and 3.7 mL of pyridine, then cooled to -25C. Stir the mixture under N2 atmosphere and add 8 mL of trifluoromethanesulfonic anhydride. After 1/2 h., warm the mixture to 0 to 10C. Add an excess of 1 N HCli stir for 5 min., then extract with 200 mL of diethyl ether. Wash the organic extract with water, 1 N NaHCO3, then again with water. Dry the or~anic solution over MgSO4, filter and concentrate to sive the title compound.

PR~9~ON 12 H2N cO2c(cl 13)3 Combine 1-amino~i-cyclohexanecarboxylic acid (10.0 ~) t butyl acetate (140 mL)~ and HCI04 (6 mL) in a sealed vessel and stir at ro~mtemperaturefor2days. Ventthevessel,pourthemixtureinto aqu~e~ous NaHGO3 and extract with EtOAc. Dry the EtOAc extract over MsSO4, filter, and concentrate in vacuo to give the title compound, FAB

.
' Using substantially the same procedure, the following compounds can be prepared:

,, - .

:~ : , I
' ' . :~

~m SIJ~STITUTE SHEET
,: .

~/0 94/(13481 PCl/l_;S93/0713, :
214~785 .

Q ~ N CO2C(CH 3)~
H2N CO2C(CH3)3 Prep1 2b Prep12a FAB MS (M+H) 15B
FAB MS (M+H) 186 PREpARATlON 1~
:~ : ``:

H2N cO2c~c~H3)3 ',' ~E A Heat a solution of cyclobutanecarboxylic acid (4 g) in toluene (60 mL) to 80C, and add N,N-dimethyiformamide di-t-butylacetal (2~.0 9), dropwise. Heat the mixture for 1 h., then cool and wa~sh~ successively with water, aqueous NaHCO3 and brine. Dry over t 0; ~ ~ ~MgSO4,~filter, and distill~off the~toluene to a residue. Chromatograph the re,sidue to~give the product <~C2C(cH3)3 lH '' lhtèt,~lH~ 2.05-Z.25 (m, 4H),~ 1.75-1.95 (m, 2H), 1.4 (s, 9H).
SteD B: ~ ~ Add a solution of the product of step A ~2.2 9) in THF (5 - mL) to Ll)~ at ~78~ and stir for 1h at -78C. Add a `solution of tri-isopropyiphenylsulforlyl azide~ (4.4 9) in THF (15 mL) and stir at -78C
for~6~h. Quench with~AcOH (2.0~mL) at -70C, warm to room temperature and~stir for ~ h. Di!ute the mixture with CH2CI2 (200 mL), wash with aqueous NaHC~O3, then with brine, dry over MgSO4, filter, and concentrate to a~ residue. Ghromatograph the residue to give the azide CO2C(CH3)3 ~`

- ~ i 20~ product ~ IN3 1 !~ " ,,,1,H NMR (CDCI3) 2.6 (m, 2H), 2.1-2.3 (m, 2H), 1.7~-2.05 (m, 2H), 1.5 (s, 9H).
Step C: ~ Hydrogenatethe product of step B (2.7 g) in MeC)H (50 mL) over 10% Pd on carbon (0~5 g) for 3 h. at 60 psi. Filter and concentrate to give the title compound, 1H NMR (CDCI3) 2.4-2.6 tm, 2H), 1.8-2.3 (m, Z5 4H), t.5 (s, 9H).

~.

:; ,:
SUBSTIl-UTE SHEET

wo ~4/034~1 PCr/~iS93/07137 7~3t~

`:
PREPARATION 14 . ~

Iv--< co2c(CH3)3 '"' - ~ NH2 ::
~ ~ .
;~ 5 Step A: Add a solution of cyclopropane carboxaldehyde (10 9) in concentrated aqueous NH40H (22 mL) to a mixture of NH4CI (8.5 9) `";
and KCN (9.S g) in~water (60 mL) at 0C, and warm to room temperature. Add Et2O (200 mL) and stir at room temperature for 30 h.
Add 200 mL more Et2O and continue stirring for 15h. Separate the ~'~'; 10 organic layer and concentrate to a residue which is extracted with I < CN ; .
CH2CI2 to give the product V NH2, IR2100cm-1 (CN).
SteD B: Heat the product of step A (13 9) and 6N HCI (80 mL) at reflux for 16h.~ Cool to room temperature and concentrate to a residue.
H~at the residue~ In~ethanol ~(200 mL) at~reflux for 2 h., co~i to room 15~ temperature~and- filter.~ ~`Concentrate thé filtrate,~ add water and NH4OH
and ~stir for O S h. Concentrate, treat with Et2O and filter to give the product ~ ~N~ , ~ FAB MS (M+H) 116.
Treat the product of'step B~(10.0 gj according to the ~ of Preparation 1, step A, to give~the product 20 ~ NHC(O) OcH2~6~4, lH NMR (CDC13) 7.3 (m, SH), 5.3 (br d, 2H), 3.8 ~t, ~1H),~ 1.2~(m,~1~H), q.3-0.7~(ml 4H).
S~ 12. ~ Treat the product of step C (10.0 9) according to the ~dur* of Prèparation 13, step A, to give the product C~C~CH3)3 ~ NHC(O)-OCH 2C6Hs, 1 H NMR (CDC13) 7.35 (br s, 5H), 5.3 (br d~
25 2W), 3!75 (t, 1~Hl, '~.45 (s, gh) 0.9-1.2 (m, lH), 0.4-0.6 (m, 4H).
SteD E: Hydrogenate the product of step D (8.0 9) in MeOH over 10 % ~Pd on carbon at 60 pri for 3h. Filter, concentrate to a residue and chromatograph the residue (silica gel, 3%MeOH in CH2CI2) to give the ff tle~ compound, ~1H NMR (CDCI3) 2.80 (d, l H), 1.6~1.75 (br s, l H), 1.45 30 ~ (s, 9H), 0.8-1.0 ~m, 1 H), 0.2-0.6 (m, 4H).

SU~STITUTE SHEET
.,, ~
,. ..

WO 9~/03481 PCI/l,;S~3/0713/
, 2~L4~L785 -3~i-H2N~ H2~ C6H5 2)4 H ~ ~ H

i-i o i~

~OH -.' ~`, - Ste~ At 0-5C, treat the product of Preparation 2 (2.0 g) in C~i2CI2 ~30 mL) with Proton Sponge~ (1.5 g) and the product of Preparation 5 (1.77 9) in CW2C12 (25 mL). Warm to roorn temperature ~as ic~ melts) and stir overnight. Dilute the mixture with Et20 and filter.
Extract the Et2O solution with water (3 times), dry over MgSO4 and - concentrate ~in ~cLlo to a residue. Chromatograph the residue on silica 10 gel (40~1, 400 mL) using 1:99 MeOH:CH2CI2 as eluant to give the ~ ~ product 6,~ t-C4Hg02C C6H5 ' ~ C6Hs~o~l~N~N~ocH3 ta]2oD--1.8o (MeOH) : 1 5 Using appropriate starting materials and substantially the same procedure, the following compounds can also be prepared~
- ~, . . .
t-C4H302C ,"C~l2CH3 : " C~oH5~,, 0~ N~OCH2C6H5 ~, ~ O 1e-A
[a]22-5D = -13.4 (MeOH) SU BSTITUTE SHEET

WO ~4/03481 ~ 3 7~3~j PCI/US93/~)7137~

7 ~ :

-36- :
t c~Hg02c Q ~ ;

O H~ lf A
[~X]22-5D~ o (MeOH) Step B At 0-5C, treat the product of tep A (12.3 9) in Me.OH (150 mL) with 4 portions of 1 N NaC)H (32 mL) over 5 min. Warm to room temperature and stir for 2.25 hr. Add acetic acid and concPntrate in - 5 vacuo. Add MeOH and concentrate in vacuo to a residu (~hrorratograph the residue on silica gel ~40 ~L, 800 mL) using - CH2CI2:MeOH:glacial AcOH 310:10:1 (2250 mL); 230:10:1 ~2500 mL) to givetheprodu~t t-C4HgO2C ~C6Hs H~
~:~ 10 [a]2D=-4.Z (MeOH).

