PEPTIDE BORONIC ACID INHIBITORS OF TRYPSIN-LIKE PROTEASES

PEPTIDES CONTENANT DE L'ACIDE BORONIQUE INHIBANT LES PROTEASES APPARENTEES A LA TRYPSINE

English 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.

Claims

96

CLAIMS:
1. A compound of the formula
R1-[(A3)q(A2)p(A1)o]n-NH-CH-B-Y3
R4




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, said chain or
ring comprising 1 to about 20 carbon atoms;
R4 is a substituted alkyl selected from the
group consisting of -(CH2)z-W2, -CH(CH3)-(CH2)2-W2,
-CH2-CH(CH3)-CH2-W2, -(CH2)2-CH(CH3)-W2, and
-(CH2)2-CH(CH3)2-W2;
W2 is Cl, Br or N3;
z is 3 to 5;
A1, A2 and A3, independently, are amino acids of
L- or D-configuration selected from the group consisting
of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu,
Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val;
R1 is a peptide comprised of 1 to about 20 amino
acids, an acyl or a sulfonyl group comprised of 1 to
about 20 carbon atoms, H, or an N-terminal protecting
group; and
n, o, p, and q are, independently, either 1 or
0.
2. A compound according to Claim 1 wherein R4
is -(CH2)z-W2 and z is 3 to 4.

Description

u~ ~ 333~

TITLE
Peptide Boronic Acid
Inhibitors of Trypsin-Like Proteases

BACKGROUND OF THE INVENTION

Field of the Invention
The present invention relates generally to C-terminal alpha-aminoboronic acid
derivatives of Iysine, ornithine, and arginine, homoarginine and corresponding
isothiouronium analogs thereof, and their use as inhibitors of trypsin-like serine proteases
such as thrombin, plasma kallikrein and plasmin.
Background
The activity of many biological systems is mediated by hydrolytic or proteolyticenzymes that cleave precurser proteins at specific locations. Four classes of these
enzymes exist, metallo, thiol, acid and serin proteases. Systems such as blood
coagulation, fibrinolysis, complement, and kallikrein-kinin are all regulated by a subclass
of serine proteases, the trypsin-like proteases, a group of enzymes that have a primary
specificity for arginyl or Iysyl residues.
Within each class, the mechanism of action and the active site residues of the
enzymes as well as their susceptibility to class specific inhibitors are similar. The ability
of a compound to effectively inhibit a particular protease or a particular subclass of
proteases, however, is strongly dependent upon the structure and composition of the
compound .
A great deal of research has been done in the area of protease inhibition, and anumber of researchers in this area have experimented with boron-containing inhibitors.

2 CA I 333~
Shenvi, U.S. 4,537,773 (1985), for example, reports that alpha-aminoboronic acidanalogs of amino acids containing aliphatic and aromatic alkyl side chains are effective
inhibitors of metalloenzymes. In addition, Shenvi et al., U.S. 4,499,082 (1985) disclose
that alpha-aminoboronic acids incorporated into peptides inhibit serine proteases whose
primary specificity requirements are met by neutral side chains, such as pancreatic and
leukocyte elastase, chymotrypsin, and cathepsin G. This latter patent discloses
tetrapeptides comprising C-terminal alpha-aminoboronic acid residues as potent,
reversible inhibitors of such proteolytic enzymes. The peptides disclosed, however, did
not include C-terminal alpha-aminoboronic acid residues of Iysine, ornithine, arginine,
homoarginine or any corresponding isothiouronium salts.
Koehler et al ., Biochemistry 10: 2477 (1971) report that 2-phenyl-ethaneboronicacid is an inhibitor of chymotrypsin. Matteson et al., J. Am. Chem. Soc. 103: 5241
(1981), describe the sythesis of (R)-1-acetamido-2-phenylethane boronic acid and its use
as an inhibitor of chymotrypsin. The authors show a Kj of 4,uM.
Lienhard in Enzvme Inhibitors as Drugs, Sandler, ed., University Park Press,
Baltimore pp.43-51 (1980) speculates that peptide analogs of alpha-aminoboronic acids
will be potent inhibitors of serine and thiol proteases.
Additional disclosures include those of Kinder et al., J. Med. Chem. 28: 1917-
1925 (1985), which describes the N-acyl and dipeptide boronic acids and difluoroborane
analogs of phenylalanine, phenylglycine, alanine, valine, and isoleucine, and Matteson,
Organometallics 3: 1284-1288 (1984) which describes the synthesis of alpha-amidogama-substituted boronic esters. The latter authors state that these compounds

