Note: Descriptions are shown in the official language in which they were submitted.
\~'0 92/1719~ Pcr/uss~/026~-
'` 2107~8~
ANTIJHROMBOTIC PEPTIDES AND PSEUDOPEPTIDES
BACKGROUND OF INVENTION
10 1. Field of the Invention
This invention relates to compounds having anti-thrombotic activity. More
par~icularly, the invantion relates to novel peptides and pseudopeptides that
inhibit platelet aggregation and thrombus formation in mammalian blood and
15 are use~ul in the pr~vention and treatment of thrombosis associated with
disease states such as myocardial infarction, stroke, peripheral arterial disease
and disseminated intravascuiar coagulation.
Haemostasis, ~he biochemis~ry of blood coagula~ion, is an extremely
20 complex and as y~t not complately undarstood phenomena wh~r~by normal
whole blood and body tissuc spontansously arrest bleeding ~rom injured blood
vessels. Effective haemsstasis requir~s the combin~d activity of vascular,
platelet and plasma factors as wcll as a controlling mechanism to prevent
~xcessive clotting. Defects, deficiencies, or excesses of any of these
25 components can lead to hemorrhagic or thrombotic consequences.
Platelet adhesion, spreading and aggregation on extracellular rnatrices
are central events in thrombus form~tion. These ev~nts are mediated by a
family of platelet adhesiv~ glycoproteins, i.e., fibrinogen, fibronec~in, and von
30 Willebrand ~actor. Fibrinogen is a co-factor for platelet a~gregation, while
~fibronectin supports pla~elet attachments and spreading reac~ions, and von
Willebrand factor is important in platelst attachment to and spr~ading on
subendothelial matrices. The binding sites for fibrinogen, fibronectin and von
Willebrand factor have been located on th~ piatelet membran~ pro~ein complex
35 known as glycoprotein llb/illa. - -
:: :
~'0 92/1719~ PCr/US92/026?i-
Adhesive glycoproteins, like fibrinogen, do not bind with normal resting
platelets. However, when a platelet is activated with an agonist such as
thrombin or adenosine diphosphate, the platelet changes its shape, perhaps
making the GPllb/llla binding sit~ aoc~ssible to ~ibrinog0n. Compounds wi~hin
the scope of th0 present invention block the fibrinogen receptor, thus inhibiting
platelet aggregation and subsequent thrombus formaRon and when
administered in the form of pharmaceutical compositions comprising such
compounds are useful for the prevention and treatment of thrombogenic
diseases, such as myocardial infarçtion, stroke, peripheral arterial diseass anddisseminated intravascular coagulation.
2. Reported Developments
It has been observed that the presence of Arg-Gly-Asp ~RGD) is
necessary in fibrinogen, fibronectin and von Willebrand factor for their
interaction with the cell sur~ace receptor (Ruoslahti E., Pierschbacher, Cell
1986, 44, 517-18). Two other amino acid sequences also seem to take parl in
the platelet attachment function of fibrinogen, namely, ~he Gly-Pro-Arg
sequence, and the dodecapeptide, His-His-L~u-Gly-Gly-Ala-Lys-Gln-Ala-Gly-
Asp-Val sequence. Small synthetic pcptides containing th~ RGD or
dodecapeptide have been shown to bind to the plateiet GPllb/llla receptor and : -
competitivsly inhibit binding of fibrinogsn, fibronectin and von Willebrand factor
as well as inhibit aggrsgation of ac~ivated platelets (Plow, et al., Proc. Natl.Acad. Sci. USA 1985, 82, 8057-61; Ruggeri, et al., Proc. Natl. Acad. Sci. USA
2~ 1986, 5708-12; Ginsberg, et al., .,t. Biol. Chem. 1985, 260, 3931-36; and
Gartner, et al~, J Biol. Chem. 1987, 260, 11,891-94).
Indoiyl compounds containing guanidinoalkanoyl- and
guandinoalkenoyl- aspartyl moi~ti~s are reported to be platelet-aggregation
inhibitors by Tjoeng, et al., U.S. Patent Nos. 5,037,808 and 4,879,313. ~ . `
.
U.S. Patent No. 4,992,463 (Tjoeng, et al.), issued February 12,1991,
discioses generically that a series of aryl and aralkyl guanidinoalkyl peptide :
mim~tic cornpounds exhibit platelet aggregation inhibiting activity and
discloses specifically a series of mono- and dimethoxy phenyl peptide mimetic
compounds and a biphenylalkyi peptide mimetic compound.
:
: .
'
,
WO 92/1719fi P
2 1 0 ~ O ~ ~ ~T/US92/026?/1
,i ;` 3
U.S. Patent No. 4,E~57,508 (Adams, et al.), issued August 15, 198~,
discloses generically that a series of guandinoalkyl peptide darivatives
containing terrninal aralkyl substituents exhibit platelet aygregation inhibiting
activity and discloses specifically a series of O-m~thyl tyrosine, biphenyl, and5 naphthyl derivatives containing a ~erminal amide functionality.
Haverstick, D.M., et al., in Blood 66 (4), 946-952 (1985), disclose that a
number of synthetic peptides, including arg-gly-asp-ser and gly-arg-gly-asp-
ser, are capable of inhibiting thrombin-induced plat~let aggregation.
1 0
Plow, E.F., et al., in Proc. N~tl. Acad. Sci. USA ~9, 3711-3715 ~19~2),
disclose that the te~rapeptide glyoyl-L-prolyl-L-arginyl-L-proline inhibits
fibrinogen bindin~ to human platel~ts.
French Application No. 86/17507, filed December 15, 1986, discloses
that tetra-, penta- and hexapaptide derivatives containing the -arg-gly-asp-
sequence are useful as antithrombotics. ~ ~
U.S. Patent No. 4,683,291 (Zimmorman, et al.), issued July 2~, 1987, ; ~ ~ -
discloses that a series of peptides, cornprised of from six to forty amino aeids,
which contain the sequenee -arg-gly-asp- are platelet binding inhibitors. - -
European Application Publioation No. 0 319 ~06, published June 7,
1989, discloses that a s0ries of tetra-, penta-, and hexapeptide derivatives
25 containing the -arg-gly-asp- s~quence are platel~t aggregation inhibitors.
Cyclic peptide analogues containing the moiety Gly-Asp are reported to
be fibr~nogen receptor antagonists in U.S. Patent No. 5,023,233.
Peptides and pseudopeptidos con~aining amino-, guanidino-, imidizaloyl,
and/or amidino- alkanoyl, and alkenoyl moieties are reported to be
antithrombotic agents in pending United States applications serial nos.
~7/677,006, 07/534,385, and 07/460,777 filed on March 28, 1991, June 7,
lg90, and January 4, 1990, respectively, as well as in U.S. Patent No.
4,952,562, and in Int~rnational Application No. PCT/US90/05448, filed
September 25, 1~90, all assi~ned to the same assignee as the present
invention.
.
`,:
,:
WO 92/1719~. P~/US92/026~,
4 (~i .
Peptides and pseuctopeptides containing amino- and guanidino- alkyl-
and alkenyl- benzoyl, phenylalkanoyl, and phenylalkenoyl moieties are
r0ported to be antithrombotic agents in pending United States application
5 serial no. 07/475,043, filed February 5, 1990, assigned to the same assignee
as the present invention.
The present invention relates to novel peptides and pseudopeptides
which inhibit platelet aggregation and subsequent thrombus formation.
1 0
SUMMARY OFTHE INVENTtON :
Compounds of the present invention are described by Forrnula I :
l 1 l3
A~CH23~ 1 ~B~ I 3~CH23~D--CH ~Z
R2 R4 ( I 2) n :-:
COOH ~ :
. ~ .
Formula I
wherein:
/~N :
~NH~ I--(NH)--R I ~O)~Rs
A Is cyano, R5, ~ x x~ or 6 ; . . .
B and D are indep~ndently -CH2-NH-, -CH2-S-. -CH2-O-, :
--C--NRg~ H3- IC C I qH-CH2~ CH-CH2-O-- ~:
: ~ x"~ Rl, R7 ~ Y1 R7 or Y~
--CH--CH
\ ~ . . .
B may also be 5-tetrazol-1-yl, -CR7=GR8-, -CC- or (CH2)q
:~ Z is -ORa, nitrogen-c~ntaining heterocyclyl, a D- or L-isomer of an a-
amino aoid bonded at the o~-nitrogen, a dipeptide bonded at the N-terminal oc- .
WO 92/1719fi 2 ~ ~ 7 Q 8 ~ PCl/VS92/026~,
j:` 5
t t~
o Rl
amino acid, or -NRaRX, where Rx is H or ` ~ x ~ , where V is
o o
8 NRg--,--8-cH2--, -(CH2)p-, -CH-CH-, -CH2NH-, -CH2-O-, or
-CH2-S-;
Re and Rf are independently H, alkyl, cycloall<yl, cycloalkylmethyl, or
-(CH2)s-Rz where Rz is nitrogen~containing heterocyclylcarbsnyl, -COORn,
NH NH
Il 11
~ORn, ~SRn-,-NRnRo.--NH-C NH2,--C--N~2, phenyl, substituted phenyl,
naphth-1-yl, naphth-2-yl, substituted napth-1-yl, substituted naphth-2-yl, 1,1-
diphenylmethyl, 1,1-di(substituted phenyl)methyl, N-P~n substituted indol-2-yl,
10 N-Rn substituted indol-3-yl, substituted ~N-Rn substituted) indol-2-yl,
substituted ~N-Rn substituted) indol-3-yl, quinolin-2-yl, quinolin-3-yl, quinolin-4-
yl, substituted quinolin-2-yl, substituted quinolin-3-yl, substituted quinolin-4-yl,
N-Rn substituted imidazol-2-yl, N-Rn swbstituted imidazoi-4-yl,N-Rn substituted
imidazol-5-yl, substitutecl N-Rn substituted imidazol-2-yl, substituted N Rn
15 substituted imida~ol-4-yl, substituted N-Rn substitutAd imidazol-5-yl, imidizol-1-
yl, or substituted irnidæol-1-yl,
R1-10. Ra, Rg, Rk, Rm.p, Rq, and Rs are independently H, alkyl, cycloalkyl,
cycloalkylmethyl, a~l, substituted aryl, aralkyl or substituted aralkyl;
Rt is -H, -COQH, -COORk, carbamoyl, N-containing heterocyclyl or
o .,
13
--C ~NRhRm;
: ~
:~: Rn is R10 orY1;
o . ,.
Y i H amino or--NH-8--Rp it being understood that Y1 may be the
same or different ~or B and D;
X~ X~l X~ xn~ and x"~ are indeperldently 0 or 1; m1 and m~ are ; ~
independently 0 to 9; h1, h2, and k are independently 0 or 1; n is 1 to 3; q is 1 ~ :
30 to 5; and p and s are ~independen~ly 0 to 6; : ~
WO ~2/1719~. PCr/llJS92/02~s7
6 ~
provided that when A is guanidino, and B and D are -C(O)NH-, then Z is
other than aralkylamino or substituted aralkylamino; and
when A is guanidino, B and D are -C(O)NH-, and Z is -NRaRX where Rx
C--R,
j5 R~ and Rt is -C(O)NHz and P~e is hydrogen, then Rf is other than ~ :
benzyl, substituted benzyl, naphthylmethyl or substitut~d napthylmethyl; and ~ :
Rl R3 .. ~. .
At~H23;;;~ 1 ~B~ ¦ ~CH23;;~D ~ CH
R2 R,, ( I 2) n
when COOH isarginyl-
glycyl-aspartyl, then Z is other than a naturally occurring amino acid or a
dipeptide composed of two naturally occurring amino acids; .;; - .
and pharmaceuticaliy acceptable salts thereof.
