Note: Descriptions are shown in the official language in which they were submitted.
1 3 ~ 7
TITLE
Peptide Boronic Ac~d
Inhlbitort of ~rypsin-Like Protea6e~
BAC~GROUND OF THE I~v~.ION
rield of the Invention
The pre6ent invention relate6 generally to
C-termlnal alpha-amlnoboronic acld derlvatlve6 of
ly61ne, ornlthlne, and arglnlne, homoarglnine and
corretpond~ng ~tothlouronium analogt thereof, and their
u6e as inhibitort of tryptin-l~ke ter~ne proteates tuch
as thrombin, pla6ma kalllkreln and platmln.
Background
The activlty of ~any biolog~cal 6y6tems i6
medlated by hydrolytlc or proteolytic enzyme6 that
cleave precur6er proteln6 at 6peclfic location6. Four
cla6te6 of the6e enzymcs ex$6t, metallo, thlol, acid and
6erlne protease6. System~ tuch a6 blood coagulation,
fibrinoly~i6, complement, and kall~krein-k~nin are all
regulated by a ~ubclat6 of 6erlne protea6e6, thc
tryp6in-~i~e protease~, a group of enzymet that have
a primary 6peclflclty for arginyl or ly6yl re61due6.
Wlthln each cla6t~ the mech~ni6~ of actlon and
the actlve ~lte re61due6 of the enzymes a~ well a6 thelr
su6ceptibillty to cla6s 6peciflc inhibltor6 are ~lmllar.
The ability of a compound to effectively inhibit a
particular protea~e or a particular subclas6 of
protea6es, however, ls strongly dependent upon the
6tructure and composition of the compound.
A great deal of research has been done ln the
area of protease inhibition, and a number of rerearcher~
ln thir. area have experimented with boron-containing
inhibitor~.
-- 1 -- *
2 1 ~ 9 7
8henvl, U.S. 4,537,773 ~1985), for example,
report6 that alpha-aminoboronlc acld analog6 of a~ino
acidc contalning aliphat~c and aromat~c alkyl ~lde
chalne are effective ~nh~b~tor6 of metalloenzy~ec. ln
add~tion, Shenvi et al., U.S. 4,499,082 (1985) dltclo6e
that alpha-am$noboronic acids ~ncorporated lnto pept~de6
lnh~bit ~erinc proteases who6e primary cpecific~ty
requlrement6 are ~et by neutral tide cha~n6, ~uch a6
pancreatic and leukocyte ela6ta6e, chymotryp6~n, and
cathep6in G. Thi6 latter patent dl6close6 tetrapeptide6
compr~ng C-terminal alpha-aminoboronic acid retidue6
ac potcnt, re~er6~ble lnhibitor~ of ~uch proteolytic
enzymec. ~The peptides dl6closed, however, did not
lnclude C-term~nal alpha-aminoboronic acid ret~duet of
lytlne, ornithine, arginine, homoargin~ne or any
corre6ponding ~60thiouron$um talts.
~ oehler et al., B~ochemi6try 10: 2477 (1971)
report that 2-phenyl-ethaneboronic acid ~6 an lnhlbitor
of chymotrypcin. Matte~on et al., J. Am. Che~. 80c.
103: 52~1 (1981), de~cribe the 6ynthesit of
(R)-l-acetamido-2-phenylethane boronic acid and ~tt u6e
a6 ~n ~nhibitor of chymotryp~in. $he author6 thow a
of 4~uM.
Lienhard ~n ~nzyme Inhibitor6 a6 Drug6,
~andler, ed., Unlver6ity Park Pret6, Baltlmore pp.43-51
(1980) ~peculatc6 that pept~de analog6 of alpha-
aminoboronic acid~ will be potent inhibitort of terine
and thiol proteases.
Additional disclo~ures include tho6e of ~inder
et al., J. Med. Chem. 28: 1917-1925 ~1985), which
describe~ the N-acyl and dipeptide boronic acids and
difluoroborane analog6 of phenylalanine, phenylglycine,
alanine, valine, and i601eucine, and Matteson,
Organometallics 3: 12~4-1288 ~1984) which de~cribes the
~ynthesi~ of alpha-amido gama-substituted boronic
esters. ~he latter author6 state that these compound~
3 13~9897
were prepared a6 po661ble preeur60r6 to boron$e aeld
analog6 of arqin~ne and prollne.
Tryp61n-llke protea6e6 are extremely important
in eontrolling a number of phy6~010qleal proee~re6. For
a dl6eu6610n of 6ueh ~etlvlty, tee ~Protea6e~ and
alologleal Control~, Releh, Rlfkln and Shaw ed6., Cold
Sprlng Harbor Pre66 (1975). Thrombln, one type of
tryp61n-like protea6e, ha6 a elear ~nd deei6ive role in
the blood eoagulation procet6. ~lood eoagulation ~ay
oeeur through either of two ea6eade6 of zymogen
aetlvatlon6. The la6t protease in e~eh of the6e
pathway6 i6 thrombln, whleh aet6 to hydrolyze flbrlnogen
to form flbrln, whieh ln turn aggregates to form a blood
elot. Thl6 thrombln eatalyzed hydroly616 i6 e66entlal
to the blood eoagulatlon proee66.
Pla6ma kallikrein, another trypsln-like
protea6e, 1~ al60 involved ~n the blood eoagulation
proee66, 6peeifieally in the initlation of one of the
blood eoagulation pathway6. Al60, kalllkrein aet6 on
klninogen to llberate the nonapeptlde, bradyklnln.
Bradyk~nin is a hypoten6ive peptide that i6 a660eiated
wlth pa~n. ~n additlon, kalllkreln i6 thought to have
other blologleal funetlon6. Reeent information ~ugge~t6
that pla6ma kallikrein ~6 lnvolved in ~nflammation.
Baumgarten et al., J. rmmun. 137: 977-982 (1986), for
example, report elevated levels of kinin and kallikrein
in allergie individual6 ehallenged with allergen.
Wachtfogel et al., Blood 67: 1731-1737 (1986) report
that pla6ma kallikrein aggregate~ human neutrophil6 and
relea6e6 neutrophil ela6ta6e. The relea6e of ela~ta6e
and accompanying elasta~e-mediated ti66ue de6truction
are event6 a6~0ciated with the proce~6 of inflammation.
The de~ign of ~pecific inhibitor~ of
tryp6in-like enzyme~ to control biological proce66e~ i6
not a new concept. Particular effort~ have been made in
the preparation of inhibitor~ of thrombin to replace
- 3 -
13398~7
heparin ln treatment of thrombo6i6 without the ~ide
effect~ ae60clated with heparln therapy, eee ~ark~ardt
TIPS 153-157 ~1980) and Green et al., ~hro~b. ~e-. 37:
145-153 (1985). Highly effect~ve peptide chloromethyl
ketone6 have been prepared for a number of tryp~ln-llke
protease6 by ~ettner et al., Methods ln Enzy~ology 80:
826-842) (1981). One ex~mple, H-(D)Phe-Pro-ArqCH2Cl, ls
highly effective ~n the ~nhibit~on of thrombin (~l ~ 37
nM), and, a6 shown by Shaw et al., U.S. 4,318,90q
(1982), is effective ~n the prevention of coronary
thrombosis in a rabbit model. Similarly, Ba~usz et al.,
~nt. J. Peptide Protein Res. 12: 217-221 (1979) report
the peptide aldehyde, H-~D)Phe-Pro-Arg-H, ~e an
effectlve lnhibltor of thrombln (~ - 75 nM) and Tremoll
et al., Thromb. Re~. 23: 549- 553 (1981), report that a
related co~pound, Boc-(D)Phe-Pro-Arg-H, reduces the ~ze
of venous thrombo~ n rats.
Sub~tituted arginine amides composed of
6econdary amines have also been shown to be effect~ve
inhib~tor~ of thrombin. ~ikumoto et al., ~ochemi6try
23: 85-90 (1984) report that (2R,4R)-4-methyl-llN~-
((3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)-ulfonyl}-~-
arginyll-2-piperidinecarboxylic acid ~6 an ~nhibitor of
thrombin (~ 9 nM). As reporte~ by Green et al.,
Thromb. Res. 37: 145-153 (198S), this lnh~bitor
~ncreases the prothrombin times of pla6ma in vitro blood
coagulation as6ay~ 2-fold at 1 ~M, and ~t ~ clai~ed a6
a fibrinolytic enhancing agent to be uced ~n combln~tion
w~th tis~ue plasm~nogen activator Yosbikun~ et al,
European Patent Application 0,181,267 (1986). Finally,
Sturzebecher et al., Thromb. Res. 29: 635-642 (1983) and
~ai~er et al., Thromb. Res. 43: 613-620 (1986) report
that N-alpha-(2-naphthyl~ulfonyl-glycyl)-4-
amidinophenyl-alanine piperidide i~ the most effective
known inhibitor of thrombin (Rl - 6 nM), and demon~trate
that i~ in vivo efficacy in mice and rat~.
1:~39897
De6plte the foregoing, new and better cla6~e6
of lnhibltor~ of thrombln and other trypsln-llke enzyme6
are needed to provlde potentlally valuabl~ therapeutlc
agents for treatment of blood coagulatlon dl~order~,
lnflammatlon and other mammalian ailment6. The preeent
~nvention 16 directed to th~t end.
SUMMARY or THE INVENT~ON
The pre~ent lnvent~on prov~de6 compound6 of
the formula
yl
R~--[ (A~ )~ (A2 )~, (Al )o ]~--NH-:H-B/
_2 y2
lFORMULA Il
wherein
Y~ and y2, lndependently, are -OH or r or,
taken toqether, form a ~olety derlved from a d~hydroxy
compound having at lea6t two hydroxy group6 6eparated by
at lea~t two connecting atom6 ~n a cha~n or ring, ~aid
chain or rlng compri61ng l to a~out 20 carbon atom6 and,
optlonally, a hcteroatom whlch can be N, S, or Ot
R2 ~6 a ~ub6tltuted alkyl 6elected from the
group con6i6tlng of -(CH2),-X, - CH(CH, )-(CH2 )2-X~
-CH2 -CH ( CH~ ) -CB2 -X, - ( CH2 ) 2 -CH ( CH, ) -X, and
-(CH2 ),-CH(CH, )2-X, where X 16 -NH2, -NH-C(NH)-NH2 or
-S-C(NH)-NH2, and z i6 3 to 5;
n, o, p, and q are, independently, either l
or O;
Al, A2 ~nd A3, independently, are amino ac~d~
of L- or D-configuration relected from the group
con~isting of Ala, Arg, Asn, Asp, Cy6, Gln, Glu~, Gly,
Hi6, Ile, Leu, Ly~, Met, Phe, Pro, Ser, Thr, Trp, Tyr
6 13~98~7
and Val~ and
Rl ~- a peptlde comprl6cd of I to about 20
amlno ac$dc, an acyl or a 6ulfonyl group comprl-ed of 1
to about 20 carbon atom6, H, or an N-term~nal protect~n~
qroup;
or a phy6iologically acceptable ~alt thereof
~ he lnvention al60 pro~lde6 compo~ltlon6
compr~61ng one or more of the foregolng Formula I
compound~, and method6 of u6ing 6uch compound6 or
compo6itlon6 ln the inhibltion of tryp61n-llke ~erine
prote~er, ~uch a~ thrombln and plasma kallikreln, and
ln the treatment of aberrant phy6iologlcal condit~on6,
~uch a6 tho6e ~nvolvlng blood coagulat~on dl60rder6 and
$nflammation, which are mediated by tryp6in-llke
protea6ec
Further, two cla66e6 of lntermediate6 to the
foregoing compound~ are provided ~he fir6t 6uch cla6
of lntermediate~ include~ compound6 of the formula
NH2-:H-B-Y3 HW
lFORMULA Il¦
wherein
Y3 15 a moiety deri~ed from a dihydroxy
compound having at lea~t two hydroxy group~ ~epar~ted by
at lea~t two connecting atoms in a chain or ring, 6aid
chain or ring compri~ing 1 to about 20 carbon atoms;
R3 iS a ~ubstituted ~lkyl selected ~rom the
group consisting of -(CH2),-Wl, -CH(CH3)-(CH2)2-Wl,
-CH2-CH~CH3)-CH2-Wl, -(CH2)2 -CH ( CH, ) _Wl ~nd
-(CH2)2-CH(CH3 )2 - Wl;
7 1~8~7
W and W~, lndependently, are Cl or Br~ and
Z ~ 3 to 5.
The second class of $ntermediates lncludec
compounds of the formul~
Rl-¦(A~)~(A2)~(Al)oln-NH-CH-B-Y3
¦FORMULA III
wherein
Al, A2, A~, Y~, Rl, n, o, p and q are as
prevlously def$ned;
R~ is a subst$tuted alkyl ~elected from the
qroup cons$st$ng of -(CH2),-W2, -CH(CH3)-(CH2)2-W~,
-CH2-CH(CH~)-CH2-W , -(CH2)2-CH(CH~)-W , and
-(CH~)2-CH(CH~)2-W ;
W2 ~s Cl, Br or N~ and
z ~ 3 to S.
BRIEF DESCRIPTION OF THE FIGURE
F$gure 1 ~hows a plot of relative clott$ng
tlmes vercus ~nh$b$tor concentratlon for two ~nhlb~tors
of the ~nvent$on, H-(D)Phe-Pro-boroArg-Cl0~ nd
Boc-(D)Phe-Phe-boroArg-Cl0Hl~. The data for F$gure 1
~as obtained from Tables 3 and 4. Relat$ve clott~nq
t$me is the activated partial thromboplastin times
(APTT) or the prothrombin times (PT), as the ca~e may
be, in the presence of inhibitor, divided by the APTT or
the PT, respectively, $n the absence of the $nhibitor.
The $nh$bitor concentration is shown in micro molar.
DETAILED DESCRIPTION OF THE INVENTION
The principal compounds of the pre~ent
invention, the Formula I compound, are N-acyl and
8 ~339897
peptide derlvative~ of alpha-aminoboronic acid~ in which
the C-terminal re~idue consi6t6 of ly~ine, ornlthine,
and arglnine, homoarginine and correspond$ng
$~othiouronlum analogs thereof. The~e compound~ are
characterized by their potency a~ lnhibitor~ of eertain
tryp61n-like proteolytic enzymes, notably human
thromb$n, pla~ma kallikrein and pla~mln.
The ac$d terminal boron of the pre~ent
compounds can optionally be in the form of an
unprotected boronic acid, that is, where Y~ and Y~ each
are -OH, or borane dlfluoride, that is, where yl and Y~
each are -F, or combinations thereof. Alternatively,
the terminal boron can be protected with a w$de variety
of protect$ng group~ wherein Y~ and Y~ are taken
together (-Y~-Y~-) to form a mo$ety.
Sultable protect$ng group6 whereln yl ~nd y2
are _y~ _y2 _ $nclude moiet$e~ derived from compound6,
~ princ$pally dlol6, having at lea6t two hydroxy group6
~eparated by at least two connecting ~toms in a chain or
ring. The term chain denotes both a branched or
unbranched moiety. The cha$n or r$ng 1~ compr$~ed of 1
to about 20 carbon ~toms and, optionally, and may
$nclude a heteroatom wh$ch can be N, 8 or O.
Contemplated compounds within the foregoing descrlptlon
$nclude, for example, pinanediol, p$nacol,
perfluorop$nacol, ethylene glycol, diethylene glycol,
catechol, 1,2-cyclohexanediol, 1,3-propanediol,
2,3-butanediol, 1,2-butanediol, 1,4-butanediol,
glycerol, diethanolamine and other amino alcohol6, ~nd
other equivalents apparent to those skilled in the art.
As used throughout the specification, the
following abbreviations for amino acid residue6 or amino
acid6 apply:
Ala - L-alanine
Arg . L-arginine
Asn - ~-a~paragine
9 1~398.~7
A6p - ~-aspartic acid
Cy6 - L-cygteine
Gln ~ L-glutamine
Glu - L-glutamic acid
Gly - glycine
Hifi - L-hi6tidine
Ile - L-i601eucine
Leu - L-leucine
~ys - L-ly6ine
Met - L-methionine
Phe - L-phenylalanine
Pro - L-proline
8er - L-6erine
Thr - L-threon~ne
Trp ~ L-tryptophan
Tyr - L-tyro6ine
Val - L-val$ne
Where prefixed by a ~D~, the foregoing abbrevlationg
~nd~cate ~n ~mino acid of D-configuration. Where
pref~xed by a ~D or L~, the the foregolng abbreviation6
~ndic~te that the ~mino acid can be of either the D- or
the L-configuratlon.
~N-terminal proteeting group,~ ~6 ueed hereln,
ref~r~ to ~arlous amino-terminal protecting groups
employed in peptide 6ynthe~ie. Example6 of ~uitable
qroupt ~nclude ~cyl protectlng group~, for ex~mple,
for~yl, ~cetyl (Ac), benzoyl (Bz), trifluoroacetyl, and
methoxy6uccinyl (MeOSuc); aromatic urethane protect$ng
qroup~, for eXample, benzyloxcarbonyl (Z); and aliphatic
urethane protecting groups, for example, tert-
butoxycarbonyl (Boc) or adamantyloxycarbonyl. Gros6 and
Mienhoffer, eds., The Peptide~, Vol. 3: 3-88 ~1981),
Academic Pre6s, New York l9B1, di6clo~e numerous
6uitable amine protecting groups.
The following represent preferred N-terminal
protecting groups Rl:
lo 13398~
~c - (cH3)3cO~-
Bz - ~ C-
Z ' ~ 2~~-
Compound6 of the lnventlon havlnq elde-ehaln
amlno group~, for example, where Al, A2 or A~ are ~ye or
Arg, e~n optlonally eonta~n eu~table N-termlnal
proteetlng groupC attached to the 61de ehalne;
el~ll~rly, ~mino acld reeldue6 havlnq aeldlc or hydroxy
e$de ehalnt ean be protected ln the form of t-butyl,
ben2yl or other su~t~ble e6ter~ or ethere.
Ae noted prevlouely, R' refer~ to an alkyl
group eomprleed of 3 to 5 earbone attaehed to an ~lno,
guanldlno, or l~othiouronlum group. Preferrably, the ~2
le -(CH2),-~. A ~ore preferred value of R2 le -(CH~ X
where ~ 16 3 to 4. Example~ of ~ore preferred ~aluee of
~' ~nclude 3-guanidlno-propyl, 3-amino-propyl, and
4-amlno-butyl. Mo~t preferred 16 3-guanidlno-propyl.
Abbre~tlon6 and terms preflxed by ~boro-
~lndle~te amino acid~ of Formul~ S whereln the ter~lnal
earboxyl group -CO~H ha~ been repl~ced by ~ boronie
functionality
--B/
\y2
~ hu6, ~boroarginine~ or ~boroArg-~ referr to
boronic ~cid ~nalog~ of ~rginine; ~borolycine~ or
-- 10 --
ll 133~37
~bo~oLy6-~ refer~ to boronic ac~d analog~ of ly~ine~ and
~boroornithine~ or ~boroOrn-~ refer6 to boronlc ac~d
analog of orn~thlne. ~he pref$x ~homo~, a~ ln
~homoboroarg~nine~ or ~homoboroArg-~, refer~ to
boroarginine analog6 in which the ~ide chain ha6 an
additional methylene group. ~Irg~ refere to the
leothiouronlum analog of arginine or homoarglnine in
wh~ch the thiouronium qroup, -S-C(NH)NH2, replace6 the
guanidino group, -NH-C(NHJ-NH2, and ~boroIrg-~ or
~borohomo~rg~ the abbreviation for the corre6ponding
boronic acid analog.
In naming compound6 of the ~nvent~on, yl and
y2 are ~impl$fied by the 6uffix ~-F~ for the
difluoroborane6 (yl - Y~ - -F), ~-0~ for the
unprotected boronic acids (yl _ Y~ _ -OH), ~-C~Hl2~ for
the pinacol e~ter~ (yl and Y~, taken together, are
-C~Hl~), and ~-CloHl~ for the pinanediol e6ters (yl and
Y~, taken together, are -CloHl~).
The pre6ent invention al60 contemplates
phy6$010gically acceptable 6alt~ of Formula I. ~he6e
6alt~ include acid addition 6alts, for example, ~alt6 of
benzene 6ulfonic acid (BSA), hydrochloric acld ~HCl),
hydrobromic acid (HBr), acetic acid, trifluoroacetic
acid (TFA), 6uccinic acid, citric acid, or other
~uitable acid addition 6alt~. When employed ~n naming
compound6 of the pre6ent invention, the~e salt6 ~hall be
introduced in the compound name by a ~
Contemplated classe~ of compounds within the
~cope of the present invention include the following
amino acids of the D- or L-configuration. A fir6t
clas~ includes compounds wherein Al i~ Ala, Pro, Gly,
Val, Leu, Ile or Met, that is, an amino acid having a
neutral side chain. A second class includes compounds
wherein Al i6 Phe, Trp or Tyr, that i~, an amino acid
having an aromatic side chain. A third class includes
compounds wherein Al is Lys or Arg, that is, a ba~ic
12 1~3~7
amlno aeld, and a fourth ela6s lnclude~ eompound6
whereln A~ le ~er or ~hr, that le, an amlno aeld wlth a
hydroxy elde eha$n. F$nally, a flfth ela~e ~neludes
eo~pounds where$n Al ~s Asp, Glu, Asn or Gln, that 1~,
an a-lno aeid wlth an ae$dle or a earboxamldo ~lde
ehaln. ~referable ~aluee of Al ~ub6tltuents ~nelude
Ly6, Phe, Pro, Ala, Leu, Gly, Glu, Val, Thr, Sle, Met,
~yr, ~rp, Arg, Aep, A~n and Gln. One preferable elas~
of 6ueh 6ubctltuent6 $neludes Ly6, Phe, Pro, Ala, Leu,
Gly, Glu, Val and Thr.
She foreqolng pr$nclpal ela66es lnelude
6ubclas6e~ eorre6pondlng to preferred values of R2, and
the6e subela66e6 are further subtended ~nto group~
deflned by preferred values for A2 and for N-termlnal
proteet$ng group ~1.
Preferred ~alues for A2 lnelude all amlno
aeld6 havlng a D-eonfiguration, ~06t preferably (D)Phe.
Other preferrable values for A2 are (D or L) Phe, (D or
L) Ala, (D or L) ~eu, (D or L) Pro, (D or L) Glu and (D
or L) Gly. Another elas~ of A2 6ub~t$tuent6 ~neludes
(~) Glu and (D) Val.