Using appropriate starting materials and substantially the same procedure, the fol!owing compounds can also be prepared: ;
t-C4Hg02C ~CH2CH3 C6H6 0~,N~l~N~OH
O H o 1e-~3 [~]22-~D = +~.6~ (MeOH) ~,~

SUBSTITUTE SHEET

WO 94/03481 P~/ US93/07 1 3 /

;~ 43 q~5 -37- ;:

t-C4Hg02C O '~
C6H5~O~N ~N~fOH
O H o 1f-B
[]22D = +14.3 (MeOH) Step C: Add EDCI ~0.46 g) to the product of Step B (0.83 g)t O-t-butyl-L-tyrosine t-butyl ester hydrochloride (0.66 9), HOBT (0.080 9) and triethylamine (0.63 mL) in DMF (~ mL) and stir the resuiting mixture at room temperature overnight. Dilute with Et2O (150 mL) and wash with water (2x50 mL), 1N tlCI (2x25 mL), water (~0 mL), 1N NaHC03 (25 ~; mL), and water (50 mL). Coneentrate the dried (MgSO4) Et2O solution in vacuo to give residue A. Neutralise the HCI extract with IN Na)tC03 and ~extract~with CH2GI2.: Concentrate the dried (MgSO4) CH2CI2 in ;1:0 K acvo~to~give~residue B. Combine residuesAand B and chromatog:raph~on silica gel plates (10, 1000~u plates) using 2% MeOH
in CH2GI2 to give the product :~
t-C4HgO2C C~H5 N~NJ~Oc4Hs t ~:: 15 [a~20 5D = -32.2 (MeOH).

Using appropriate starting materials, and substantially the same procedure, the following compounds can also be prepared:
; 1`:

~'~ ',.
~ ' , SUB~;TITUTE SHEET

WO 94~034~1 PCT/US93/07137 .~
...
7~35 t-C4Hg02C C6H5 C6HS--O`b'N~`N~N`Jl`oC~CH3 OC4Hg-t 1 a-C
[a]24 5D = -23.0 (MeOH) C3h5 0~,,N~N~N--D`OC H

OC4Hs~t 1 c-C
a]20-5D = -30.5 (MeOH) O ~

~a]22~5D 31 1'(M OC,Hg-t 1d-C

t-C4Hg02C CH2CH3 ~

OH 1 e-C
a]20 5D = -18.2 (MeOH) ;~

,, ~ , ; ~
, .

SUBSTITVTE SHEET

~ . , ~J('~ /v7 wo 94/034~
7~3S

t-C4~902C O

C6Hs O~
O H O

~ OH 1f-C
[a]2~ 2~.6 ~MeOH).

CH3CH202C C6H5 , ;:
HJ~ J~H ~

o H o ~NHSO2CH3 , ~ ~
[a]2~D = -28.2 (MeOH) ~, .
t C4Hg02C CH2CH3 C6Hs~o~l~NJ~N~Nv`ocH2cH3 O H o ~¢~,NHSO2CH3 1h-C
a]20D = ~12.3 (MeOH) St~D D: Hydrogenate the Product of Step C (0.5 9) with 10% Pd/C
(0.15 9) in MeOH (25 rnLj at 50 psi for 3 hr. Filter and concentrate the ~: filtrate in vacuotogivetheproduct ~ , .
~:
"

~,, ~ ~ SUBSTITUTE SHEET ;-3~1 PCr/US93/07137 t-C4H90 ~C C6Hs H2N ~ N ~N~JI~ oC~g t OC4Hg-t [a]2Q 5D - -36.3 (MeOH).
.~
Using appropriate starting materials and substantially the 5 same procedure, the following compounds can also be prepared:
:
t-C4Hg02C ~ C6H5 : Ht~ OGH3CH3 ~ OC4Hg-t l a-D
a]2~-5D = -40.3 (MeOH) ~ OC~119-t l~D
[aJ20 5 5 = -25.6 (MeOH).

; ~ ~ ' ' ', `', , .
~' -: ,:''' :; ' - - .

.
-.
SUBSTITUTE SH~ET - ;

: ~ :

wo 94/03481 4~-q~

? - ;
C6H5CH2CH202C C6H5 :' H2N ~` N ~N I~ OC4Hg-t ~: ~
Il ,1 ~, OC4Hg-t 1 d-D
la]23D = -24.0 (MeOH) t-C4Hg02C CH2CH3 H2N~lNJ~N~J~OCH3 H O

~ OH 1 e-D
;~ a]2o-5D =~-37.5 (MeOH) t-(~4HgO2C

H~

OH l f-D
a]20-5D = -43~0 (MeOH) C~CH2O2C C6H5 H~
OH 1g-D
[a]23D = -32.0 (MeOH) SUBSTITUTE S~EET
, , .

~'O 94/034Xl PCI`~US93/07137 . ~. ,` , 4~ 785 , ,~.
t-C4Hg02C C H2CH3 ~ H
H2N ~ N~N--~` OCH2CH3 H O --~,~ NHSO2CH3 ~ 1h-D ~`
l~]20D = -12.0 (MeOH).

steD E: Add EDCi (0.11 9) to N6-benzyloxycarbonyl-N2-metharlesulfonyl-L-lysine (0.19 9), the product of Step D (0.255 9), HOBT (0.014 9), and triethylamine (0.085 mL) in DMF (1 mL) and stir ovemight at room temperature. Concentrate the reaction mixture in 5 ~cuo and dilute with~ Et2O (50 mL). Wash the Et2O solution with 1 N
NaHCO3 (75 mL, then 50 mL), water (2x50 mL), and brine (2~25 mL).
Concentrate:~the~dried~ (MgSO4j~Et2O solution in vacuo to a residue. `-Ghromatogràph~:the residue~on ~silica~gel:(40 ~, 500 mL) using MeOH:CH2CI2 1:99 (2000 mL) and then 1.5:98.5 to give the product CbzH~ t-C~HgO~C G6Hs CH~so2~N~N~N~N~J~oc~H9-t ; H ~O ~ H ~ O -OC4H9-t [a]21.5D i-i -33 7 (MeC~H) ~. .
Usjnglappropriate starting! materials and substantia!ly jthq same procedure, the following compounds can also be prepared:

" ~
.; , , "~

SUBSTIl UTE S~EET

~VO 94/03~81 I'(~ /Ui l~ , Z~ 785 CbzHN\ t-C4Hg02C C6Hs . :.
. (CH2)4 H ~ ~ H
C~SO2~ N'~ N~l~ N~N~` OCH2CH3 O H
I
~ oC41-19-t 1 a-E
a]25D = -36.6 (MeOH) CbZHN~CH3CH O C C6H5 Cl~502~ NJ~ ~N~
- i ~ H O ~ O ~ -~

0~4Hs-t 1 c-E
[~215D = -32.5 (MeOH).