3 ~h ~ 333~

were prepared as possible precursors to boronic acid analogs of arginine and proline.
Trypsin-like proteases are extremely important in controlling a number of
physiological processes. For a discussion of such activity, see "Proteases and Biological
Control", Reich, Rifkin and Shaw eds., Cold Spring Harbor Press (1975). Thrombin, one
type of trypsin-like protease, has a clear and decisive role in the blood coagulation
process. Blood coagulation may occur through either of two cascades of zymogen
activations. The last protease in each of these pathways is thrombin, which acts to
hydrolyze fibrinogen to form fibrin, which in turn aggregates to form a blood clot. This
thrombin catalyzed hydrolysis is essential to the blood coagulation process.
Plasma kallikrein, another trypsin-like protease, is also involved in the blood
coagulation process, specifically in the initiation of one of the blood coagulation
pathways. Also, kallikrein acts on kininogen to liberate the nonapeptide, bradykinin.
Bradykinin is a hypotensive peptide that is associated with pain. In addition, kallikrein is
thought to have other biological functions. Recent information suggests that plasma
kallikrein is involved in inflammation. Baumgarten et al., J. Immun. 137: 977-982
(1986), for example, report elevated levels of kinin and kallikrein in allergic individuals
challenged with allergen. Wachtfogel et al., Blood 67: 1731-1737 (1986) report that
plasma kallikrein aggregates human neutrophils and releases neutrophil elastase. The
release of elastase and accompanying elastase-mediated tissue destruction are events
associated with the process of inflammation.
The design of specific inhibitors of trypsin-like enzymes to control biological
processes is not a new concept. Particular efforts have been made in the preparation of
inhibitors of thrombin to replace

4 ~A I 333~0B

heparin in treatment of thrombosis without the side effects associated with heparin
therapy, see Markwardt TIPS 153-157 (1980) and Green et al., Thromb. Res. 37: 145-
153 (1985). Highly effective peptide chloromethyl ketones have been prepared for a
number of trypsin-like proteases by Kettner et al., Methods in Enzymology 80: 826-842)
(1981). One example, H-(D)Phe-Pro-ArgCH2CI, is highly effective in the inhibition of
thrombin (Kj = 37 nM), and, as shown by Shaw et al., U.S. 4,318,904 (1982), is
effective in the prevention of coronary thrombosis in a rabbit model. Similarly, Bajusz et
al., Int. J. Peptide Protein Res. 12: 217-221 (1979) reportthe peptide aldehyde, H-
(D)Phe-Pro-Arg-H, is an effective inhibitor of thrombin (Kj = 75 nM) and Tremoli et al .,
Thromb. Res. 23: 549-553 (1981), reportthat a related compound, Boc-(D)Phe-Pro-Arg-
H, reduces the size of venous thrombosis in rats.
Substituted arginine amides composed of secondary amines have also been shown
to be effective inhibitors of thrombin. Kikumoto et al., Biochemistry 23: 85-90 (1984)
report that (2R,4R)-4-methyl- 1 [N2-{(3-methyl- 1,2,3,4-tetrahydro-8-quinolinyl) sulfonyl}-L-
arginyl]-2-piperidinecarboxylic acid is an inhibitor of thrombin (Kj = 19 nM). As reported
by Green et al., Thromb. Res. 37: 145-153 (1985), this inhibitor increases the
prothrombin times of plasma in vitro blood coagulation assays 2-fold at 1 ,uM, and it is
claimed as a fibrinolytic enhancing agent to be used in combination with tissue
plasminogen activator Yoshikuni et al, European Patent application 0,181,267 (1986).
Finally, Sturzebecher et al., Thromb. Res. 29: 635-642 (1983) and Kaiser et al., Thomb.
Res. 43: 613-620 (1986) report that N-alpha-(2-naphthylsulfonyl-glycyl)-4-amidinophenyl-
alanine piperidide is the most effective known inhibitor of thrombin (Kj = 6 nM), and
demonstrate that is in vivo efficacy in mice and rats.