Preferrably, the rormula I eompound~ have a
total of two to four a~lno aeid sub6tltuent6, ~nelud$ng
the boro amlno aeld analog. A three am$no ae$d eompound
wh$eh has Pro $n the Al poslt$on and boroArg as the boro
amlno aeld analog, ~uch as
R -(D)Phe-Pro-boroArg ~
are particularily suited as inhibitors of thrombin,
having an IC 50 of significantly less than SnM.
- Obvious equivalents of the foregoing compounds
include compounds comprising less common or modified
amino acids, for example, norleucine, hydroxyprollne,
1~39~7
13
pyroglutamlc ac~d or other der~vatl~e6, lncludlng
re~due~ with ~lde chaln protecting group~, capable of
~ncorporatlon lnto the alpha-~m~no~oron~c acld peptlde~
of the pre6ent lnvent$on.
Spec~f~c compounds ~l~h~n the ~cope of the
~nvent~on, named ~n accordance w~th the conv~ntlon~
de6crlbed above, ~nclude the following examples:
Ac-(D,L)Phc-boroArq-CI0Hl~ BSA
Ac-Phe-boroOrn-C~0Hl~ BSA
Ac-Phe-boroArg-ClOHl, E~
H-(D)phe-pro-boro~rg-cloHl~-HBr-H
80c-(D)Phe-Pro-boroIrg-C10~-HBr
Ac-Phe-boroIrg-CIOHl~ ~Br
Ac-Ala-~y~(Boc)-boroOrn-C~ BSA
Ac-Ala-Ly~(Boc)-boroIrg-C10~l~-HBr
Boc-(D)Phe-Pro-boroArg-Cl0~ ~-BSA
Boc-(D)Phe-Phe-BoroIrg-C10~l~-HBr
~-(D)phe-pro-boroArg-cloHl~-Hcl
Boc-(D)phe-phe-boroorn-clo~l~ BSA
Boc-(D)Phe-Phe-boroArg-Cl0Hl~ BSA
Ac-Ala-Ly~(Boc)-boroArg-Cl0Hl~ BS
Ac-( D)Phe-Pro-boroArg-Cl~6 ~HCl
Ac-(D)Phe-Pro-boroArg-OH ECl
Boc-Leu-Gly-Leu-Ala-boroIrg-Cl0Hl~ HBr
Boc-~eu-Gly-Leu-Ala-boroOr~-ClOHl~-BSA
Boc-Leu-Gly-Leu-Ala-boroArg-ClOHl~ BSA
Bz-Pro-Phe-boroOrn-Cl0Hl~-BSA
Bz-Pro-Phe-boroArg-Cl0Hl~-BSA
Boc-Ala-Phe-(D,L)boroIrg-C6Hl 2 ~HBr
Bz-Glu(oBu)-Gly-boroIrg-cloHl6~ HBr
Bz-G1u-G1y-boroArg-Cl0Hl6-~SA
Bz-Glu~OBu)-Gly-boroOrg-ClOHl 6 ~BSA
Bz-Glu~OBu)-G1y-boroArg-ClOHl6-BSA
Bz-Pro-Phe-boroI rg-Cl o Hl 6 -~Br
Z-Phe-Gly-Gly-boroIrg-C~0~l 6 HB
Boc-Ala-Phe-(D,L)borohomolrg-C6Hl2 ~HBr
- 13 -
lq 1 ;~ 3 7
Bz-Pro-Phe-boroArg-OH-HCl
Bz-Pro-Phe-boroArg-r
~-(D)phe-pro-boroArg-cloHl~-2H
H-(D)phe-phe-boroArg-cloHl~-2
Ac-Al~-Lys-boroArg-cloHl~ 2HCl
H-Leu-Gly-Leu-Ala-boroArg-Cl0H~-HCl-BSA
Boc-Ala-Phe-(D,L~boroLys-C~H1~-HCl
~-Al~-Phe-(D,L)boroLys-C~Hl~-2HCl
Boc-(D)Val-Leu-boroLys-C~ Hl 2 ~HCl
Ac-Phe-boroLy~-C~H~ HCl
Bz-Glu-Gly-boroArg-Cl0Hl~-BSA
H-(D)Phe-Phe-boroIrg-C1OHl~-2HBr
H-Leu-Gly-Leu-Ala-boroIrg-ClOHl~-2HBr
~-Ala-Phe-(D,L)boroIrg-C~Hl~-2~Br
Bz-Glu-Gly-boroIrg-Cl0HI~-HBr
H-Al~-Phe-(D,L)boroHomoIrg-C6Hl~-2HBr
Ac-Al~-Lys-boroIrg-C10HI~-2HBr
Bz-boroIrg-C~Hl 2 ~HBr
Bz-boroOrn-C~H1~-BSA
Bz-boroArg-C~Hl~-BSA
Ac-Leu-Thr(OBu)-boroOrn-Cl0Hl~-BSA
Ac-Leu-~hr(08u)boroArg-CloH~BSA
Ac-Leu-Thr-boroArg-ClOH~BSA
Ac-Lys(Boc)-pro-boroorn-cloHl6.BSA
Ac-Lys(Boc)-Pro-boroArg-C~0H~ 6 ~BSA
Ac-Lys-Pro-boroArg-ClOHl~ BS
Ac-Al~-Glu~OBu)-boroOrn-ClOHl 6 ~ BSA
Ac-Ala-Glu(OBu)-boroArg-ClOHl~-BSA
Ac-Ala-Glu-boroArg-ClOHl~ BSA
Boc-Val-Val-boroLys-C6 Hl 2 ~BSA
H-Val-Val-boroLy~-C6 Hl 2 ~BSA-TFA
Boc-(D)Phe-Phe-boroLys-C6Hl 2 ~ BSA
H-(D)Phe-Phe-boroLys-C6H1 2 ~ BSA-TFA
BoC-Glu-Phe~b~r~LYs~C6 Hl 2 BSA
PyroGlu-Phe-bo roLys-C6 Hl 2 ~ BS~
-- 14 --
' 13~98~
~ he lnvention al~o provide~ compo61tlone and
method~ for lnhibiting trypsin-llke ~erlne proteaee~,
1ncludlng but not llm1ted to thrombln, pla-ma kalllkreln
~nd pla~ln, ~nd for treatlng aberrant phy~lologlc~l
conditlons, lncluding but not llmited to blood
coagulation and lnflammatlon ln mammal~. The
compositlon~ of the present lnventlon comprl6e an
effectlve amount of a compound of Formula I and a
phy~ioloqlcally acceptable carrler or dlluent. In
practlcing the method of the inventlon, the compounds or
comporltlon6 can be u~ed alone or in comblnatlon wlth
one another, or ln combination with other therapeutlc
agents. ~hey can be admini~tered orally, parenterally,
lntravenously, subcutaneously, lntramuscularly,
colonlcally, rectally, na~ally or intraperitoneally in a
variety of do~age form~. The u~eful do~age to be
admini6tered and the mode of adminlstration wlll vary
dependlng upon the age, weight and mammal treated, and
the partlcular compounds employed. Typlcally, therapy
16 lnltlated at lower do~age level6 with do~age belng
lncrea~ed until the de~ired effect 1~ acheived.
The pre~ent invention further contemplate~ two
clas~es of critical intermediates to compound~ of
rormula I, the compounds of rormula~ II and III. The
Formula II lntermediate~ include~ compound~ of the
formula
NH~-Cs-s-Y~ sW
~3
[FORMULA II]
wherein
Y~ i~ a moiety derived from a dihydroxy
- 15 -
16 13 3g~7
eompound havlng at lea~t two hydroxy group~ ~eparated by
at least two eonneetlng atomr ln a ehaln or r~ng, cald
ehaln or rlng eomprlslng 1 to about 20 earbon ~tomcJ
R3 lt a eub~tltuted alkyl 6eleeted from the
group eonc~tlng of -(CH, ),_Wl, -CH(CH, )-(CH2 )2-Wl,
-CH2 -CH ( CH, ) -CEI, _Wl, - ( CH, ) 2 -CH ( C}l, )_Wl and
--(CH2 12--CH(CH3 )2--Wl
W and Wl, lndependently, are Cl or Brs and
2 ~ 3 to 5.
A partleularly preferred eompound of rormula II ~c one
wherein R3 ~ -(CH2),-WI and z 16 3 to 4.
A ~eeond ela6s of ~ntermediate~ ~nelude~
eompoundc of the formula
Rl--t (A3 )~ (A2 )~ (Al )o 1.l--NH-CH-B-Y3
¦ FORMULA I I I
whereln
Al, A2, A~, Y3, Rl, n, o, p and q are.a~
prevlously deflned;
R~ ~c a cubctltuted alkyl celeeted from the
group eoncl~tlng of -(CH2)~-W2, - CH(CH, )-(CH2 )2-W
--CH2--CH ( CH3 )--CH,--w2, --( CH2 ) 2 -CH ( CH3 )--w2, and
--(CH2 )2-CH~CH3 )2--W2;
W2 1~ Cl, Br or N3 and
Z ~s 3 to 5.
Contemplated elac~e~ of eompounds within the ~cope of
Formula III are ac de6cribed for the analogou~ Formula I
compound~. A particul~rly preferred compound of Formula
III is one wherein R is -(CH2)~-W2 ~nd z ic 3 to 4.
17 ~ 89
Prepar~tt~on of Snh~b~tore
Te~peratYre6 are ln 'C. ~he numbered
co~tpound6 chown ln the echem~t~c entltled ~ynthe~16
8che~te~, lllu6trated below, are referred to ln the text
accordlng to thelr re6pect~e number~. "NM~, a~ u6ed
here~n, ~qn~fie6 proton nuclear ~gnetlc re60nence.
- SYNTHES I S SCHEME
~..C~-c~-C~ H-B ~ _ Br-CH~-CH~-CH~-B/
Plc -~d ol 0
Br-CH~-CH~-CH~-B~
2 ~ /
CHCI~-LI- Br-CH~-CH~-CH~CHCI-80~-C,OH~
t(CH,), Sl
~CH,),5112Ntl-
8rCH~CH~CH~CHBO~.C,OH"
H~CI
~qu Ha
Elr~(CH~)~-C'H-BO~-C~OH"
mp 14~-14S-C
peptlde P~ptlde.NH.CH-~o,-C,OH"
(CH~)~
6Br
No-N~ P-ptlde-NH-CH-BO~-C,OH"
(CH~
N~
18 ~3~38~
H~ F~C P-ptide Illl C'H-BO~-C~oH~
O-SO~H ~CH~)~
NH~ OSO~-
cyon-mld- Pepl do IIH CH-80~-C~oH~
elh~nol 100 C (CH~)~
~H
C =NH
NH~- OSO~-
OH
lon F~cl~-n~- Peptld~llll ÇH-B~
or BCI~ ¦ OH
~CH~
NH
C=NH
NH~- Cl
~queou~ HF Pepl do ~IH-CH-B~
(CNHH~)~
C=NH
NH,- cr
6 Ihicu~e- Peplide-NH-CH-BO~C~0H
(CH~)~
C=NH
NH~- 8~-
-- 18 --
13~8~
Followlng the procedures 6et forth hereln, the rormula I
compounds of the present inventlon are obtalnable in a
hlgh Yceable purlty, that i6, an 80-100% pure form.
Startlng ~aterial6 are avallable ln hlgh
purlty from chemlcal supplier6 or can be readlly
synthcslzed by procedurc6 known to those 6klllcd in the
art. The Synthesls Scheme show6 the general order in
which the compounds of thi6 invention were synthe61zed.
Compounds 1-~ are prepared a6 descr$bed by Matteson et
al., Organometallics 3: 128q-1288 (1984), except that
the procedure was modlfied to allow large scale
preparation.
Compound 1 i6 prepared by hydroboration of an
alkene hallde w~th catechol borane. The components are
heated in tetrahydrofuran or some other lnert 601vent
and the product ls i601ated by distlllat~on. The
halo-6ubstituted alkyl boronic acid-catechol cster ~6
transe6terified by allowing it to react with a suitable
diol (alpha-pinanedlol, p$nacol, 2,3-butandiol, etc) in
tetrahydrofuran. (~)-Alpha-plnanedlol ls preferred in
~iew of the ob~ervations ~n Matte~on et al., J. Am.
Chem. Soc. 103: 5241 (1981) that steric restraint6 ~n
the molecule allow the 6tereo 6peciflc addltlon of the
-CHCl- group ln formatlon of Compound 3 and the
6ub6equent ~ntroduct~on of an amino group in the ~L~
configuration. Structures 3-9 in the Synthesi6 Scheme
are shown with the p~nanedlol protectlng group. For
large scale preparations, the removal of catechol, a
product of the esterification reaction, is achieved by
crystallization from hexane, a solvent in which catechol
has limited ~olubility. Compound 2 is then purified
either by chromatography on silica gel, by dictlllation~
or is used without additional purification. Compound 2,
as the pinanediol e6ter i6 obtained in close to
analytical purity by the removal of solvent. Additional
purification can be achieved by ~ilica gel
-- 19 --
i~39~
chromotography. ror the plnacol e~ter of Compound 2,
flnal purlflcatlon by dl~tlllatlon 1~ preferred.
Compound 3 1~ prepared by the homologatlon of
2 u~lng C~C12-Ll~. Thlc reagent 1~ made by treatlng
~othylene chlorlde wlth n-butylllthlum ln
tetrahydrofuran at -100-. To Compound 2 i~ added 0.65
equlvalent~ of z~nc chlorlde at -100-. The mlxture 1
allowed to ~lowly warm to room temperatu~e and 16
~tlrred over night. Compound 3 i 6 obtalned after
evaporatlng solvent, then dl~olvlng the rerldue ln
hcxane, followed by wa~hlng the organic pha~e wlth
water, dry$ng ~t with magne~ium ~ulfate, and flnally
evaporatlng the hexane. Compound 3 1~ u~ed wlthout
further purlflcation when lt i~ protected a~ the
pinanediol e~ter and alternately, lt can be dl~tllled
~hen lt 1~ protccted a~ a pinacol e~ter.
Compound 4 ~ prepared by treating the
alpha-chloro-~ub~tltuted boronlc acld e~ter, Compound 3,
wlth [(C~ Sil2N Li~. Hexamethyldlsllazane ~
di-~olved ~n tetrahydrofuran and an equlvalent of
n-butylllthlum ~ added at -78-. The mlxture ~ allowed
to ~arm to room temperature and then, after recoollng to
-78-, an equlvalent of 3 18 added ln tetrahydrofuran.
The ~ixture i~ allowed to ~lowly come to room
temperature and to ~tlr over nlght. The
alpha-bl~[trlmethyl~llanel-protected amine 1~ ~olated
by evaporating ~olvent and adding hexane under anhydrous
condition~. In~oluble re~idue 1~ removed by flltratlon
under a nitrogen blanket yieldlng a hexane 601utlon of
Compound 4.
Compound 5 i~ obtained by cooling the hexane
~olution of Compound 4 to -78~ and adding three
eguivalent6 of hydrogen chloride. The ~olut$on
~lowly allowed to warm to room temperature and 16
~tirred for 1.5-2 h. Compound 5 i~ then i601ated by
filtration and i~ purified further by di~olvlng ln
- 20 -
21 1~3~8~7
chloroform and removing in601uble material. Compound 5i~ obtained a~ a white cry6talllne ~olid by removing the
chloroform by ~vaporation and cry~tallizing the r-~idue
for ethyl acetate.
The above proce~6 of converting Compound 3 to
Compound 5 6urpr~6ingly re6ult6 ~n analytically pure
preparation6 of Compound 5 wh$ch then allow6 Compound 6
to be obtalned without the difficulty normally
encountered ln coupling heterogenou6 material. The art
teache6 or rtrongly 6ugge6t6 that Compound ~ ha6 to be
purified prior to conver6ion to Compound 5 ~n order to
obtain pure 6ample~. The only known procedure for the
preparation of pure alpha-am$noboronlc ac$d6 1~ that
di6clo6ed in Shenvi U.S. q,537,773 and u6ed ~n 8henvi et
al., U.S. q,q99,082. In the Shenvi et al. di6clo6ure,
compounds analgou6 to Compound 4, except that they have
aromatic and alkyl 6ide chain~, are purified by
di6t$11ation. Compound 4 it un6table to the Shenvi et
al. dl~tillation and ~n altered product i6 obtained.
Compound 6, the N-acyl or N-peptidyl form of
Compound 5, can be prepared by two different route6.
The fir6t i6 a modification of the procedure de6cribed
by Matte6cn et al., Organometallics 3: 1284-1288 tl984)
in which Compound 4, prepared in ~itu (without
evaporation of ~olvent and removal of ~alt~ by
filtration), i6 treated wlth an equivalent of acetic
acld and an exce~6 of acetic anhydride to yield
N-Acetyl-NH-CHt(CH~)~BrlsO~-pinanediol. ~hi6 method i6
applicable to the coupling of highly reactive acid
chloride of N-Acetyl-phenylalanine (Ac-Phe-Cl) with the
modification that prior treatment with acetic acid i6
omitted. When ~cetic acid i5 added in conjunction with
Ac-Phe-Cl, extremely low yield are obtained which appear
to be due to the formation of the a mixed anhydride of
Ac-Phe and acetic acid and the subsequent chemically
preferred coupling which results in N-acetyl-NH-
1:~398~7
CH[(CH2)3Br]BO2-pinacol. Application of the mixed anhydride
procedure to the preparation of Compound 6 resulted in low
yields of the desired product and extensive problems in
purification. Thus, it appears that this method is
applicable to the coupling of alkyl, aryl, and N-protected
amino acids to Compound 4 by using the acid chloride method.
However, it should be noted that there are limitations due
to the requirement of the acid chloride coupling procedure.
First, the procedure is not readily applicable to peptide
coupling because of side reactions such as oxazolinone
formation limiting its application to a single amino acid
residue. Second, an acid stable protecting group is
required due to excess HCl generated during formation of the
acid chloride. Finally, racemization of amino acid residue
is inherent in the procedure.
The second method for the preparation of Compound 6 is
the coupling of an acyl group or N-protected peptide with
suitable side chain protection to Compound 5. This method is
clearly superior to the first since it is sufficiently
versatile to allow the synthesis of any peptide within the
limits normally encountered during peptide synthesis such as
insufficient solubility. Acid chlorides or other active
forms of acyl groups can be coupled. For peptides, the mixed
anhydride procedure of Anderson et al., J. Am. Chem. Soc. 89:
5012 (1967) is preferred. The mixed anhydride of N-protected
amino acids or peptides varying in length from a dipeptide to
tetrapeptide with suitable side chain protecting groups is
prepared by dissolving the given peptide in tetrahydrofuran
and adding one equivalent of N-methylmorpholine. The
solution is cooled to -20~ and an equivalent of isobutyl
chloroformate is added. After 5 min, this mixture and one
equivalent of triethylamine (or other stericly hindered base)
are added to a solution of Compound 5
23 1339~9~
dl~eolved ln ~lther cold chloroform or tetrahydrofuran.
The reactlon mlxture 16 routinely ~tlrred one hour at
-20' followed by 1-2 h of 6tirrlng at room tempe~ature.
Sn601uble ~aterlal ls removed by flltratlon, the ~olvent
removed by evaporatlon, and the re~due dls601ved ln
ethyl ~cetate. The organlc 601utlon lt wa~hed ~lth 0.20
N hydrochlorlc acld, 5% agueous todlum blcarbonate, and
~aturated aqueou6 60dlum chlorlde. The organlc phaee 18
then drled over anhydrous codlum culfate, flltered, and
~ub~ected to evapor~t~o~ to y~eld a partlal 6011d ln
~ott carec. For a number of compound~, further
pur~flcatlon of Compound 6 wa~ dee~ed unnece66ary.
Method6 which are appllcable for the purlflcatlon of
Compound 6 are 6illca gel chromatography,
cryttalllzatlon ln ~ome ca~e~, and gel permeatlon
chromatography uslng 8ephadexS~ LH-20 and methanol a~ a
eolvent. The latter method 16 preferred. Typically,
NM~ tpectra lnd~cated the -CH2-Br band at delta 3.45 and
a 6harp 61nglet band ~t delta 0.80-O.9S for one of the
~ethyl group ln the pinanediol protect$ng group or
~inglet at delta 1.3 for the p~nacol group.
The pept~de alkyl hallde, Compound 6, 16 then
converted to the alkyl azlde, Compound 7, by treatment
wlth two equlvalent6 of eodlum ~zlde in
dlmethylformamlde at 100- for 3 h. In all caset, thl6
reactlon appeared to go ~moothly wlthout alterlng
reaction condit~on6. ~he NMR 6pectrum of Compound 7 in
CDCl3 typically lndicated a delta 0.1-0.2 ppm upfield
6h~ft of the -CH2-Br on conver~lon to the azide.
Further purlfication can be obtained by LH-20
chromatography, but it 16 not neces~ary for a many of
the peptide~.
The boroOrnithine peptide~, Compound 8, are
prepared routinely by catalytic hydrogenation of the
alkyl azide~, Compound 7, $n the pre~ence of 10% Pd/C
and one equivalent of benzene ~ulfonic acid in alcohol.
24 13398~7
Hydrogenatlon~ are run on a Parr apparatu6. Alternate-
ly, the hydrogenation6 ean be run at ~tmo~pherle
pre~-ure and mlneral aeid6 can be ~ub~tituted for
benzene ~ulfonie acld It ehould be noted that lt
nece~ary to u~e peptlde proteetlnq group6 ~b~eh are
etable to eatalytle hydrogenation. 8ueh peptlde
protecting group~ are known to tho~e ekllled ~n the art
and are dl6cu~ed ln The Peptide~ (E Gro~6 ~nd J.
Melenhofer ed~ ) vol 3, Academlc Pre~, New York,
(1981). The preferred protecting group~ are the
t-butyloxycarbonyl qroup for amino group6, and t-butyl
ether~ and e6ters for hydroxy and carboxyllc ~eld ~ide
eha~n~. Other ~uitable protecting group6 include
dli60propylmethyloxycarbonyl, t-amyloxycarbonyl,
adamantyloxycarbonyl, biphenyli~opropyloxycarbonyl and
to~yl. St i6 expected that converslon of the azlde to
the amine by reductlon with other redueing agent6 ean be
achieved u~ing reagent6 ruch a6 ~tannou6 ehlorlde and
trlalkyl phosphlte6 a6 de6cribed by Maltl et al ,
Tetrahedron Lett , 27: 1423-1424 (1986) and ~oziara et
al , Synthe6i6, 202-204 (1985) The6e reagent6 are
expected to be compatible with peptlde protectlng group6
whleh are labile to eatalytlc hydrogenatlon. The
boroOrnlthlne peptlde6 are routlnely ehromatogramed on
SephadexS~ ~H-20 and are white amorphou6 601id6 after
trituration with ether.