Cb~zHN--(cH2)42 1~ ~C6Hs -CHg502~N~N N~N--~`OC H t ~'~

OC4Hs-t 1 d-E
a]21-5D = -26.3 (MeOH~ ~

t~C4Hg02G C~CH3 `.. !
CbzHN--(CH2)4 , ~ f H3 2~ I~N~N~N .--OCH3 H O H O ~ :

~ OH l ~E
`~ []215D = -22.3 (MeOH) ~.

~ , - - SU BSTITUTE SH EET
,, , , WO 94/03~ ` PCI'lUS93/0713/, ....
Z1417~3~ ^ f~
i -44- ;~
.

t-C4Hg02C ~) CbzHN--(CH2)4 H ~ ~ H
C ~gso2~ N~n, N ~ ~ N ~N ~J~ OCH
O O
Il .~
1~ ~ OH lf-E
~a~22D = -32.1 (MeOH) GbzHN--(CH2)4 H ~ ~ H
CH3SO2~N, ~ ,N J `N ~ N--Il`OCH2CH3 O O ~ NHSO2C~

[a]20D - -28.9 (MeOH) t-C4Hg02C C6H~
GbzHN--~CH2)4 H : 1 ~ O
3` 2 ~N~ ~N ~ ~ocH2C~b O n o ~ ~ NHSO2CH3 l h-E
a]22D = -22.8 (MeOH) Ste~ F; Cool the product of Step E (2.0 9) in C~12CI2 (10 rnL) to 0-5C, add TFA (10 mL), stir for 30 min, then warm to room temperature andstirfor2hr. Concentratethe reaction mixture in vacuo to a residue.
5 :Oissolve the~residue in Et20 (115 mL), add 15% NaOH, and extract with E~. Acidify the aqueous solution with glacial AcOH to pH 5 and :extractwith~EtOAc(3x50mL). Concentratethedried(MgS04)EtOAc soi~tion in Yacuo to give the product ~ , SU BSTI~UTE SH EET

WO 94/03481 PCI`/US93/07137 .
7~5 -45- :

CbzHN\ HO2C C6Hs (C ~ H~ ,H~

~a]22 D = -17.4 (MeOH).
:
Using appropriate starting materials and substantially ~he -~
5 same procedure (leaving out the NaOH treatment), the following compaunds can also be prepared:

C~5O2~N~:N~lN~n~ocH~cH~

a~26D = 24.8 (MeOH) bzHN\ ; HO2C ; fC6Hs~

H~ N~ .--~OCH~CH C H

'; ~ ,: OH i1b-F~
MS: m/e 860 (M~) . ~, . . . .

.: . :~ ~ . . :

~ .~

SU8STI~UTE SHEET

~0 94/03481 PCl/US93/07137 , 4~ 3 -~6-CbzHN\ CH3CH2O2C C6H5 CH3SO2~N~N~N~N~I~oH

~OH 1c~F
[a322-5D = -21.0 (MeOH) G6HsCH2CH202c C6H5 CH3S02`N~N~I~N,~N~oH

~ OH 1d-F
[a]23D = -16.3 (MeOH) HO2C ,C~CH3 :~ CbzH~ (CH~4 H I H :~
: C 3 2 N'~ J~N~ ~OCH~

OH 1 ~F
x]21 5D = -13.8 (MeOH) , ~ ~

; HO C ~ J ``
. ~CbjzHN--(CH2)4 IH ` l ~ `~r H"
3 2`N~N~I~N~N~J~OCH3 b~OH 1f-F
[a~2~D _ -17 8 (MeOH) : : ~
; ~ SUE~STITUTE S~EET

~v~ 94/03481 YCr/~;S93/~)713l .

7~5 H~ N~ OCH2CH3 OH O ~,NHSO2cH3 ~ 1 h-F
:~ [a]22 ~D = -9.5 (MeOH) .

: Ste~ G: Hydrogenate the product of step F (û.5 g) in MeOH ~30 mL) .
with 10% Pd/C (0.5 9) at 50 psi for 2 hr. Filter through celite and . concentrate the filt~rate in vacuo to give the title compound, m.p.j 150C
(decomp), [~]22 5D = -20.0O (MeOH?.

Using~appropriate s~arting materials, the following ~''r `~ compour!ds Carl be prepared usirig substarltially the same procedure~ ,~

(C~b)4 H ~ H
C~bSO2~ N~N~l , ~ J~ oCH2C~b H ~O: H O ~

`OH 1a a]22 5D = -1g.6 (MeOH) (C\H ` ~ H~;)2~; ~ G6Hs ! ' `:

CH3SO2~N~N~l~N~N~J~ocH2cH2cr~Hs H o H o ~OH 1b [~23D = 13.9 (MeOH) ~ . ~

SUBSTI~UTE SHEET

. "j 21~177 CH3CH2 02C ~6H5 '` .

CH3502~ ,,~N~N~ ~OH

~a323D = -21.1 (MeOH) C6Hs CH2CH2 C)2C C~6Hs H2N - (CH2)4 ~ ~

O '~

a]23D = -t8.3 (MeOH) :~ -HO2C ~ C~CH3 H O H

[a321-5D = -15~3 (MeOH).

':

SU BSTITVTE SH~ET
.

WO~4/~)34~1 ~CI/I !j~3/()713 ;~1417~5 -49- :

H2N--(CH2)4 H ~ 1 H
H~ H~ _ OCH3 ~OH lf M: m/e 628 (M+) CH3CH202C C6H5 ' H2N--~CH2)4 H ~ ~ H
CH3S02`N~N~I~N~N`J~oCH2C~3 "
o H O -- ,~NHSO2CH

cL]22D = -31.1 (MeOH) HO2C C~CH3 ~- ~ H2N--(CH2)4 H ~ H
C~3SO2~ N,l~ N~ N~NJI`OCH2CH3 H o ~ NHSO2CH3 al2~-5D = -9.~ (MeOH) I i, : . , , ! `

~, :

,, ' ``' ~ ~ SUBSTI~aJTE S~EET `~
. .

I'CI / US93/07 1 37 ~ ~3 7~

: -50- :
~' CH SO (,~H ,~ ~H~

S~ A: Add EC~C:I (1.50 9) to the product P10a of Preparation 10 (2.60 g), L-tyrosirle methyl ester (t.80 9), HOST (0.27 g) and tri~thylamine (1.80 mL) in DMF (10 mL) and treat the reaction mixture as d~scribed in Exarnple 1, Step C to give a residue. Chromatograph the residue on silica gel (40 ~L, 300 mL) using 1% MeOH in CH2C12 to give 10 the product ~6H5CH~CH202C ( 6H5 C6Hs 0~N~l NJ~N I`OOH~

la]22 5D = -29.3 (MeOH).

Step B: Hydrogenate the product of Step A (2.1 9) with 10% Pd/C
~: 15 (0.50 9) in MeOH (60 mL) at 50 psi for 3 hr. Filter and concentrate the ~; . filtrate in va o to give a mixture of products H
~: ~ ~ N~ J~ OCH3 ~ ~ O ~

~ OH
and . .