~JA 1 333~0-8
Despite the foregoing, new and better classes of inhibitors of thrombin and other
trypsin-like enzymes are needed to provide potentially valuable therapeutic agents for
treatment of blood coagulation disorders, inflammation and other mammalian ailments.
The present invention is directed to this end.

SUMMARY OF THE INVENTION
The present invention provides compounds of the formula




[FORMULA 1]

whereln
Y' and y2, independently, are -OH or F or, taken together, form a moiety derivedfrom a dihydroxy compound having at least two hydroxy groups separated by at least
two connecting atoms in a chain or ring, said chain or ring comprising 1 to about 20
carbon atoms and, optionally, a heteroatom which can be N, S, or O;
R2 is a substituted alkyl selected from the group consisting of -(CH2)z-X,
-CH(CH3)-(CH2)2-X, -CH2-CH(CH3)-CH2-X, -(CH2)2-CH(CH3)-X, and -(CH2)2-CH(CH3)2-X,
where X is -NH2, -NH-C(NH)-NH2 or -S-C(NH)-NH2, and z is 3 to 5;

n, o, p, and q are, independently, either 1 or 0;

A', A2 and A3, independently, are amino acids of L- or D-configuration selected
from the group consisting of Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, lle, Leu, Lys,
Met, Phe, Pro, Ser, Thr, Trp, Tyr

~A i 333208
and Val; and
R' is a peptide comprised of 1 to about 20 amino acids, an acyl or a sulfonyl group
comprised of 1 to about 20 carbon atoms, H, or an N-terminal protecting group;
or a physiologically acceptable salt thereof.
The invention also provides compositions comprising one or more of the foregoingFormula I compounds, and methods of using such compounds or compositions in the
inhibition of trypsin-like serine proteases, such as thrombin and plasma kallikrein, and in
the treatment of aberrant physiological conditions, such as those involving blood
coagulation disorders and inflammation, which are mediated by trypsin-like proteases.
Further, two classes of intermediates to the foregoing compounds are provided.
The first such class of intermediates includes compounds of the formula




[FORMULA ll]

whereln
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, said chain or ring
comprising 1 to about 20 carbon atoms;
R3 is a substituted alkyl selected from the group consisting of -(CH2)z-W',
-CH(CH3)-(CH2)2-Wl,-CH2-CH(CH3)-CH2-W1,-(CH2)2-CH(CH3)-W1 and-(CH2)2-CH(CH3) 2-
W';

7 l A i 333208

W and W1, independently, are C1 or Br; and
z is 3 to 5.
The second class of intermediates includes compounds of the formula




[FORMULA lll]

wherein
A1, A2, A3, Y3, R', n, o, p and q are as previously defined;
R4 is a substituted alkyl selected from the group consisting of -(CH2)z-W2,
-CH(CH3)-(CH2)2-W2,-CH2-CH(CH3)-CH2-W2,-(CH2)2-CH(CH3)-W2, and-(CH2)2-CH(CH3)2-
w2;
W2 is Cl, Br or N3 and
z is 3 to 5.
BRIEF DESCRIPTION OF THE FIGURE
Figure 1 shows a plot of relative clotting times versus inhibitor concentration for
two inhibitors of the invention, H-(D)Phe-Pro-boroArg-C1OH,6 and Boc-(D)Phe-Phe-boroArg-C,OH,6. The data for Figure 1 was obtained from Tables 3 and 4. Relativeclotting time is the activated partial thromboplastin times (APTT) or the prothrombin
times (PT), as the case may be, in the presence of inhibitor, divided by the APTT or the
PT, respectively, in the absence of the inhibitor. The inhibitor concentration is shown in
micro molar.