BoroArg~nine peptlde6, Compound 9, ~re
prepared by allowing the corre~ponding boroOrnithine
peptide, Compound 8, to react with at lea6t a 4-fold
exces6 of cyanamide (50 mg/mL) in ab601ute ethanol at
100~ Initially the component~ are allowed to react 2-3
day6 under a blanket of nitrogen with a water cooled
conden6er in place Water cooling i6 di~continued and
the reaction mixture i6 allowed to concentrate 610wly
over a period of 6everal days ~he completion of the
reaction i~ determined by the progre~6ive increa~e in
2s 1~39897
the intensity of material stalning wlth Sakaquchi ~tain
for the guanidino group of the boro~rglnine moiety and
the disappearance of ~aterial staining pos~tl~e ~ith
ninhydrin ~tain for the amino group of the boroOrn~thine
moiety on rever6e phace th$n layer plate~ run ln
methanol:water (B5:15). Typically, the boroArglnlne
peptides streaked from the oriqin of the plate, the
boroornithine peptides traveled as di6crete cpots in the
middle of the plate, and cyanamide traveled with the
col~ent front allowing each component to be ldent~fled.
Specific stains for the guanidino group and the amino
group are co~monly w ed in peptide cynthesis. Compound
9 wa~ purified by gel permeation chromatoqraphy u~lng
Sephadex~ LH-20 and ~ethanol a~ a 601~ent. ~his
chromatographic step readily separates the boroArg$nine
peptides from low molecular weight byproducts and
unreacted cyanamide. In ~06t cases, no further
purification is needed. However, it is essential that
the quanidation reaction of Compound 8 be permitted to
run to completion since it is difficult, if not
impos6ible to separate a mixture of Compounds 8 and 9.
rinal products are obtained as amorphous white solids by
trituration with ether and, in most case, are of
analytical purity as determined by NMR, ~ass ~pectral,
and combustion analyses.
It should be noted th~t guanidation of
Compound 8 with cyanamide ha6 been found to be very
dependent upon reaction conditions. First, as discus6ed
above, it is important that the reaction be run
sufficiently long to result in relatively complete
conversion of Compound 8 to Compound 9. Reaction times
of up to 7 days and accompanying concentration of
reagents by slow evaporation of solvent~ are often
required. In an initial survey of reactions to
quanidate Compound 8, Compound 8 as the hydrogen
chloride salt, was refluxed with cyanamide in ethanol
- 25 -
26 133~7
for ~everal hour6. The de6ired product, Compound 9, wasnot detectable. Attempts to guanidate Compound 8 uc~ng
thc eucces6ful conditlon6 noted above except that
tetrahydrofuran was 6ubst~tuted for absolute ~thanol
failed to yield detectab~e product. Slmilarly, when ~n
~ttempt wa6 made to guanldate Compound 8 u6ing the6e
condit~on~, except that the benzene ~ulfon~c acid 6alt
of the am$no group of the boroOrnithine pept~de wa~
neutralized prlor to guanidation, Compound 9 wa6 present
only at a barely detectable level. The preferred
conditlon6 ~nvolve reaction6 with the benzene eulfonic
acid ~alt of Compound 8 (unneutrallzed). ~ucce~eful
reaction6 have al~o been run wlth the corre6pondlng
hydrogen chlor~de 6alt.
U~ual methods of guanidat~on of ornithine
peptide~, to yield the corre6ponding arginlne peptide6,
used by those 6killed in the art of peptide 6ynthesi6
are the neutralization of the amine of the ornithine
peptide and coupling with either ~-~lkyl or O-alkyl
l~ourea6 or guanyl-3,5-dimethylpyrazole nitrate, ~6
de6cribed by Barany et al., ~n The Peptide6 (E. Gro66
~nd J. Meienhofer eds) vol 2, pp. 169-175, Ac~demic
Pres~, New York,(1980). Bannard et al., Can. J. Chem.
36: l5q~-1549 ~1958) have surveyed different ~cthod6 of
guanldation of amine6 and found that guanyl-3,5-dimethyl
pyrazole is 6uperior to the u6e of S-methyl ~60urea and
conclude~ that guanidation with cyanamide ls
unacceptable although it i6 described in the early
literature. Reactions run with S-methyl isourea
hydrogen ~odide in ethanol and guanyl-3,5-dimethyl
pyrazole under a variety of conditions failed to
guanidate the boroOrnithine peptide. ~he lack of
reactivity in this case is probably due to the formation
of an internal Lewis acid base complex between the amino
group of the ornithine side chain and the boronic acid
ester. Synthesis of Compound 9 by the treatment of
- 26 -
2, 1339~
Compound 6 w~th guanldine ln ethanol was also an
unacceptable ~ethod of ~ynthe~is. Compound 6,
~pproxlmately 50% pure, was lcolated from the reactlon
of guanldlne wlth 6 ~n les6 than 1% yield.
~ he guanldlno group of boroArglnlne-plnanedlol
behave~ in a fashlon sl~ilar to the guanld~no group of
the natural amlno acld arglnine once it i6 lncorporated
lnto the ~olecule. For example, the alpha-amlno group~
can be ~electl~ely acylated with an anhydrlde wlthout
effecting the guanldino group of boroArginlne. Thu-, lt
lc our expectatlon that Compound 9 can be prepared by
the synthes$~ of H-boroArglnine-plnancdlol and
sub6equentially adding the N-protected form of the
peptlde portion of the molecule uslng the m~xed
anhydride procedure and ~imilarily, di-, tri-, etc.
peptide analogs contalning boroArginine can be extended
ln length by coupling additional amino acid6 or
peptldes.
Addltlonal purlficatlon of the protected
boroArginine peptldes can be achleved by lon exchange
chromatography on SP SephadexSn. The peptides ~re
di6solved ~n 20~ acetic acid and applied to the column
in lt6 H~ form. After wa6hing the column with 20%
acetic acid, product i6 eluted by running a gradient
from 0-0.3 N hydrochlorlc acid in 20% acetic acid. ~he
product 1~ eluted as a mixture of pinanediol ester and
free peptide boronic acid. A homogenous preparatlon ls
obtalned by treatlng the ~ixture with plnanediol under
anhydrous conditions and trituration of the product with
ether.
Two procedures have been developed for the
removal of the pinanediol protection group to yield the
free boronic acid, Compound 10. The first is a
modification of the above purification procedure in
which a mixture of the free boronic acid and pinanediol
ester are co-eluted from the ion exchange column. These
133~8g7
28
compound6 are readlly 6eparated by chromatography on
LH-20. Thc ~econd procedure 1~ a modlflcatlon of the
method of ~lnder et al., J. Med. Chem. 28: 1917-1925
(1985). The boronic acld ester i6 treated wlth a 2-3
fold excer6 of boron trichlorlde ln methylene chlorlde
for 5 mln at -78- and tbe mlxture 16 allowed to ~tlr 15
~ln ~n a 0- ~cc bath. Water 16 ~lowly added to
hydrolyzc exce6~ boron trlchlorlde to boric acld and
hydrochlorlc acld. The reaction 16 further dlluted wlth
20% acetlc acld to ~leld a final concentratlon of
hydrochlorlc ac~d of 0.05 M. The concentratlon of
hydrochlor~c ac~d ~6 ba6ed on the ~nitial quantlty of
boron trichlorlde u6ed ~n the reaction. The aqueou6
pha6e ~6 applled to a SP-Sephadex~ column and product
~6 eluted a6 the hydrochloride 6alt a~ de6crlbed above.
The free boronlc acld peptlde6 were obtalned a6 whlte
amorphou~ ~olld6.
Compound 10 can be converted to the
difluoroborane, Compound 11, u6ing a modlflcatlon of the
procedure of ~lnder et al., J. Med. Chem. 28: 1917-1925
(1985). The peptide boronlc ac~d ~6 treated wlth a
5-fold molar exce~ of 0.50 N aqueou~ hydrofluorlc acld
at room temperature. Exce~6 hydrofluorlc ac~d and water
are removed by lyophlllzatlon and the rerultlng colld 16
tr~turated wlth ether to yleld de~lred product a8 a
wh~te amorphou~ colld.
In the foregoing descrlptlon, the preparatlon
of the free boronic acid, Compound 10, i6 from
boroArginine-plnanedlol ester and the preparatlon of the
difluoroborane ~cid, Compound 11, i~ from Compound 10.
The procedure for the removal of the e~ter protectlng
group 6hould applicable to acyl peptides of
boroornithine, boroLy~ine, and boroHomoarginlne
protected a~ either pinanediol, pinacol, or other ester
protecting group. Similarly, the corre~ponding free
boronic acid~ can be converted to difluoroboraner..
- 28 -
13~8~7
29
The prcferred 6~ de chaln protect~ng group6 andN-termlnal protectlng group6 of the peptlde portlon of
~olecule~ are tho6e ~table to catalytlc hydrogenatlon
and llable to anhydrou~ hydrogen chlorlde or
trlfluoroacetlc acld. The6e criteria ~re readlly met by
the t-butyloxycarbonyl amino protectlng group and
t-butyl ether6 and e~ter6 for hydroxy and acldlc ~lde
chaln6. To remove there group~, the peptlde6 are
treated w~th 4 ~ hydrogen chloride ln dloxane at room
.temperature. The deprotected peptlde 16 1601ated by
cither evaporatlng 601vent or by preclpltatlon with
ether. Partlcular care rhould be taken wlth peptlde6
containinq ~n acidic 6ide chain to remove all hydrogen
chlorlde by evaporatlon. Thi6 ln~urer that the
boroArgln$ne peptlde lr maintalned a6 benzene ~ulfonlc
acld ~alt. Other peptlde can be l~olated ~ elther a
mixed hydrogen chlorlde-benzene ~ulfonlc acld salt or
~ost can be converted to the hydrogen chlorlde ~alt by
pa~6age through a anion exchange column ~n the Cl-~on
form.
I60thiouronium derivative6 of Compound 6 are
prepared by treatment of Compound 6 wlth thloure~ ~n
ab601ute ethanol to yield Compound 12, analoq6 of the
peptlde boroArglnlne e6ter6, Compound 10. Routlnely,
the alkyl halidc6 were allowed to rtlr wlth a 4-5 fold
exce~ of thiourea for ~everal day6 at roo~ temperature.
The product ~6 6eparated, when nece66ary, for unreacted
Compound 6 by trituration with ether. Compound 6 1~
readily 601uble ln ether for mo~t peptide6 whlle the
product i6 insoluble. Final purification, removal of
exces~ thiourea, i6 achieved by chromatography on
SephadexSn LH-20 in methanol and trituration with ether
to yield final product~ a~ hydrogen bromide ralt~. Side
chain and N-terminal protectinq group~ are removed by
treatment with anhydrou~ hydrogen bromide or other
anhydrous acid.
- 29 -
1~398~7
Bioloqical ActivitY
The biological activity of compounds of the
present invention is demonstrated by both in vitro
and ln vivo data pertaining to inhibition of synthetic
substrate hydrolysis by the trypsin-like enzymes, human
thrombin and plasma kallikrein, and inhibition of
physiological reactions catalyzed by such enzymes such
as blood coagulation and inflammation.
In the Examples which follow, the hydrolytic
activity of each enzyme is measured in both the
presence and absence of inhibitor and the percent
enzyme activity determined. It has been found that the
most effective inhibitors of both plasma kallikrein and
thrombin are slow-binding inhibitors whose
effectiveness progressively increases with time until a
steady state is reached. A steady state is reached
fairly rapidly and nears completion within 5 min.
Activity is evaluated between 10-20 min after the
components are mixed to insure that reaction components
are at equilibrium. The lowest concentration of
inhibitor tested is determined by the estimated
concentration of enzyme. An inhibitor concentration 5-
fold in excess of enzyme concentration is the lowest
maintained concentration so that pseudo-first-order
reaction conditions are observed. The maintenance of
pseudo-first-order reaction conditions and the
sensitivity of the respective assays sets the lowest
limit level of inhibitor tested at 10 nM for kallikrein
inhibitors and 5 nM for thrombin inhibitors.
Usually, reversible inhibitor effectiveness is
evaluated by measuring Ki's, the dissociation constants
for the enzyme-inhibitor complex. This value, by
definition, is the concentration of inhibitor required
to inhibit the enzyme 50% in the absence of substrate.
But the substrate has a protective effect, therefore
higher concentrations of inhibitor are required to
achieve 50% inhibition. Nevertheless, a conservative
estimate of the Ki can be obtained from the percent
activity (inhibition) data and the concentration of
31 1~39~7
lnhlbltor. ~ level o~ inhlbitor of about 20-fold hlgher
than ~l ls requlred to lnhlblt a reaction 95% and a
level of $nhlbltor of about 50-~old higher than
requlred for 98% $nhibitlon.
Platma kallikrein preferentially hydroly~es
and liberates bradykinin. BoroArgln$ne pcptide~
containing Phe adjacent to the boroArginine are the most
ef~ect$ve lnhibltort of this enzyme. ~or example, 10 nM
H-(D)Phe-Phe-boroArg-C~OHl~ lnh$blts kalllkreln greater
than 95%. No tigniflcant differencet are obterved
between the effectivene6s of the boroArglnlne p~nanedlol
etter~ and the corre~pond~ng $~othiouron~u- analog-
(boro~rg-). ~n addition, no differences are obter~ed $n
the effectivencst of the unprotected boronic acld and
corretponding difluoroborane.
Retultt timilar to those with kallikrein are
obtained for thrombin in ast~yt with tynthet$c
~ubttratet, except that thrombin has a ~uch hlgher
afflnlty for lnhlbltort wlth proline in the ~lte
adjacent to the boroArginine. The ~ott effective
inhib~tor it Ac-(D)Phe-Pro-boroArg-CIOHl~ wh$ch $nhib~tt
thrombin 99% at a concentration of 5 nM. The ~06t
potent lnhib~tor reported ln the llterature it
N-alpha-(2-naphthylsulfonyl-glycyl)-4-amldlnophenyl-
~lanine plperididc, which has a ~l of 6 nM. It wat
reported by B. ~alser et al., Thromb. Res. 43: 613-620
(1986) and Sturzebecher et al., ~hromb. Res. 29: 635-642
(1983). ~he relationship between inhibitor
concentration, ~l~ and percent inhibition, as previously
described, suggests that the Kl of Ac-~D)Phe-Pro-
boroArg-Cl O Hl 6 iS in the picomolar range. Furthermore,
the effectiveness of inhibitors having a (D)Phe-Pro-
boroArg- sequence appear relatively insensitive to the
precence or absence of, or the nature of an amino
terminal protecting group. Such compounds having a Boc
and an Ac protecting group and having no protecting
32 133~897
group lnhlblt thrombin ~lmilarlly, eacb ehowing an I.C.
50 of les6 than 5 nM.
~ he effectlvene6s of $nhibltor6 $n reactlon6
$n wh~ch they compete w$th natural sub6trates for targ-t
enzyme6 ls mea6ured ~n vltro In blood coagulat~on
a66ays. Two dlfferent as6ay6 are u6ed, the ~PTT
(act$vated partlal thrombopla6tln tlmes) and PT
(prothrombin time6) a66ays. The6e a66ay6 mlmic the
blood clott~ng proce66 in vivo. Blood coagulat$on
occurs through elt~er of two pathway6, each con~l6tlng
of a ca6cade6 of zymogen actlvation step6. ~he pathway6
are termed the $ntr$n6$c and the extrinsic pathway6 ~see
L. Lorand, Method6 ln Enzymology 45: 31-37 (1976). The
$ntrln61c pathway i6 $nit$ated by negatively charged
surfaces ln which plasma kalllkreln, factor XII and
factor IX are act$vated and then factors IX and X and
prothrombin are actlvated ln calclum dependent ~teps.
Thrombln, the lact protea6e ~n the ca6cade, hydroly6e6
fibr$nogen to f$br~n which re6ults $n clot format$on.
In the APTT assay, pla6ma components are act~vated by
expo6ure to negatively charged surfaces and then
clottlng t~me6 are mea6ured ~fter calclum i6 added to
the sy6tem. In the extrln61c pathway, tlssue
thromboplastln actlvate6 factor VII wh~ch then act$vate~
factor ~ leading to the actlvatlon of thromb~n. The6e
events are mea6ured ~n the prothrombln t~me6 ae~ay.
Peptides of boroArginine and the corre6pond$ng
isothiouronium analog~ effectively $nhibit blood
clotting in both of the6e a6say~. The most effective
inhibitor6 of the pre~ent ~nvention for thrombin are the
mo6t effective for both a66ay~. On the other hand,
inhibitor6 of kallikrein, while le6~ potent clotting
inhibitor6, inhibit the APTT a6say (kallikrein i6
involved in the initiation of this as~ay) more
effectively than the PT a6say. Thi~ i~ clearly 6hown in
Figure 1 by the effect of H-(D)Phe-Pro-boroArg-ClOHl6
- 32 -
~3~9897
33
~thrombln lnhlbltor) on the relatlve clottlng tlme6 o~plaema. It demon~trate6 the 6electivlty whlch can be
achleved by ~arylng a elngle amino acld ln the
trlpeptlde lnhlbltor ln a rather complex blologlcal
~y6tem. The effectlve levels of thrombin inhlbltor~ are
ln the ~ame ~olar range a6 heparln. U6ually, 0.2-O.q
unlt~ of heparln per mL of pla6ma increa6e6 clottlng
tl~e~ 2-2.5 fold. If one ~66ume6 an average molocular
welght of 15,000 for heparln ant ~peclflc actlvlty of
150 unlt6/mg, lt~ molar concentration i6 86-170 nM. The
concentratlon of the boroArginine peptlde6 requlred to
lncrea~e clottlng tlme6 ln the APTT a66ay are ln the
range of 170-230 nM. It 6hould be noted that heparln i6
a cofactor for the hlgh molecular welght protea6e
lnhibltor, antl-thrombin I~I.
The 6tablllty of the boroArglnlne peptide6 ln
human platma 16 ~hown by incubatlng them wlth pla~ma at
a concentration effective to delay the clotting proce66.
~ample~ of the lnhlbltor6 are removed at lncrea6ing time
lntervals and thelr ability to delay clottlng lr
mea6ured at each lnterval. No change ln the clotting
tiae ~ndicate6 no change ln the lnhlbltory activity of
the lnhibltor6 durlng lncubatlon ln pla6ma. No
~lgnlflcant change ln lnhlbltor actlvlty wa~ obtcrvod
oxcopt for ~-(D)Phe-Pro-boroArg-ClOHl~, whlch lo~t
actlvlty after 2q h. The lnhibitor6 of thl6 lnvention
are al60 ~table for 2~ h ~n pho6phate buffer at pH 7.5
except for H-(D)Phe-Pro-boroArg-ClOHl~, which lost
lnhibitory activity within one hour. The greater
ln6tability of thl6 lnhibitor in buffer 6ugge6t6 that
phorphate buffer playc a role in de6tabilizing the
compound.
The ~n vivo data 6upplied clearly indicates
the eff~ciacy of the 6ubject compound6 a~ inhibltltor6
of blood coagulation in mammalian 6yrtem6.
Compound6 of the precent invention are ~lro
3~ 13~8'~7
effectlve anti-lnflamm~tory agents as ~hown by the
~nhlbltion of rat ear edema when the compounds are
applled toplcally along with ~lth an lrrltant. The
molecular ba6i~ for this pharmacologlcal actlvlty 16
unknown, slnce multiple events occur durlnq
lnflammatlon. However, proteases whlch lncrease
vascular permeabllity, ~uch as plasma kall$kreln whlch
llberate~ kinins and enzymes of the complement eystem
whlch llberate the anaphylatoxln peptldes, are thought
to be lmpllcated 1~ the inflammatory proces6.
F~nally, peptldes of boroLy~ine were ehown to
effectlvely lnhiblt pla~min, an enzyme whlch plays a key
role ~n hemo~ta~
Util~ty
N-Acyl and N-peptide alpha-amlnoboronic acid6
which are analogs of ornlthine, arglnine, ly6ine and
homoarglnine of the present lnventlon represent a novel
clas6 of potent, rever~ible inhlb~tors of trypsin-llke
enzymes. Trypsln-like enzymes are a group of protease6
whlch hydrolyze peptide bonds at baslc residues
liberatlng elther a C-termlnal arginyl or lysyl residue.
Among these enzymes are the proteases of the blood
coagulatlon system (factors, ~IIa, ~Ia, IXa, VIIa, Xa,
and thrombln), the flbr~nolytlc ~ystem (plasmlnogen
activators and plasmin), the complement system (Cls,
Clr, C3 con~erta~e, factor D, etc.), pancreatlc tryp~ln
(whlch as a digestive functlon), and acrosln, (whlch ~s
a protease a~oclated with ~perm and required for
fertilizatlon).
The ability of the compound~ of this invention
to inhibit trypsin-like protea~es ha~ been determined by
inhibitinq two different trypsln-llke enzymes, human
thrombin and plasma kallikrein. Compounds of the
present lnvention are much more potent inhibitor~ of
both of the~e enzymes than other known revereible
~nhibitors. For example, the most effective inhibltor
13~3g8~7
of thrombln reported to date 16 N-alpha-(2-naphthyl-
~ulfonyl-glycyl)-~-amidinophenylalanine plperldlne ~hlch
a ~1 of 6 nM. Compound6 of the pre6ent lnvontlon almo6t
completely lnhlbit thrombln at a concontratlon of S nM
lndlcating a ~ of ~ l nM, and thus provide excellant
candidate6 for the control of thrombin medlated
proce66e6 6uch a6 blood coagulatlon. ~he mo6t effectlve
boroArglnlne peptide lnhlbit6 blood clottlng a6
demon6trated by thc increa6e ln the APT time6 and PT
time6. It6 level of effectivene66 i6 6imilar to that of
heparln on a molecular ba61~. In addltion, the
compound6 are 6table ln human pla6ma. ~he compound~ can
be used a6 anticoagulantc in thc preparation of pla~ma
for protein i601atlon as well a6 for clinical te~tinq.
An addltional example it the protea6e,
pla6min, which ha6 a plvotal role $n the ly~l6 of blood
clot6. Peptldes containing boroly6ine were prepared and
te6ted and found to be active inhibitor6 of pla6min.