- SUE3STtTUTE SHEET

wo 94/034~ a~
2,~.417~5 C H32C ~6H5 H2N~l N~N--U`OCH

Il l .`
~OH

Step C Add EDCI (0.76 9) to the product mixture frorn Step B (2.00 9), N6-benzyioxycarbonyl-N2-methanesulfonyl-L Iysine (1.42 9), HOBT
(0.14 9), and triethylamine (0.76 mL) in DMF (10 mL) and treat as described in Example 1, Step E to give a residue. Chromatograph the residue on sllica gel (40 Il, 35û mL) using MeOH:CH2CI2 1:99 to give :~
Fractions A and B. Chromatograph Fraction A on silica ~el plates (10, ~
1000 ~,1) using CH2CI2:MeOH:NH4OH 110:10:1 to give product 2-C-I :
~: 10 C6H~CH2cH202~ C6Hs ~; CHSO (~H~l ~H~ :

- ,~ ~q ;:
--I` OH 2-C-I
~22.5D - 23 8O (MeOH).
, ,~
Chromatograph Fraction B on silica gel plates (10, 1000 ~L,) using : : 15 CH~Cl2:M~OH:NH40H 110:10:1 togiveproduct2-CII ~:
~:: C~bO2C C6H5 CbzHN~(cH2)4 H ~ H
N~ ~N~ I`OCH~

a]20 5D = -26.6 (MeOH).
;~ ' , SUBSTI~UTE SHEET

WO ~4/034X I PCl~/ Us93/07 1 37 ~4178~5 -)~i .; "

Ste~ D Hydrogenate compound 2~C-I from step C (0.45 9) in DMF
(18 mL) with 10% Pd/C (0.41 g~ at 50 psi for 5.~ hr. Filter thr~ough celite and concentrate the filtrate in vacuo to give a residue. Chromatograph the residue on silica gel piates ~6, 1000 Il) using CH2C12:MeOH:NH40H
70:10:1 as eluant to give the title compound, [a]21D = -24.3 (MeOH).

Using MeOH as the solvent and following substantially the same procedure, hydrogenate compound 2-C-II from Step C to 9iVP
compound CH3SO~ N~1 J~H~D~

OH 2a al21D = -29.6~(MeOH).

15 ~ ~EXAMPLE 3 H2N--(CH2)4 H~ ~ ~ H
CH3SO2~N,~N~'~N,~N~D~o~, HCI OH

Stir the compound of Example 1e (0.70 9) in acetone (1 imL)~with 1 N NaOH (2!m'L) I~or 1~5 hr. Acidify with 2N HCI (4 mL) and ; allow to concentrate under nitrogen to a residue. Triturate the residue u ~yith Et20 and filter. Stir the residue with cold MeOH (2x2 mL) then hot : MeOH (4 mL) and fi!ter. Concentrate the MeOH solution in vacuo to give the title compound, la]22D = -6-9 (MeOH).

, ~

SUBSTIT~ITE St~EET

WO 94/03481 PCTi i,iS~3/U7 1 3~
æ~4~7~5 I~:

Using appropriate starting materials and substantially the same procedure, the following compounds were also prepared-HO2C [~
CbzHN--(CH2)4 H ~ ~ H
C H3SO2~ N '~ N~ N ~N Jl~ OH
O O ~q '' ~ OH 3a [a~22D = -10.2 (MeOH3 .

HO2C ~ J , .
H2N--(G~H2)4 H ~ H
CH3502`~N~NJ~N~N--I~oH

~: ~ []22D = -4.8 (MeOH) HO2C C6Hs CH3SO2~N~N~N~ O
oH o -~,NHSO2CH3 W 3c `
22~5b = ~1g~2 (MeOH3 " ~ .-.~ , .
. , SlJBST17 UTE SHEET -~, 4/u~ 417~~ PCr/US93/07137 -54- -:

HO2C CH2CH3 . ;,,~
H2N--(C~)4 H ~ ~ H ~ . :
2 N~ N ~ N ~N ~JI` OH :
O H o ~NHSO2CH3 3d ~]22D - -3.6 (MeOH) ~02C CH2CH3 CbzNH--(CH2~4 H ~ ~ H

HN~ ~N~ J~OH

~ OH 3e ~ :;
a]Z 5 D =~-6.2 (M2011) -~

, : .
CH3CH2HNrNCH2C~b TFA

H o HJ~
.
~ 0 teD A. Hydrogenate thè pro.ducS of Example 1,~ Step E (3.0 9) in MeOH (3~ mL) with 10% Pd/C (0.83 9) at 50 psi for 3 hr. Filter and ~: ~: concentrate the filtrate in vacuo to giYe the product .
SlJB5Tll~UTE SHEET

.

~0 g4/034~ S~31~713, ~i .
1417~35 .
.
-5~

t-C4Hg02C ( ~6H5 ; ;

CH3SO2~N~N~N~N~J~OC"H9-t ~OC4Hg-t ~itep ~: Heat the product of Step A (1.3 9), N,N-diethylcarbodiimide ~:
(0.46 9), diisopropylethylamine (0.21 9), and p TSA (0.25 9) in dioxane 5 (3~ mL) under reflux for 6 hr. Concentrate the mixture in vacvo to a r~sidue and dissolve the residue in CH2CI2 (100 mL~. Wash the CH2Cl2 solution with 1% NaOHlbrine (3x50 mL); brine/water; and brine.
Concentrate the dried (MgS04~ CH2CI2 in vacuo to give a r2sidue.
Ghromatograph this residue;on silica gel plates (11, 1000 ~L) using 10 CH2CI2:MeOH:AcOH 70::iO:1 as eluanttogive the product G~bCH2HN~ NCH2CH

HN\ t-c4H9o2c C6H~

~: CH3so2~ ~N~l ~DfH

~ OC4Hg-t ` `
[a~2 5D = -19.6 (MeC)H).
` ~
$teD~ ; Underla dr~ nitrQgen atmosphere, treat the product of Step ~:
B (0.28 9) with TFA (3 ml ) and stir overnight. Concentrate the reaction under nitrogen, twice add CH2CI2 then concentrat~ in vacuo to a ~ :
residue. Traurate the residue with Et20 to give the title compound, [al23D = -14.5 (MeOH).

S-IBSTITUTE SHEET
'`~

WO94/03481 PCTlUS93/0713, ;Z:1417~5 ~7''' HO2C C6Hs ~:

H2N(CH2)s~N~N~NJ~OH
o H o - .
OH `~;

. 5 Stee A- Add EDCI (1.00 9) to 6-benzyloxycarbonytaminohexanoic aoid (1.20 9), the product of Example 1, Step D (2.15 9), and HO~T ~:
(0.067 9) in DMF (7 mL), and treat as described in Example 1, Step E to give a residue. Chromatograph the residue on silica gel (40 Il, 300 mL) using MeOH:CH2CI2 0.7~:99.25; 1:99 to give the product t-C4HgO21 ~C6 s O

H2~s~N~l~N~N--D`oc H t ~ OC4H9-t .: :~
[ot]24-5D =-19.0 (MeOH).