Additionally, this invention rslates to pharmaceutical compositions . .
comprising such compounds, and to methods of treatment of abnormal :
thrombus formation in mammals comprisinç~ the administration of such
compounds and pharmaceutical compositions
DETAILED DESCRIPTION OF THE INVENTION .:
As used above, and throughout the description of this invention, the
following terms, unless otherwise indicated, shail be understood to have the . .:
~ol!owing meanings:
. :
"Nitrogen-containing heterocyclyl" means about a 4- to about a 15-
rnemberad nitrogen-containin~ monocyclic or multicyclic ring system in which . :.
one or more of the other atoms in the ring or rings may be an elament other
than carbon, for axampie nitrogen, oxygen or suHur and further that the
het~rocycie is bound~at the nit.ogen atom. Preferred nitrogen-containing ;~
heterocyclyl groups include pyrrolidin-1-yl, piperidin-1-yl, homopiperidin-1-yl,.::;
. .
. .
: :,'- . ~'
WO 92/17196 PCI/US92/0~6~,
f` 2~0708~ 7
morpholin-4-yl, 1,2,3,4-~etrahydroisoquinolin-2-yl, piperazin-1-yl. In the case of
piperazin-1-yl, the nitrogen at the 4-position preferably may be substituted by
alkyl, cycloalkyl, cycloalkylrnethyl, aryl, substituted aryl, aralkyl or substituted
arakyl.
"a-amino acid" rneans a synthetic or naturally occurring amino acid.
Preferred c~-amino acids are the naturally occurring amino acids, i.e, glycine,
alanine, valine, leucine, isoleucine, s~rine, threonine, phenylalanine, tyrosine,
tryptophan, cysteine, methionine, proline, hydroxyproline, aspartic acid,
aspargine, glutamine, glutamic acid, histidine, arginine, ornithine, and Iysine.
"Dipeptide" means a-aminoacyl-a-arninoacid.
"Carboxy" means a -COOH group.
1i5 o
Il . . .
nCarbamoyln means a--C--NH2 group.
"Alkyl" means a saturated aliphatic hydrocarbon group which may be
straight or branched and having about 1 to about 20 carbon atoms in the chain.
20 Branched means that a lower alkyl group such as methyl, ethyl or propyl is
attached to a iinear alkyl chain. Prafcrred straight or branohed alkyl groups are
the "lower alkyl" groups which are those alkyl groups having from 1 to about 6
carbons.
.
"Cycloalkyl" maans a saturated carbocyclic group having about 3 to
about 8 carbon atoms. Preferred cycloalkyl groups include cyclopropyl,
cyclobutyl, cyclopent~l, and cycloh~xyl.
: : . NH ~::
Il
"Guanidino" means an--NH-C NH2 ~roup -
"Aryl" means a phenyl or naphthyl group.
.-
"Substituted aryl" means a phenyl or naph~hyl group substituted by one
or more aryl group substituents which may be ~he same or different, where "aryl
35 group substituen~" includes alkyl, alkenyl, alkynyl, aryl, aralkyi, hydroxy, alkoxy,
' ' ,'
~'O 92/ 1 7 ] 9() PCl`tUS92/026
aryloxy, aralkoxy, hydroxyalkyl, acyl, formyl, carboxy, alkenoyl, aroyl, halo,
nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxyoarbonyl, aralkoxycarbonyl,
acylamino, aroylamino, carbarnoyl, alkylcarbamoyl, dialkylcarbamoyl,
arylcarbamoyl, aralkylcarbamoyl, alkylsulfonyi, alkylsulfinyl, arylsulfonyl,
5 arylsulfinyl, aralkylsulfonyl, aralkylsulfinyl, or-NRR' where R and R' are
independently hydrogen, alky~, aryl, or aralkyl.
"Substitutedn- phenyl, naphth-1-yl, naphth-2-yl 1,1-diphenylmethyl, 1,1-
di(substituted phenyl)methyl, indol-2-yl, indol-3-yl, quinolin-2-yl, quinolin-3-yl,
10 quinolin-4-yl, imida,701-2-yl, imidazol-4-yl, in idazol-5-yl, and imidazol-1-yl
means that these groups are substituted by an ~ryl group substituent.
Preferred aryl group substitutents for these groups are hydrogen, halo, nitro,
trihalomethyl, phenyl, alkyl, nitrogen-containing heterocyclyl carbonyl,
nitrogen-containing heterocyclyl carbonylalkyl, amidino, guanidino, ~NRqRs~
1l ~':
o - C--N--Rq
15 ~SRq~ ~COORq,~NHSO2Rq~ NH-c--Rq,or Rs where Rqand Rs
are independently H, alkyl, cycloalkyl, cycloalkylrnethyl, aryl, substituted aryl,
aralkyl or substitut~d aralkyl.
... . .
"Aralkyl" means an alkyl group substituted by an aryl radical. Preferred
20 aralkyl gr~ups includa benzyl and phenethyl.
:~',~-''
"Substituted aralkyl" means an aralkyl group substituted on the aryl
portion by one or more aryl group substituents.
.: .
A preferred class of compountls of the present invention is described by
Formula I wherein:
B and D are independently -CH2-NH-, -CH2-S-, -CH2-O-,
0 19
8--NRg~H~ Cl C~ H_CH2_N-- ~--CH-CH2-O~ :
x"" Rt1 R7 , Y, R7 or Yl ;
CH--/CH~
B may also be -CR7=CR8-, -CC- or (CH2)q
~ ,
.:'
WO 92/1719f PCl/US92/026~-
~-` 21~7(3~8 9
and Rz is nitrogen-containing heterocyclylcarbonyl, -COORn, ~ORn~ -SRn-,
NH NH
Il 11 .
-NRnRo~--NH-C--NH2, or--C~NHz
Another preferred class of cornpounds of the present invention is
described by Formula I wherein:
B and D are independently -CH2-NH-, -CH2-S-, -CH2-O-,
0 19 0
--8--NR~H~- I C I I H-CH2- 1-- CH -CH2-O
x"~ R,l R~ ~ Y1 R7 or Y-
''
--CH--~CH - :
B may also be -CR7=CR8-, -CC- or (CH2)q ; ~;:
and Rz is nitrogen-containing heterocyclylcarbonyl, -COORn, -ORn, -
SRn-, -NRnRo, NH-C NH2, or C NH2; and
:
Z is -ORa, pyrrolidin-1-yl, piperidin-1-yl, homopiperidin-1-yl, morpholin-4-
yl, piperazin-1-yl, a D- or L-isomer of an a-amino acid bonded at the a-
nitrogen, a dipeptide bonded at ~he N-t~rminal oc-amino acid, or -NRaRX wh~re
l e , ~.
- tl J I ;
RxisHor x~ ,
~o .~ ;. .Another preferred class of compounds of ~he present invention is
described by Formula ll - -
NH O O O
NH2--C--NH ~CHz~;;;;C NH ~CH2--C--NH-CH-C ~Z
1 2
COOH ..
~5 Formula ll .
:
~ ~ .
~'0 92tl 7196 PCT/I~S92/026~
~,3 1 o ~ ;
wherein:
m1 is2to9;and
Z is phenethylamino or 1,2,3,4-t~trahydroisoquinolin-2-yl.
Still another preferred class of compounds of the present invsntion is
described by Formula I wherein:
, ' ' '''''
B is 5-tetrazolyl-1-yl; and
R;~ is
~W~
15 -@W"~W2 ,~ 6~W" ~
~\~W1 /---, ~W1
N~ ~ ~ --N~N
Nl l .:
Rn , R ` or W1
wher~in: W1 and W2 are independently hydrogsn, halo, nitro,
20 trihalomethyl, phenyl, alkyl, nitrogen-containing he~erocyciyl carbonyl,
nitrogen-containing heterocyclyl carbonylalkyl, amidino, guanidino, ~NRqRS,
Il . ::".'
O --C--N--R~
~SRq, ~COORq,-NHSO Rq,--NH-C--Rq,~r Rs ; and
Rn, P~q and Rs ar~ independently H, alkyl, cycloalkyl, :
25~ cycloalkylmethyl, aryl, substituted aryl, aralkyl sr substituted aralkyl.
.
~ ~ .
WO 92/1719~ 2 1 ~ 7 0 8 8 PCT/US92/026~-
i ';-:,'~ 1 1
A more preferred class of cornpounds of the present invention is
described by Formula lll
A~CH2);~B~Hz~C-NH--CH ~;Z
CH2 . ' .
COOH
Formula lli
wherein: .
/~ N ~:
-N~ J ~: ~
A is guanidino or R5; . .
1 0
mlis1to9; ~
- m2isOor1;and ~ '.
O ~
Il
--C--NRg - ,H~
B is -CH-CH- or I x~
A still more preferred class o~ compounds of the present invention is . .:
described by the more preferred class of cornpounds wherein A is guanidino. ..
ZO
A most pr~ferred class of compounds of the present invention is
described by the still more preferred class of compounds wherein Z is a D- or .1 ~
L-isomer of an a-amino acid bonded at the -nitrogen, or Z is a dipeptide : -
bonded at the N-terminal a-amino acid.
2~
Another mo~st preferred class of compounds of the preser!t invention is
described by the still more preferred class of compounds wherein Z is a D- or : :~:
L-isomer of an -amino acid bonded at the a-nitrogen.
. :
\~'0 92/1719fi PC~T/US92/()26?i/
.
A particularly preferred special embodiment of the present invention is
described by the still more preferred class of compounds wherein Z is a D- or
L-isomer of an a-amino acid bonded at the a-nitrogen, said o~-amino acid
being selected from the group consisting of glycine, alanine, valine, leucine,
isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, cysteine,
methionine, proline, hydroxyproline, aspartic acid, aspargine, glutamine,
glutamic acid, histidine, arginine, ornithine, and Iysine.
:
A most preferred special embodiment of lhe present invention is
described by the still more preferred class of compounds wherein Z is a D- or
L-isomer of an o~-amino acid bonded at the a-nitrogen, said a-amino acid
being selected from the group consisting of valine, leucine, isoleucine, and
arginine. .
Representative compounds of the present invention include:
5-guanidinopentanoyl-N-~ethyl)-glycyl-L-aspartyl-L-leucine;
.. . j
6-guanidinohexanoyl-N-~ethyl)-glycyl-L-aspartyl-L-leucine; . .
;
6-guanidinohexanoyl-N-(ethyl)-glycyl-L-aspartyl-L-isoleucine; . :
. .
6-guanidinohexanoyl-sarcosyl-L-aspartyl-L-leucine;
6-guanidinohexanoyl-N-(ethyl)-glycyl-L-aspartyl-L-valine;
6-guanidinohexanoyl-sarcosyl-L-aspartyl-L-valine;
~-guanidinovaleroyl-sarcosyl-L-aspartyl-L-Yaline;
~-guanidinopentanoyl-N-(ethyl3-glycyl-L-aspartyl-L-arginine;
8-guanidinooct-2-enoyl-L-aspartyl-L-valine;
9-guanidinononanoyl-L-aspartyl-L-isoleucine-4-guanidinobutyl amide;
9-guanidinononanoyl-L-aspartyl-L-leucine;
.
'~
WO 92/1719~ 2 1 ~ ~ ~ 8 8 PCr/US92/026:~,
", . .
13
9-guanidinononanoyl-L-aspartyl-L-arginine;
9-guanidinononanoyl-L-aspartyl-L-arginine-isobutyl ester;
9-guanidinononanoyl-L-aspartyl-L-leucyl-arginine;
9-guanidinononanoyl-L-aspartyl-L-valyl-arginine;
1 0 N-~N-(9-guanidinononanoyl-L-aspartyl)-2-aminobutanoyl]-L-arginine;
.
9-guanidinononanoyl-L-aspartyl-L-alanyl-arginine;
9-guanidinononanoyl-L-aspartyl-L-norleucyl-arginine;
9-guanidinononanoyl-L-aspartyl-D-homoisolsucyl-L-arginine;
9-guanidinononanoyl-L-aspartyl-L-phenylalanyl-L-arginine; or . - ~:
~:
2û N-(9-guanidinononanoyl-L-aspartyl)-3-amino-2-sec-butylpropionyl-L-
arginine of the ditrifluoroacetate salt th0reof; or
.
pharmaceutically acceptable salts thereof.
Compounds of the prasent invention contain asymmetric centers. These
asymmetric centsrs may indep~ndently be in either the R or S eonfiguration.