Compound6 of the present ~nventlon are
effectlve in controlllng proteolyrl6 ~n vivo and ~hould
be phar~aceutically effective in the troatment of
dl6eare6 in ~ammal6 arl6ing from uncontrolled protea6e
actlvlty. Notable among the6e are condltlon6 at60clated
wlth thrombosl6 and con6umptlve coagulopathy. Coronary
thro~bo6i6 play~ an lmportant contributlng role ~n
myocardial infarction. Con~umptive coagulopathy, a
conditlon marked by decrea6e6 in blood coagulation
factor6 and plasma protea6e inhibitor, i6 ob6erved in
p~tient~ with acute pancreatiti6 and di66eminated
intrava~cular coagulation (D~C). It i~ expected that
compounds of the pre~ent invention can be u6ed in place
of heparin with the advantage that heparin~ pla6ma
cofaetor, anti-thrombin III, i~ not con6umed in the
reaction. Al60, thrombocytopenia, a 6ide effect of
heparin treatment, 6hould not be ob~erved. rurthermore,
compounds of the pre6ent invention are expected to be
- 35 -
36 ~3~897
valuable ln the treatment of dl6ea6e6 ln whlch there 16
a deflclency of natural lnhlbltor~ of tryp61n-llke
enzyme~ euch a- herltary edema. Thl~ dl60rder arl~ec
fro~ a deflclency of Cl lnhlbitor, the ma~or lnhlbltor
of pla6ma kalllkreln.
Flnally, eompound6 of the pre6ent lnventlon
have demon6trated effectlve antl-lnflammatorle6 actlvlty
ln vlvo.
Synthe616 Example6
The exampIe6 whlch follow lllu6trate
partlcular embodlment6 of the lnventlon. All ~eltlng
polnt~ reported are uncorrected. All partg are by
welght and all temperature6 are reported ln degree6
Celtlu6. Proton nuclear magnetic re60nance (NMR or lH
NMR) report6 chemlcal shlfts ln delta unlt6, part6 per
~llllon downfleld from the lnternal tetramethyl611ane
6tandard. Varlou6 abbrevlatlon6 employed throughout
lnclude: TFA - trlfluoracetlc acld; DMr -
N,N-dlmethylformamides MS - ma66 spectrometry; TLC -
thln layer chromatography; RP-TLC - rever6e pha6e thln
layer ehromatography. The e6ter protecting group6 for
the boronlc acld6 are abbrevlated: -C~Hl 2 - the plnacol
group and -C10~l~ - the plnanediol group. ~rg~ 16 the
~bbrevlatlon for the 160thlouronlum analog of arglnlne
(Arg) ~nd the prefl~ ~ho~o' ~nd~cate6 structure6 ln
whleh the clde ehaln eontalns an addltlonal ~ethylene
group. All amlno acld re61due6 are ln the
eonflguratlon unles6 speclfled.
TLC and RP-TLC were conducted on E. Merk
Slllca Gel 60 Plates (Cataloq ~ 5534, E. M. Science~,
G$bb6town, NJ) and Whatman RC18F Rever6e Pha6e Plates
(Catalog t 4803-600, Whatman Co., Cllfton, NJ),
re6pecively. Neutral compounds were visualized under Uv
light and after exposure to iodine vapors. Compound~
with free ~mino group6 were 6tained with ninhydrln and
compound6 with guanidino groups were 6tained with the
13~9897
8akaguchi ~tain. The Sakaguchi ttain exhlblt~ a
conelderable ~peclficlty for the mono~ub~tituted
guan~dine6 tuch a6 those precent ln the boroArg~n~ne
pept~des (~ee Chemistry of the Amino Ac~d~, 3: (198~)
Greenctein and Wlnitz, ed6., Robert E. ~rleger
Publi6hing Co., Malabar, FL).
Example la
l-Amlno-4-bromo-butyl boronate pinanedlol-hydrogen
chlorlde, NH2-cH~(cff~)3srlBo2-cloHl~ HCl
4-Bromo-l-chlorobutyl boronate p~naned~ol was
prepared by the method in Matte60n et al.,
Organometallic6 3: 128q-12B8 (1984), except conditlon6
were modlfled for large scale preparatlon6. ~n a
typ~cal experiment, allyl bromlde (173 m~, 2.00 mole6)
wac hydroborated with catechol borane (240 mL, 2.00
~ole~) by the addltion of the borane to allyl brom~de
and then heating the react~on for 4 h at 100- under a
nitrogen atmo6phere. The product, 3-bromopropyl
boronate catechol ~bp 95-102-, 0.25 ~m) wa6 ~solated in
a yield of 49% by di6tillation. The catechol etter (124
g, 0.52 moles) wa6 tran6e6terif$ed w~th
(~)alpha-pinanedlol (88 g, 0.52 mole6) by mixlng the
component in 50 mL of tetrahydrofuran (THr) and allowlng
them to ~tlr for 0.5 h at 0- and 0.5 h at room
temperature. Solvent wa6 removed by evaporatlon and 250
mL of hexane wa6 added. Catechol wac removed a6 a
crystalline sol~d. Quant~tative re~oval wa~ achieved by
tuece6~ive dilution to 500 mL and to 1000 m~ with hexane
and re~oving cry~tals at each dilution. Product (147 g)
wa6 obtained a~ an oil by evaporating ~olvent.
Analy~it for Cl 3 H2 2 ~2 BrB
Calculated: C-51.85%, H-7.38%, and Br-26.54.
Found: C-52.85%, H-7.30%, and ~r-26.5B%.
- 37 -
38 133~7
~ -~romo-l-chlorobutyl boronate pinanedlol wa6
prepared by homologation of the corre6pond~ng propyl
boronate. Methylene chloride (34.8 mL, 0.540 ~ole6) wa6
di6601ved ~n 500 mL of THF, 1.54 N n-butyll~th~u~ ~n
hexane l350 mL, 0.540 mole6) and wa6 610wly added at
-100-. 3-Bromopropyl boronate p~nanedlol (14a g, 0.490
~ole6) wa6 di~601ved in 500 ~L of SHF, coolcd to the
freezing point of the ~olution, and added to the
reaction mixture. Zlnc chloride (33.5 g, 0.246 mole6)
wa6 dic601ved in 25~ mL of THF, cooled to 0-, and added
to the reaction mixture in 6everal portion6. Thc
re~ct~on mixture, wh~le 6tirrinq, was allowed to warm
610wly overn~ght to room temperature. Solvent wa6
evaporated and the re6~due was d~6601ved in hexane and
washed with water. ~fter drying over anhydrous
magne61um 6ulfate and filtering, 601vent was removed to
yield the de6ired product (140 g).
l-Am~no-4-bromobutyl boronate plnanediol was
prepared fir~t by dl6601v$ng hexamethyldi6ilizane (28.0
g, ao . o mmoles) in 30 mL of THF, cooling the solution to
-78-, and adding 1.62 N n-butylllthium ln hexane (49.4
~, 80.0 mmole6). The 601ution was allowed to 610wly
warm to room temperature and wa6 then recooled to -78-
and 4-bro~o-1-chlorobutyl boronate pinancdlol (28.0 g,
ao.o ~mole6) in 20 mL of THF wa~ added. The mixture was
allowed to ~lowly warm to room temperature and to ~tir
overnight. Solvent wa6 removed by evaporat~on ~nd dry
hexane (400 mL) was added to yield a precipitate which
was removed by filtration under an nitrogen atmosphere.
The filtrate was cooled to -78~ and 4 N hydrogen
chloride in dioxane (60 mL, 240 mmole~) was added. The
reaction was allowed to warm slowly to room temperature,
at which temperature it was stirred for 2 h. The
resulting product (20 g) was isolated as a 601id by
filtration. After drying in vacuo, the crude product
wa~ dis~olved in chloroform and insoluble material was
- 3B -
39 1339~97
removed by f~ltr~tlon. Thc f$1trate wa~ evaporated and
the re~idue di~olved ~n ethyl acetate. The product
cry~talll~ed from ethyl acetate to y~eld 15.1 g (mp
1~2-144.5~ ~1D25 - ~16.7 ~ 0.80, C-l.0 ~n ~b-olute
ethanol.
Analy6i~ for Cl~H2~NO~BrClB:
Calculated: C-45.~7%, H-7.16%, N-3.82%, and ~-2.95%.
Found: C-45.76%, H-7.21%, N-3.79%, ~nd ~-3.04%.
Example lb
(D,L)l-Amino-4-bromobutyl boronate pinacol HCl
(D,L)NH2-C8[(CH2)~Br¦BO2-C~Hl2-HCl
4-bromo-1-chlorobutyl boronate plnacol wa6
prepared by the method dercrlbed for the correspondlng
pinaned$ol (Example la) except pinacol ~a~ ~ub~tltuted
for pinanediol ant 3-bromopropyl boronate pinacol (bp
60-64~, 0.35 ~m) and 4-bromo-1-chlorobutyl boronate
pinacol (bp 110-112~, 0.20 mm) were d$stilled.
Analy~i~ for ClOH~O2BrClB:
Calculated: C-40.3B~ and H-6.45%.
Found: C-40.70% and H-6.37%.
l-Amlno-4-bromobutyl boronate plnacol-hydrogen
chloride ~a~ al~o prepared by the procedure ln Example
la. The flnal product was crystalllzed for ethyl
acetate:hexane in y~eld of 52%.
Analy~i~ for C~oH2 2 NO2BrClB:
Calculated: C-38.19%, H-7.05%, N-4.45%, Cl-11.27% and
Br-25.~1%.
Found: C-38.28%, H-7.39%, N-4.25~, Cl-11.68% and
Br-26.00%.
Example lc
l-Amino-q-chlorobutyl boronate pinacol-hydrogen chloride
(D~L)NH2-CHl ~CH2 )~ Cl ]BO2 -C6 Hl ~ ~HCl
_ ~9 _
1~39~7
3-Chloropropyl boronate catechol ~bp 80-85O,
0.30 ~m) and 3-chloropropyl boronate plnacol(bp 63-,
0.20 ~m) were prcpared by the method ln ~xample la
except ~llyl chloride wag 6ub~t~tuted for ~llyl bromlde
~nd plnacol wa~ ~ub6t~tuted for pinanediol.
Analy~l~ for C~Hl~ ~2 ClB:
C~lculatcd: C~52.85, H-8.89%, and Cl-17.33~.
round: C-53.~1%, H-8.15%, and Cl-16.81%.
Homologat$on wa6 al60 conducted by the
procedure in Example la and the product wa~ i601ated by
di~t~ tion ~bp 95-, 0.25 mm) ~n ~ y~eld of 65%.
An~ly~i~ for CloRl~O2Cl2B
C~lculated: C-47.47%, H-7.58%, and Cl-28.02%.
round: C-47.17%, H-7.45%, ~nd Cl-27.75.
l-Am$no-4-chlorobutyl boronate pinacol HCl wa~
prepared by a procedure ~dent$cal to Example la. The
product cry~tall~zed from ethyl acetate to y~eld 8.8 9
~mp 132-135.5-) ~nd 2.2 g ~mp 145- lq7~). The product
melt~ng 145-147~ wa~ u~ed for analy~e~.
Analy~i~ for ClOH22NO2Cl2B
C~lcul~ted: C-44,47%, H-8.23%, N-5.19%, and B-4.00%.
round: C-q4.01%, H-8.23%, N-4.77%, and B-3.80%.
Example ld
(D,L)l-Am$no-5-bromopentyl boronate-p$nacol-HCl
'L)NH2-CHl~cH2)<BrlBO2c6Hl2~Hcl
4-bromobutyl boronate pinacol was prepared by
the method de~cribed for 3-bromopropyl boronate
p$nanediol (Example la) except 4-bromo-1-butene was
~ub6tituted for allyl bromide and pinacol wa~
~ubrt$tuted for pinanediol. The product was i~olated as
~n o$1 (bp 77~, 0.3 mm). Homologation yielded 5-bromo-
l-chloropentyl boronate pinacol.
- 40 -
41 1339g97
MS(C~) for CllH2~02BrClB:
Calculated - ~: 310.47.
round: 310. .~
The final product, l-amlno-5-bromopentyl
boronate plnacol-HCl, wa6 prepared by the procedure in
Example la ~n a yield of 35%.
Analy6i6 for cllH2~No~srBcl:
Calculated: C-40.22~ 7.36%, N~4.26%, Cl-10.79%,
Br-2~.32%, and B-3.~9%.
Found: C-39.23%, H'7.18%, N-4.04%, C1-15.21% and
Br-25.66%, and B-3.75
Example 2
Boc-(D)Phe-Pro-NH-CBl~CH2)~BrlBo2-clOHl 6
Boc-(D~Phe-Pro-OH wa~ produced by first
preparinq the d~peptide benzyl ester and then removing
the ester by catalytic hydrogenation. Boc-(D)Phe-OH
(10.0 g, 37.7 mmoles) was dis601ved in 50 ~ of THF and
N-methylmorpholine (4.14 m~, 37.7 mmolec) wa~ ~dded.
The eolution wa6 coDled to -20- and 160butyl
chloroformate (4.90 m~, 37.~ mmole6) wa6 added. After S
~in, H-Pro-OBzl.HCl (9.11 ~, 37.7 mmoles), di6601vcd in
50 ~ of chlorofor~ and co~led to -20~, was added.
Triethylamine (5.25 mL, 37.7 ~mole6) was ~dded and the
mixture was stirred for 1 h at -20~ and 2 h at room
temperature. The reaction mixture wa~ filtered and the
f~ltrate evaporated. The residue was dissolved in ethyl
acetate and was washed with 0.2 N hydrochloric acid, 5~
aqueous sodium bicarbonate, and saturated aqueous sodium
chloride. The orqanic phase was dried over anhydrous
sodium sulfate, filtered, and evaporated to yield 15.2 g
of ~oc-(D)phe-pro-oszl as an oil. The benzyl ester
(15.2 g) wa6 dissolved in 100 mL of methanol and it was
hydrogenated at an initial pressure of 40 psi on a Parr
- 41 -
13398~7
apparatus in the presence of 0.5 g of 10% Pd/C. The
reaction solution was filtered through CeliteTM and
evaporated to yield a solid. This solid material was
isolated and was washed with ethyl acetate and then by
ether to yield 10.0 g of the desired product (mp
176.5-177~).
AnalySiS for ClgH26N2Os:
Calculated: C= 62.95%, H= 7.24%, and N= 7.73%
Found: C= 62.91%, H= 7.15%, and N= 7.53%.
Boc-(D)Phe-Pro-NH-CH[(CH2)3Br]sO2-Cl0Hl6 was
prepared by coupling the dipeptide to the corresponding
amine using the mixed anhydride procedure. The mixed
anhydride of Boc-(D)Phe-Pro-OH was prepared by
dissolving this acid (4.94 g, 13.6 mmoles) in 30 mL of
THF and adding N-methylmorpholine (1.50 mL, 13.6
mmoles). The solution was cooled to -20~ and isobutyl
chloroformate (1.77 mL, 13.6 mmoles) was added. After
stirring for 5 min at -20~, the mixture was added to the
amine as in Example la, NH2-CH((cH2)3Br)BO2-clo
(5.0 g, 13.6 mmoles) dissolved in 10 mL of cold
chloroform. Cold THF (10 mL) and triethylamine (1.90
mL, 13.6 mmoles) were added and the mixture was stirred
for 1 h at -20~ and approximately 2 h at room
temperature. The mixture was filtered and the liquid in
the filtrate was evaporated. The residue was dissolved
in ethyl acetate and washed with 0.2 N hydrochloric
acid, 5% aqueous sodium bicarbonate, and saturated
aqueous sodium chloride. The organic phase was dried
over anhydrous sodium sulfate, filtered, and the solvent
evaporated to yield 9.0 g of an oil. This material was
dissolved in methanol and chromatogramed on a 2.5 x 50
cm column of LH-20. Fractions containing the desired
product were pooled and evaporated to yield 5.8 g of a
solid. TLC with methanol:chloroform (1:9) indicated a
single spot, Rf 0.70.
~r
8 ~ 7
MS(FAB) for C~H~N~O~BBr:
Calculated ~ H: 674.30
round: 674.30
~xample 3
Boc-(D)phe-pro-NH-cHt(cH~)~N~lso2-cloRl~
Boc-(D)Phe-Pro-NH-CHl(CH~)~BrlBO~-ClOHl~, the
product of Example 2, (4.4 9, 6.54 mmoles) wa~ d~6solved
~n 7 mL of DMr ~nd 60dium azide ~0.919 g, 14.1 mmole~)
wa~ ~dded. Thc ~ixture was heated ~t 100- for 3 h.
Ethyl ~cetate (100 ~L) was added to the reaction mlxture
and lt wa~ washed with water ~nd w~th caturated aqueou~
sodium chloride. ~he organic phase was dr~ed over
~nhydrou~ sodium ~ulfate, filtered, ~nt 6ub~ected to
evaporation. A y~eld of 4.1 g of ~ ~olld re~ulted.
This mater~l was chromatographed on ~ 2.5 X 50 cm
column of ~H-20 ~n ~ethanol. rractiOns contain~ng the
det$red product were pooled, liquid was evaporated to
y~eld 2.3 g of the az~de. TLC ~n ~ethanol:chloroform
(1:9) ~ndlcated ~ ~ngle spot, ~f 0.76.
Analysis for C" H" N~O,B:
Calculated: C-62.35%, H-7.63%, N-13.33%, and B-1.70%.
Found: C-63.63~, H-8.02%, N-11.58%, and B~1.80%.
MS(FAB) for C" H~,N,O~B
Calculated ~ H: 637.39.
round: 637.49.
Example 4
goc-~D)phe-pro-NH-cHl(cH2) 3 NHll ~~2 -Cl o H~ 6 benzene
~ulfonic acid
The azide of Example 3, (8.80 q, 13.8 mmoles)
was dissolved in 150 mL of methanol and was hydrogenated
on a Parr apparatus at 40 psi in the presence of 0.50 g
of 10% Pd/C and benzene sulfonic acid ~2.19 g, 13.8
~4 133~i897
~mole~). After 1 h, cataly6t wa6 removed and the
~olution wa6 evaporated to yield a 6011d which wa6
triturated w$th hcxane to y~eld 9.9 g of the de~ired
product. RP-TLC in methanol:water (85:15) indlcated
W ~pot, RF 0.91, and a ninhydrin po6iti~e ~pot, ~F
0.52.
Example 5
80c-(D)Phe-Pr o-NH-CHt (CH~ )3--NH-C(NH)NH2 ¦BO2--C, oHl,
benzene 6ulfonic ~cid.
Boc-(D)Phe-Pro-boroArg-CIOH~-benzene ~ulfon~c acid
Boc-(D)Phe-Pro-boroOrn-ClOHl~-benzene 6ulfonic
acid, Example 4, (4.6 g, 6.11 ~mole6) wa6 refluxed at
100- in 20 mL of ab601ute ethanol contain~ng cyanamide
(50 ~g/mL). ~he proqre6s of the reaction wa~ ~onitored
by RP-TLC in methanol:water (85:15) ln which the
di6appearance of the ninhydrin 6pot for the amine
6tarting material (Rf 0.54) ~nd the ~ppearance of the
Sakaguchi 6treak of the product (Rf 0-0.13) wa6
obser~ed. Product could be detected after refluxing 18
h and it6 level progrec6ively increa6ed with time.
After 7 day~, ~mine could not be detected and the
reaction 601ution was concentrated to an approximate 50%
~olution through pa66ive evaporatlon. ~he reaction
eolut~on wa~ filtered, concentrated, and chromatographed
on a 2.5 ~ 10~ ca colu~n of LH-20 in ~ethanol.
Fraction6 containing the desired product were pooled and
subjected to evaporation to yield 3.7 g of the de6ired
product. A portion (2.3 g) was crystallized for ethyl
acetate:hexane to yield 0.89 g and the residue (1.2 g)
wa~ obtained as a solid by triturating with ether. In
separate experiments.
MS(FAB) for C34Hs~N6~65B
Calculated ~ H: 653.42
Found: 653 . 38
i33~897
Analysis for C4oHsgN6OgSB~H20
Calculated: C=57.95%, H=7.43%, N=10.14%, and B=1.30%.
Found: C=57.20~, H=7.14%, N=10.94%, and B=1.01%.
Example 6
H-(D)Phe-Pro-boroArg-Cl0H16-2HCl
Boc-(D)Phe-Pro-boroArg-Cl0Hl6-benzene sulfonic
acid, the product of ExamPle 5, (1.17 g, 1.54 mmoles)
was reacted with 5 mL of 4 N hydrogen chloride in
dioxane for 15 min at room temperature. The product was
precipitated by the addition of ether, isolated, and
washed with ether and dried in vacuo. It was then
dissolved in 10 mL of water and applied to a 5 mL anion
exchange column of BIO-RAD AG1 X8TM (C1 form, BIO-RAD
Co., Richmond, CA) and the column was washed with water
(approximately 30 mL). The effluent was evaporated in
vacuo and the residue was triturated with ether to yield
the desired product (0.80 g).
MS(FAB) for C29H45N6O4B:
Calculated + H: 553.37.
Found: 553.40 and 538.40 (unidentified).
Analysis of H-(D)Phe-Pro-boroArg-Cl0Hl6-lBSA-TFA:
Found: 553.4
Examples 7 - 8
Ac-(D)Phe-Pro-boroArg-Cl0Hl6-HCl (Example 7)
Ac-(D)Phe-Pro-boroArg-OH-HCl (Example 8)
Boc-(D)Phe-Pro-boroArg-Cl0Hl6 -benzene sulfonic
acid, the product of Example 5, (0.86 q, 1.13 mmoles)
was reacted with anhydrous TFA (approximately 5 mL) for
15 min at room temperature. Excess TFA was removed by
evaporation and the residue was triturated with ether to
yield 0.76 g. This product (O . 70 g, 0.91 mmole) was
dissolved in a mixture consisting of 2 mL of dioxane and
1 mL of water. Acetic anhydride (0.47 mL, 5.0 mmoles)
46 1339897
and codlum bicarbonate (0.42 g, 5.0 mmoles) were addcd.
~he m~xture wa6 ~t~rred for 20 min at room temperature.
Fthyl acetate (50 mL) and water (5 mL) were ~dded. The
phace6 were teparated and the organic pha6e ~a~ drlcd
over ~nhydrou6 60dium ~ulfate, f$1tered, and ~ol~ent
removed by evaporatlon to y~eld 0.56 g of a partlal
~olld.