Cool the product:of S~ep A~(2.20 9) in CH2CI2 (15 mL) to 5 ~ 0~5C,~add~TFA~ (1$: m;L), and stir overnight. Concentrate the reaction ``
mixture ~in ~Yacuo~to :give~ a residue. Triturate the residue with Et20 and ''.
: ~ filterto give the product " ~ O2C ~ ~C6Hs .
:~ : H ~ ~ H
~N~ ~ OH

OH
m.p. 98-102C, [a]22-~D = -9.9 (MeOH). .
~ ~ , SUBSTITUTE SHEEl~ :

., : :

WO 9~/034~1 PCI/US93/071 3 / ' r ~ il4~785 -57 ::

Step C: Hydrogenate the product of Step B (1.00 9) in M~eOH (30 mL) with 10% Pd/C (0.37 9) at $0 psi for 2 hr. Filter and concentrate the filtrate m vacuo to give the title compound, la]23D = -7.2 (MeOH).

:; CbzHN\ t-C4HgO2C C6Hs CH3SO2~N,.~N~I~N~NJ~OCH~CH2C6Hs OC4Hg-t Treat product 1a-E of Example 1, Step: E ~3.3 g) in acetone ~5~mL)~portionwise with 1N NaOH (7.5 mL) over 1 min., and stir for 30 -~
min.: Add~1Q% citric acid to pH~5-6 and extract with EtOAc (3x25 mL).
Ext~ract the::EtOAc solution with brine. Concentrate the dried (MgSO4) ::
5 ~ ~EtO~Ac~solution~in vacuotogive the product CbzHN tC ~H ~ C C6Hs ,-(C~)4 H ~ f H

OC4Hg~t ,[aJ26D = ~30.Q ~MeOH).
~, ~
steD B: Stir the product of Step A (1.5 g) in DMF (5 mL) with 20 cesium carbonate (0.56 9) and phenethy! bromide (0.75 mL) at room temperature ovemight. Add glacial AcOH and concentrate in vacuo to ;
- g n~ a residue. Dissolve the residue in EtOAc (t00 mL), extract with :~: SUBSTITUTE SHEET :
~ .

~O 94/034~1 PCI/US93/07137 7~5 water (2x35 mL) and then brine. Concentrate the dried (MgSO4) EtOAc tn vacuo to give a residue. ChromatQgraph this residue on-silica gel (40 ~, 300 mL) using MeOH:CH2CI2 99:1 (2000 mL), then 98.75:1.25 to give the title compound, [a]26D - -32.8 (MeOH).

, ,:

\ HOOC \/
CH3SO;~N,~N~I~N~N~I~oH
O ~ -~

~ OH

~ Treat 10.0 9 of the product of Preparation 1 1 in 100 mL of CH2C12, at~0 to~5C, with 8.0 g of ~Proion Sponge~ and a solution of t7.0 9 of Yallne~t-butyl~ester in 1~0Q~mL of GH2CI2. Warm the mixture to room~temperature and stir~overnight. Dilute the mixture with ether, filter, thèn~sh th~e~filtrate~with~water. Concentrate to a residue, which is 5~ -chromatographed on silica gel (1:3 ethyl acetate/hexane) to give the product N3~N~OC(CH~ ~
H U
F~B MS (M+H) 30 Thefollowing compounds can be prepared using - - substantia!ly the same procedures:

. .

SU BSTITUTE SI~ EET - :

WO 94/03481 PCI/US93/07137 `
;~ 7~5 :.

. . . _ _ :-`

N3~ ~OC(CH3)3 CH300C ~ J : ~ ~
O N3~ ~OC(CH 3) 3 ~:
7a-A o FAB MS (M ~ H) 273 7b-A
FAB MS (M+H) 327 ~:
:~ :;, CH,OOC ~/--\ CH300C <~
¦ N ~ X D~oc(CH3)3 ~ ~ N~

7C-A 7d-A ~ -~
FAB I\AS (M+H~ 313 ~ FAB MS (M+H) 299 : CH300C ~ ~ 7 : ~><~OC(CH3)~ ~CH300C ~ r N ¦3~N~OC(CH3)3 ¦ ~;

~ :FAB MS~ (M+H): 285 isomer A .`:
:~: : : : :~ ~: 7~-A :~:
- : ~ FAB MS (l~ H~ 299 ~, " ~ : ~
: :
CH30QC ~
~: ~ OC(CH3)3 ~ o !~ !
isomer B
: 7g-A
: ~ ~ FAB MS (M+H) 299 ,, ~- :
., ,~ . ~
teD B: Dissolve 5.0 9 of the product of step A in 20 mL of CH2CI2 and treat with ~20 mL of trifluoroacetic acid (TFA) at room temperature ;~

SUBSTITUTE SI~EET

wos4/0348l 21417~5 PCr/US93/07137 -60- ;

overnight. Concentra~e to a residue, add diethyl ether and concentrate again several times to ~ive the product:

CH3 OOC y N3~ ~ C)H

O ~.
1H NMR (200 MHz, CDt::13) 4.12 ~dd, J= 5.0, 7.0 Hz, 1H); 4.22-404 ~;
(2dd, J= 12.5, 5.0 Hz and J_12.5, 7.0Hz, 2H); 3.90 (s, 3H); 2.50 (m, 1 H);
0.95-0.9B (2d, 5.6 Hz, 6H).

Substantially the sarne procedure is used to prepare the . .
following compounds: ~ ~

: ~ ~ ~' 7a-B H
FAB MS (M+H) 217 7b-B
FAB MS ~M~H? 271 CH3QOC ~ ~ ~ CH300C <>
N3~ ~ rOH ¦ N3~`N~

" ~ !~ 7Cj-B , . 7d-B
FAB MS (M~H) 257 FAB!MS (M+H) 243 '; ' ' ~':
;: .
~ : . .
SU BSTITUTE SH EET ~:
' L4~ 785 .
-61- ~
., __ _ . . . ~ ~

N3~ ~ ~OH

7e-B o FAB MS (M~H) 229 isomer A :

¦ FAB MS (M+H) Z43 . ~7 '~':.-, CH300C ~' :.

N
H ~
isomer B ~ ~ _ 79-B ~ r ; ~FAB~MS (M+H~ 243 ~ :

Add 2,85 9 of ~EDCI to a mixture of 3.30 9 of the product of Step ~C:
step jB, 4.459 of O-t-butyl-L~tyrosine-t~butyl ester hydrochloride, 1.82 9 of HOBT,`5~mL of triethylamine and 15 mL ot DMF, and stir ov~rnight at room temperature~ :Dilute the mixture with 500 mL of diethyl ether, wash -- with water ~(2 X 100 mL), dry over MgSO4, filter, and concentrate to a rssidue.~ ~Chromatograph the residue on silica gel (30:70 e~hyl :~
acetatelhexane) to give~the~ produc~

CH300C \~ -N3~ N~ .--OC(CH3)3 ,.-',:~.'`~ : 11 1 ' 0 ~OC(CI~1~3 .;