The present invention comprises the individual stereoisomers and mixtures
lhereof.
; ~ 30 The oompounds of the present invention may be useful in the form of the
free base or acid or in the form of a pharmaceutically acceptable salt thereof. `
All forms are within the scope of the invention.
Where the compound of the present invention is substitut~d with a basic
;: ~ 35 moiety, aoid addition salts may be formed and are simply a more convenient
form for use; and in practice, use of the salt form inherently amounts to use oft he ~free base forrn. The acids whioh can be used to prepare the acid addition
~ , :.
"
U'() 92/1719~ PCr/US92/026~,
1 4
salts include preferably those which produce, when combined with the free
base, pharmaceutically acceptable salts, that is, salts whose anions are non-
toxic to the animal organism in pharmaceutical doses of the salts, so that the
beneficial antithrombotic properties inharent in the free base are not vitiated by
5 side effects ascribable to the anions. Although pharmaceutically acceptable
salts of said basic compounds are preferred, all acid addition salts are useful
as sources of the free base form even if the particular salt, per se, is desiredonly as an intermedia~e product as, for cxample, when the salt is formed only
for purposes of purification, and identification, or when it is used as
10 intermediate in preparing a pharmaceutically acceptabls salt by ion exchange
procedures. Pharmaceutically acceptable salts within the scope of ~he
invention are those derived from the following acids: mineral acids such as
hydrochloric acid, sulfuric acid, phosphoric acid and sulfarnic acid; and organic
acids such as acetic acid, citric acid, lactic acid, tartaric acid, malonic acid,
15 methanesufonic acid, ethanesulfonic acid, benzenesulfonic acid, p-
toluenssulfonic acid, cyclohoxylsulfamic aeid, quinic acid, and the like. The
corresponding acid addition salts comprise the following: hydrochloride,
sulfate, phosphate, sulfarnate, acetate, citrate, lactate, tartarate, malonate,
methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,
20 cyclohexylsulfamate and quinate, respectively.
The acid addition salts of the compounds of this invention are prepared
either by dissolving the free base in aqueous or aqueous-alcohol solution or
other suitable solvents containing the appropriate acid and isolating the salt by
25 evaporating the solution, or by reactin~ the free base and acid in an organicsolvent, in which case the salt separates directly or can be obtained by
concentration ol the solution.
Where the compound of the invention is substituted with an acidic
30 moiety, base addition salts may be formed and are simply a more convenient
form for use; and in practice, use of the salt form inherently amounts to use ofthe free acid form. The bases which can be used to prepare the base addition
salts include preferably those which produce, whan combinecl with the free
acid, pharmaceutically acceptable salts, that is, sal~s whose cations are non-
35 toxic to the animal organism in pharmaceutical ~oses of the salts, so that the
beneficial antithrombotic properties inherent in the free acid are not vitiated by -~
side effects ascribable to the cations. Pharmaceutically acceptable salts within
:..' '
~'O 92/ ~ 7 1 96 Pcr/l~ls92/026~s
`. 21070~i~ 15
the scope of the invention are those derived from the following bases: sodium
hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide,
lithium hydroxide, magnesium hydroxide, zinc hydroxide, ammonia,
ethylenediamine, N-methyl-glucamine, Iysine, arginine, ornithine, choline,
5 N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-
benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-
aminomethane, tetramethylammonium hydrexide, and the like.
Metal salts of compounds of the present invention may be obtained by
10 contacting a hydroxide, carbonate or similar reactive compound of the chosen
metal in an aqueous solvent with the fr~e acid form of the compound. The
aqueous solvent employed may be water or it may be a mixture of water with
an organic solvent, preferably an alcohol such as methanol or ethanol, a
ketone such as acetone, an aliphatic ether such as tetrahydrofuran, or an ester
15 such as ethyl acetate. Such reactions are normally conducted at ambient
temperature but they may, if desired, be conducted with heating.
Amine salts of compounds of the present invention may be obtained by
contacting an amine in an aqueous solvent with the free acid form of the
20 compound. Suitable aqueous solvents include water and mixtures of water
with alcohols such as me~hanol or ethanol, ~thers such as tetrahydrofuran,
nitriles such as acetonitrile, or ketones such as ac~tone. Amino acid salts may
be similarly prepared.
. . .
Compounds o~ this invention may bs prepared in accordance with the
reaction sequonces described below, or c~n be pr0pared by methods known in
the art. The starting materials used in the preparation of compounds of this
invention are known or are commercially available, or can be prepared by
known methods or by specific reaction schernes described herein.
The cornpounds of the pres~nt invention may be readily prepared by
standard soiid phase or solution phase peptide synthesis using starting
materials and/or readiiy available intermediates from chemical supply
companies such as Aldrich or Sigrna, (H. Paulsen, G. ~Aerz, V. Weichart, "Solid-Phase Synthesis of O-Glycopeptide Sequencesn, Angew. Chem. Int. Ed. Engl.
27 (1988); H. Mergler, R. Tanner, J. Gosteii, and P. Grogg, "Peptide Synthesis
by a (:;ombination of Solid-Phase and Solution Methods l: A N~w Very Acid-
,
WO 92/1719fi PCr/~lS9?/026~.
Q~ 1 6
Labile Anchor &roup for the Solid-Phase Synthesis of Fully Protected
Fragments. Tetrahedron letters ~i, 4005 (1988); Merrifield, R.B., "Solid Phase
Peptide Synthesis after 25 Years: The Design and Synthesis of Antagonists of
Glucagon", Makromol. Chern. Macromol. Symp. 1~, 31 (-i988)).
A preferred method of preparing compounds of the present invention is
by the solid phase method schematically represented as tollows:
solid support--X1--N P Deprotect
P R
solid support ~X1--NH Coupling
....
solid support --X1 NH ~--X2--N--P
P R P R
1 0 '"
wherein: the solid suppor~ may be, but is not limited to, p-alkoxy benzyl resin, --I n~ I--P
and P R iS a protected amino acid.
In the synthetic process of making the desired compound the amino acid
15 derivatives are added cne at a time to the insoluble resin until the total
sequence has been built up on the resin. The functional groups of the amino ;
acid derivatives are protected by blocking groups to prevent cross reaction
during the coupling procedura. These blocking groups include N-a~tertiary
butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ), benzyi, t-butyl, 9-fluor-
20 enylmethyloxycarbonyl (FMOC), 2-(trimethylsilyl3ethyl, and 4-methoxy-2,3,6-
trim~thylbenzenesulfonyl. Upon completion of the coupling reaction a
functional group is deprotected by standard methods to give an active a-amino `
function which, in turn, is reacted with a protected amino acid derivative having
a free carboxyl function thereon. This procedure is repeated until the desired
25 peptide or pseudopeptide is formed. The compound is then deprotected and
WO 92/1719( PCr/US92/026?i-~
` :` 21070~7
removed from the solid support by standard procedures to obtaln the final
product.
In another preferred method, the compounds of the present invention may
5 be prepared in solution, i.e., without using a solid support. In a manner that is
similar to the solid phase synthesis the protected amino acid derivatives or
analogs are coupled by using standard procedures, then deprotected to yield
the desired final compound.
It may also be desirable or nacessary to prevent cross-reaction between
other chemically active substituents on reactants. The substituents may be
protected by standard blocking groups which may subsequently be removed or
retained, as required, by known methods to afford the desired prvducts or
intermediates (see, for example, Green, "Protective Groups in Organic
1 5 Synthesis", Wiley, N~w York, 1981). Selective protection or deprotection mayalso be necessary or desirable to allow conversion or removal of existing
substituents, or to allow subsequent reaction to afford the final desired product.
The invention is further explained by the following illustrative examples.
20 In the examples, when the carboxyl terminus of a co npound ends in an amino
acid other than valine, ~he synthetic process starts with the use of an
appropriate commercially available N-a-FMOC-arnino acid p-atkoxybenzyl
alcohol resin ester. When such is not available, the appropriate N-a-FMOC
protected amino acid p-alkoxybcnzyl alcohol resin ester is prepared by the
25 procedure of E. Givalt, ~ ~. (Int. J. Peptide Protein Res. 1989, 33, 368).
Subsequent treatment of the stàrting materials is described in Example 1.
EXAMPLE 1
;
L-Ar~invl-L-Aspa~l-L-valine
,~
1 g of N-(9-~luorenylmethyloxycarbonyl)-L-valine p-alkoxyben~yl alcohol
resin ester (containing 0.56 mmole of amino acid) is shaken with 20 ml of 20%
(v/v) piperidine in methylene chloride ~or 1 hQur to remove the FMOC group.
35 The mixture is filtered and the resin washed with methylene chloride. The
deprotected resin is treated with 0.929 of N-FMC)C-L-aspartic acicl-B-t-butyl
ester in 15 ml of dimethylformamide in the presence of 0.43g 1-(3-
WO 92/ 1 7 1 9~ PCl /US92/026~ ~ .
a~ ~ 18 ~r~ ~
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 0.31 ml
triethylamine, and 0.30g 1-hydroxybenzotriazole (HOBT), for 1 1/2 hours. This
is fiitered, washed with methylene chloride, and the resulting resin treated with
20% piperidine in methylene chloride as above to remove the FMOC group.
5 The resulting resin derivative is then treated as above with 1.369 N-a-FMOC-
N~ (4-methoxy-2,3,6-trimethylben~enesulfonyl)-L-arginine in the presence of
triethylamine, EDC, and HOBT. The FMOC group is removed as above. The
peptide is removed frorn the resin by treating with 20 ml of 95% trifluoroaceticacid for two hours. The arginine rssidue is deprotectsd by overnight treatment
10 with concentrated trifluoroacetic acid. The resultin~ solution is diluted with
0.5% acetic acid, washed with 3 portions of ethyl acetate, then Iyophilized to
give L-arginyl-L-aspartyl-L-valine as the ditrifluoroacetate salt; m.p. 90-95C.
EXAMPLE 2
L ~partyl-~-!sQ.~utyl~m~e . ,
A. 1.16g of 1-(3-dimethylaminopropyl)~3-ethylcarbodiimide
hydrochloride (EDC) and 0.93 mt of triethylamine ar0 stirred together in 50 ml
20 of methylene chloride for 10 minutes. 2.5g N-a-(FMOC)-L-aspartic acici 13-t-
butyl ester, 0.60 ml isobutylamine and 0.82g hydroxyb~nzotriazole (HOBT) are
added and the solution stirred at room temperature overnight. The solution is
diluted with ethyl acetate, washed twice with water and dried over magnesium
sulfate. The filtered solution is evaporated in va~o ~o give 2.2g N-a-~FMOC)-
25 L-aspartic acid isobutyl amide 13-butyl ester.
B. The amide obtained in E~xample ~A is dissolved in 20% (V/V) : .
piperidine in me~hylene chloride and stirred at room temperature for 2 hours. ~ -
The solution is evaporated in ~cuQ and the residue dissolved in ethyl acetate
30 and this solution is washed with 10% sodium bicarbonate solution, dried ov~r
sodium sulfate, filtered and evaporated to give 1.7g L-aspartic acid-a-isobutyl
amide-13-t-butyl ester.
C. 0.679 N-a-FMOC glycine and 0.559 of ~he amlde obtained in 2B
35 are treated under the conditions of Example 2A to give N-a~(FMOC)-glycy!-L-
aspartic acid isobutyl arnide~l3-butyl ester.
:
V~'O 92/1719~i Pcl/us92/O~
: 2~ ~7Q~8
D. The product obtained in Example 2C is ~reated as in Example 2B
to remove the FMOC protecting group to give glycyl-l -aspartic acid isobutyl
amide~ butyl ester.
E. 0.40g of the prûduct of Example 2D and 0.789 N-a-t-BOGN-c~(4-
methoxy-2,3,6-trimethylbenzanesulfonyl)-L-arginine are treated as in Exarnple
2A with 0.29g EDC, û.17g HOBT an~ 0.18 ml triethylamine to give N-a-BOC-N-
(4-cD-methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginylglycyl-L-aspartic acid
isobutyl amide-B-butyl ester.