The 6ample wa~ di~601ved ~n 4 mL of glaclal
~cet~c acid and diluted wlth 16 m~ of water. It was
~mmediately applied to a column containing 15 mL of
SP-SephedexS~ (H~ form) and equillbrated w~th 20% acetic
~c~d. The column was washed with 300 ~L of 20% acet~c
ac~d and thcn a linear grad~ent from 100 mL of 20%
~cet~c ~cid to 100 mL of 20% ~cet~c ac~d ad~u~ted to
0.30 N hydrochloric acld was run. Fract~ons collected
from 0.08 to 0.17 N hydrochlor~c ac~d contalned the
N-~cetyl peptide (0.29 g) a6 a mlxture of the free
boronic acid and pinanediol e~ter.
~ he pinanediol ester and the free boronic acid
were ceparated by chromatography on ~ 2.5 X 100 cm
column of L~-20 ~n methanol. The fract~on ~lze was 8.2
m~. The p~nanediol e6ter ~102 mg) eluted ~n fractlon
41-43 while free boronlc acid (131 ~9) 610wly eluted ln
fract~on~ 45-129.
MS(FAB) (Example 7):
Ac-(D)phe-pro-boroArg-cloHl6) for C3lH~7N~05B:
Calculated I ~: 595.33.
Found: 595.33.
MS (FAB) (Example ~):
~c-~D)Phe-Pro-boroArg-OH HCl) for C2lH" N60s:
Calculated ~ H: 449.60.
Found: 579.2Ç-581.24.
The latter result could not be interpreted.
However, NMR was consistent with the structure of the
free boronic acid ~ince definitive bands for pinanediol
group 6uch as the methyl groups singlet6 observed at
- 46 -
133~98~7
delta 0.85~3H), 1.30(3H), and 1.36(3H) were abrent. A~
addcd proof of ~tructure, a 6ample of the free boronlc
W~6 re-e~ter~ficd to g~ve the product ~n Exampl~ 7. ~n
~nalytical ~ample (20 ~g) wa~ treated w$th a 2-fold
excer~ of pinaned1ol (14 ag) in 3 ~ of methanol for 5
ain. Solvent wa6 evaporated and exce~ pin~nedlol wa~
removed by trituration of the 6ample with ether to yield
the product ~26 mq).
MS(FAB) (Found: 595.3B) and NMR were con~$~tent with
that expected for the e~terified product and were almoct
~dentical to the pinanediol product of Example 7.
Exa~ple 9
Ac-Phe-boroArg-Cl0H~HCl
Ac-Phe-NH-CHl(CH2)~BrlBO~-C~0Hl~ w~ prepared
~y the procedure deccrlbed in Example 2. The ~ixed
anhydride of Ac-Phe-OH (0.565 g, 2.73 mmole~) wa~
prepared ~n 10 mL of THF and coupled to
NH~-cHl(cHl)~BrlBo2-cloHl~-Hcl (the product of Example
la, 1.00 g, 2.73 ~moles) di~sol~ed ln 10 ml of cold THF
to yield ~.47 g of a wh~te foam. Thl~ ~aterlal war
stirred with hexane overn~ght to yield a 601id, 1.01 g
(~p 106.5-109~).
Analy~6 for C2 SH~N2O~BrB
Calculated: C~57.81, H-7.00%, N-5.40%, ar-15.~0%,
B-2.0~%. Found: C-5~.33%, H-7.33%, N~4.76%, Br-14.18~,
B-1.80%.
Ms(rAB) for C2sH~N2O~BrB:
Calculated ~ H: 519.20.
Found: 519.23.
Ac-Phe-NH-CHl (CH2 )~N~ ¦BO2-CloH~ wa~ p p
by treating Ac-Phe - NH-CH[ (CH2)~Br¦BO2-Cl0Hl~ (3.22 9,
6.20 mmole~) with ~odiu~ azide by the procedure
de~cribed in Ex~mple 3. Product from the reaction ~3.03
48 13398~7
g) wac chromatographed on LH-20. Fractlon6 contalnlng
the de~lred product were pooled and evaporated. The
re~ldue ~ac trlturated wlth hex~ne to yl~ld 2.21 g of
the azlde.
~ c-Phe-boroOrn-ClOH~ enzene ~ulfonlc acld
was prepared from Ac-Phe-NH-CH[(CH2)~N3lBO~-ClO~ (2.21
g, 4.59 mmole6) by the procedure ln Exa~ple 4 except
hydroqenatlon was performed at atmospherlc pre~cure.
After filtratlon, and the evapor~tlon of 601vent, the
de61red product (2;22 g) was obtalned by trlturatlng
wlth ether.
Ac-Phe-boroArq-ClOHl~-benzene ~ulfonic ac~d
wa6 prepared by treat$ng Ac-Phe-boroOrn-~10~l~ benzene
~ulfonlc acid (2.0 g, 3.26 mmole~) wlth ~ 10 mL colutlon
of cyanamide (100 mg/mL) in ethanol. The guanldatlon
procedure in Example 5 was used except ~he reaction time
was 3 day6 and the reaction mixture contained a mixture
of starting materlal and product. Thir. requlred an
addltlonal purlflcatlon ctep which mort probably could
have been eliminated by a longer reactlon tlme. The
colution wa6 concentrated and chromatogramed on 2.5 X
100 cm column of LH-20 in meth~nol. The fraction6
containlng the de61red product, detected by Sakaguchi
ctain, were pooled and ~ubjected to e~apDratlon to yield
1.4 g. The reculting material (1.2 g) wac diccolved ~n
6 mL of acetic acld and dlluted wlth 30 ~L of water to
yield a mil~y ~olution. It wa6 applled to a 30 ~L
column of SP-Sephedex~ C-25 (H~form) equllibrated ln
20% aqueou~ acetic acid. The column wa~ washed with 240
mL of 20% acetic acid and then a linear gradient form
250 mL of 20% acetic acid to 250 mL of 20% acetic acid
containing 0.30 N hydrochloric acid was run. rraction6
eluted from the column from 0.12 N to 0~16 N hydro-
chloric acid were pooled to yield O.q2 ~ of the desired
peptide as a mixture of the free boronic acid and
pinanediol ester. The mixture wa~ dis6Dlved in methanol
- 48 -
49 133~97
(10 ml) and B0 mg of plnanedlol was added to esterify
the free boronic acid. After ~tlrr1ng for 30 min,
sol~ent w~ e~aporated and the residue was trlturated
with ether to yleld 0.28 9 of the deeired product.
Analy61s for C2~H~oNsO~B~HCl~2H~O:
Calculated: C-54.78%, H-8.15%, N-12.30%, and ~-1.90.
Found: C-55.34, H-7.83, N-11.66, and B~l.99.
MS(rAB) fo~ C2~H~oNs~-B
Calculated ~ H: 498.32.
rOund: 498.31.
Example 10
Ac-(D,L)Phe-(D,I.)boroArg-C~Hl ~
The l~termediate, Ac-(D,~)Phe-(D,L)-N~-
[(CH2)~Br¦BO2-C~Hl2, was prepared by a modif~cation of
the procedures of Examples lb and 2. The acid chlorlde
of Ac-Phe-OH wac prepared by react$ng Ac-Phe-O~ ~30 g,
0.145 molec) with pho~phorou~ pentachlorlde (30 g, 0.144
moles) ln 175 mL of THF at -10-. The reaction was
stirred at 0- for approximately 1 h, then dlluted to a
volume of 350 m~ with cold ether. The product was
lsolated as a solid, washed with cold ether, and dried
ln ~acuo to yleld 21 g. The activated Ac-Phe deri~atlve
(14.8 g, 65.6 ~molet) was d~ssolved ln 40 ~ of T~F and
added to the product of the reactlon of 4-bromo-1-
chlorobutyl boronate pinacol and hexamethyldisilizane
(prepared on a 20 mmole scale) at -78~. The reaction
mixture was allowed to warm to room temperature then
stirred overnight. The solvent was removed by
evaporation. The residue was dissolved in ethyl acetate
and washed successively with water, 5% sodium
bicarbonate solution and a solution of saturated aqueous
sodium chloride. The organic phase of the resulting
mixture was dried over anhydrous sodium sulfate and
concentrated to yield the desired product ac a
- 49 -
1~3~897
ery~talllne ~ol~d (1.37 g, mp 146.S-148-). In a
eeparate exper~ment, the following ~naly~6 wac
obtalned.
Analy8i 6 for C~lH~N2O~BrB:
Caleulated: C-53.98%, H-6.92$, N-6.00%, Br-17.10%, and
B-2.31%.
Found: C-54.54%, H-6.78%, N-5.89%, ~r-16.46%, and
B-3.40%.
The alkyl bromide was converted to the
eorregpond~ng az~de by the procedure ~n Example 3. The
produet erystall~zed from ethyl acetate (mp 143-144-).
~nalysis for C~lH~2N5O~B:
Caleulated: C-58.74%, H-7.53~, N-16.31%, and B-2.53%.
Found: C-58.85%, ~-7.48%, N-16.53%, and B-2.93%.
The az~de was eonverted to Ac-(D,~)Phe-
(D,L)boroOrn-C~HI2-benzene eulfonlc aeid by the
proeedure in Example 4 exeept hydrogenation wa6
eondueted at atmospher~e pre~ure.
Ae-tD,L)Phe-(D,L)boroOrn-C6H12-benzene
~ulfon~e ae~d (0.243 ~, 0.433 mmoles) was reaeted w~th
eyanam~de (0.020 g, 0.476 mmole~) at 100- ~n 2 mL of
ab601ute ethanol overnlght. The solutlon was
eoncentrated and tr~turated wlth ether to yield 0.21 g
of a wh~te 601~d. RP-TLC of the material lnd~eated the
eharaeterist~e streak staining positive with the
Sakaguchi stain for the boroArginine peptide~, ~f
0-0.55, and a discrete spot, Rf 0.68, corresponding to
unreacted ~tarting material. The product (81 mg) was
retreated with 2 mL of cyanamide (10 mg/mL) overnight by
the above procedure to yield 71 mg after trituration
w~th ether.
MS(FAB) for C22H37Nso~s:
Calculated ~ H: 446.30.
Found: 446.23 and 404.19 (corresponding to the
-- 50 --
51 13~897
unreactcd boroOrn peptlde).
Note that the ~ethod of Example 5 ~c a
cuperlor ~ethod for prcparing the boroArgin~ne pept~de6
and d$fferc ln that a larqer cxces6 of cyanamlde and
longer reactlon tl~e6 are u6ed.
~xample 11
~oc-(D)Phe-Phe-boroArg-ClOHl~-benzene 6Ul fonlc acld
Boc-(D)Phe-Phe-OH wa8 prepared by the method
de~crlbed for Boc-(D)Phe-Pro-OH ~n Example 2. Followlng
hydrogènatlon of the benzyl e6ter, ~ateria~ cry~tall~ed
from chloroform:hexane yielding the deslred pept~de (mp
133-133.5-).
Analysi6 for C23H2~N2O~:
Calculated: C-66.96%, H-6.B6%, and N~6.79%.
Found: C-66.75%, R-6.79%, and N-6.56%.
Boc-(D)Phe-Phe-NH-CH[(CH2)~BrlBO2-Cl0Hl 6 wa~
prepared by coupling Boc-(D)Phe-Phe-O~ (6.00 q, 14.5
~ole6) to NH2-CH[(CH~)~BrlBO2-ClO~ HCl (Examplc la,
5.33 q, 14.5 mmole6) u6ing the procedure descrlbed ~n
~xa~ple 2 except that the LH-20 chromatography 6tep wa6
ellmlnated. The product crystalllzed from ethyl acetate
to yield 2.47 g (mp 132-134~) ln the f$r6t crop ~nd
5.05 g (mp 133-135-) ln ~ second crop. RP-TLC ~n
methanol:water ~B5:15) indicated a single spot, Rf 0.29.
Analy6i~ for C3 ~ HS 1 N, O~ BrB:
Calculated: C-61.32%, H'7.11%, N-5.80%, Br~11.03%.
Found: C-61.21%, H-7.02%, N-S.59%, Br~10.22%.
Boc-(D)Phe-Phe-NH-CH[(CH2)~N~]BO2-ClOHl 6 was
prepared by treating the corresponding alkyl bromide
(7.15 q, 9.87 mmoles) with sodium azide using the
procedure in Example 3, except the LH-20 chromatography
rtep was not needed for purification. ~he product
S2 133~897
emerged from an ethyl acetate:hexane ~olut$on a~ a gel
and wa~ 16Olated and washed w$th hex~ne to y$eld 3.0 9
ln the f$rrt crop and 2.9 g $n a 6econd crop.
Boc-(D)Phe-Phe-boroOrn-C~0RI~-benzene ~ulfon$c
ac$d w~ prepared ~rom the azide (5.37 g, 7.82 ~mole~)
by the procedure $n Exa~ple 4 to yield 5.33 g. RP-~LC
~ethanol:water (8S:lS) $nd$cated an $nten~e n~nhydr$n
po~$t$~e ~pot, Rf 0.q2, and a weak ultrav$olet ( W )
l$ght rpot, 0.92. tThe W gpot at Rf 0.92 i6 typic~l of
am$ne~ or guan$dino compounds wh~ch are benzene ~ulfon$c
ac$d ~altc.)
MS(FAB) for C~H5~N~O~B:
Calculated + H: 661.76.
round: 661.14.
Boc-(D)Phe-Phe-boro~rg-Cl0H1~ wa~ obtained by
the procedure $n Example 5. The boroOrnith$ne pept~de
(4.83 g, 5.90 mmole~) wa~ treated w$th cyanam1de ~50
~g/~L) ~n 20 m~ of absolute ethanol for 7 day6. A
port$on of the reaction m$xture corre~pond$ng to 1.0 g
of ttart$ng ~ater$al wa6 removed and heated ~cparately
~n the absence of a reflux conden~er overn~ght to obt~ln
complete conver~$on of the amine to the guanld$no
compound. Following chromatography on ~H-20 ~nd
tr$turat$on of tbe product with ether, 0.52 g of the
de6$red product were obtained.
Analy~$~ for c~H~lN~o~ss:
Calculated: C-61.38%, H-7.16S, N-9.76S, B-1.25%.
Found: C-5g.69%, H-7.41%, N'9.82%, ~-1.26%.
MS~FAs) for C" H55N6O6B
Calculated + H: 703.43.
Found: 703.49.
Ex~mple 12
H-~ D )Phe-Phe-boroArg-Cl0Hl 6 ~ 2HCl
133~8~
~ oc(D)Phe-Phe-boroArg-ClOHl~-benzene ~ulfonic
~cid (Example 11, 0.59 g, 1.25 mmole~) war debloc~ed by
the procedure in Example 6 except that the ~mple war
applied to the ~on exchange column ln 20% ethanol ~nd
the column was eluted w~th 20% ethanol. The product
(0.424 g) was obta~ned a6 a white 6011d.
MS(FAB) for C"H~N~O,B:
Calculated ~ H: 603.38.
Found: 603.41.
Example 13
~c-Ala-~y~(Boc)-boroArg-C1OHl~-benzene ~ulfonlc ~cld
Ac-Ala-Ly~(Boc)-OH wa~ prepared by coupling
the N-hydroxysuccinimide e6ter of Ac-Ala-OH, prepared by
the method of Ander~on et al., J. Am. Chem. Soc. 86:
1839, (1964), to H-Ly6(Boc)-OH. The N-hydroxy-
~ucclnlmide of Ac-Ala-OH (6.25 g, 27.4 ~mole~) wa~
di6solved ln 30 m~ of dloxane and wa~ added to a
601utlon of H-Ly~(Boc) - OH (7.50 g, 30.4 ~moles)
die601ved in a ~olut~on con~i~ting of 30 ~L of 1.0 N
~odium hydroxide and triethylam~ne (2.12 mL, 15.0
mmole~). The reaction mixture was ~tirred overnight,
then acidified with hydrochloric acld. Sufflcient dry
eodium chloride to nearly saturate the 601ution wa~
added. The product was extracted into ethyl acetate and
lt wa8 washed with 0.2 N hydrochloric acid prepared ~n
saturated aqueous ~odium chloride. The organ~c phase
was dried over anhydrou~ ~odium sulfate and filtered.
Solvent was removed by evaporation. The product was
crystallized from ethyl acetate:hexane to yield 7.3 g
(mp 86-89~).
Ac-Ala-Lys(80c)-NH-CHI(CH2)~Br]BO2-ClOHl6 was
prepared by the procedure of Example 2 except that the
product was purified by fractional crystall~2-ation from
ethyl acetate. The product (1.13 g) obtained in the
54 1~3~837
~econd and thlrd crops exhibited a ~nglc spot on RP-TLC
ln methanol:water ~85 15), wlth an Rf 0 51. ~he TLC
plate was exposed to hydrochloric acid fume~ wherein the
result~ng am~ne was detected after the add~t~on of
ninhydrin stain
Ac-Ala-Lys(~oc)-NH-CH[(CH~)~N31BO2-ClOHl~ ~as
prepared from the correeponding alkyl bromide (1 95 g,
2 90 ~mole~) by the procedure ~n Example 3 except that
the product was purified by cry~tallizing it from ethyl
acetate rather than L~-20 chromatography Crude product
(1 60 g) crystall~zed to yield 0 55 g (mp 79-8~-) and
0 96 g of re~idue The ~naly~ of the cry~tall~ne
product follows
Analysi~ for C~oHS2N7O7B
Calculated: C-56 86%, ~-8 29%, N-15 48%, and B-1 71%
Found: 56 76%, H-8 26%, N-15 89%, and B-1 65%
Ac-Ala-Lys(Boc)-boroOrn-C~0Hl~ b-nz-ne
~ulfonic acid was prepared from the corresponding alkyl
azide (0 433 g, 0 683 mmoles) uelng the method descr~bed
~n Example 4 The catalyst ~nd eolvent were removed,
then the product (0 45 g) wa~ obtained by trituration
w~th ether
MS(FA8) for C~oHs NSO7B
C~lculated + H 608 42
Found: 608 49
Ac-Ala-Lys(Boc)-boroArg-Cl0Hl~benzene
~ulfonic acid wa~ prepared by reacting the corresponding
boroornithine peptide with cyanamide using the method
de~cribed in Example 5 The chromatography fractions
containing the desired product were triturated with
ether to yield 0 83 g as a white solid
Analysis for C~7H62N7OloBS
Calculated C-55 00%, H-7 75%, N'12 14%, and B-1 34%
Found C~54 09%, H-7 53%, N-12 22%, and B-1 34%
1~.3~ i37
Example lq
Ac-Ala-Lys-boroArg-C~OHl~ 2HCl
Ac-Ala-Lys~Bocl-boroArg-C~0HI~-benzene
~ulfonic acid (0.200 g, 0.248 mmoles) was deblocked by
the procedure $n Example 6. Following ion exchange,
evaporatlon of tol~ent, drying in ~acuo, and triturating
with cther, 0.14 g of materlal were obtained.
MS(FAB) for C2~H<~N7OSB:
Calculated ~ H: 550.39.
Found: 550.42.
Example 15
Boc-Leu-Gly-Leu-~la-boroArg-C~0H~-benzene sulfonic ac~d
Boc-Leu-Al~-O~zl was prepared by the procedure
for dipeptide ~ynthesis in Example 2. Boc-Leu-Ala-OBzl
(23.7 g, 57.7 ~moles) wa~ dissolved in 40 mL of
~nhydrous trifluoroacetic acid. After 15 min, exces6
trifl~oroacetic acid was removed by evaporation and the
~e~ldue was treated with ether to yield H-Leu-Al~-
OBzl.tr~fluoroacetic acid as a crystalline product
(22.8 g).
~naly~ for C~,H, 5 N2OSF~:
Calculated: C-53.19~, H-6.21%, and N-6.89%.
Found: C-53.37%, H-5.68~, and N-6.84%.
aoc-Gly-Leu-Ala-OBzl was prepared by coupling
Boc-Gly-OH (5.70 g, 32.6 mmoles) to H-Leu-Ala-08zl using
the mixed anhydride procedure described in Example 2.
The product (13.8 g) was obtained as an amorphous solid.
Boc-Gly-Leu-Ala-OBzl was deblocked with trifluoroacetic
acid by the procedure described for the preparation of
H-Leu-Ala-OBzl except that the trifluoroacetate salt was
soluble in ether. The preparation was dissolved in
- 55 -
13~837
56
ethyl ~eetate and treated with anhydrou~ hydrogen
ehloride. The re~ulting produet wa~ pree~pitated by the
addition of ether to yield 7.7 g of H-Gly-Leu-Ala-OBzl
~~Cl ln a flr~t erop.
Boe-Leu-Gly-Leu-Ala-OBzl wa~ prepared by
eoupllng Boe-Leu-OH (2.62 g, 10.5 ~mole~) to
H-Gly-Leu-Ala-OBzl u~lng the mixed anhydrlde proeedure
de~erlbed ln Example 2. The re6ulting produet wa~
erygtallized from ethyl aeetate:hexane to yield 2.7 g
(mp 9S-96~) ln the fir~t erop.
Analyri~ for C2 ~ H~N~O7:
Caleulated: C-61.89%, H-8.26%, and N~9.96.
round: C-62.00%, H-8.40%, and N-9.83%.
Boe-Leu-Gly-~eu-Ala-OH wa~ prepared by the
eatalytie hydrogenat~on of tbe benzyl erter (2.6 g, 4.62
~mole6) by the proeedure deseribed ln Example 2 to yield
2.1 g. The re6ulting produet wag ery~talllzed fro~ hot
ethyl aeetate to yleld 1.4 g.
Analy~i~ for C~H~oN~O7:
Caleulated: C-55.90%, H-8.SS%, and N-11.86%.
Found: C-55.42%, 8-8.47%, and N-11.73%.
Boe-Leu-Gly-Leu-Ala-NH-CH[(CH2)~Br¦BO2-Cl0R~
wa6 prepared by eoupling Boe-Leu-Gly-Leu-Ala-OH (1.40 g,
2.96 ~mole~) to the ~mine from Example la. Thi~ wa~ done
u~$ng the proeedure in Example 2 exeept that the
ehromatographie ~tep was eliminated. The produet
erystallized from ethyl acetate:hexane to yield 1.17 g.
TLC ~n ~ethanol:chloroform tl:9) indicated ~ cingle cpot
Rf 0.68.
Analy6i6 for C~6H6 3 NsO~BrB:
Caleulated: C-55.10%, H-8.11%, N~8.93%, and B-1.38%.
Found: C-5S.96%, H-8.30%, N-8.7q%, and B~1.33~.
The corre6ponding azide wa6 prepared by the
- 56 -
1 ~ 3 ~ 7
procedure de6cr$bed in Examplc 3 ~n a yield of 97% and
~t was converted to ~oc-Leu-Gly-Leu-Ala-boroOrn-Cl0H
by the method descrlbed ~n Example 4. An ~nalytlcal
eample was prep~red by precipitating the product with
ether and then chromatoqr~phing lt on LH-20, and
reprec1pitatlng ~t from chloroform with hexane.