SUBSTITUTE SHEET

2~7~5 .-:
H NMR ~200 MHz, CDCI3) 7.50 t1H); 6.90-7.10 (2d, J= 8Hz, 4H); 4.72 (dd, J=~.0, 7.0 Hz, l H); 3.74 (s, 3H); 3.~8 (d, J=4.0 Hz, 2H); 3.42 (t, J=4.0 :~ ~
Hz, 1 H); 3.00-3.05 (2xdd, J= 13.8, 7.09 Hz and J= 13.8, 5.0 Hz, 2H); 2.94 . .~:
(d, J=4.5 Hz, 1H); 2.0 (m, 1H); 1.32-1.42 (2s, 18H); 0.84-0.92 (2d, ~.5 Hz, 6H)-Using substantially the same procedures, the following ~.
compounds can be prepared.
. : .
1 H NMR (200 MHz, CDCI3) ~ :
- CH300C 7.6 (br d, l H); 6.92-7.1 (2 :~
H ll br. d, ~=8.5 Hz, 4H); 4.68 Na N~N~`~ OC(CH3)3 (br. q, J-7.0 Hz, 1 H); 3.75 H O ~ (s, 3H); 3.55-3.60 (2dd, J=15.0,6.00HzandJ= ~ ~
(CH3)3 15-0, 6.~ Hz, 2Hj; 3.37 .;
7a~C ~ : (dd, J~.00, 6.50 Hz, 1H); ~:
3.10 (m, 1 H); 3.00-3.05 (2 dd, J=~14.5, ?.0 Hz and : ~-J=t4.5, 7.0 Hz and J=14.5, 6.5 Hz, 2H); 1.32-1.45 (2s, 1 8H); 1 .Z8 (d, : `
J=7.0Hz. 3H). _ ;

GH300C ~ ~ O

¦ ~ ,~OC(CH3)3 `1, ~ ' ,' ; ~ O I ~ : ,;, ~ , : ! I

OC(CH3)3 7b-C :~
~. ~ :: _ .
- ~ . :

; :

SUBSTITUTE S~EET `~

WO 94/03481 PCI/ljS93/0713 / ,.
, `~ -63- `~

. ~:
_ _ _ _ _ C~1300C ~
FAB MS (M~H~ 532 H~ _ ( 3)3 :~
O ~ ,:~

~OC(C~3)3 '~

. __ _ _,,.`,~

FA3 MS (M+H) 518 :;
11 ~ I
OG(CH3)3 7d-C: ; ~ - -00C~ : :
FAB~ MS (M~H) 504 N~ OC(GH3)3 ~ ~ ~.

: ~ ~ : ~' : ~CH300C ~ ~
~ ~ r H FAB MS (MIH) 518 j:-~N~OC(CH3)3 ~ !

r A 7~ C
i, "

SU BSTITUTE SH EET

: ; ` :.. ~`;:

WO 94/03481 ;~L4~L7~'~; PCI/US93/07137 -~i4-; - .~ . ._, ~
CH300C ~ ~ -.
N~ N~N~I~OC(cH3)3 FAB MS (M+H) 518 isomer B 7a-C
. ., . , _ ~ :
CH300C \,~/
¦ ~ FAB MS (~A+H) 445 . .

Ste~ D; The product of step C (1.0 9) is combine~ with 0.50 9 of 10% Pd on carbon and ~0 mL of ethanol and hydrogenated at 60 psi for 4 h. Filter and concentrate the filtrate to a residue. Dissolve the residue in 2 mL of DMF, then add EDGI (0.45 9), N6-benzyloxycarbonyl-N2-methane-sulfonyl-L-lysine (0.75 9), HOBT (0.159) and triethylamine, and stir the mixture overnight at room temperature. Dilute the mixture with 20 mL of diethyl ether, wash with water (2 X 50 mL), dry over MgSO4, ~llter and concentrate to a residue. Chromatograph the residue on silica 10 gel to giva the product:

CbzNH~ CH300C V

CH 50 NH~N~I~N~ .~OC~CH3)3 o O

~OC(CH3)3 ~ ~ SUBSTITUTE SHEET

wo ~4/03481 pcrlus93/o7137 -65~
-Cl MS (M+H) 834 ~
~ .
Using a similar procedure, the ~ollowing compounds can be prepared~
;
CbzNH~ cH300C
¦ (C l~ D~H

:: ~0 ~ Q ~ ;
OC(CH3~3 7a-D
: FAB MS (M+H)_Q6:
.
: ..
CbzNH~ ~ CH300C~

¦ ~ G ~ W~
OC(CH3)3 : ~ :~

FAB~MS(M+H~860 ~ ~ :
~ i ~ `
: ~ CbzNH~ 1300C: ~`
:~ (CH 4~; ~ Q~ O ~i CH3502NH r N ~r - OC(CH3)3 ~i:

(CH3)3 : ~ 7c-D
: ~ FAB;MS (M+H) 846 ;~;

, : ~:, , : ~ :
,, ~ : : ~ :~:
: ; SUBSTITUTE SHEET ;~

:`, :
, ~
.ç~

WO 94/03481 ~l'~ pcr/Us93/07137 -66~

._.~ .
CbzNH~ CH3OOC ~ :

CH350zNH J~ ~ ~ N$ ~ oo(CH 3)3 OC~C~3) 7d-D
FAB MS (M+H) 832 ~:
, _ , ~ . , ~:
:~ - ` CbzNH~ CH3 OOC

oZ~ OC(~.H~

7e-D
FAB MS ~M+H) 818 _ ~:

(CH2)~ y o CH3~3 . . - . ~F, AB MS (M+H) 832. .

SUBSTI~UTE SHEET ~`~

, .
..~, .

WO 94/03481 PCI/US93/~7137 ~4~7~
-67~

r~ ~ O
CH SO NH~ ~N N N~

isomer B 7g-D
_ _ FAB MS (M+H) 832 _ _ :

(CH ~ ~ y O ¦
CH3sql~HJ N N~N~

¦ 7h-D ¦ ~.

: ',';
Cornbine a solution of the product of step D (1.0 g) in meth:anol with a solution of LiOH (0.~ 9) in water and stir the mixture at ~:
0:to 25C for 2 h. Add 3N HCI to bring the mixture to pH= 7 and remove - -5 : the methanol under :vacuum. Adjust the pH to 3 with additional acid and ~. ;`;
extract with 3 X 100 mL of ethyl acetate. Dry the extracts over MgSO4 : and concentrate to give the product:
CbzilH I ii Hàoc ! ' ONH~N~I`Nxlr .~OC(CH3)3 : ~ ~OC(CH3)3 Cl MS (M+) 820 .
: ~: SUBSTI~UTE SHEET

.

W~:) 94/03481 PCr/US93/07137 ~1417~5 .-Using substantially the same procedure, the following compound can be prepared from compound 7j, of step G: ~ ;

CbzNH~ HOOC \/ ; -;

CH3SO2NH~ ~N~N~J~
o O ~ '' ~OH 7m - .
FAB MS (M+H) 708 ~: Combine 0.5 9 of the product of step E, 0.20 9 of 10% Pd on carbon and 30 mL of ethanol,~ and hydrogenate at 60 psi for 4 h.
Filter and concentrat the filtrate to give the product~

O O
OC(CH3)3 ., CI MS (M+H) 686 Using subst ntially:the same procedure, the following 5 :~ ~ compound can;be from compound 7j, of step G: . ;

NH2~ CH300C \~ /
(CH~ ot ~

CH3502NHJ~ --`N~ ~OH

~OH 7k FAB MS (M+H) 588 ~.

SUBSTITUTE SHEET ``

WO 94/03481 PCl'/lJS93/07137 ~ , , ~4~ l5 Step G:Cool a solution of 0.2 9 of the product of step F in 2 mL of ~;~
CH2CI2 to 0C, add 2 mL of TFA and stir for 1/2 h. Warm the mixture to room temperature and stir overnight. Concentrate to a residue and wash the residue with several portions of diethyl ether. Evaporate the :.
5 residual ether under vacuum to give the title compound, Cl MS (M+H) 574.
Using substantially the same procedure, the following compounds can be prepared: ~:
from compound 7-1:), of step D: ;

CbzNH~ CH3 OOC \/ `
H~ ~ H~l~

OH 7j : FAB MS (M+H) 722 Using substantially the same procedure as described in steps E, F and G, compound 7a-D, of ster D, can be converted to: ~.
H2N~ HOOC
(CH~)4 ~ ~ C~H3 H FAB MS
Cl-13S:O2NH~N ~ N~N~Jl~oH ~M+Na) 568, o H o ~ (M+H) 546 7a.