F. 0.35g of the product obtain~d in Example 2E is traated with
concentrated trifluoroacetic acid in the presence of two drops of ethanedithiol
overnight. The solution is diluted with 0.5% acetic acid and washed with 4x100
ml of ~thyl acetate. The aqueous solution was Iyophilized to 0.199 of a white
solidl L-arginylglycyl-L-aspartyl-a-isobutylamid~ as th~ ditrifluoroacetate salt; -
rn.p. 90-95C.
EXAMPLE 3
1 O~ v~VV L~KD~ na
A. 1.27g L-valine t-butyl ~ster and 2.5g N-oc-FMOC-L-aspartic acid ~-
t-butyl ester are treated as in Example 2A in the presence of 1.160 EDC, û.93g
triethylarnine and 0.829 hyclroxybsnzotriazole. The resulting product is then
deprotect~d as in Example ~B to giv~ L-aspartyl-B-t-butyl ~ster-L-valine-a-t-
butyl ester.
B. 1.1g of the product obtained from Example 3A is treated with N-a-
FMOGglycine in the presence of 0.609 EDC, and 0.43~ of triethylamine in
methylene chloride as in Example 2A and the resu~ting product deprotected in
20% piperidine in methylene chloride as in Example ~B to give 0.65g glycyl-L- :aspartyl-B-t-butyl ester-L-valine-a-t-butyl ester.
. , .
C. 0.259 of the product ~rom Example 3B is treated with 0.23g N-a-t-
BOC-N-~CBZ orni~hine in 5ml of m~thyl~ne chlorW~ in th0 presence of 0.1 2g
EDCI Q.8g HOBT and 0.09 ml trie~hylamine as in 2A to give 0.459 N-a-t-BOC-
N-~-CBZ-L-ornithyl-glycyl-L-aspar~yl-13-t-butyl sster-L-valine-a-t-butyl ester.
. '
'': ., '
. ..
U'O 92/1719f PCT/IJS92/02~
~l,~05Q 20 ~-`
D. The benzyloxycarbonyl protecting group on the product compound
of Example 3C is removed by dissolving 0.459 of the protected compound in
20 ml of cyclohexene and adding 0.10g 10% palladiurn on carbon and heating
5 at reflux, under nitrogen, for 2 hours. The resultin3 solution is filtered,
evaporated, and chromatographed on silica gel in chloroform/methanol/water
90:10:3 to give 0.2~g N-a-t-BOC-L-ornithyl-glycyl-L-aspartyl-B-t-butyl ester-L-
valine-t-butyl ester.
E. 0.23g of the product obtained in Exampl~ 3D is dissolved in ~ ml
trifluoroacetic acid with 3 drops of ethan~dithiol added. The solution is stirred
for 7 hours, evaporated, and the residue partitioned between ethyl acetate and
0.5M acetic acid. The aqueous portion was separated and Iyophilized and the
resulting solid purified by HPLC to giv~ L-ornithyl-glycyl-L-aspartyl-L-valine as
15 the ditrifluoroacetate salt; m.p. 122-25C.
EXAMPLE 4
: .
L-Ar~iinylsar~osyl-L~ -Va!in~
N-a-FMOC-sarcosine is substituted for N o~-FMOC-glycine and the
resulting product is treated with piperidine in rnethylene chloride as in Example
I to remove the FMOC group. The corrasponding product is obtained.
Treating this product with the arginin~ derivative of Fxample 1, cleaving the
25 resulting peptide from the resin and d~prot~cting as in Example 1 gave L-
arginylsarcosyl-L-aspartyl-L-valine as the ditrifluoroacetate s~lt; m.p. 145C
(dec.).
EXAMPLE 5
L-Ar~invl~L-L-A~artyl-L-tN-Methyl~Valine . , .
A. 1g of p-alkoxyben~ylalcohol resin (0.~-1 mmole/g of resin), 0.706g
of N-FMOC-N-methyl-L-valine, 0.382g EDC, 0.270g HOBT, and 0.28 ml
3~ triethylamine are csmbined in 15 ml of dimethylformamide arld shak~n for 2
hours. The mixture is filtered and the resin washed with DMF. The resin is -~
treated as above for a second time, then shaken with 0.?8 ml glacial acetic
~ .
WO 92/1719( PCT/US92/026~7
2~ ~7~38 21
acid, 0.955g EDC, and 0.7 ml triethylamine in DMF and deprotection effected
with 20% piperidine in methylene chloride as in Example l. This gives N-
Methyl-L-valine-p-alkoxybenzyl resin es~er.
B. L-aspartic acid, glycine and L-arginine are coupled and
deprotected, sequentially, as in the previous examples and the peptide
removed from the resin to give L-arginyl~lycyl-L-aspartyl-L-(N-me~hyl)valine as
the ditnfluoroacetate salt which decomposes at 1 53C.
EXAMPLE 6
L-Ar~invl~lvcyl-LA~Q~,rtyl ~l~çin~
Starting with N-a-FMOC-glycino-p-alkoxy benzyl resin ester, sequentially
15 coupling L-aspartic acid, glycine and arginina, deprotecting and removing thepeptide as in the above examples, L.-arginylglycyl-L-aspartyl glycine is
obtained as the ditrifluoroacetate salt; m.p. 85-90C.
! - -
EXAMPLE 7
2 0
N-(L-~r~inyl-?-Aminoethvl~-L-As,Qarlvl-L-V~llne
A. 1.18g EDC and 0.86 mi of triethylamine are combined in 20 ml of
methylene chloride and stirred for 10 minules. 2 g N-a-CBZ-L-aspartic acid B-t-
butyl ester, 0.83g HOBT, 1.3 g L-valine-t-butyl ester and 0.86 ml triethylamine ~ ;
w~re added and the solution stirr~d ovcrnight. Th~ solution is diluted with ethyl
acetate and washed with 10% citric acid solution, 10% sodium carbonate
solution, wa~er, th~n dried over sodium sulfalte, evaporated to give 1.9g N-a-
CBZ-L-aspartyl-t-butyl ester-L-valine-t-butyl ester.
B. 2.2g of N-a-CBZ-glycine methyl ester is dissolved in 50 ml of
anhydrous toluene and cooled to -78C, under nitrogen. To this is added 13 ml
of 1.5M diisobutyl aluminum hydride in toluene over a period of 1 hour. The
solution is stirred for an additional hour at -78C, then quenched by addition of
50 ml 5% hydrochloric acid solution. The solution is extracted with ethyl
acetate which is washed with water and dried over sodium sulfa~e, evaporated
to give 1.~59 N~ CB:Z-2-aminoacetaldehyde.
.
.
. .
WO 92/1719~i PCT/US92/026?i
22
C. The product from Example 7A is deproteoted as in Example 3D to
give L-aspartyl-t-butyl ester-L-valine-t-butyl ester.
D. 1.S5g of the aldehyde from Example 7B, 3.4g of the product from
Example 7C, 1.64g sodium acetate, 1.239 sodium cyanoborollydride and lg of
3 Angstrom molecular sieves are stirred together in 100 ml methanol fcr 3
days. The solution is fii~ered and 5 ml of 5% hydrochloric acid is addccl. The
solution is dilu~ed with water and adjusted to pH 9 wi~h 10% sodium carbonate,
10 ~hen extracted with water, and dried over sodium sulfate. The solution is
evaporated and the residue purified by flash chromatography in ethyl
acetate/hexane, 1:1, to give 1.1g N-CBZ-aminoethyl-L-aspartyl~ t-butyl ester-
L-valine-t-butyl ester.
E. The CBZ group is rsmoved from the product of Example 7D as in
Example 3D to give N-aminoethyl-L-aspartyl-t-butyl ester-L-valine-t-butyl ester.
F. The product frorn Example 7E is coupled with N-c~-t-BOC-N-e~-(4-
methoxy-2,3,6-trimethylbenzenesulfonyl)-L-arginine as in Example 2D and the
resulting product daprotected as in 2E to give N-(L-arginyl-2-aminoethyl)-L-
aspartyl-L-valine as the tritrifluoroacetate salt; m.p. 91-5C. ~ -:
EXAMPLE 8
L-~nylgly~ -A~p~rtic ~sis;L o~-B~n~L Ester
A. 1~ of N-t-BOC-L-aspartic acid a-benzyl ester is treated with 0.366g
of 2-(trimethylsilyl)e~hanol in the presence of 0.5929 EDC, 0.4199 HC)BT and
0.43 ml triethylamina in 20 ml of ma~hyiene chloride ~or 2 hours. The product is3û isolated as in Example 2A to yive N-t-BOC-L-aspartic acid a-benzyl ester-~-2- (trimethylsilyl)ethyl ester.
B. The product of Exampie 8A is deprotected by trsating with 10 ml of
trifluoroacetic acid in 30 ml of methylene chloride for 2 hours at roorn
35 temperature. The mix~ure is cooled to 0C and 20 ml of saturatsd sodium
carbonate solution is added dropwise. The iayers are separated ancl the
-.
~:
\l;'O 92/171 9fi PC~/USg2/026~ ~
~` 2~07~
organic layer dried over magnesium sulfate, filtered, evaporated to give L-
aspartic acid-a-benzyl ester-B-2-(trimethylsilyl)ethyl ester.
C. The product of Example 8B and N-t-BOC glycine are coupled in a
manner similar to that described in the previous axamples to give BOC-glycyl-
L-aspartic acid-oc-benzyl ester-B-2-(trimethylsilyl)ethyl ester.
D. The BOC group is ramoved from the product of Example 8C as in
Example 8B to give glycyl-L-aspartic acid-a-benzyl ester-B-2
(trimethylsilyl)ethyl ester.
~
E. The product from Example 8D is coupled to N-a-BOC-N-~-(4-
methoxy-2,3,6-trimathylben~cnesulfonyl)-L-arginine as in 2D to ~ive N-~-BOC-
N-~-(4-methoxy-2,3,6-trimathyl-benzenosulfonyl)-L-arginyl-glycylaspartic acid
a-benzyl ester-13-2-(trimethylsityl)athyl ester.
F. 0.30g of the product obtain0d in Exarnple 8E is stirred with 5 ml of
trifluoroacetic acid at room temperatur~ for 24 hours. The reaction mixture is ~ -
then stirred with 0.5 N acetic acid aind washed with ethyl acetate. The aqueous
layer is Iyophilized to ~ive ~-arginylglycyl-L-aspar~ic acid a-benzyl ester :
ditrifluoroacetate; m.p. 85-7C. `
EXA~APLE 9
~ .
N-(Ç-AmlnQ~exc~Lk~
A. 1 9 of N-(9-fluorenylrnethoxycarbonyl)-L-valine p-aikoxybenzyl
alcohol resin ester (containing approximately 0.5B mmol of amino acid) is
deprotected by shaking with 10 ml of a solution of 20% piperdine in
30 dimethylformami~e for 1.5 hours. The mixture is fiHered and the resin
derivative washed with methylene chloride to give L-vaiine p-alkoxybenzyl
resin ester.
B. The product ~rom Example 9A is shaken with 0.9~g of N-a-FMOC-
35 L-aspartic acid 13-t-butyl ester, 0.3 9 of 1-hydroxybenzutriazole (HOBT), 0.43 9
of 1-(3-dimethyl-aminopropyl)-3-ethylcar~odiimide hydrochloride ~EDC) and
0.32 ml of triethylamine in 10 ml o~ dimethylformamide for 2 hours. The mixture
WO 92/1719~ PCr/US92/026~'s,
2 4
is filtered and the resin washed with mathylene chloride. The resin derivative
is then deprotected as in Example 1 to give L-aspartyl-B-t-butyl ester-L-v~line
p-alkoxybenzyl resin ~ster.
C. 2 g of 6-aminohexanoic acid and 3.23g of sodium carbonate are
dissolved together in 30 ml of water. The solution is cooled in an ice bath and
3.329 of di-t-butyldicarbonate in 15 ml of tetrahydrofuran is added. The mixtureis stirrecl at room temperature for 5 hours, then diluted with 400 ml of water and
extracted with ether. The aqueous solution is acidified to pH 2 with
hydrochloric acid and extracted with ethyl acetate. The ethyl ac~tate layer is
dried over magnesium sulfate, filtered and evaporated In ~LQ to give N-o~-
tert-butoxy-carbonyl-6-aminohexanoic acid.