MS~FAB) for C" H~5N,O,B:
Calculated + H: 721.50.
Found: 721.55.
Boc Leu-Gly-Leu-Ala-boroArg-Cl0H16
~ulfonic acid was prepared by the method descr~bed ~n
Example 5. The corresponding boroOrnithine peptide
(0.695 g, 0.791 mmoles) was reacted with 5 ~L of ~
cyanamide solution (50 mg/m~) in absolute eth~nol. The
above m$xture was chromatographed and tr~turated w~th
ether, wherein 0.41 q of the desired product was
obtained.
MS(FAB) for C~H~N~O~B:
Calculated ~ H: 763.53.
Found: 763.8.
Example 16
H-Leu-Gly-Leu-Ala-boroArg-C~0Hl 6 HCl-benzene ~ulfonic
acid
Boc-Leu-Gly-Leu-Ala-boroArg-Cl0Hl~-benzene
sulfonic acid (Example lS, 0.050 g, 0.0543 mmoles) was
reacted with 2 mL of 4 N hydroqen chloride in dioxane
for 5 min at room temperature. Solvent and excesb
hydrogen chloride were removed by evaporation. The
6ample was dried over potassium hydroxide in vacuo, over
night, and then triturated with ether to yield the
product (46 mg) as a mixed salt.
MS~FAs) for C~2Hs9N~O6B:
Calculated + H: 663.47.
- 57 -
sa 1339~
round: 663.50.
~xample 17
Bz-Glu(OBu)-Gly-boroArg-C~0Hl~-benzenc ~ulfon~c ac~d
Bz-Glu(OBu)-Gly-NH-CH~(CH2)~ Lr ILO2 - C~ o Hl~ was
prepared by coupllng Bz-Glu(OBu)-Gly-OH to the amine
according to the method described in Example 2. The
corresponding azide wa~ prepared by the method described
ln Example 3 and bo~oOrnithine peptide were prepared by
the method described in Example q.
MS(FA~) for C~2H~N~O~B:
Calculated ~ H: 613.38.
Found: 613.60.
The final product was obtained by the method
de~cribed in Example 5.
MS(FAB) for C~H5~N~O~B:
Calculated I H: 6S5.40.
Found: 655.37.
An~lysis for C~H57N6O10SB
C~lculated: C-57.62%, H-7.08%, N-10.34%, and B~1.33%.
Found: C-57.43%, H-7.25%, N~ 9.91~, ~nd ~-1.23%.
Example lB
Bz-Glu-Gly-boroArg-C~0Hl~-benzene sulfonic acid
sz-Glu(osu)-Gly-boroArg-cloHl~ benzene
sulfonic acid (0.13 g, 0.16 ~oles) was dissolved ~n
5 mL of dioxane, benzene ~ulfonic acid (0.10 g, 0.66
mmoles) wa~ added, and the 601ution was stirred
overnight at room temperature. The solution was then
concentrated to approximately 1 mL by evaporation and
then it was triturated with ether to yield a solid (0.14
g). The material wa~ chromatographed on a 2.5 X 50 cm
column of LH-20 in methanol. Fraction~ containing the
ss 13393g~
desired product were subjected to evaporation and the
residue ~a6 triturated with ether to yie~d 53 ~g of the
teslred product.
MS(FAB) for C~H~N~07B:
Calculated ~ ~: 599.34.
Found: 599.35 + 613.36 (unidentified).
Example 18a
Bz-Glu-Gly-boroArg-C~O~l 6 benzene eulfonic acid
Bz-Glu(OBu)-Gly-boroArg-ClOHI~-benzene
~ulfonic acld (Example 17, 0.20 Q, 0.246 ~moles) war
treated with anhydrous hydrogen chloride by the
procedure described in Example 6 for 45 min. After the
~aterial was triturated with ether, NMR lndicated that
approximately 30% of the t-butyl protecting group was
still present. The product was then reacted wlth
anhydrou~ ~FA for 45 ~in at room temperature. TFA was
removed by evaporation and the re6idue was triturated
with ether to yield 143 mg.
MS(FAB) for C2~H~3N6O~B:
Calculated ~ H: 599.34.
Found: 599.35.
Example 19
Bz-Pro-Phe-boroArg-C~0Hl 6 ~benzene eulfonic acid
Bz-Pro-Phe-OH (mp 200-201~) was prepared by
the ~ethod described in Example 2 for dipeptide
synthesis.
Analysis for C2lH22N2O~:
Calculated: C-68.82~, H-6.06%, and N-7.65%.
Found: C-68.91%, H-6.09%, and N-7.47%.
sz-pro-phe-NH-cHl(cH2)~srlso2-cloHl~ ~a~
prepared by coupling Bz-Pro-Phe-OH to the amine using
- 59 -
~33~g7
the general method described in Example 2 except the
chromatography ~tep was eliminated. TLC ln
methanol:chloroform (1:9) indicated ~ ma~or ~pot ~t ~f
0.72 and a trace at Rf 0.86.
MS(FAB) for C3sH~sN3~sBBr:
Calculated ~ H: 678.27.
Found: 677.95.
The alkyl halide wa6 converted to the azide
and to the boroOrnithine peptide by the procedure6
described in Examples 3 and 4.
MS(FAB) for (Bz-Pro-Phe-boroOrn-CI0Hl6) C~sH~7N~O5B:
Calculated + H: 615.37.
Found: 615.42.
Bz-Pro-Phe-boroArg-Cl0H~-benzene ~ulfonic
acid was prepared by the method described in Example S.
Ms(rA~) for C3~,N~O~B:
Calculated ~ H: 657.39.
Found: 657.13.
Analy6i6 for C~ 2 HssN6O~SB
Calculated: C-61.90%, H-6.82%, N-10.31%, and B-1.33%.
Found: C-60.16%, H~7.27%, N- 9.79%, ~nd s-1.44%.
Example 20
~z-~ro-Phe-boroArg-OH HCl
Bz-Pro-Phe-boroArg-C10Hl~-benzene sulfonic
acid (Compound of Example 19, 0.64 g, 0.79 mmole~) was
dis~olved in 4 mL of methylene chloride ~nd cooled to
-78~. It was added to flask containing 4 mL of 0.50 N
boron trichloride, which had been prepared by diluting
1.0 N boron trichloride (Aldrich Chemical Co.,
Milwaukee, WI) 50% with dry methylene chloride, in a dry
ice bath. The solution was stirred for S min at -78~,
then the flask was transferred to a O~ ice bath where
the solution was stirred for lS min. Cold water (5 mL)
- 60 -
61 1~3~
wa6 added 61Owly then the 601utlon wa6 diluted to 120 mL
with 20% acet~c acid. The organ1c pha~e which 6eparated
war removed and d~rcarded. The aqueouc pha6e Wa6
~pplied to a 20 mL column of SP-SephedexS~ whlch wa~
equilibrated with 20% acetic acid. The column wa~
washed with approximately 150 mL of 20% ~cet~c acid then
~ub~ected to a linear gradient from 200 mL of 20% acetic
acid to 200 mB of 20% acet~c acid containing 0.30 N
hydrochloric ac~d. The product eluted when the
concentration of h~drochloric ac~d was between 0.08 and
0.15 N. The de6ired product (0.19 g) waC obtained after
evaporating the ~olvent, drying the re6~due ~n ~acuo,
and triturating it with ether.
MS(FAB) for C2~H~SN6OsB:
Calculated + ~: 523.29.
Found: 579.34 ~unidentified).
Analy6i~ for C2,H,~N~O5ClB.
Calculated: C-53.29%, H-6.55%, N-14.3q%, and B-1.8q%.
Found: C-S3.27%, H-6.58%, N-13.25%, and B-1.89%.
E6terification of the product with pinanediol
as de6cr~bed ~n Example 8a gave a product who~e NMR and
MS propert~e6 were con~i~tent with the ~tarting e~ter of
Example 19.
Ms(rAB) for C~H~N~O~B:
Calculated + H: 657.40.
Found: 657.39.
- 61 -
~33~8~7
62
~xample 21
Bz-Pro-Phe-boroArg-F-hydrogen chlorlde
Bz-Pro-Phe-NH-CHl(CH2)~NH-C(NH)NH2¦BF2~HCl
Bz-Pro-Phe-boroArg-r was prepared by a
modlflcatlon of the procedure descrlbed by ~lndcr et
al., J. Med. Chem., 28: 1917-1925, (1985). Free ~oronic
~cid (Compound of Example 20, 0.100 g, 0.179 mmoles) was
dissolved ln 2 mB of water. To it, 0.040 mL of ~B~
hydrofluoric acld was added at room temperature. A
gummy prec~pltant formed almost in6tantly. The reactlon
was ttirred for 10 m~n, then the mixture was frozen ~nd
exces6 hydroflur$c ~cld ~nd water were removed ln v~cuo.
The resldue was di~601ved in methanol, concentr~ted, ~nd
triturated with ether. ~ yield of 0.093 g was obtained.
Ms(FAB) for C26H33N6O3BF2
Calculated H: 527.29.
Found: 527.31 and additional masses characterlstic
of the free boronlc acid.
Analysl6 for C2~H~N6O3BF2Cl H2O
Calculated: C-53.47~, H-6.25%, N-14.47%, B-1.86%, and
F-6.54%.
Found: C-54.00%, ~-6.40%, N'13.48%, B-1.95%, and
F-7.06%.
Exampl e 22
~oc-(D)Phe-Pro-boroIrg-ClOH~ 6 ~Hsr
BoroIrg- ls the abbreviation for
-NH-CH[tCH2)~S-C~NH)NH2]so2- in which the isothiouronium
group replaces the guanidino of boroArginine.
Boc_(D)phe-pro-NH-cHl(cH2)3BrlBo2-cloHl 6 ( CompoUn
Example 2, 1.00 q, 1.61 mmoles) was dissolved in 4 mL of
absolute ethanol and thiourea (0.37 9, 4.82 mmoles) was
added. ~he mixture was stirred overnight at room
temperature. ~he solution was concentrated and the
- 62 -
63 133~8.97
residue wac triturated with ether to yield O.S8 g of
colid. ~he resulting solid wa8 chro~atographed on a 2.5
X 50 cm column of LH-20 in methanol. Pooled fractlon6
contalning the deslred product were sub~ected to
e~aporatlon to y~eld 0.26 g of product. The ~ample
wa~ triturated with ether to y~eld O.lS0 g of an
~morphou~ rolid.
MS(FAB) for C~Hs3N50~BS:
Calculated ~ ~: 670.38.
Found: 670.39.
Analy6is for C~H5~N50~SBrs.
Calculated: C-54.40~, H-7.13~, N-9.33%, and a-1.44%.
Found: C-54.10%, H-7.39%, N-9.27%, and ~-1.47%.
~ he ether ~oluble re~idue obtained from thi 6
reaction cons$6ted mainly of etarting material which was
converted to i60thiouronium 6alt by longer reaction
period6.
Thi6 general procedure was u6ed to prepare
other lsothiouronium salt~ except ln some case6 a 4-fold
exces6 of thiourea and 3-4 day reaction t~me6 were used.
Example 23
H-(D)Phe-Pro-boroIrg-C~OH~ HBr,HCl
Loc-(D)Phe-Pro-boro~rg-ClOHl~-HBr (Compound of
Example 22, 0.050 g, 0.067 mmolec) wa6 reacted wlth
~L of 4 N hydrogen chloride in dioxane for 15 min at
room temperature. Solvent was evaporated and the
rebidue was triturated with ether to yield 0.040 g of a
white 601id.
MS(FAB) for C2~H~sNs~~SB
Calculated ~ H: 571.29.
Found: 570.47.
- 63 -
64 13398~
Example 24
Ac-~la-Ly~(Boc)-boroIrg-ClOHI~ HBr
Ac-Ala-Ly6(Boc)-NH-CHI(CH2)~Br]BO2-ClOHl~
(from Example 13, 0.700 g, 1.04 umolc6) wa6 reacted w~th
thiourea (0.320 g, 4.00 mmole6) for 4 day~ ln 4 mL of
abco1ute ethanol. Thc product wa6 pur~fied by the
procedure descr1bed ln Example 22. rollow~ng
chromatography, 0.2B g of the de~red product were
obtained. ~rlturat~on w~th ether y1elded 0.173 g of the
product as an amorphou6 white 601ld.
MS(FAB) for C~H5sN~O~SB:
Calculated + H: 667.8.
Found: 667.
Analysi~ for C~ HS ~ N~ 07 SBrB:
Calculated: C-49.79%, H'7 . 56%, N-11.24%, and B-1.44%.
Found: C-49. io%, H-7 . 62%, N'11.31%, and B-1.36%.
Example 25
Ac-Ala-Ly6-boroIrg-C~OHl~2HBr
Ac-Ala-~ys(Boc)-boroIrg-clo~l~ Hsr ~t
compound of Example 24, 0.050 g, 0.067 ~mole6) wa6
d~6solved ~n 1 mL of methanol and hydrogen brom~de gac
was bubbled though thc 601utlon of 10 ~n. 801vent wa6
removed by evaporat~on and the re61due wa~ tr~turated
wlth ether to yleld the dec~red product a6 a sol~d
(49 ~g).
MS(FAB) for C~ H4 7 N6 ~s SB
Calculated I H: 567.35.
Found: 567.41.
Example 26
Ac-Phe-boroIrg-ClOHl 6 ~HBr
Ac-phe-NH-cH[(cH2)~BrlBo2-cloHl 6 ( from p
- 64 -
6s 1339~97
9, 1.00 9, 2.41 ~moles) wa~ reacted with a 3-fold exce~6
of thiourca ~n 5 mL of ab~olute ethanol followlng the
procedure de~cribed $n ~xample 22. The product (0.284
g) wa~ obta~ned a6 a white amorphou6 ~ol~d. Add~t~onal
product wa~ obtained by aga$n reacting any rema1ning
ether ~oluble material with thiourea and repeating the
purification procedure.
MS(FAB) for C2~H"N~O, SB:
Calculated ~ H: 51S.29.
Found: 515.29.
Analysi~ for C2~H~N~O~SB.
Calculated: C-52.44%, H-6.79%, N-9.41%, ~nd ~-1.82%.
Found: C-52.83%, H-6.89%, N-8.47%, and s-1.85%.
Example 27
Bz-Pro-Phe-boroIrg-C~OHl~-HBr
BZ_pro-Phe-NH-CH 1 ( CH2 ) 3 BrlBO2ClOHl~ (produ
from Example 19, 0.500 g, 0.737 ~moles) wa6 used to
prepare the product of this example by following the
procedure descr$bed ~n Example 22. Product (0.358 g)
wa~ obtained as a white ~olid.
MS(FAB) for C3~H~NsO5SB:
Calculated + H: 674.35.
Found: 674.27.
Analysis for C~H~sOsSBBr.
Calculated: C-57.29%, H-6.56%, N-9.28%, and B-1.43%.
Found: C-57.46%, H-6.q5%, N-B.78%, and B-1.38%
Example 28:
Boc-Leu-Gly-Leu-Ala-boroIrg-ClOHl6-HBr
BOC-LeU-Gly-LeU-Ala-NH-CH[ ( CH2 ) 3 BrlBO2-C~OH~6
(product from Example 15, 0.770 9, 0.980 mmoles) was
used to prepared the i60thiouronium analog of ~his
example u6ing the procedure described in Example 22.
- 65 -
66 13 3g 8~ 7
Following chromatography o~ the rcaction product~, the
final product t0.400 g) wa~ obtalned ar a white colld by
trituration with hexane.
MS~FAB) for c~7H~N7o~ss:
Calculated: ~ H: 780.48.
round: 780.52.
~nalycl~ for C~7H~N5O~SBrB.
Calculated: C-51.62%, H-7.~6%, N'11.39%, and B-1.26%.
Found: C-51.03%, H-7.86%, N-11.14%, and B-1.18%.
Example 29
H-Leu-Gly-Leu-Ala-boro~rg-cloHl~-zHBr
Boc-Leu-Gly-Leu-Ala-boroIrg-Cl0Hl 6 ~HBr
(compound of Example 28, 0.100 g, 0.12 mmoler) war
di~olved in 1 mL of methanol and 1 mL of 0.7 N hydrogen
bromide in methylene chloride wa~ added. The mixture
wa~ ~tirred for 15 min at room temperature. Solvent and
exce~ hydrogen bromide were removed by evaporation and
the re~idue wa~ triturated with ether to yield the
de~ired product ~n almost quantitative y~eld.
MS~FAB) for C,2H5,N7O~SB:
Calculated ~ H: 680.43.
Found: 680.50.
Example 30
Bz-Glu(oBu)-Gly-borolrg-cloHl 6 HBr
Bz-Glu(oBu)-Gly-NHl(cH2)~srlso2-cloHl~
(product from Example 17, 0.293 q, 0.433 mmole~) was
u~ed to prepare the i~othiouronium analog (0.220 g)
u~ing the procedure de~cribed in Example 22.
MS~FAB) for C~Hs0N5o75s
Calculated + H: 672.36.
Found: 672.3.
Analysis for C~ HslN507SBBr:
- 66 -
67 1339~7
Calculated: C-52.66%, H-6.84%, N-9.31%, and B-1.44%.
round: C-52.38%, H-6.76%, N~8.81%, and B-1.46%.
Example 31
Bz-~lu-Gly-boroIrg-CIOHl~NBr
~ z-Glu(OBu)-Gly-boroIrg-CIOHl~-HBr (the
product of Example 30, 0.050 g, 0.066 ~moles) wa~
di6~01ved in 1 mL of ~FA and ~tirred for 1 h at room
temperature. Hydrogen bromide ln methylene chloride
(O.35 mmolcc) wa~ added and the liquld of the re~ultlng
~olution wa6 evaporat~d. The re~ldue wa~ triturated
wlth ether to yleld 47 mg.
MS(FAB) for C2~H~2N5O~SB:
Calculated + H: 616.30.
Found: 616.34.
Example 32
Boc-(D)Phe-Phe-boroIrg-ClOHl6 HBr
Boc-(D)Phe-Phe-NH-CH[(CH~)~ e rlBO~-ClOH~
(compound from Example 11, 1.50 g, 2.07 ~ole~) wac used
to prepare the i~othiouronium analog (0.90 g) uslng the
procedure de~cr$bed in Example 22.
MS(FAB) for C~Hs~N5O6SB:
Calculated + H: 719.84.
round: 720.
Analy~is for C~HssNsO6SBBr
Calculated: C-56.99%, H-6.94%, N-8.75%, and e-l . 3S% .
Found: C-55.89%, H-6.87%, N-8.59%, and B-1.18%.
Example 33
H-(D)Phe-Phe-boroIrg-Cl0Hl 6 2HBr
Boc-( D ) Phe-Phe-boroIrg-ClOH~ 6 ~HBr (compound of
Example 20, 0.20 g, 0.25 mmoles) was reac~ed with
- 67 -
68 ~39~7
hydrogen bromide by the procedure descrlbed in Example
29 to yield 188 mg of the desired product.
MS(FAB) for C~ 3 H, 6 NS ~, SB:
Calculated + H: 620.34.
round: 620.40.
Ex~mple 34
Z-Phe-Gly-Gly-borolrq-C~OHl~-HBr
z-phe-Gly-Gly-NH-cH[(cH2)~B~lBo2-cloHl 2 wa~
prepared by coupling Z-Phe-Gly-Gly-OH to the ~mine
(Example la) us~ng the procedure de~crlbed ln Example 2.
Analy~ic for C~H~N~07BBr:
Calculated: C-57.93%, H-6.40%, N-7.72%, ~nd B-1.49%.
round: C-58.42S, H-6.83~, N-7.74%, ~nd B-1.96%.
The alkyl halide (1.00 g, 1.38 mmole~) was
converted to the isothiouronium analog by the method in
Example 22 to yield product (0.87 g) as a white
~morphous 601id.
MS(FAB) for C,~H" N60~SB:
Calculated + H: 721.36.
Found: 721.32.
An~ly~i8 ~or C~ 6 HSoN~ 07 SBBr:
Calculated: C-54.00%, ~-6.31%, N-10.50%, and B-1.35%.
Found: C-53.17%, ~-6.50%, N-10.03%, ~nd B-1.25%.
Example 35
Boc-Ala-Phe-(D~L)boroIrg-C6Hl2 Hsr
Boc-Ala-Phe-OMe was prepared using the mixed
anhydride procedure described in Example 2.
Analysis for C1~H26N20s:
Calculated: C-61.70% H 7.48% N-7.99%.
Found: C-61.51% H-7.56% N-7.92%.
- 68 -
1~3~7
69
~ he methyl ester was hydrolyzed w~th ba6e to
yleld Boc-Ala-Phe-OH ~n a y~eld of 56~.
Boc-~la-Phe-NH-CH[(CH 2 ) ~ BrlBO2-C~Hl 2 wa 6 p p y
coupllng ~oc-Ala-Phe-OH to NH2-CHl(CH2)3Br]BO2-C~H~-HCl
(Example lb) u~ing the method de~cr~bed ~n Example 2,
except LH-20 chromatography was not used.
Boc-Ala-Phe-NH-CHt(CH2) 3 BrlBO2-C~Hl 2 (1.00 g,
1.72 ~mole~) was reacted with th~ourea ur.~ng the
procedure descr~bed ~n Example 22 to y~eld the
~othlouron~um anal~g (0.485 g) as a wh~te sol~d.
MS(FAB) for C2~H~N50~SB:
C~lculated +H: 592.33.
Found: 592.60.
Analysi6 for C2 ~ H~7Ns0~SBBr:
Calculated: C-50.00%, H-7.06%, N-10.41%, and B-1.61%.
Found: C-49.50%, H-7.2~%, N-10.22%, and B-1.41%.
Example 36
H-Ala-Phe-(D,L)boroIrg-C~Hl 2 ~2HBr
Boc-Ala-Phe-boroIrg-C~Hl 2 ~ HBr (Example 35,
0.10 g, 0.149 ~moles) was reacted w~th hydrogen brom~de
by tbe procedure de~cribed ~n Example 29 to y~eld the
desired product ln almo6t quantitative y~eld.
MS(FAB) for C2~H~N506SB:
Calculated ~H: 492.28.
round: 492.26.