,~, ~ . .

- ::

:
SUBSTI~UTE SI~EET `

. l/U~i~3/()7137 : ~:
7~5 70~

EXAMP~E 8 H2N~ HOOC ~, ~H~H~

O ~

~OH ~ ~

S ~SteD A A solution Qf 0.5 g of compound 7b-D, from Exarnple 7, -st p D, in 5 mL of CH2CI2 is cooled to 0C. Add 5 mL of TFA and stir at ~:
0~ for 1/2 h., then at roem ~emperature overnight. Concentrate to a residue and wash the residue with several portions of ether. Remove he~residual ether under vacuum to give Zhe product:

bzNH~ ~ CH3 00 CH,SqNH~ ~l~N~J~

~ OH

Hydrolyze the product of step A according to the procedure ~ `dèscribed: in Example 7, step E to give the compound ` -. .

2)4 H~, O
CH3SO2NH J~-- HN , OH
, ~

OH
: FAB MS (M+H) 734 `
:.`
~ ~ SUBSTITUTE SHEET

WO 94/03481 PCl~/US93/07137 ;............................................................................... .
- - ;~ 14~7~5 Ste~ C: Hydrogenate the product of step B according to.the procedure described in Example 7, step F to give the title compound, FAB MS (M+1) 600.
llle following compounds can be prepared by substantially the same procedures as in steps A, B and C: ~;
from compound 7c-D, of Example 7, step D: ~:
, ~;
.
H2N~ HOOc 8a FAB MS
H o -~ (M+H) 586 ; , : '```'.
from~ compound ~7d-D, of: Example 7, step D: .-H~,~ <~> H 8b CH~502NH~ N~N~Jl~oH FAB MS
H o ~ (M+Na) s94 ., from compound 7e-D, of Example 7, step D:

H2N~ HOOC 8C ;
H~ ~7 H
CH3SO2NH ~ N~N~J~oH FAB MS
H o _ (M+H) 558, (M+Na) 580 OH `;` `

: 1 0 , ~ .

~ : SUBSTITUTE SHEET
:

WO 94~03481 PCI`/US93/07137 ' `
7~5 from compound 7f-D~ of Example 7, step D:
~ isomer A
H2N~ HOOC ~7 8d (C~2)4 H ¦ '~ H ~:
CH3SO~NH~N~ N~N~II~oH FAB MS
H O - (M+H) 572, :
(M+Na) 594 ~OH
; ' from cornpound 7g-D, of Example 7, step D: `. .
isomer B
H2N~ HOOC
(CW2)4 ~ Y H 8e C;H3SO2NH~N~N~ J~OH FAB MS
H O ~ (M+H) 572, ; ~-(M+Na) 594 from ~compound 7h-D, of Example 7, step D: ~:

~N~ HOOC \ : ~ 8f ` `

C:H35~NH~ ~1N~OH FAB MS
0 ~ : H O~ M+H) 597 N

.
h ~ ~ : 5 Using the methods described above, the following activity data were obtained ~for compounds of the formulae shown in the following iables. Changes in BP are expressed as mm Hg (dose) and : A1 :Challenge data are expressed as % inhibition (dose). ;
, :

, ~
~, - ~. ,.
: ~ - ~ :, -, - ~ ~ SUBSTI~UTE SHEET -q ~ .

` - .. ..
I ~ ~ o 1 ' 1~
~ ~D tD ~5) ~ ,.. ,:., ~io lo ; ~o ~ ~ 1 ~n _ . _ OC o o ô o O O ~: ~
~c ~ I 1~11--1 1 I~

=~ r Q ~ D D o~ D O D ~ ~
. u~ IZ O _ _ _ _ _ _ _ _ :::
= ~ a : ~ ~ ~ ~ o ~ o .
: ~ _ _ .
o ~ Z~ ~ ~ ~ : o~ o C~ _ ~
~ ~
aC ) c _ I_ ! ~ . . , ~ I O Z ~ t~ 0 o o o ~I z I c I z I ~ I z ~ ~ I ~ I ~ . ~
_ ~ . ._ O O _ -~
1~1 G O O I T O

. i, , I` ~ j ; . V O !
¦ ~ ¦ ¦ T I I ¦ O

S~;8STITUTE S~EET
., "

~VO ~4/03481 PCI'/ US93/07 137 . ~` `. :, 7~

-74- :

loô lo lo ~TTTl -~- ~ ~ -_ ' :
_ . __ . .... . _ _ . _ .'`'~:' ~ ~) C~ ~ `'``~' -- O O O l ~ '' ~
0~ 10 O . ~
... _ . . . '`,'' c~ : c~ C) Q c~ c~ : ~ O '.i;
0 : O _ O O O _ :~ O) _ ~C ~q - ~q O) ~q ~D :~

U~ C~ O ~ ô ~ O Q O O O `~.j : : ~ 0 ~ - - ~ 0 O O ;,:, o~ ~ o~ ~0~ ~ ~ --~ _ : "~.

,..~
': _ _ o ~: __-- ''~

Z: ` ~ ~ ~ ~ ~ I ~ ~ ~ ~ I I

. O o~ ~ - C) ---- ~;
~T: ~ ~N ~o ~ oO~ O T ~;
~' ~! ~ ~

; ¦~ oN ¦ N ¦ N ~ ¦ ¦ N N

I I I I T

SUBSTITUTE SHEET

WO 94/03481 PCrtUS93/07137 ;~417~5 .: ., . -,. `,-~= C~l _ `~

O G O : ~
_ _ O : ~

: ` ~ ~ : . , .
: ~:
~: ~
~:: ~ , _ T u~ I
~ I ~Z ~` ,~
Z ~ ~
, : ~

I I O
O, .~ . ~ ' ~
~ , O . ' ' ;.'~.:
= ~ I 6 ~ ~: . : T N Z
O O O `
u~ (n u~ ,.
~: ~ I I

SU8STITUTE SHEET ; -"`~

WO ~4~034~1 P~/US93~07t3?
17~5 . .;

IN ~) O O . O O O :`~
O C~ O N O ¦ -_ . . _ ., ~., o ~ l lo ~ o 1~ 1 c _ _ _ `,~

:Z O : O C~ . ~
=O ~ ~

~¦ I ¦ Z ¦ I I N ~;
~1 1 1 IT II I
I N I O O
~o L~ ~ ~ .
D I I I I
c~ ~ ~ o o o o _ 1~ ~ ~L ~ ~
~, _ 1~ = ~ ~ ., ---- ¦ T S ~ I II
~ ~ I Z N NZ N
~ O ~n O u~ ~n I I I I I

SUBSTIl-UTE SHE~T :`

WO 94/03481 PCr/US93/07137 - -`; t ~ 1417~S5 _ _ ~, C~OOO 10 1ô :~
C~ T O o ~, o o : cq c~) ~ `

1~11 ô l ô O ¦ ~, O ¦ O
=~ g~
~ I~IIN 1~ IN ¦A

Z I
~ ~ e~ 1 ,~ ~ . ~: ~c, ~ l~. la. ~

T T T ~

SUBSTITUTESHEET

W O 94/03481 t PC~r/US93/07137 3~
Z~4i7~5 -78- ~

;~;, '., ~ ~ O Q O ~) o c~ ~ O _ ~ I~ C~. ~ O
_ ~ ~ _ O O _ ~ O _--. O o O O O .