D. The product from Example 9B is shaken with 0.52g of N-b~BOC 6-
aminohexanoic acid, 0.3 9 of HOBT, 0.439 of EDC and 0.32 ml o~ tricthylamine
in 10 ml of dim~thylformamide for 17 hours. The mixture is filtered and the
resin derivative washed with rnethylene chloride. Th~ peptide derivative is
deprotected and cleavsd from the resin by treating with 10 ml of 9~%
trifluoroacetic acid for 2 hours. The r~sin is ~iltered off and the filtrate diluted
with 50 ml of 0.5 N acetic acid. The aqueous solution is washed with 4x25 ml
of ethyl acetate, filtered, then Iyophilized to give N-(6-aminohaxanoyl)-L-
aspartyl-L-valine as the trifluoroac~ate sa~; m.p. 75-85C.
EXAMPLE 10
N-~7-Amin~hQ~L L-Aspartyl-L-Valine
A. When 7-aminoh~ptanoic acid is substitut~d for 6-aminohexanoic
acid and treated in a manner similar lo that in Example 9C, N-~ter~-
butoxycarbonyl-7-aminoheptanoic acid is obtained.
B. L-aspartyl-13-t-butyl ester-L-valine p-alkoxy-benzyl resin es~er
(prepared from 1 g of N-FMOC-valine p-alkoxybenzyl resin ester as in
Fxamples 1A and B) is treated with 0.559 of N-BOC-7-aminoheptanoic acid,
with 0.3 9 of HOBT, 0.439 of ED(:: and 0.32 mi of triethylamine in 10 ml of : ~ -
dimethylformamide in a manner similar to that in Exampl~ 9D to give N-(7-
aminoheptanoyl)-L-aspartyl-L-valine as the trifluoroacetate salt.
'
' ',
WO 92/171~f PCT/US92/026?~/ ~
- 2 ~ 07~882~
.
EXAMPLE 1 1
~r3y~ ~;
A. 7-Guanidinoheptanoic acid is prepared essentially by the method
of Miller, et al, Svnthçsis, 777 (1986), which is incorporated herein by
reference. 0.50g of 7-aminoheptanoic acid is dissolved in a solution of 0.475g
of potassium carbonate in 3.5 ml of water. 0.4279 of
10 aminoiminomethanesulfonic acid is added portionwise over 1O minutes and
the mixture stirred at room temperature for 24 hours. The resulting solid is
collected by filtration. The guanidine is dissolved in diluted hyclrochloric acid
and the solution evaporat0d lQ~. Two portions of 2-propanol are ~;
evaporated from the residue to giv~ 7-guanidinoh~ptanoic acid hydrochloride.
B. L-aspartyl-13-t-butyl ~ster-L-valine p-alkoxy-benzylalcoholester ~ -
(prepared from 1 g of N-FMOC-L-valine p-alkoxybenzylaleohol ester resin as in
Examples 9A and B) is treatsd with 0.50 of 7-guanidinoheptanoic acid
hydrochloride, 0.3 9 of HOBT, 0.439 of EDC and 0.32 ml of triethylamine in 10
20 ml of dimethylformamide in a manner similar to that in Example 1 D to give N-~7-guanidino-heptanoyl)-L-aspartyl-L-valine as the trlfluoroacetate salt; m.p.
75-80C.
..
EXAMPLE 12 `
N-~:Guan din~o~anoYI!-L-A~r~-L-Valin~
A. 8-guanidinooctanoic acid hydrochloride is prepared from 8- ~ ;
aminooctanoic acid in a mannar similar to the process used in Example 11 A.
~0 :.... .
B. 0.4 g of 8-guànidinooctanoic acid hydrochloride, L-aspartyl 13-t- :
butyl ester-L-valine p-alkoxybenzyl resin estar (prepared in the same rnanner
as in Example 1), 0.22g of HOBT, 0.329 of EDC and 0.24 rnl of triethylamine
are shaken in 1 Oml of dimethylformamide and treated as in Example 9D to giYe
N-(8-guanidinooctanoyl)-L-aspartyl-L-valine as the trifluoroacetate salt. : ~
; ~ ~ : . :.. '
. . .:
WO 92/17196 P~/US92/026~
26 (-
EXAMPLE 13
If 6-guanidinohexanoic acid hydrochloride is substitute~ for 7-guanidino-
heptanoic acid hydrochloride in Example 11B, N-(6-guanidinohexanoyl)-L-
5 aspartyl-L-valine is prepared.
EXAMPLE 1 4
A. If 8-aminooctanoic acid is substituted for 6-aminohexanoic acid in
10 Example 9C, N-tert-butoxycarbonyl-8-aminooctanoic acid is prepared.
B. If N-cl)-BOC-8-aminooctanoic acid is substituted for N-~-BOC-6-
aminohexanoic acid in Example 9D, N-~8-amino-octanoyl)-L-aspartyl L-valine
is prepared as the trifluoroacetate salt.
EXAMPLE 15
A. 4g of 6-amino-1-hexanol is dissolvecl in 50 ml of 1 Q% aqueous
tetrahydrofuran and th~ solution ~oled to 0C. 7.469 of di-tert-
butyldicarbonate in 25 ml of tetrahydrof-uran is added dropwise and the
resulting mixture stirred for 3 days at room temperature. The solvent is
evaporated in ~Q and the residue dissolved in ~thyl acetate. The ethyl
acetate solution is washed with watsr, dri~d over magnesium sulfate and
evaporated in ~Q to giv~ 7.49 of N-tert~bu~oxycarbonyl-6-amino-1-hexanol.
B. To a solution of 8.8g of pyridinium chlorochr~rnate in 250 ml of
methylcne chloride is added 8.8g of 3 Angs~rom moleoular ~ieves. A solution
of 7.4~ of N-tert-butoxycarbonyl-6-amino-1-hexanol in S0 ml of methylene
chloride is added dropwise and the mixture stirred at room temperature for 2
hours. The reaction rnix~ure is fil~r~d through silica 9~1, washing with 40%
ethylacetate in hexane, and the filtrate evaporated In Y~LQ to give 6-N-tert- -
butoxycarbonylaminohexanal.
C. 1g of 6-N-tert-butoxycarbonylaminohexanal and 1.549 of
methyl~triphenylphosphoranylidene) acela~e are combined in 25 ml of
':
\~lo 92/171~6 PCT/US92/026~-~
2~ 07~88
.
chloroform and the solution refluxed for 2 hours. The solvent is then removed
In ~Q and the residue taken up in ether and allowed to stand in the freezer
overnight. The resulting suspension is ~iltered, the filtrate evaporated and theresidue flash chromatographed in ~0% ethyl acetate in hexane to give methyl-
5 8-1~1-tert-butoxycarbonylamino-2-octenoate.
D. A solution of 3.2g of methyl 8-N-tert-butoxycarbonylamino-2-
octenoate in 25 ml of methanol and 25 ml of 1 Normal aqueous sodiurn
hydroxide is heatsd at reflux for 2 hours. The methanol is removed in ~Q
10 and the aqueous so!ution acidified with 1 N hydrochloric acid. The resulting
mixture is extracted with ethyl acatate. The organic solution is dried over
magnesium sulfate and evaporated to give 8-N-tert-butoxycarbonylamino-2-
octenoic acid. :
E. 3g of 8-N-tert-butoxycarbonyl-amino-2-octenoic acid is dissolved
in 30 ml of trifluoroacctic acid and the solution stirred at room temperature for 1
hour, then evaporated ~Q ~Q to 9iv9 8-amino-2-octcnoic acid as ~he ..
trifluoroacetate salt. `
.
F. 3.1g of 8-amino-2-octenoic acid trifluoro-ac0~ate is added to 30 ml
of water and the pH adjusted to 7 with 1 N sodium hydroxide solution. 1 .9g of
potassium carbonate is added, then 1.75~ of aminoiminomethane-sulfonic acid
is added, portionwise, over 10 minutes. The mixture is stirred for 5 hours at
room temperature and the rosultin~ solid collected by filtration. The solid is
dissolv~d in diluted hydrochloric acid and the solution evaporated and two
portions of 2-propanol evaporàted from the residua to give 8-guanidino-2-
octenoic acid hydrochloride.
G. L~aspartyl~ t-butylester-L-valine p-alkoxybenzyl resin ester
(prepared from 0.69 of N-FMOC-valine p-alkoxybenzyl resin ester as in
Examples 1A and B) is tr0ated with 0.33g of 8-guanidino-2-octenoic acid
hydrochloride in the presence of 0.184g of HOBT, 0.26g of EDC and 0.19 ml of
triethylarnine in 10 ml of dimethylformamide in a manner similar ~o that in
Fxample 1 D to give 8-guanidinooct-2-enoyl-L-aspartyl-L-valine as the ~ ,
trifluoroacetate salt. -;
Wo 92/ 1 7 1 9~ PCr/US92/026
28 '~
EXAMPLE 16
A. If 6-aminohexanoic acid is substituted for 7-aminoheptanoio acid in
Example 11A, 6-guanidinohexanoic acid is prepared.
B. To 14.8 g of a 50% aqueous solution of glyoxylic acid is added 50
ml of water. The resulting solution is cooled to 0C and treated with 10 ml of a10 70% solution of ethylamine in water added by dropwise addition over 15
minutes. The reaction mixture is transfsrred to a Parr bottle, then 1~%
palladium on carbon is added and the r~action vessel is shaken under
hydrogen at 44 psi for 24 hours. The raaction mixture is filtered through a cefite
pad and the filtrate is concentrat~d i~Q to giv~ a tan oil. The oil is troat6d
15 with 1 N aqueous HCI and concentrated ~Q to giv~ a solid which is
recrystalized from acetic acid.
3.65 g of N-ethyi glycine hydroohloride is stirred in 35 ml of water. This is
treated with 8.31 g of sodium carborlate and cooled ~o 0C, ~ollowed by ~he
20 dropwise addition of 6.77 9 of 9-fluor~nylmethyl chloroform~te in 15 ml of
tstrahydrofuran (THF). Th~ r~action mixture is allowed to slowly warm to room
tempcrature and stirred for 24 hours. The T,HF is r~moved ~L!Q and the
rcsidue is diluted with water and axtract,sd with ether. The aqueous ~raction isacidified to pH ~ 2 with 1 N aquoous HCI and extractod with ethyl acetate. The
2iS or~anic extracts (ethylacetate) are dried, filtered and conGentrated to give N-a-
FMOC-N~ ethyl glydne as a white solid. All FMOC proteoted substituted
glycines are made by this procedure simply by substituting tha appropriate
amine for ethyl amine in this procedure.
C. N-a-FMOC-N-~-ethyl glycine is substituted for N'-o~-FMOC glycine
and the resulting product is treated with piperidine in methylene chloride as inExample 1 to remove ~he FMOC groups. N-~x-Ethyl glycyl-L-aspartyl-~-t-butyl
est,sr-L-valine-p-alkoxybenzyl atcohol resin ester is obtained.
':
D. A solution of û.44 g of 6-guanidinohexanoic acid hydrochloride in
10 ml of DMF is ~re~ted with 0.23 9 of triethylamine. The solution is cooled to
0C and 0.~7 g of N,N-bisi2 oxo-3-oxzoiinyl]phosphorodiamidic chloride (i30P~
. .
,
.
,
~vo 92/171 9h PC~/US92/02G~
i ` 2~ 0708~ 29
Cl) is added in a single portion. The reaction mixture is stirred at O~C for 5
minutes and then 1 9 of N-a-Ethyl glycyl-L-aspartyl-13-t-butyl ester-L-valine-p-alkoxybenzyl alcohol resin ester is added. The reaction mixture is shaken ~or 2
hours at room temperature. The procedure for removal of the peptide from the
5 resin is the same as that described in Example 1. The trifluoroacetate acid
solution is diluted with 0.5% acetic acid, washed with 3 portions of ethyl
acetate, then Iyophilized to give 6-guanidinohexanoyl-1~1-ethyl glycyl-L-
aspartyl-L-valine as a white powder.