Example 37
Boc-Ala-phe-(D~L)boroHomo~rg-c6Hl 2 ~ HBr
Boc-Ala-Phe-NH-CHf(CH2),-S-C(NH)NH2IBO2-C6Hl2-HBr
soc-Ala-Phe-oH (from Example 35) was coupled
to the a~ine (Example ld) to yield Boc-Ala-Phe-NH-
CHl(CH2)~Br]BO2-C6Hl2. The procedure in Example 2 was
u6ed except the LH-20 chromatography step wa6 not needed
- 69 -
1339897
for purification. An analytical sample was obtained by
chromatography on silica gel using ethyl acetate as an
eluent.
MS(FAB) for C28H4sN3O6BrB:
Calculated + H: 610.27.
Found: 610.24.
AnalySiS for C2sH4sN3o6BrB-
Calculated: C=55.19%, H=7.28%, N=6.90%, Br=13.11%, and
B=1.78%.
Found: C=55.30%, H=7.39%, N=6.40%, Br=12.07%, and
B=1.95%.
The alkyl bromide (0.537 q, 0.883 mmoles) was
reacted with thiourea using the procedure in Example 22.
The product (0.23 g) was obtained as an amorphous white
solid after trituration with ether.
MS(FAB) for C29H49N5O6S:
Calculated + H: 606.35.
Found: 606.38.
Analysis for C29H49N5O6SBBr.
Calculated: C=50.73%, H=7.21%, N=10.20%, and B=1.57%.
Found: C=50.22%, H=7.46%, N=9.74%, and B=1.55%.
Example 38
H-Ala-Phe-(D,L)boroHomoIrg-C6Hl2-2HBr
Boc-Ala-Phe-(D,L)boroHomoIrg-C6Hl2 -HBr
(compound of Example 37, 0.050 g, 0.073 mmoles) was
allowed to react with hydrogen bromide by the procedure
described in Example 29 to yield 44 mg of the desired
product.
MS(FAB) for C24H4oN504SB:
Calculated + H: 506.30.
Found: 506.39.
Example 39
.~
71 1~3~97
Boc-Ala-Phe-(D,L)boroLy6-C~Hl2-HCl
Boc-Ala-Phe-NH-CH[(CH2)~N~]BO2-C~H~2-benzene ~ulfonlc
acld
Boc-Ala-Phe-NH-CH[~CH2)~Br]sO2-C~H~ (from
Example 35) was converted to the alkyl azlde u~lng the
procedure ln Example 3 except the LH-20 chromatography
etep wac not needed for purlflcatlon. The azlde wa6
hydrogenated uslng the method descrlbed ln Example 4
except 2 equivalent6 of benzene sulfonlc acld were used
and the hydrogenation time wa~ 2 h to yleld the flnal
product in a y~eld of 40% (mp 154-160~, dec).
MS(FAB) for C2~H~N~O~B:
Calculated I H: 547.38.
Found: 547.43.
Example 40
H-A~a-Phe-(D,L)boroLys-C6H~2-TFA-benzene sulfon~c acld
Boc-Ala-phe-(D~L)boroLy~-c6Hl2-benzene
culfonlc acid (compound of Example 39) wa6 reacted wlth
trifluoroacet~c ac$d for 1 hr at room temperature.
~olvent wac e~aporated and the residue was trlturated
wlth ether to yield a sol~d.
MS(F~B) for C~3H" N~O,B:
Calculated ~ H: 447.31.
Found: 447.31.
Anal- for C3lH,6N~OgSF3B 2H2~
Calculated: C-49.34%, H-6.68% N-7.42%, and B-1.43%.
round: C-49.26% H-5.94%, N-7.12% and s-1.34%.
Example 41
soc-(D)val-Leu-boroLys-c6H~ 2 ~benzene sulfonic acid
Boc-(D)Val-Leu-OH was prepared by the method
described in Example 2. The benzyl ester was obtained
- 71 -
72 133~7
ln a yield of 76%.
MS ( FA~ ) for C2,H,~N2 05:
Calcul~ted + H: 421.27.
round: 421.38.
Following hydrogenation, the free acld wa6
obtalned in a yleld of 100% a~ a whlte crystalllne
601 ld.
Analy~is for Cl~H29N2O5:
Calculated: C-59.34%, H-8.87%, and N-8.50%.
Found: C-59.34%, H-8.87%, and N-8.50~.
Boc-(D)Val-Leu-OH wa~ coupled to the am~ne
(~xample ld) u~ing the method descrlbed ln Example 37
for the coupllng of Boc-Ala-Phe-OH to yield
Boc-(D)Val-Leu-NH-CH[(CH2)~Brl~O2-C~HI2 ~n a yleld of
97% .
MS(FA~) for C27HSlN~~6BBr:
Calculated ~ H: 604.31.
round: 604.31.
~ he alkyl bromide wa~ converted to the
corresponding az~de ln a yield of 85% by the ~ethod
described ln Example 3, and the azlde was hydrogenated
by the method de~cribed ln Example 39 to y$eld the flnal
product a~ a white ~olid ln a yield of 62%.
MS(FAB) for C2~HS~N~~6B:
Calculated + H: 541.41.
Found: 541.46.
Analysis C~Hs~N~OgSL~1.5 H2O:
Calculated: C-54.62%, H~8.61%, N-7.73%, and B-1.49%.
Found: C-54.SB%, H-8.59%, N'7.92%, and B-1.9B%
Example 42
Ac-Phe-boroLys-C6H~2-benzene sulfonic acid
13 3 9 8 9 l
Example 42 was prepared according to the
procedure de6crlbed in Example 39.
Ac-Phe-N~-CHt(CH~)~Br]B0~-C~Hl2 was prepared in a yield
of 72%.
MS(FAB) for CllH3~NlO~B~r:
Calculated + H: 481.00.
Found: 481.21.
The azide was obtained in a yield of 57%. The
final product was obtained in a yield of 50%.
MS(FAB) for C22H,7N,0,8:
Calculated: + H: 418.29.
Found: 418. 31.
Analyti6 for C2~ N~O~SB H20:
Calculated: C-56.66%, ~7. 47%, N-7. 08%, and 8-l.B2%.
Found: C-56.8~%, B-7.43%, N-7.22%, and B-1.53~.
Example 43
Bz-(D~L)boroIrg-C~B12 ~~Br
Bz-(D~L)NH-cHl(cH2)~BrlBo2-c6Hl2 wa6 prePared
by react~ng the amine (Example lb, 5.0 g, 15.9 mmole6)
with an equivalent of benzoyl chloride and two
equ~alent6 of sodium bicarbonate ~n a mixture
con6isting of 4 m~ of dioxane and 4 m~ of water at 0~.
After ~nitially mixing the reagents, the react~on wa6
diluted with 6 ~L of 50% dioxane:water and ~t was
allowed to warm to room temperature. ~he reaction
m~xture was stirred approximately 30 mln at room
temperature and then the product wa~ extracted into
ethyl acetate and washed with water, 0.2 N hydrochloric
acid, 5% aqueous sodium bicarbonate, and 6aturated
aqueous 60dium chloride. The organic phase wa~ dried
over anhydrous ~odium ~ulfate, filtered, and evaporated
to yield a crystalline product. After i~olation and
washing with ethyl acetate, 3.26 g of compound l~p
- 73 -
74 1~3~9~
176-177-) were obtained.
~naly~i~ for CI~H25NO~BrB:
Calculated: C~53.44~, H-6.59%, N~3.67~, and B-2.83%.
Found: C~54.50%, H-6.76~, N~3.68%, and B.2.84%.
~ he alkyl halide (1.00 g, 2.62 mmole~) wa6
converted to corre~ponding i~othiouronium ~alt by the
procedure de6cribed in Example 22. The product, 0.84 g,
was obta~ned a~ a white ~olid.
MStFAB) for CI~H2~N~O3SB:
Calculated ~ H: 378.20.
Found: 378.21.
Analy~1~ for Cl~H~N~O3SBBr:
Calculated: C~47.18%, H-6.38%, N-9.17%, and 8~2.36%.
Found: C-46.11%, H'6.71%, N-8.97%, and B-2.22%.
Example 44
Bz-(D,L)boroArg-C~H~ 2 ~benzene 6ulfonic acid
The ~lkyl h~lide (Example 43, 2.0 g, 5.25
Dmole~) wa~ converted to 0.97 g of the azlde (mp
138-139~) u~ing the procedure ~n Example 3. The azide
wa~ converted to Bz-boroOrn-C6H~ 2 .benzene ~ulfonic acid
~n almo~t quantitative yield u~ing the procedure ~n
Exa~ple 4.
~S(FAB) for Cl~H27N2O~B:
Calculated ~ H: 319.22.
Found: 319.26.
Bz-boroOrn-C6Hl 2 ~benzene ~ulfonic aeid
(0.90 q, 1.84mmole~) was allowed to react with cyanamide
u~ing the procedure in Example 5 to yield 0.65 g of
crystalline product (mp 242-244~).
FAs(MS) for C1~H29N~O3B:
Calculated + H: 361.24.
Found: 361.24.
- 74 -
1~3g~7
Analysit for C2~H~5N~O~SB:
Calculated: C-55.59%, ~-6.82%, N~10.81%, and B-2.08%.
Found: C-54.60%, H-6.70%, N-11.24%, ~nd B-1.87%.
Example 45
Ac-Leu-Thr(OBu)-boroArg-C~OH~-benzene sulfonic acid
Ac-Leu-Thr(O~u)-OH wa~ prepared by coupling
~c-Lcu-OSu to H-Shr(OBu)-OH using the procedure ln
Example 13 for dipeptide ~ynthe~i~ except the final
product was obt~ined ac an amorphou6 white solid after
chromatograpby on ~-20. Ac-Leu-Thr(OBu)-OH (3.29 g,
9.90 moole~) wa~ coupled to the amine (Example la) using
the mixed ~nhydride procedure ~n Example 2 except the
~H-20 chromatography 6tep wac not needed.
Ac-Leu-~hr(OBu)-NH-CH[(CH2)~Br¦BO~-C~ wa~
a6 ~n amorphou~ white 601id, 5.39 g. The ~lkyl halide
wa~ con~ertet to the corresponding Azide in ~ yield of
82% u~ing the procedure in EYample 3 except a
chromato~raphy 6tep wa~ needed for further purificatlon.
The azide ~3.88 q, 6.42 mmoles) was hydrogenated by the
procedure in Example 4. ~he product,
Ac-Leu-Thr(OBu)-boroOrn-ClOHl~-benzene ~ulfonic acid,
wa~ obtained in a yield of 74% after chromatography of
the product on LH-20 ~nd trituration with ether.
MslrAB) for C~o~ssN~~6B
CalculAted ~ H: S79.43.
round: S79.48.
The boroOrnithine peptide was converted to the
final product in a yield of 86% by the procedure in
Example S.
MS(FAB) for C,lH57N6O~B
C~lculated ~ H: 621.qS.
Found: 621.S0.
Analysi~ for C37H6~N65O9~:
- 75 -
76 ~L~3~8~7
Calculated: C-57.05%, H-8.17%, N-10.79%, B-1.39%.
round: C-56.47%, H-8.01%, N~10.93%, and B-1.34%.
Example 46
Ac-Leu-Thr-boroArg-Cl0Hl~ benzene ~ulfonic acid
Ac-Leu-~hr(OBu)-boroArg-C~0H1~benzenc
6ulfonic acid tExample 45, 0.200 g, 0.257 ~mole~) wa~
dlc~olved ln a mixture of 2 mL of methylene chloride ~nd
2 mL of 4 N HCl:dioxane and wa~ allowed to 6t~r for 30
min at room temperature. Solvent wa~ evaporated and the
re6idue ~a~ dried under high vacuum. The de6ired
product wa6 obtained a~ a white 601id in a yield of 97%
by tr~turat~ng with ether.
MS(FAB) fo~ C27H~SN6O~B
C~lculated + H: 565.39.
round: 56S.48.
.
Example 47:
Ac-Ly~(Boc)-Pro-boroArg-Cl0Hl~-benzene ~ulfonic acid
Ac-~y6(Boc)-Pro-OH wa~ prepared by the method6
de6cribed in Example 13. It wa6 obta~ned a~ ~ wh~te
~olld (mp ~60-161.5-) after cryctallizat~on from ethyl
~cetate. Ac-Ly~(Boc)-Pro-OH (3.15 g, 8.1B mmole6) wa~
coupled to tbe amine (Example ~) u6inq the procedure ln
Example 2. The product, ~.8 g, wa~ u~ed without further
purif$cation. It wa~ converted to the azide ~n a yield
of 73% by the method in Example 3 after chromatography
on ~H-20. Hydrogenation by the method in Example 4,
chromatography on LH-20, and trituration of the ~ample
w~th ether gave Ac-Ly~(Boc)-Pro-boroOrn-ClOHl~-benzene
6ulfonic acid in a yield of ~1%.
MS(FAB) for Cl2HSsNsO7~
Calculated + H: 634.43.
~ound: 634.46.
- 76 -
8 g 7
The boroOrnithine peptide (2.0 g, 2.53 mmoles)
reacted with cyanamide by the procedure in Example 5 to
yield 1.8 g of the desired product as a white solid.
MS(FAB) for C33H57N707B:
Calculated + H: 676.46.
Found: 676.41.
AnalySiS for C39H63N7OloBS:
Calculated: C=56.23%, H=7.64%, N=11.77%, and B=1.30%.
Found: 56.06%, H=7.48%, N=11.75%, and B=1.22%.
Example 48
Ac-Lys-Pro-boroArg-ClOHl6-2HCl
Ac-Lys(Boc)-Pro-boroArg-Cl0Hl6-benzene
sulfonic acid (Example 47, 0.30 g, 0.360 mmoles) was
reacted with a 50:50 mixture of glacial acetic and 4 N
HCl:dioxane for 15 min at room temperature. Solvent
was evaporated and the residue was dried in vacuo.
The residue was dissolved in water and passed through
a 5 mL column of AG1-X8 (Cl form). The sample was
evaporated and the residue was triturated with ether
to yield the desired product as a white solid (230
mg).
MS(FAB) for C28H4gH705B
Calculated + H: 576.40.
Found: 576.45.
Example 49
Ac-Ala-Glu(OBu)-boroArg-Cl0Hl6-benzene sulfonic acid
Ac-Ala-Glu(OBu)-OH was prepared by coupling
Ac-Ala-OSu to H-Glu(OBu)-OH using the procedure in
Example 13. The product crystallized from ethyl
acetate:hexane (mp 147.5-148~).
Analysi S for Cl4H24N2O6:
Calculated: C=53.14%, H=7.66%, and N=8.85%.
X
1~3!~7
Found: C=53.28%, H=7.53%, and N=9.08%.
Ac-Ala-Glu(oBu)-NH-cH[(cH2)3Br]Bo2-cloHl6 was
prepared by the method in Example 2 except chloroform
was used instead of ethyl acetate for the organic phase
during the initial workup of the reaction and
chromatography on LH-20 was not used. The desired
product was obtained in a yield of 87% as partially
crystalline solid after evaporation of the organic
phase. The alkyl bromide was converted to the azide by
the procedure in Example 3. The desired product (mp
163.5-166~) was obtained in a yield of 50% by
crystallizing the crude reaction product from
chloroform.
analysis for C28H47N6O7B:
Calculated: C=53.51%, H=7.55%, N=6.69% and B=1.73%.
Found: C=55.51%, H=7.50%, N=6.50%, and B=1.66%.
The boroOrnithine peptide was prepared by the
method in Example 4 to yield the desired product in a yield
of 79%.
MS(FAB) for C28H49N4O7B:
Calculated + H: 565.38.
Found: 565.51.
The final product was obtained as a white
amorphous solid in a yield of 70% using the procedure in
Example 5.
MS(FAB) for C29H5lN6O7B:
Calculated + H: 607.40.
Found: 607.41.
Analysi S for C35H57N6OloBS:
Calculated: C=54.96%, H=7.53%, N=10.99%, and B=1.41%.
Found: C=54.36%, H=7.71%, N=11.27% and B=1.21%.
ExamPle 50
Ac-Ala-Glu-boroArg-C10H16-benzene sulfonic acid
~,~
~393~7
Ac-Ala-Glu(Bu)-boroArq-C~OH~-benzene ~ulfonic
acid (Example 49, 0.10 g, 0.131 umole6) wae dl~olved
ln 10 mL of acetic acid and anhydrou6 HCl ~a6 bubbled
through the ~olution for 20 min. The 601ution ~a6
~tlrred at room temperature for 1.5 h and colvent wa6
evaporated to yleld an oll. The de6ired product wae
obtained a6 a whlte solid (82 mg) after drying ~n ~acuo
and trituration with ether.
MS(FAB) for C~sH~N607B:
Calculated I H: 551.34.
round: 551.41.
The following compound6 were al60 prepared
u6ing ~ub~tant~ally the 6ame procedures a6 ln ~xample6
39 and 40 above:
Boc-Val-Val-boroLys-C~Hl~-BSA;
8-Val-Val-boroLy6-C~H1 2 ~BSA TFA;
80c-(D)Phe-Phe-boroLy~-C~Hl 2 ~ BSA;
H- ( D ) Phe-Phe-boroLy6-C~H1 2 ~ BSA-TFA
Boc-Glu-Phe-boroLyt-C~H~ 2 ~BSA
PyroGlu-Phe-boroLys-C~Hl~-BSA
Biological Example6
In the following examples, ~ denote6 micro.
Example6 S1 - 71
Inhibition of Human Plasma Rallikrein
Human plasma kallikrein was obtained from
Protogen AG (Switzerland). The 6pecific activity as
described by the 6upplier is 15 units per mg. A unit i6
defined as the quantity of enzyme required to hydrolyze
1 ~mole of substrate, H-(D)Pro-Phe-Arg-p-nitroanilide
(Kabi S2302), per min at a substrate concentration of
0.50 mM at 25~ in 50 mM potassium phosphate buffer, pH
8Ø
_ ,9 _
133~897
A rtock colut~on of enzy~e (1 ~n~t/~L) ~ac
prepared ~n 50% glycerol-0.lOM codlum phorphate buffer,
pH 7.5, conta~n~ng 0.20 M 60d~um chlor~de and 0.1~ P~G
6000 (polyethylene glycol). In et~ndard atr~y-, 10 ~L
of thc ~tock ~all~kre~n 601utlon ~ere added to 990 ~L of
a ~olut~on con~i~t~ng of 0.20 ~M S2302 ~n 0.10 DM ~od~um
pho6phate buffer, pH 7.5, conta~n~ng 0.20 M ~odlum
chlor~de and 0.1~ PEG at 25-. ~he effect of ~nhlbltor-
were e~aluated by mon~toring enzymat~c act~v~ty
determlned by ~e~ur~ng the ~ncrea~e ~n ab~orbance at
405 nm w~th time both ~n the pre~ence and ab6ence of
~nhlb~tor~. Table 1 chow6 ~nhib~tor level~ and the
actlv~ty remalnlng ~ea~ured ~n the tlme lnterval from 10
to 20 mln followlng ~nitlatlon of the reactlon.
~ct~v~ty of the control~ werc 0.0092 ~ 0.0095 mln~l.
Table 1
Inh~bit~on of Human Pla~ma ~alllkreln
Conc. Percent
Ex Inhlbltor (nM) Act~vlty
51 Boc-(D)Phe-Phe-boroIrg-Cl0Hl~-HBr 10 2
52 H-(D)Phe-Phe-boroArg-Cl0Hl~-2HCl 10 2.6
53 Boc-(D)Phe-Phe-boroArg-Cl0Hl~BSA 10 5.2
54 Boc-Ala-Phe-~D,L)boro~rg-C~Hl 2 ~HBr 10 15
55 Bz-Pro-Phe-boroArg-Cl0Hl~-BSA 10 15
56 Bz-Pro-Phe-boroArg-OH HCl 10 16
57 Bz-Pro-Phe-boroArg-F 10 18
58 Boc-Leu-Gly-Leu-Ala-boroArg-Cl0Hl6 ssA 10 30
59 Ac-Ala-Lys(soc)-boroArg-Cl0Hl6 ssA 10 34
60 Ac-phe-boroArg-cloHl6 HCl 10 q8
61 Ac-(D)-Phe-Pro-boroArg-Cl0Hl6-HCl 10 56
- 80 -
8~ 8 ~ ~
(Table 1 Continued)
Cone. Pereent
Ex Inhibitor (nM) Aetivity
62 H-(D)phe-pro-boroArg-cloHl~2 HCl 10 61
63 Ae-Ala-Ly~(~oc)-borolrg-CIOH~-HBr 50 1.4
64 Bz-Glu(OBu)-Gly-boroArg-ClOHl~;~SA 50 11
65 Ae-Phe-boroIrg-ClOHl~ H~r S0 17
66 8z-Glu-Gly-boroA~g-Cl~H1 6 ~ BSA 50 39
67 Ac-Ala-Ly~-boroArg-ClOHl 6 ~ 2HC1 50 39
68 Boe-Ala-Phe-(D;L)borohomoIrq-C~Hl 2 ~ Hsr 100 38
69 Boc-~la-Phe-(D,L)boroLy6-C~Hl2-HCl 1000 17
70 Boc-(D)Phe-Phe-boroOrn-ClOH16 BSA 10000 39
71 Boe-(D)Phe-Pro-boroOrn-C~OHl6-BSA 10000 100
Examples 72 - 110
Inhibit$on of ~hrombin (Esterase Aetivity)
Human thrombin (speeifie aetivity 2345 NIH
units/mg) was obtained from R.Q.P. Laboratorles, ~outh
Bend, IN) (Lot HT102). A stoek solution of thrombin wa~
prepared in O.OlOM PIPES Buffer, pH 6.0, eontaining 0.75
M 60dium ehloride. A~say~ of thrombin were run
aeeord~ng to. the proeedure of Green ~nd Shaw, ~nal.
~ioehem., 93: 223 (1979), in sodium phosphate buffer, pH
7.5, eontaining 0.20 M sodium ehloride ~nd 0.1% PEG
6000. The $nitial eoneentration of ~ubstrate was 0.10 ~M
and the eoneentration of thrombin was 1.0 nM (based on
weight). Table 2 shows inhibitor levels and the
activity remaining measured in the time interval from 10
to 20 min. following initiation of the reaction. The
activity of thrombin for the controls was 0.0076 +
0.0005 min~l.
