CA1339897C - Peptide boronic acid inhibitors of trypsin-like proteases - Google Patents

Abstract

Peptides comprising C-terminal boronic acid derivatives of lysine, ornithine, and arginine, homoarginine and corresponding isothiouronium analogs thereof, are reversible inhibitors of trypsin-like serine proteases such as thrombin, plasma kallikrein and plasmin.

Description

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 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$de ehalnt ean be protected ln the form of t-butyl, ben2yl or other su~t~ble e6ter~ or ethere.
Ae noted prevlouely, R' refer~ to an alkyl group eomprleed of 3 to 5 earbone attaehed to an ~lno, guanldlno, or l~othiouronlum group. Preferrably, the ~2 le -(CH2),-~. A ~ore preferred value of R2 le -(CH~ X
where ~ 16 3 to 4. Example~ of ~ore preferred ~aluee of ~' ~nclude 3-guanidlno-propyl, 3-amino-propyl, and 4-amlno-butyl. Mo~t preferred 16 3-guanidlno-propyl.
Abbre~tlon6 and terms preflxed by ~boro-~lndle~te amino acid~ of Formul~ S whereln the ter~lnal earboxyl group -CO~H ha~ been repl~ced by ~ boronie functionality --B/
\y2 ~ hu6, ~boroarginine~ or ~boroArg-~ referr to boronic ~cid ~nalog~ of ~rginine; ~borolycine~ or ll 133~37 ~bo~oLy6-~ refer~ to boronic ac~d analog~ of ly~ine~ and ~boroornithine~ or ~boroOrn-~ refer6 to boronlc ac~d analog of orn~thlne. ~he pref$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 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 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~

' 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 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~-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 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 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 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 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 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..

13~8~7 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.

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 ~3~9897 ~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 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%.

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 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.

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 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) 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$1tered, and ~ol~ent removed by evaporatlon to y~eld 0.56 g of a partlal ~olld.
The 6ample wa~ di~601ved ~n 4 mL of glaclal ~cet~c acid and diluted wlth 16 m~ of water. It was ~mmediately applied to a column containing 15 mL of SP-SephedexS~ (H~ form) and equillbrated w~th 20% acetic ~c~d. The column was washed with 300 ~L of 20% acet~c ac~d and thcn a linear grad~ent from 100 mL of 20%
~cet~c ~cid to 100 mL of 20% ~cet~c ac~d ad~u~ted to 0.30 N hydrochloric acld was run. Fract~ons collected from 0.08 to 0.17 N hydrochlor~c ac~d contalned the N-~cetyl peptide (0.29 g) a6 a mlxture of the free boronic acid and pinanediol e~ter.
~ he pinanediol ester and the free boronic acid were ceparated by chromatography on ~ 2.5 X 100 cm column of L~-20 ~n methanol. The fract~on ~lze was 8.2 m~. The p~nanediol e6ter ~102 mg) eluted ~n fractlon 41-43 while free boronlc acid (131 ~9) 610wly eluted ln fract~on~ 45-129.
MS(FAB) (Example 7):
Ac-(D)phe-pro-boroArg-cloHl6) for C3lH~7N~05B:
Calculated I ~: 595.33.
Found: 595.33.
MS (FAB) (Example ~):
~c-~D)Phe-Pro-boroArg-OH HCl) for C2lH" N60s:
Calculated ~ H: 449.60.
Found: 579.2Ç-581.24.
The latter result could not be interpreted.
However, NMR was consistent with the structure of the free boronic acid ~ince definitive bands for pinanediol group 6uch as the methyl groups singlet6 observed at 133~98~7 delta 0.85~3H), 1.30(3H), and 1.36(3H) were abrent. A~
addcd proof of ~tructure, a 6ample of the free boronlc W~6 re-e~ter~ficd to g~ve the product ~n Exampl~ 7. ~n ~nalytical ~ample (20 ~g) wa~ treated w$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 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$ng Ac-Phe-O~ ~30 g, 0.145 molec) with pho~phorou~ pentachlorlde (30 g, 0.144 moles) ln 175 mL of THF at -10-. The reaction was stirred at 0- for approximately 1 h, then dlluted to a volume of 350 m~ with cold ether. The product was lsolated as a solid, washed with cold ether, and dried ln ~acuo to yleld 21 g. The activated Ac-Phe deri~atlve (14.8 g, 65.6 ~molet) was d~ssolved ln 40 ~ of T~F and added to the product of the reactlon of 4-bromo-1-chlorobutyl boronate pinacol and hexamethyldisilizane (prepared on a 20 mmole scale) at -78~. The reaction mixture was allowed to warm to room temperature then stirred overnight. The solvent was removed by evaporation. The residue was dissolved in ethyl acetate and washed successively with water, 5% sodium bicarbonate solution and a solution of saturated aqueous sodium chloride. The organic phase of the resulting mixture was dried over anhydrous sodium sulfate and concentrated to yield the desired product ac a 1~3~897 ery~talllne ~ol~d (1.37 g, mp 146.S-148-). In a eeparate exper~ment, the following ~naly~6 wac obtalned.
Analy8i 6 for C~lH~N2O~BrB:
Caleulated: C-53.98%, H-6.92$, 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 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 13~837 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 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.

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 ~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) 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.

~33~8~7 ~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 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.

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 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.

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:

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 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%.

1~3~7 ~ 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 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 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 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.

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~:

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.

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 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.

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 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) 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

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) 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.

88 ~ 89 7 ~able 7 Stab~lity of Inhib~torr. ln Human Plasma Example Number 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.

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 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.

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 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.

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.

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 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 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 This patent application is a division of Canadian Application No. 568224 filed 1988-05-31.

Claims (2)

1. A compound of the formula wherein Y3 is a moiety derived from a dihydroxy compound having at least two hydroxy groups separated by at least two connecting atoms in a chain or ring wherein the oxygen atoms of two of said hydroxy groups are bonded to B, said chain or ring comprising 2 to about 20 carbon atoms;
R3 is a substituted alkyl selected from the group consisting of -(CH2)z-W1, -CH(CH3)-(CH2)2-W1, -CH2-CH(CH3)-CH2-W1, -(CH2)2-CH(CH3)-W1 and -(CH2)2-CH(CH3)2-W1;
W and W1, independently, are C1 or Br; and z is 3 to 5.

2. A compound according to Claim 1 wherein R3 is -(CH2)z-W1 and z is 3 to 4.