EXAMPLE 17
, ~...
:
A. A solution of 10 g (51 mmol) of 6-bromohexanoic acid in 100 ml of
15 m0thanol is treated with anhydrous HCI gas for 5 minutes at room temperature. Concentration in vacu~ giYes the methyl ester.
A 50 mL round bottom flask is charged with 8 9 (38.29 mmol) of 6-
bromohexanoic acid methyl aster, 5.7 9 (84.24 mmoi) of imidazole and 20 rnL
20 of THF. The resultin~ mixturo is heatcd at reflux for 24 hours. S~lvent is
removed iQ~Q and the residue is purified by flash chromatography using
5% methanoltethyl acetate.
.: '
6-(imidazol-1-yl)-hexanoic acid methyl ester is treated with 1 N aqueous
25 HCI for 24 hours at reflux to provide, after concentration in va~uo, 6-~imida~ol-
1-yl)-hexanoic acid hydrochloride.
All corresponding compounds are prepared in a similar ~ashion starting
from the appropriate ~bromohexanoic acid.
B. When 6-(imidæol-~-yl)-hexanoic acid hydroehloride is substituted
for 6-guanidinohexanoic acid hydrochloride and treated in a manner similar to ;
that in Example 16D, 6 (imidazol-1-yl)-hexanoyl-N-ethyl glycyl~L-aspartyl-L-
valine is obtained. `~
.
~ :,
'.; '
wo 92/1719~ PC~/US9~i/026~7
EXAMPLE 18
rtvl-L-V~lin*
A. N-Phthalyl-5-Aminopentanoyl-Glycine, Methyl Ester
To a stirred suspension of 5.02 9 (20.3 mmol~ of N-phthalyl-5-
arninop~ntanoic acid and 3.04 g (24.2 mmol) of glycine methyl ester
hydrochloride in 1 C0 ml of dry tetrahydrofuran is added 3.~7 g (24.3 mmol) of
10 HOBT, 4.6~ g (24.3 mmol) of EDC, and 4.94 9 (48.8 rnmol; 6.80 ml) of tri~thylamine in that order. The resulting suspension is stirred for 18 hours, then
partitioned between 250 ml of ethyl acetat~ and 100 ml of water. The layers
are separated and the aqusous phas~ is extractQd with 2i50 rnl of ethyl acetate
The combined organic lay~rs are washed with ~ 60 ml of 10% aqueous sodium
15 carbonate, then with two 125 ml portions of saturated aqueous sodium
chloride. The organic phase is dried (MgSO4) and concentrated ~ to
give 7.39 g of a white solid. Recrystallization of the product from
dichloromethane-hexane gives 6.22 9 (96%) of the title compourld as a fluffy
solid.
B. [4-(N-Phthalyi-4'-Aminobutyl)Tetrazol-1-yl]-AceticAcid, Methyl
Ester.
' .
To a stirred suspension of 3.7 g (11.6 mmol) bf the protect~d dipeptide in
65 ml of banzene i8 added 3.~ 9 (16.8 mmol) of phosphQrus pentachloride in
one portion. The suspension is stirred for 2 hours during which time the solids
had dissolved. The r~action mixtura is concentratod ~Q to give a light
brown oil which is dissolved in 60 mi of a hydrazoic acW solution in benzene.
This~solu~ion is stirred for 24 hours, cnncentrated ~ and the rasidue is
purified by extraction and chromatography as described before to give 2.89 g
(73%) of the desired tetrazole ester as an oil which crystalliæes on standing. ~:
C. ~4-(N-Phthalyl-4'-Aminobutyl)Tetrazol-l-yl]-Acetic Acid.
To a stirred suspension ef 2.5 g (7.29 mmol~ of the above ester in 99 ml of
methanol, cooled in an icewater bath, is added 1.4 9 ~33.7 mrnol) of lithium ~ -
hydroxide monehydrate in 33 ml of water. The suspension is warmed to 30C
. .: .
~ .. ~';
U'0 92/1719fi 21 Q 7 0 ~ ~ PCT/US92/026~,
31 ~.
to dissolve the solids and the solution is stirred at room temperature for 1.5
hours. Acidification of the solution with 12 ml of 6M aqueous hydrochloric acid,followed by work-up as described previously affords 2.39 9 (100%) of the
tetrazole acid as an unstable white foam.
D. [4-(4'-Aminobutyl)Tetrazol-1-yl3-Acetic Acid, Hydrochloride.
To a solution of 2.3~ 9 (7.14 mmol) of the tetrazole acid in 29 ml of
ethanol is added 463 mg (4~0 ml; 7.94 mmol) of 55% aqueous hydrazine. The
suspension is heated under reflux ~or 2.5 hours, cooled to room temperature,
and diluted with 30 ml of water. The solution is acidified with 1.36 9 (22.7
mmol) of acetio acid, stirred for 2 hours, then boiled for 30 minutes. After
cooling the solution in a refrig~rator ovemight, the precipitate is flltered andwashed with 20 ml of cold water. The combined filtrate and washings are
concentrated to dryness and the rcsidue (1.g8 9~ is dissolved in 20 ml of 50%
aqueous ethanol and 515 mg (16.1 mmol; 5000 ml) of 55% aqueous hydrazine
is added. This sblu~ion is heated for 2.5 hours, cooled to room temperature
and concentrated to dryness. The residu~ is stirr~d with 15 ml o~ 2N aqueous
hydrochloric acid for 13 hours, then the suspension is heatsd under reflux for
30 minutes. The cooled suspension is filter~d and the precipitate is washed ::
with 15 ml of water. The filtrate and washings are concentrated ~Q to
give 3.05 ~ of the crude amin~ hydroGhloride as a colorl~ss semisolid. Further
solid impurities are removed by filtration of the ethanol-soluble material; thisgives 1.93 9 of a pale y9110w oil.
~5
E. [4-~4'-Guanidinobutyl)Tetrazol-1-yl~-Acetio Acid, Hydrochloride.
'",-;' .' .
To a stirred solution of 1.85 g (~.92 mmol) of ths t~trazole amino acid
hydrochloride in 2Q ml of water is added 2.3 9 ~16.6 mmol) of potassium
carbonate. To this solution is added 1.18 9 (9.52 mmol) of aminoimino-
methanesulfonic acid in small portions over a 10 minute period. This solution
is stirred for 35 hours and worked up as before to give 1.4 9 o~ the crude ;;
guanidino tctrazole acid as an off-white solid. The solid is converted to lhe
hydrochloride by evaporation of a dioxane-aqueous hydrochloric solution of
35 this material to d~yness.
:
WO 92/17196 ~ PCI/~JS92/026~-
~ 32
F. The peptide coupling of [4-(4-guanidinobutyl)tetrazol-1 yl]-acetic
acid hydrochloride to L-aspartyl-B-t-butyl ester-L-valine p-alkoxybenzyl alcoholresin ester, the cleavage of the resulting peptide fronn the resin, and the
subsequent isolation steps is performed as describeld in Example 1. The [4-~4'-
5 guanidinobutyl)tetrazol-1-yl]-acetyl-L-aspartyl-L-valine is obtained as the
trifluoroacetate salt.
EXAMPLE 19
1 0 ~5-~5~-@uanidin~pentvl~T~zQ~l-Ac~tyl-L~u3G~y3~
The desired product is prepared by the procadure describsd for the
preparation of [4-(4'-guanidinobutyl)t~trazol-1-yl]-acetyl-L-aspartyl-L-valine, by
substituting N-phthalyl-6-aminohexanoic acid for N-phthalyl-6-aminopentanoic
15 acid in Example 18A.
,
EXAMPLE 20
9-G~anidinonQnanoyl-~-~i~artvl-a-~enzy!ph~nylalianine - .
A. BOC-L-phenylalanine ~4.74~, 17.9 mrnol), paraformaldehyde
(1.6i5g, 54.9 mmol), and p-toluenesulfonic aeid (0.38g, 2 mmol) are dissolved
in totuene (100 ml) and heated at reflux for ~wo hours while removing water
with a Dean-Stark trap. The mixture is aliowed to cool, diluted with ether and
25 the organic phase washed with saturated sodium bicarbonate solution, brine,
dried over magnesium sulfate, then conc0ntrated ~Q to give the crude
oxazolidinone. The oxæolidinone (4.389, 15.8 mmol) is dissolved in
tetrahydofuran (Tl IF) (40 ml) and the solution cooied to -78C under a nitrogenatrnosphare. A solution of 1 M sodium bis(trimethylsilyl)amide (23 ml) in THF
30 is added and th~ mixture stirred at -7~C for 30 minut~s. -Benzyl bromide
(2.8~g, 23.7 mmol) is added and s~irring continued for 1.i5 hours at -78C. The
mixture is quenchsd with ammonium chloride solu~ion and diluted with ether.
The ether solution was washed wi~h saturated sodium bicarbonate solution,
brine, dried, and conc0ntrated ~ lo giva the crude dibenzyl
35 oxazolidinone. The dibenzyl oxazolidin~n& (6.349) is dissolved in 85%
ethanollwater (100 ml) and sodiurn hydroxide (1.359) is added. The mixture Is
haeated at reflux for 1 hour, cooled, concentrated ~, and the residue
- .
:
WO 92/1719~ PCT/US92/026~
,"
21 07 D ~ 33
~iluted with water and extracted with ethyl acetate. The aqueoLIs layer is
acidified with 3N HCI and extracted with ether/ethyl acetate (1:1). The organic
solution is washed with brine, dried over magnesium sulfate, filtered, and
concentrated in vaç~Q to give BOC-2,2-dibenzyl glycine (3.29g).
B. BOC-2,2-dibenzyl glycine (3.29g, 9.25 mmol) is dissolved in a
solution of methanol (1~ ml,l and water (2 ml) and the pH adjusted to 8 with
2û% cesium carbonate solution (11 ml). The solution is concentrated in va~uo
to dryness and the residue dissolved in dimethylformamide ~DMF) (25 ml3 and
10 re-concentrated twice and dried under high vacuum. The cesium salt is taken
up into DMF (25 ml), benzyl ~romide (1.74g, 10.2 mrnol) is added and the
mixture stirred at room temperature ~or 16 hours. The mixture is concentrated
in vacuo and the residue diluted with ether. The organic phase is washed with
water, brine, dried ov0r magnesium sulfat~, and concentrated in v~c~o . The
15 crude product is purified by flash chrornatography, eluting with 10% ethyl
acetate in h~xane to give BOC-2,2-dibenzyl glycine bsnzyl ester.
G. BOC-2,2-dibenzyl glycine benzyl ester is depro~ected
(trifluoroacetic acid) as in Example 8 and coupl~d (BOP-(::I) to N-BOC-L-
20 aspartic acid-~-benzyl ester essentially in the manner of Example 16. The
resulting dipeptide is, in turn, deprotect6d ~TFA) and coupled (EDC) ~ssentiallyin the manner of Example 2 to 9-nitroguanidinononanoie acid. Subsequent
hydrogenation ~H2, Pd/C) gives ~he desired product, which is isolatad as the
trifluoroacetate salt, 9-guanidinononanoyl-L-aspart~ benzylphenylalanine,
25 M.S., Cal'd: 568, Found: 568.
. . .
EXAMPLE 21
.. . .
9-~uan~i~Qnonaooyl:L ~pa~l ~-iso~L~i~
3~ -
A. BOC-L-leucine is eonverted to rac~mic BOC-(2-isobutyl~-allyl
~Iycine benzyl ester using essentially the procedures of Example 20, Steps A
and B, substituting allyl bromide in the alkylation step. The benzyl ester (2.07g,
~:.73 mmoi) is dissolYed in THF (40 ml), under nitrogen, 0.5 M 9- ;
35 borabicycio[3.3.1]-nonane (9 BBN) in hexanes (46 ml, 23 mmol) is added and
mixture stirred at room temp~rature for 16 hours. The r~action mixture is
quenched with water (1 ml) and a mixture of 1 N aqueous sedium hydroxide
.