~ _ _ _ ~ _ _ '~"
O OO Q Q OQ Q Q OQ -O O . O _ O O O O O .
_ _ . ~ _ _ ~ _ _ _ . ` '."
a) CU N O O U~ C~l ~
~ c~l . c~l . . c~ ~.
-- _ `
Cq 1.7 ~o ~ ~ ~ u~
¦A ~ ~ IA
; N N I ~ ~ ~ . ~ ~ I I N
r~ ~ ~ ~T~ ~ C~ I I I I I C~ ~ ~ I

~ _ __ ¦T~ ~ 1 ¦~T~ r lo lo ~ ¦o ¦ ¦o 1~ ~ ~ ~ ~ 1 ~ -------- --r r r j I I I~!, I ~ ~! . ~ ~ ;

1~ ~ N 1' ~
I II I I I ~C I I I I
Z Z Z Z Z: Z Z Z Z Z Z
~: : ~: - : ~ N C~l C~ C~l C~J C`J C~l C~J C~l ' ~ ' O ' O O O O O O O O O O ''' :. ~ ~ u~ ~ncJ)CJ3 tJ~) (I)cl~ ct~ cl~ cl~ :~
$ : T T T T T T T T T T
. .:
- .;
SUBSTITUTE SHEET

~, W O 94/03481 P ~ /US93/07137 ~

., .. j ., :

-79~
~r ..~
~ ~ . O

~;~ ~ ~ ~ ~
.

~; ..
I I : I

~ ~ ~ ' ~ I 'C ~ m ! ~ :

: ~ ~ ~. Kl i~

T T T . ~
~: ~ ~ ~n u~
T T T : `
, ~
: . : :.
SUBSTITUTE SH EET ~

WO 94/03481 PCI/US93/û7137 r,~l41q~5 -80- ~:

~.."rR~ ~

I Z~ ~ ~ ~ : :
_ Z
~: : ~Z~

, o: la.

:~ ~ SUBSTITUTE SHEET

Claims

We Claim:

1. A compound of the formula wherein Z is amino, lower alkylamino, di-(lower alkyl)amino R9C(O)NH- or a guanidino group of the formula R11R12NC(=NR13)N(R14)- or R11R12NC(NR13R14)=N-, wherein R11, R12, R13 and R14 are independently hydrogen or lower alkyl, or wherein R11 and R12, or R13 and R14, taken together with the nitrogen atom to which they are attached, comprise a 5- or 6-membered ring;
R1 is a group of the formula R7R8N-;
R2 is hydrogen, lower alkyl, cyclolower alkyl, aryllower alkyl or heteroaryllower alkyl; and R3 is hydrogen, lower alkyl or cyclolower alkyl; or R2 and R3, together with the carbon to which they are attached, comprise a 3-7 membered carbocyclic ring;
R4 is hydrogen, lower alkyl, aryl lower alkyl or heteroaryllower alkyl;
R5 and R6 are independently selected from the group consisting of hydroxy, lower alkoxy, amino, aryllower alkoxy, lower alkylamlno and di-(lower alkyl)amino;
R7 is R9C(O)- or R10SO2-; and R8 is hydrogen, lower alkyl, aryllower alkyl or aryl; or R7 and R8, together with the nitrogen to which they are attached, comprise a 5-7 membered ring;
R9 is lower alkyl, aryllower alkyl, aryl, heteroaryllower alkyl, heteroaryl, lower alkoxy, aryllower alkoxy, amino, alkylamino or dialkylamino;
R10 is lower alkyl, aryl lower alkyl, aryl, heteroaryl lower alkyl, amino, lower alkylamino, di-(lower alkyl)amino or heteroaryl;

n is 1,2,3,4 or 5;
m is 1,2,3,4 or 5;
or a pharmaceutically acceptable addition salt thereof.

2. A compound of claim 1 wherein n is 4, m is 1, and R5 and R6 are independently selected from the group consisting of hydroxy, lower alkoxy or aryllower alkoxy.

3. A compound of claim 2 wherein R4 is aryllower alkyl.

4. A compound of claim 3 wherein R4 is 4-hydroxyphenyl-methyl or 3-methanesulfonylamidophenylmethyl.

5. A compound of claim 3 wherein: Z is selected from the group consisting of amino, R9C(O)NH- and R11R12NC(=NR13)N(R14)-, wherein R9 is aryllower alkoxy, R11 and R13 are alkyl, and R12 and R14 are hydrogen; and R1 is lower alkyl-SO2-NH-.

6 . A compound of claim 5 wherein Z is amino, benzyloxycarbonylamino or C2H5NC(=NC2H5)NH-.

7. A compound of claim 5 wherein R3 is hydrogen and R2 is aryllower alkyl, cyclolower alkyl or lower alkyl.

8. A compound of claim 7 wherein R2 is methyl, iso-propyl, n-propyl, benzyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

9. A compound of claim 5 wherein R2 and R3, together with the carbon to which they are attached, comprise a 3-6 membered carbocyclic ring, 10. A compound of claim 1 having the structural formula wherein R1 to R6 and Z are as follows:

11. A pharmaceutical composition useful for treating hypertension, congestive heart failure or renal insufficiency comprising an effective amount of a compound of claim 1 in a pharmaceutically acceptable carrier.

12. A method of treating hypertension, congestive heart failure or renal insufficiency comprising administering to a mammal in need of such treatment a pharmaceutical composition of claim 11.

13. A method for preparing a pharmaceutical composition comprising admixing a compound of claim 1 with a pharmaceutically acceptable carrier.

15. The use of a compound of claim 1 for the manufacture of a medicament for treating hypertension, congestive heart failure or renal insufficiency.

16. A process for the preparation of a compound of the formula wherein R1, R2, R3, R4, R5, R6, Z, m and n are as defined in claim 1, selected from the following processes A, B, C or D:
(A) coupling an amine of the formula wherein R2, R3, R4, R5, R6 and m are as defined in claim 1, with a carboxylic acid of the formula wherein R1, Z and n are as defined in claim 1, to form a compound of claim 1;
(B) hydrogenating a compound of the formula I, wherein Z is Cbz-NH-, and R1, R2, R3, R4, R5, R6, m and n are as defined in claim 1, by treating with H2 in the presence of a Pd catalyst to form a compound of claim 1, wherein Z is NH2;
(C) hydrolyzing a compound of formula I, wherein: R6 is lower alkoxy or aryllower alkoxy; R5 is hydroxy; and R1, R2, R3, R4, Z, m and n are as defined in claim 1, by treating with NaOH to form a compound of claim 1, wherein R5 and R6 are both hydroxy;
(D) reacting a compound of formula I, wherein Z is NH2 and R1, R2, R3, R4, R5, R6, m and n are as defined in claim 1, with an N,N-dialkylcarbodiimide, diisopropylethylamine and p-TSA, to form a compound of claim 1 wherein Z is a guanidino group as defined in claim 1;
wherein each process is followed by isolation of the preferred isomer, if desired, and removal of the protecting groups, if necessary, to yield the desired product and if desired, preparation of a salt thereof.