- 81 -
82 ~ 3~
Table 2
Inh~b~t~on of Thromb~n
- Conc. Percent
Ex. Inh~bitor (n~) Act~vlty
72 Ac-(D)phe-pro-boroArg-c~oHl~-Hcl 5
73 Boc-(D)Phe-Pro-boroIrg-C~0Hl~ HBr 5 3
74 Boc-~D)phe-pro-boroArg-cloHl~ BSA 5 3
75 Ac-(D)Phe-Pro-boroArg-OH HCl 5 3
76 H-(D)Phe-Pro-boroIrg-ClOHl~-HBr-HCl 5 4
77 H-(D)Phe-Pro-boroArg-ClOHl~-2HC1 5 7
78 Boc-(D)Phe-Phe-BoroIrg-ClOHl~Br 5 ~8
7g H-(D)Phe-Phe-boroArg-Cl0~l~ 2HC1 10 10
~-~eu-Gly-Leu-Ala-boroArg-C~0Hl~-HCl-BSA10 25
81 Boc-(D)Phe-Phe-boroArg-C10Hl~-BSA 10 32
82 H-Leu-Gly-Leu-Ala-boroIrg-C1OHl~-2HBr 10 37
83 Boc-Leu-Gly-Leu-Ala-boroArg-Cl0Hl~ BSA 10 38
84 H-(D)Phè-Phe-boroIrg-ClOHl~-2HBr 10 49
Bz-Glu(OBu)-Gly-boroArg-Cl0Hl~-BSA 10 52
86 Bz-Glu(OBu-Gly-boroIrg-Cl0H~ HBr 10 59
87 Boc-Leu-Gly-Leu-Ala-boroIrg-cloHl~ HBr 10 66
88 Boc-(D)Phe-Pro-boro~rn-ClOHl~ BSA 100 18
89 Ac-Ala-Lys(Boc)-boroArg-Cl0~l~-BSA 100 18
Z-Phe-Gly-Gly-boroIrg-ClOHl~ H 100 46
91 Bz-Glu-Gly-boroArg-ClOHl~-BSA 100 46
92 Ac-Al~-Ly~(Boc)-boroIrg-ClOHl~-HBr 100 55
93 Bz-Pro-Phe-boroArg-OH-HCl 1000 18
94 Bz-Pro-Phe-boroArg-F 1000 18
Bz-Pro-Phe-boroIrg-ClOHl~HBr 1000 21
96 Boc-(D)Val-Leu-boroLys-C6Hl2~HCl 1000 21
97 Bz-Pro-Phe-boroArg-ClOHl6~BSA 1000 24
98 Boc-Leu-Gly-Leu-Ala-boroOrn-Cl0Hl 6 ~BSA 1000 24
99 Boc-Ala-Phe-(D,L)boroIrg-C6Hl2 HBr 1000 28
100 BZ-Glu-Gly-boroIrg-cl O Hl 6 H 1000 39
01 Ac-Ala-Lys-borOArq-Cl O Hl 6 ~2HC1 1000 45
- 82 -
B3 13 398~
~Table 2 Cont~nued)
Cone. Pcrccnt
Ex. Inhibltor (nM) ~ctlvlty
102 Ac-Phe-boroArg-C~OHl~-HCl 1000 53
103 Ac-Phe-boroIrg-ClOHl~Hsr 1000 6~
104 Ac-Ala-Ly6-boroIrg-cloHl~-2Hsr 1000 68
105 H-Ala-Phe-(D,L)boroIrg-C~Hl2 2HB~ 10000 23
106 Boc-Ala-Phe-(D,L)borohomoIrg-C~Hl2 HBr 10000 32
107 Loc-Ala-Phe-(D;L)boroLy6-C~Hl2 ~Cl 10000 46
108 H-Ala-Phe-(D,L)boroHomo$rq-C~Hl2 2HBr 10000 47
109 H-Ala-Phe-D,L)boroLys-C~Hl2-2HCl 10000 89
110 Ac-Phe-boroLys-C~Hl 2 HCl 10000 97
~xamples 111- 124
Inh~bltlon of Blood Coagulat~on As Shown by APTT and PT
Determlnatlon
She effect of protea~e lnhlbitors on blood
coagulat~on in vltro was determined by measurlng thelr
effects on two different cllnlcal parameter6, the
activated partlal tbromboplastln tlme6 (AP~) and
prothrombln ti~e~ (PT). Reagents for each of the~e
as6ay~ were supplled by General ~iagno~t$c~, Je6eup MD.
Stock ~olutlons of lnhlbitors were prepared ln 25 ~M
~EPES buffer, pH 7.5, contalnlng 0.10 M sodlum chlorlde.
ror the APTT assay, the lnhlbltor solutlon (0.100 mL)
was lncubated with normal human plasma (0.100 mL) and
automated APTT reagent (0.100 mL). After lncubation for
5.0 min at 37~, calcium chloride (0.100 mL) wa~ added
and the clotting time6, measured in ~econds, waC
determined on a fibrameter. ~he effects of the varying
concentrations of inhibitor on blood clotting time6
compared with the clotting times of controls run in the
absence of inhibitor, are 6hown in Table 3.
For PT assays, inhibitor solution6 (0.100 mL)
- 83 -
84 ~3~89~
were ~ncubated wlth nor~al human pla~ma (0.100 m~ ~d~
~ln at 37- Slmpla~tln re~gent (0 200 mL) wa~ t~en
added and clottlng tlme~ mea~ured a~ ~ho~n ln T~ble 4
Table 5 prov~dec a ~ummary of the re~ult~ ~n
T~ble~ 3 and 4, ~howlng the approx~mate concentratlon~
of lnhlb~tor requlred to lncrea~e the Actlvated Partlal
Thrombopl~tln Tlme~ t2X APTT), and the Prothrombln
Times (2XPT) two fold
Compound
De61gn~t~on In
Table~ 3-5, 7-10 Name of Inhlb~tor
A ~oc-(D)Phe-Pro-boroArg-ClOH~
B H-(D)Phe-Pro-boroArg-ClOH
C Boc-(D)Phe-Pro-boroIrg-ClOH
D Boc-(D)Phe-Phe-BoroIrg-C~OH
E Boc-(D)Phe-Phe-boroArg-ClOH
F Ac-Phe-boroArg-ClOH
G Ac-Phe-boroIrg-ClOH
Table 3
Actlvated Partlal Thrombopl~stln T~me~
(mea~ured ~n ~econd6)
Example Number
111 112 113 llq 115 116 117
Conc Compound
(n~) A s C D E F G
0 35 3 32 32 8 34 7 34 7 33 7 33 8
35 8 36 8 35 35 2
62 5 36 5 39 2
125 36 6 44 2 46 9 45 2 40 5 35 2 36 2
200 40 2 71 5 67 46 2 43 6 36 7 39 3
250 81 2 158 7 160
275 60 7 52 8 44 8 58 2
300 113 3 169 7 197 8
350 128 7 249 7 301 8 75 7 54 8 58 2 66 7
550 94 B 61 3 144 2 98 4
13 3g 8~7
Table 4
Prothromb~n T~me6
(me~ured ln ~econd~)
Example Number
llB 119 120 121 122 123 124
Conc. ' Compound
(nM) A B C D E F G
0 15.5 15.8 14.4 15.8 16.7 15.7 15.3
12.5 16.4 20.1 16.8
250 17.1 22.5 20.8
500 21.5 33.8 27.2 15.7 19.8 13.7
625 46.5
750 26.7 85.3 44 17.2 22 14.9
875 40.1
1000 >200 >250 152.7 19.9 29.3 16.2 19.7
2000 23.4 39 20.8 43.6
4000 49.2 70.8 51.5
Table 5
~nh~b~tion of ~lood Co~qul~t~on
C~lc. Concentr~t~on
Compound 2X APTTtnM) 2XPT(nM)
A 230 800
B 170 460
C 200 650
D 370 1200
E 375 2600
F 325 2500
G 625 1200
- 85 -
86 1339897
Example~ 125 - 127
Inhlbltion of Blood Coagulatlon A6 Shown ~y TT
Determination6
The effect of the protea6e inhlbltor
Ac-~D)Phe-Pro-boroArg-OH (Examplc 8) on blood
coagulation in vitro wa~ determined by mea6uring it6
effect on thrombin time~ (TT). A mixture of 0.2 ml of
normal rabbit pla6ma and 0.05 ml of buffer containing
the inhlbltor at 6 time~ the de~lred f$nal concentratlon
wa6 warmed to 37~C. Clotting wa6 lnltiated by addition
of thrombin (0.05 ml at 6 tlmes the f~nal
concentration). The thrombin u~ed wa~ purcha6ed from
Sigma Chemical Company (No. T-6634, activity 1190 NIH
unit6 per mg protein) and prepared ~n buffer. The
buffer employed for both the inhibitor and the thrombln
wa~ 0.1 M Tris buffer (12.10 g/L), containing 0.154 M
NaCl (8.84 g/L) and 2.5 mg/ml bovine 6erum album~n, pH
7.4. The clotting times, measured ln second6, were
determlned u~ing a fibrometer. The effect6 of the
inhibitor on blood clotting times compared with the
blood clotting time~ of control6 run in the absence of
inhibltors, are 6hown in Table 6. Value6 repre6ent the
average of at lea6t three determination~. If clotting
did not occur within 300 6econds, the reaction wa~
termlnated.
Table 6
Thrombin Times
~x. Thrombin Conc. Inhibitor Conc. ~hrombin Times
(~/ml) (nM)
--- 0.75 0 > 300
--- 0.83 0 226.9 ~ 14.8
87 133989~
--- 1 0 1~7.2 ~ 9.1
--- 1.2 0 121.1 + 0.8
--- 2 0 51.8 + 0.6
--- 3 0 40.0 + 1.9
--- 4 0 24.4 + 0.3
125 4 150 > 300
126 4 100 62.4 + 7.2
127 4 S0 32.7 + 0.8
the mean time needed for clotting, measured in
seconds, ~ the standard dev$ation
Examples 128 - 132
Stab$1ity of Inhibitors in Human Plasma
As Measured 8y APTT
The stability of inhibitor6 in plasma was
determined by their ability to $nhibit blood
coagulation. Fir~t, a stock ~olutions (1.0 ~M) of the
inhibitors to be tested in 25 mM HEPES buffer, pH 7.5,
containing 0.10 M sodium chloride were diluted 50% with
normal human plasma. The ~i~ture~ were ~ade at 0-, then
aliquots (0.200 ~) were re-oved ~nd incubated for 2 min
at 37-. An equal volume of automated APTT reagent was
added and clotting times were measured as described in
Examples 111-117. The final concentration of inhibitor
during the clotting assays was 250 nM. The incubation
t$mes (shown in hou~s) and clotting time (measured in
seconds) for individual inhibitors are chown in Table 7.
Values for compounds E and F were determined
simultaneously with the Control. Values for compounds
A, ~ and C were obtained on a different day.
- 87 -
88 ~ 89 7
~able 7
Stab~lity of Inhib~torr. ln Human Plasma
Example Number
128129 130 131 132
Compound
Control F E A B C
Incubation
Time (h) Clotting Time (sec)
0 41.5 76.3 63.2 81.2 152.2 203.2
0.5 42.7 76.4 73.2 84.7 157.7 207.2
1 42.7 76.7 66.2 79.7 163.7 214.2
2 42.7 79.6 67.7 86.7 152.8 203.7
3 44.8 77.8 61.7
4 44.2 81.8 58.2 98.2 157.7 209.7
45.7 80.8 61.3
6 45.2 79.3 5~.3
24 35.2 73.9 64.7 92.2 109.3 248.7
48 4?.2 49.3 58.7
Examples 133-136
Stabillty of Inhibltor6 in Buffer
Inhibitor~, each at a concentration of 1.0 ~M,
were incubated at room temperature ln 0.20 M ~odium
phosphate buffer, pH 7.5, containing 0.20 M ~odium
chloride and 0.10% PEG. Aliquots ~4.0 ~L) were removed
and assayed in the thrombin assay as de~cribed in
Examples 72-110. The percent of thrombin activity
reaaining after incubation and the lengths of time the
inhibitors were in the sodium phosphate buffer is
reported in Table 8. With inhibitors A and C, there is
little los6 of inhibitor activity. Inhibitor B loses
its biological activity over a period of an hour.
- 88 -
89 ~33~7
Table 8
~tabillty of Inhibitor6 in Buffer
Percent Thrombin activity
Example No. Compound 0 hr 6.5 hr 24 hr
133 A 3.3 1.6 0.8
13q C 3.0 0.9
135 C 65 77
0 hr 0.5 hr 1 hr
136 B 2.0 15 100
Examples 137 - 142
Inhib~tion of ~lood Coagulation
Following In Vivo Oral Dosing
Male rat6 (Sprague Dawley CD Rat6, 130-140 g,
6upplied by Charles River ~abs, Inc., Wilm~ngton, MA)
were ~ne6thet~zed with sodium pentobarbital (50 mg/kg,
l.p.). A ~idline inci6ion wa6 made on the ventral
eurface of the neck, and a polyethylene catheter was
~nserted ~n one of the earotid arterie6 and exter~orlzed
at the back of the neck. After recovery from
anesthesia, control blood 6ample6 were taken from the
carotid artery catheter, anticoaqulated with sodium
citrate, and centrifuged (2000 x g, 10 minutes). Plasma
was transferred to plastic tubes and kept on ice until
it was as6ayed. Thrombin times were measured using a
fibrometer, a6 de~cribed in Examples 125-127.
Rats were given either the protease inhibitor
Ac-(D)Phe-Pro-boroArg-OH in a vehicle, or the ~ehicle
alone, by oral gavage in a volume of less than q ml.
The vehicle employed was 5% dimethylsulfoxide in saline.
- B9 -
o 133~8~7
Blood camples were taken at various time6 after oral
dosing and as6ayed as described above. The resultc,
shown ~n clotting times ln ceconds, are g~ven ~n Table
9, below. When clotting t~me excceded 300 ~econd-, lt
i~ reported below a6 ~300. The remainlng data ~how the
mean time needed for clottlng, measured in seconds,
the ctandard deviation.
Table 9
Inhibition of Blood Coagulation
Following In Vivo Oral Dosing
Ex. Time Control Inhibitor Concentrat~on
(hr) 1 mg 2 mg 10 mg
137 .5 68 ~ 18 > 300 ~ 300 > 300
138 1 52 ~ 26 > 300 ND > 300
139 2 55 1 11 > 300 ND > 300
140 3 3~ + 12 > 300 ND > 300
141 4 41 47 ~ 4 54 ~ 29 ND
142 6 50 46 ~ 3 44 ~ 4 ND
ND - not determined
Example 143
In Vivo Inhibition of
~Io~ar~oagulatlon Following Oral Dosing
To further demonstrate the ability of this
compound to inhibit blood coagulation in vivo, rats were
ane6thetized with 60dium pentobarbital (50 mg/kg, i.p.),
a jugular vein catheter was inserted, and the incision
was clo~ed. After recovery from anesthesia, rats were
treated orally with either 5 mg/kg of the protease
inhibitor Ac-(D)Phe-Pro-boroArg-OH dissolved in water,
or an equal volume of water. Thirty to sixty minutes
later, all rats received an infusion of 500 unitb/kg
-- 90 --
91 1~39897
thrombin over a period of one minute. All fourteen rat
gi~en only water died within ten minute6 of the thrombin
infu6ion. In contra~t only 8 out of 17 rat6 treated
with the ~nhibitor-containing water died with~n ten
mlnute6, and the rema~nder turvived one hour, ~t whlch
time they were euthanized.
Examples 144 - 162
In Vivo Inhibition Df Blood Coagulation
FolI~ng Oral, Colon~c and Rectal Admini~tration
General Proceduret:
Male Lewit rat6 weighing between 300-350 g
were ane6thetized with todium pentobarbitol (50 mg/kg,
i.p.) and the ~ugular vein wa6 cannulated u6ing a
sila6tic tubing attached to a polyethylene 50 tubing.
The tubing wa6 exteriorized at the back of the neck and
attached to a tyringe through a ~top cock. Blood
tamplet (0.5 ml) were withdrawn before and at different
time ~nterval6 after dosing with the protea6e lnhibitor
Ac-(D)Phe-Pro-boroArg-OH, into the 6yringe that wa6
flushed with citrate buffer prior to each collection.
The blood samples were then tran6ferred into vacuta~ner
containing citrate buffer. Also, after each collcction
the cannula wa6 flu6hed with saline. The blood ~ample6
were then centrifuged ~mediately (2500 rpm for 15 min)
and 0.2 ml of the p~a6ma 6ample6 were u6ed for clotting
time ~eaturement6. ~he clotting time measurement6 were
carried out using a fibrometer as follow6. Fir6t,
pla6ma (0.2 ml) was placed in a fibro cup, and pH 7.4
Tri6 buffer (50 microliters) was added. The pla~ma
buffer 601ution was incubated at 37~C for 1 min, 50
microliters of a 2~ ~/ml thrombin solution in Tri~
buffer was then added, and clotting time in teconds was
measured. When clotting time exceeded 300 seconds, it
is reported below as >300.
-- 91 --
92 13 398
Oral dosing:
The ~ugular veln-cannulated rats were allowed
to recover from ane~the~a before they were do6ed
orally. The protease ~nhib~tor Ac-(D~Phe-Pro-boroArg-OH
aqueous 601ut~0n, con6i6ting of 3 mg of ~nhibitor per kg
weight of rat (approximately 1 mg/rat) in a ~olume of
0.75 ml of water per kg of rat, was admini6tered by
gavage. ~he result6 are reported ~n Table 10, below.
Colonic admin~6trat~0n:
A 3 cm ~nc~6ion wa6 made ~n the abdomen of the
jugular vein-cannulated rat6 wh~le they were still under
anesthe6~a. The colon wa6 located and was tied off at
both the beginning and the end. The protea6e ~nhibitor
Ac-(D)Phe-Pro-boroArg-OH aqueous 601ution, cons~6ting of
3 ~g of ~nhibitor per kg we~ght of rat (approxlmately 1
mg/rat) ~n a volume of 1 ml of water per kg we~ght of
rat, was injected at the beginn~ng ~nto the colon
cavity. The ~nci6$0n wa6 clo6ed u6ing wound cl~ps. The
re6ult6 are reported in Table 11, below.
Rect~l administration:
The procedure for rectal administration ~n the
~ugular vein-cannulated rats wa6 a6 de6cribed by ~am~ya
et al., J. Pharm. Sci., 71: 621 (1982). ~n brlef, a
device was made consi6tlng of a 0.89 cm and a 0.71 cm
silicon rubber septa connected by a 2 cm length of w~re.
Thi~ device was inserted into the rectum of the rat, the
large septum first, and glued to the annus using
suitable glue. Dosing was accomplished by injection
through the exposed septum. The rectal dose was 3 mg of
of the protease inhibitor Ac-(D)Phe-Pro-boroArg-OH per
kg weight of rat (approximately 1 mg/rat) in a volume of
0.6 ml of water per kg of rat. The results are reported
in Table 12, below.
- 92 -
1~3S8~7~
Table 10
In Vivo Inh~bition of
Blood Coagula~lon Following Oral Admln~6tration
Ex. ~ime(hr) Control Inhibitor
144 0.00 49.7 57.3
145 0.25 67.4 ~300
146 0.50 51.8 ~300
147 1.00 43.6 ~300
148 2.00 42.5 ~300
lq9 3.00 58.4 >300
150 4.00 42.7 >300
. data repre~ent~ the average for 2 rat6
data repre~ents the mean for 3 rat~
Table 11
In Vivo Inhibition of
Blood Coagulat~n Follow~ng Colonlc Adm~ni~tration
Ex. T~me(hr) Control Inhlbitor
151 0.0 59.9 59.4
152 0.5 42.7 ~300
153 1.0 42.7 >300
154 2.0 52.1 ~300
155 4.0 54.2 >300
156 5.0 57.9 ~300
data represents the average for 2 rats
data repre6ents the mean for 3 rats
- 93 -
l339897
Table 12
In Vivo Inhibition of
Blood Coagulat~ron Following Rectal Adminlstration
Ex. Time(hr) Control Inhibitor
157 0 S3.9 66.4
158 0.25 ~2.3 ~300
159 0.5 43.1 >300
160 1.0 52.7 ~300
161 2.0 42.5 >300
162 4.0 75.6 >300
. data obtained from 1 rat
data repre~ents the mean for 3 rats
Examples 163-168
In Vivo Inhibition of Croton Oil Induced Inflammation
Two solutions were prepared, the first
consisting of 5% croton oil, ~ known inflammatory agent,
ln an acetone carrier (Croton Solution) and the second
cons~sting of 5~ croton oil in an acetone carrier to
which 10 mg/mL of a compound of the ~nvention was added
(Compound Solution). The Croton Solution (10 ~L), or
alternatively the Compound Solution (10 ~L), was applied
to the right ear of each animal (Sprague Dawley CD Rats,
130-140 g, cupplied by Charles River Labs, Inc.,
Wilmington, MA). The acetone carrier alone (Acetone
Solution) (10 ~L) was applied to the left ear of each
animal. At l h following treatment, the animals were
sacrificed, their ears removed and 1/4 inch diameter
dicks punched out and weighed. Swelling was measured as
the difference in weight between the Croton Solution
treated right ear and the Acetone Solution treated left
ear. The results are compared with indomethacin, a
- 94 -
133~97
~nown non-~terold anti-inflammatory (Indomethacin
~olution), which wa~ prepared ~nd applled in ~ manner
cub~tantially ~dentlcal to the Compound solution. Mean
data are ~hown ~n T~ble 13 for Compound r,
Ac-Phe-boroArg-C~08l~. The term ~do~e~ ~ u~ed below,
~ndicate~ the ~mount of active ~nti-inflammatory
lngredlent ln ~9 (Compound6 A, C, D, E, r or G, or
Indomethac~n, a~ the ca~e may be) ~n the 601ut~0n
applied to each right ear, ~nd ~n~ lndicate~ the number
of rat6 u~ed ~n each te~t. ~SE~ denote~ ctandard error.
~xample~ 16q-168 ~n Table 14 ~how the ~nt~-inflammatory
actlvity for Co~pound~ A, C, D, E, F and G which were
run under e~entially the ~me condit~on6 (do6e - 100
~g).
Table 13
Inhibit~on of Croton Oil Induced Inflammation
Example 163
Doce Mean Mean Mean
Right carR$ght ear ~eft ear Swelling Percent
Soln. (~g/ear) Wt. (mg) Wt. (mg) (mg+SE) Inhibition n
Croton 0 27.4 16.3 11.1+ 1.5 0 B
Indometh. 100 20.6 15.6 5.0 + 2.8 55 8
Cmpd. F 100 18.9 16.6 2.3 + 0.7 79 8
Table 14
Inhibition of Croton Oil Induced Inflammation
Example No. Compound Percent Inhibition
164 G 69
165 E 82
166 D 93
167 A 59
168 C 76
This patent application is a division of Canadian Application
No. 568224 filed 1988-05-31.