WO 92/171 9fi PCr/US92/026:~ /
~ ~a~ 34 ~ .,
solution (51 ml) and 30% hydrogen peroxide solution (18 ml) is added
dropwise. The mixture is stirred for 1 hour at room temperature, saturated with
solid sodium chloride, then extracted with ether. The organic layer is washed
with saturated sodium bicarbonate solution, brin~, dried over magnesium
5 sulfate, filtered, and conc~ntrated in vac~ . The cmde product is purified by
flash chromatography, eluting with 20% ethyl acetatle/hexanes to give BOC-(2-
isobutyl)-3-hydroxypropyl glycine benzyl ester.
B. BOC-(2-isobutyi)-3-hydroxypropyl glycirle benzyl ester (0.17g, 0.45
10 mmol) is dissolved in pyridine (2 ml), cooled to 0C, and p-tolu~nesulfonyl
chloride (0.259, 1.31 mmol) is added. The mixture is then stirred at room
temperature for 16 hours, dilut~d with ether, and the organic layer washed with
1 N HCI, 10% copper sulfate solution, brine. The organic layer is dried over
magnesium sulfate, filtered, concentrated ~ to givo the tosylated
1~ product. The primary tosylate ~0.239, 0.43 mmol) is dissolved in DMF/water
(10:1 ) (2 ml) and sodium æide (0.2gg, 4.46 mmol) is added. The mixture is
heated at 90C for four hours, cool~d, diluted with eth~r and poured into brine. The organic lay~r is washed with wat~r, brine, dried over magn~sium sulfate,
filtered, and concentrat0d in va~o . The c~ude product is purified by flash
20 chromatography, eluting with 10% ethyl aceate in hexan~s to give BOC-(2-
isobutyl)-3-azidopropyl glycine benzyl ester.
C. Using esssntially the procedure of Exampl~ 20C, 9-
guanidinononanoyl-L-aspartyl-(R,S)-a-isobutylornithine is prepared trom BOC-
2~ (2-isobutyl)-3-azidopropyl glycine b~nzyl ester, and isolated as the
ditrifluoroacotate salt, M.S., Cai'd: 501, Found~
EXAMPLE 22
~-~hQxano
A. Vsing essentially the procedlJre of Example 20B, BOC-2,2-diethyl
glycine benzyl ester is prepared from BOG-2,2-diethylglycine.
35 ~ B. U~ing essentially the procedures of Examples 2, 8, 16, and 20,
BOC-2,2-diethyl ~Iycine benzyl es'er is coupled (EDC) to N-BOC-L-aspartic
acid-,B-benzyl ester, to N-BC)C-N-ethyl glycine (BOP-CI,~, and finally to 6-nitro
.
,' :,
,'",.. ,, ,.,;.~. /.,. " .......
WO 92/1719~ PC~/US~2/026?s,
;; 210~ 35
guanidinohexanoic acid to give, after hydro~enation and deprotection as
described above, 6-guanidinohexanoyl-N-ethyi-~lycyl-L-aspartyl-2,2-
diethylglycine, which is isolated as the acetate salt.
EXAMPLE 23
~-Gu~nidinon~nanovl-L-asp inin~ methyl ester
A. (S)-(2-benzyl)-allylglycine (prepared acc:ording to the method of
10 Zydowski, et al., J. Org. Chem. 1990, 5~, 5437) is protected (di-t-
butyldicarbonate, sodium carbonate, THF/water, 7 days) and esterified (methyl
iodide, DMF, sodium carbonate, 2 days) to give (S)-BOC-(2-benzyl)allyl glycine
methyl ester.
B. Using essentially the procedure of Exampl~ 21 A, (S)-BOC-(2-
benzyl)allyl glycine methyl ester is converted to (S)-BOC-(2-ben~yl)-3-
azidopropyl glycine methyl ester.
. ~ -
C. (S)-BC)G(2-b0nzyl)-3-azidopropyl glycine me~hyl ester ~0.19g,
20 0.52 mmol) is dissolved in rnsthanol/chloroform (15:1) (8 ml) and hydrogenated
at atmospheric pressure over 10% palladium on carbon for 5 hours. The
mixture is fi~teredt concentratsd iQ~Q, and the residue tri~urated with
ether/benzene and reconcentrated to give (S)-BOC-(2-bonzyl)-ornithine methyl
ester hydrochloride.
D. (S)-BOG(2-benzyl)-ornithine me~hyl ester hydrochloride (0.2 g,
0.52 mmol) is dissolved in ethanol ~10 ml) and triethylamine (0.2 ml, 1.43
mmol) and S-methylisothiourea (0.1 g, 0.74 mrnol) are added and the mixture
heated at reflux for 16 hours. The solvent is removed !QY~Q and the cn~de
30 product purified by flash chromatography ~0% ethyl acetate in hexane) to give (S)-BOC-t2-benzyl)-nitroarginine methyl ester.
. .
E. Using procedures analagous to those describ~d her~inabove, (S)~ -
BOC~(2-benzyl)-nitroar~inine methyl ester, is coupled to N-BOC-L-aspartic acid
35 ,B-benzyi ester and the resulting dipeptide coupled to 9-
nitroguanidinononanoic acid. The resu~ing product is hydrogenated and
deprotec~ed to give 9-~uanidinononanQyl-L-aspartyl-~S)-a-benzyl arginine ~ ~
':-
.
WO 92/1719fi PCr/lJS92/0~6~/
~a~ 36
~I
methyl ester, which is isolated as the ditrifluoroacatate salt, M.S., Cal'd: 591,
Found: 591.
E)(AMPLE 24
~ ine ~,nQ~I ~
A. Using essentially the procedure of Es~ample 23, Step D, (S)-BOC-
(2-isobutyl)-nitroarginine methyl ester is prepared from L-leucine.
: ~,
B. Using essentially the procedures of Example 23, Step E, 9-
guanidino-L-aspartyl-(S)-a-isobutylarginine methyl ester is prepared from (S)-
BOC-(2-isobutyl)-nitroarginin~ methyl sster, and isolat~d as the
ditrifluoroacetate salt, M.S., Cal'd: 557, Found: 557.
EXAMPLE 25
,,,~ "
A. Sodium hydride (2.52g 60% suspension in mineral oil, washed
with hexane, 63.1 mmol) is suspan~ed in THF (200 ml) and cooled to 0C
under nitrogen. Triethylphosphonoacetate (12.5 ml, 63.1 mrnol) is added over
30 minutes and the mixture cooled to -78C, and BOC-L-leucinal in THF ~0
ml) is added ov~r 30 minut~s. ~ter stirring at room temperature for 1 hour the
excess NaH is quenched by addition of saturated ammonium chloride solution.
The mixture is extracted with ethyl acetate and the organic layer washed with
satruated sodiurn bicarbonate solution, brine, th~n dri~d over magnesium
sulfate, ~iltcred, and concentrated ~Q . The crude product was purifed by
flash ohromatography, eluting with 10% ethyl ace~te in hexanes ~o give the
corresponding BOC-L-leucine-a"B-unsaturated ethyl ester. The ester tl.5 g,
~.26 mmol) is dissolved in ethanol (20 ml) and hydrogenated at atmospheric
pressure over 10% palladium on carbon (0.169~ for 24 hours. The mixture is
concentrated ~~Q, diluted with ether, filtered, and the filtrate dried over
magnesium sulfate, filtered, and concentrated ln Va~ÇUQ to give ~R)-BOC-4-
amino-4-isobutylbutyric acid ethyl ester.
. .
,
.
-: .
,
~ '0 92/ 1 7 1 96 PCr/US9~/0 6~-
-;` 2~0~ 37
B. (R)-BOC-4-arnino-4-isobutylbLItyric acid ethyl ester is coupled
sequentially to N-BOC-L-aspartic acid-~-benzyl estel, and to 9-
guanidinononanoyl as using procedures described hereinabove. The
resulting pseudotripeptide ethyl ester (0.16g, 0.25 mmol) is dissolved in
5 methanol/water (2:1) (6 ml), cesium carbonate (0.33~, 1.Q1 mmol) is added,
and the mixture stirred at room t~mperature for 18 hours. The solvent is
removed in-v~uQ and the r~sidue dissolved in ethyl acetate. The ethyl acetate
is stirred with 1 N HCI, then the organic layer is washed with brine, dried overmagnesium sulfate, filtered, and concentrated ~tlQ . This produc~ was
10 deprotected under hydro~enation conditions as described hereinabove to give
N-(9-guanidinononanoyl-L-aspartyl)-(R)-4-amino~4-isobutylbutyric acid, as the
trifluroacetate salt, M.S., Cal'd: 472, Found: 472.
EXAMPLE 26
N-[IY:~9.-(3uanidin~non~-as~ar~ R)-4~mino-4-iso~utyl~utvrvl]-L-
~ '"' .
(R)-BOC-4-amino-4-isobutylbutyrici acid ethyl ester is treatad with
20 sodium hydroxide in ethanol, followed by acidification to prepare (R)-BOC 4-
amino-4-isobutylbutyric acid which is ccupled, using essentially the procedure
of Example 2, to NG-nitro-L-arginine-benzyl ester p-tosytate, to give the
corresponding dipeptide which is, in turn, sequentially coupled to N-BOC-L-
aspartic acid-~-b~nzyl ~ster and 9-guanidinononanoic acid, then deprotected
25 as described hereinabove to give N-~N-(9-Guanidinononanoyl-L-aspartyl)-(R)-
4-amino-4-isobutylbutyryl]-L-arginins, as the ditrifluoroacetate salt, M.S., Cal'd:
628, Found: 628.
.
EXAMPLE 27
N-[N-(9-.GuanidinonQnanovl-L-~!):~-(R!-~eç-b~tyi-3-a~inobutyrvl]-L- ' ~.
~ ~' .~.
Vsing essentially the procedures of Examples 2~ and 26, th~ desired .
35 product is prepared frorn BOC-D-isoleucinal, and isolated as the
ditrifiuoroacetate salt, M.S., Calld: 628, Found: 628.
: .
WO 92/17~9~i PC~/US92/026~i
3 8 t
~,ia~
EXAMPLE 28
l R)-4-~N~ ;uan~ r~onQn~-n~ s~-o~l~ty~ ne
A. (R)-BOC-4-amino-4-isobutylbu~yric acid e~hyl ester (0.849, 2.92
mmol) is dillosved in THF (~ ml) and lithium chloride (0.27g, 6.3 mmol) and
sodium borohydride (0.23g, 6.3 mmol) and ethanol (9 ml) are added. The
mixture is stirred at room temperature for 16 hours, the mixture is cooled to 0C,
and 10% aqueous citric acid solution is added. The mixture is concentra~ed In
10 y~Q and the residue partitioned betwesn ethyl acetate and water. The
organic solution is washed with saturat~d sodium bicarbonate solution, brine,
dried over magnesium sulfate, filtered, and concentrated ~~Q . The crude
product is purified by flash chromatography, aluting with 20% ethyl ~ ;
acetate/hexane, to give (R)-BOC-4-amino-4-isobutylbutanol.
B. Using essentially the procsdures of Examples 21 and 23
(mesylation, azid~ displacoment, redLIction, and nitroguanylation), (R)-BOC-4-
amino-4-isobutylbutanol, is converted to (R)-BOC-N-(4-amino-4-
isobutylbutyl)nitroguanidine.
C. Subsequent coupling and deprotection as described
hereinaboYe oonverts (R)-BOC-N-(4-amino-4-isobutylbutyl)nitroguanidine to
(R)-4-[N-~9-guanidinononanoyl-L-aspartyl)-amino~-4-isobutylbutylguanidine,
which is isolated as the ditrifluroacetate salt, M.S., Cal'd: 479, Found: 479.
EXAMPLE 29 ;
f
Using procedures analagous to those described hereinabove, the
desired product is prepared from (Fl)-BOC-N-(4-amino-4-
isobutylbu~yl)nitroguanidine, and isolated as the acatate salt, M.S., Cal'd: 542,
Found: 542.
3~
. -,
~ '
WO 92/1 71 96 PCI /US92/~)26~i /
~` 2~070~8 3~3
E3(AMPLE 30
:~ q~ nidinQn~nanoyl-L-~L~m~h~u~
A. Using essentially the procsdures of Example 28, (S)-BOC-N-(4-
amino-4-sec-butylbutyl)nitroguanidine, is prapared from (S)-BOC-4-amino-4-
sec-butyibutyric acid ethyl ~ster.
B The desired product is prspared from (S)-BC)C-N-(4-amino-4-sec
butylbutyl)nitroguanidine, using sequential coupling and deprotection
procedures as describ~d hereinabove, and isolated as the ditrifluoroacetate
salt, M.S., Cal'd: 499, Found: 499.
EXAMPLE 31
9-Gu~nidinvnonanoyl-L:~ar~vl-(R~-6-aminQ-6-s~ tv!b~xanQ~L-L-~
A. Using essentially the procedure of Example 25, Step A, (R)-BOG
6-amino-6-sec-bu~ylhexanoic acid ~thyl ester is prepared from BOG-L-
isoleucinal and triethyl-4-phosphonocrotonate.
. .
B. Using essentially th~ procedures of Example 26, the desired
product is prepared ~rom (R)-BOC-6-amino-6-sec-butylhexanoic acid ethyl ~:
ester, and isolated as the ditrifluoroacet~te salt, M.S., Cal':d: 656, Found: 656.
~
EXAMPLE 32 ~ :
~ ' ,.
q~ A. Using essentially the procedures of Example 28, (R)-BOG(6- :
amino-6-sec-butylhexyl)nitroarginine is prepared from (R)-BOC-6-amino~6~sec~
butylhexanoic acid ethyl ester. ~;
: :
B. Using ess~ntially the procedures of Fxample 28, the desired : :
35 product is prepar~d from (R)-BOC-(6-amino-6-sec-butyihexyl)nitroarginine, andisolat~d as the ditrifluoroacetate salt, M.S., Cal'd: 527, Found: 527.
:
WO 92/171gfi PClr/US92/026~s,
sQ~ 40
Gompounds within the scope of the present invention inhibit platelet
aggregation by inhibiting fibrinogen binding to activated platelets and other
adhesive glycoproteins involved in platelet aggregation and blood clotting and
are useful in the prevention and treatment of thrombosis associated with
certain disease states, such as myocardial infarction, stroke, paripheral arterial
disease and disseminated intravascular coagulation in humans and other
mammals.
Compounds within ~he scope of the present invention exhibit activities ~ -
10 which interfere with adhesive interactions betwoen abnormal cells and the
extracellular matrix and, therefore, are believed to be useful in the treatment of
disease conditions, in humans and other animals, characterizad by abnormal
cell proliferation which have been shown to be dependent on such adhesive
interactions (see, for example, Joum. of Biol. Ch~m. 262 (36), 17703-1771 1
15 (1987); Sci~nce 233, 467-470 (1986); and Cell57, 59-69 (1989)).
The compounds of this invention can normally be administared orally or
parenterally, in the treatment or prevention of thrombosis associated disease
states. -
The compounds of this invention may be formulat~d for administration in
any convenient way, and the invantion includes within its scope
pharmaceutical compositions containing at least one compound according to
the invention aclapted for use in human or veterinary m~dicine. Such
2i5 compositions may be formulated in a conventional mannor using one or more
pharmaceutically acceptable carriers or excipients. Suitable carriers include
dilu~nts or fillers, sterile aqueous media and various non-toxic organic
solvents. The compositions may be formulated in the form of tablets, capsules,
lozenges, troches, hard candies, powders, aqueous suspensions, or solutions,
injectable solutions, elixirs, syn~ps and the like ~nd may contain one or more ~ `
agents selected from the group including sweetening agents, flavoring agenls,
coloring agents and pressrving agents, in order to provide a pharmaceuticaliy
acceptable preparation.
The partJcular carrier and the ratio of platelet aggregation and thrombus
inhibiting compound to carrier are det~rrnined by the solubility and chemical
properties of the compounds, the particular mode of administration and ;
:;,. :.
WO 92/1719~ 2 ~ ~ 7 ~ ~ 8 PCT/US~2/026~-
. .
41
standard pharmaceutical practice. For example, excipients such as lactose,
sodium citrate, calcium carbonate and dicalcium phosphate and various
disintegrants such as starch, alginic acid and certain complex silicates,
together with lubricating agents such as magnesium stearate, sodium lauryl
5 sulphate and talc, can be used in producing tab!ets. i-or a capsule form,
lactose and high molecular weight polyethylene glycols are arnong the
preferred pharmaceutically acceptable carriers. Where aqueous suspensions
for oral use are formulatad, the carrier can be emulsifying or suspending
agents. Diluents such as ethanol, propylene glycol, glycerin and chloroforrr
10 and their combinations can be employed as well as oth~r materials.
For parent~ral adminis~ration, solutions or suspensions of these
compounbs in sesame or peanut oil or aqueous propylene glycol solutions, as
well as sterile aqueous solutions of tho solublc pharmac~utically acceptable
15 salts described h~rein can be ~mploy~d. Solutions of the salts of these
compounds are especially suited for intramuscular and subcutaneous injection
purposes. The aqueous solutions, including those ~f the salts disso~ved in
pure distilled water, are also useful for intravenous injection purposes,
provided that their pH is properiy adjusted, thsy are suitably buffered, they are
20 made isotonic with suffici~nt salin~ or gluco~ and st~rilized by heating or
microfiltration.
The dosage regimen in carrying out the method of this invention is that
which insures maximum therapeu~ic response until improvement is obtained
25 and thereafter the minimum effective l~vel which gives relief. In general, the
oral dose may be between aboùt 1 mg/kg and about 200 mgtkg, prefarably
between about 2 mglkg to 100 mg/kg, and most praferably between about 10
mg/kg and 100 mg/kg, and the i.v. dose abou~ 0.1 mglkg to about 20 mg/kg,
preferably between about 0.5 mg/kg to 10 m~<g, bearing in mind, of course,
30 that in selecting the appropriate dosago in any specific case, consideration
must be given to the patient's weight, general health, age, and other factors
which may influsnce response to the drug. The drug may be adrninistered
orally 1 to 4 times -per day, preferably twice daily.
.
The following pharrnacologic tests evaluate the inhibitory activily of
compounds of the present invention on fibrinogen-mediated platelet
aggregation and fibrinogen binding to thrombin stimulated pl~telets, and
::
. : .. ~ , . ., . . ~ ..... .. .
WO 92/1719fi PCI/VS92/026~7
42 ~
results of these tests correlate to the ~Q inhibitory properties of compounds
of the present invention.
The Platelet Aggregation Assay is based on that described in Blood 66
(4), 946~952 (1985). The Fibrinogen-Binding Assay is essentially that of
Ruggeri, Z.M., et al., Proc. Natl. AGad. Sci. USA83, 5708-5712 (1986) and
Plow, E.F., et al., Proc. Natl. Acad. Sci., lJSA B2, 8057-8061 (1985).
Platelet A~grq~iQn Assay
.
Pre .~aration of Fixed-Activate~Pla~lets
Platelets are isolated from human platelet concentrates using the gel-
filtration technique as d0scribed by Marguerie, G.A., et al., J. Biol. Chem. 254,
15 5357-5363 (1979) and Ruggeri, Z.M., et al., J. Clin. Invest. 72, 1--12 (1983).
The platelets are suspended a~ a concentration of 2 x 1 o8 cells/ml in a modified
calcium-free Tyrode's buffer containing 127 mM sodium chloride, 2 mM
magnesium chloride, 0.42 mM Na2HPO4, 11.9 mM NaHGO3, 2.9 mM KCI, 5.5
mM glucose, 10 mM llEPES, at a pH of 7.35 and 0.~% human serum albumin :
20 (HSA). These washed platelets are activated by addition of human a-thrombin
at a final concentration of 2 units/mll followed by thrombin inhibitor i-2581 at a ;
~inal concentration of 40~1M. To the ac~ivated platelets is added
paraformaldehyde to a final concentration of 0.50% and this incubated at room
temperature for 30 minutes. The lixed activa~ed platalets are th0n collected by
25 c~ntrifugation at 650 x g for 15 minutes. The platelet pellets are washed four
times with the above Tyrode's-d.35% HSA buffer and resuspended to 2 x 1 o8
cells/ml in the same buffer.
e
The fixed astivated platelets are incubated with a selec~ed dose of the
compound to be tested for platelet aggregation inhibition ~or one minute and
aggregation initiated by addition of human fibrinogen to a final concantration of ::
250 llg/ml. A pla~ele~ aggregation profiler Model PAP-4 is used to record the
platelet aggregation. The extent of inhibltion of aggregation is expressed as
the percentage of the rate o~ aggregation observed in the absence of inhibitor. ~ :
IC~o, i.e., the amount o~ inhibitor required to reduce the aggregation rate by
~ ~ , '
\~'O 92/1719~ PCI/US92/026?i-
~ 21~7~g
50%, is then calculated for each compound (see, for example, Plow, E.F., et al.,Proc. Natl. Acad. Sci., USA 8~, 8057~8061 (198$)).
Fi~y
Platelets are washed free of plasma constituents by the albumin density-
gradient technique of Walsh, P.N., et al., Br. J. Haematol. 281-296 (1977), as
modified by Trapani-Lombardo, V., et al., J. Clin Invest. 76, 1950-1958 (1985).
In each experimental mixture platelets in modified Tyrode's buffer (Ru~lgeri,
10 Z.M., et al., J. Clin. Invest. 72, 1-12 (1983)) are stimulated with hurnan a-thrombin at 22-25C ior 10 minutes (3.125 x 1011 platelets per liter ancl
thrombin at 0.1 NIH units/ml). HinJdin i~ then added at a 25-fold excess
(uniVunit) for 5 minutes before addition of the 1251-labeled fibrinogen and the
compound to be tested. After th0se additions, the final platelet count in the
15 mixture is 1 x 1011/liter. After incubation for an additional 30 minutes at 22-
25C, bound and free li~and aro s~parated by centrifuging 50,ul oF tha mixture
through 300~1 of 20% sucrose at 12,000xg for 4 minutes. The plat~let pellet is
then separatad frem the rest of the mix~ur~ to det~rmine platelet-bound
radioactivity. Nonspecific binding is measured in mixtures containin~ an :
20 exoess of unlabsled ligand. Wh~n binding curves a~ analyzed by Soatchard
analysis, nonspecific binding is d~rived as ~ fitted parameter from the binding
isotherm by means of a compu~crized program (Munson, P.J., Methods
Enzymol. 92, 542-576 (1983)). To d~termine the concentration of each
inhibitory compound necessary to inhibit 50% of fibrinogen binding to
25 thrombin-stimulated platelets (ICso), ~ach compound is tested at 6 or more
concentrations with 1251-labelèd fibrinogen h~31d at 0.176~1moltliter (60~1g/ml).
The IC50 is derived by plotting resiclual fibrino~en binding against ~he
logarithm of the sample ccmpound's concentration.
Compounds of the present invention exhibit marked activity in lhe
foregoing tests and are considered useful in the prevention and treatment of
thrombosis associated with certain disease states. Results of testing of
compo~nds of the present inventi~n by the above methods are presented in
the Table I below.
The comp~unds listed in Table I are prepared by the methods described
herein, by analogous rnethods, or by methods known in the art. Mass spectral
.
;,'. ;~, 1 .. . .
WO 92/1719~ PCI/USg2/026~/ .
~ 3~ 44 ~`
analysis, where provided, is by Low Resolution Fast Atom Bombardment with
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One skilled in the art will readily appreciate that the present invention is
well adapted to carry out the objects and obtain the ends and advantages
mentioned, as well as those inherent therein. The compounds, compositions,
and methods described herein are presented as representative of the preferred
5 embodiments, or intended to be exemplary and not intended as limitations on
the scope of the present invention. Changes therein and other uses will occur
to thoise of skill in the art which are encompassed within the spirit of the
invention or defined by the scope of the appended claims.