DETAILED DESCRIPTION OF THE INVENTION
The principal compounds of the present invention, the Formula I compound, are N-acyl and

8 "~ i 3332~

peptide derivatives of alpha-aminoboronic acids in which the C-terminal residue consists
of Iysine, ornithine, and arginine, homoarginine and corresponding isothiouronium analogs
thereof. These compounds are characterized by their potency as inhibitors of certain
trypsin-like proteolytic enzymes, notably human thrombin, plasma kallikrein and plasmin.
The acid terminal boron of the present compounds can optionally be in the form of
an unprotected boronic acid, that is, where Y' and y2 each are -OH, or borane difluoride,
that is, where Y' and y2 each are -F, or combinations thereof. Alternatively, the terminal
boron can be protected with a wide variety of protecting groups wherein Y' and y2 are
taken together ( y' y2_) to form a moiety.
Suitable protecting groups wherein Y' and y2 are _y1_y2_ include moieties derived
from compounds, principally diols, having at least two hydroxy groups separated by at
least two connecting atoms in a chain or ring. The term chain denotes both a branched
or unbranched moiety. The chain or ring is comprised of 1 to about 20 carbon atoms
and, optionally, and may include a heteroatom which can be N, S or 0. Contemplated
compounds within the foregoing description include, for example, pinanediol, pinacol,
perfluoropinacol, 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 alcohols, and other equivalents apparent to those skilled in the art.
As used throughout the specification, the following abbreviations for amino acidresidues or amino acids apply:
Ala = L-alanine
Arg = L-arginine
Asn = L-asparagine

g ~A 1 333208

Asp = L-aspartic acid
Cys = L-cysteine
Gln = L-glutamine
Glu = L-glutamic acid
Gly = glycine
His = L-histidine
lle = L-isoleucine
Leu = L-leucine
Lys = L-lysine
Met = L-methionine
Phe = L-phenylalanine
Pro = L-proline
Ser = L-serine
Thr = L-threonine
Trp = L-tryptophan
Tyr = L-tyrosine
Val = L-valine
Where prefixed by a "D", the foregoing abbreviations indicate an amino acid of D-
configuration. Where prefixed by a "D or L", the the foregoing abbreviations indicate that
the amino acid can be either the D- or the L-configuration.
"N-terminal protecting group," as used herein, refers to various amino-terminal
protecting groups employed in peptide synthesis. Examples of suitable groups include
acyl protecting groups, for example, formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, and
methoxysuccinyl (MeOSuc); aromatic urethane protecting groups, for example,
benzyloxcarbonyl (Z); and aliphatic urethane protecting groups, for example, tert-
butoxycarbonyl (Boc) or adamantyloxycarbonyl. Gross and Mienhoffer, eds., The
Peptides, Vol. 3: 3-88 (1981), Academic Press, New York 1981, disclose numerous
suitable amine protecting groups.
The following represent preferred N-terminal protecting groups R1:

CA ~ 333208

~c - C~23

~oc- (cH3)3cx~_
. .

- Bz - ~8_

. .. .
Z ' (~2~-


Compounds of the invention having side-chain amino groups, for example, where
A', A2 or A3 are Lys or Arg, can optionally contain suitable N-terminal protecting groups
attached to the side chains; similarly, amino acid residues having acidic or hydroxy side
chains can be protected in the form of t-butyl, benzyl or other suitable esters or ethers.
As noted previously, R2 refers to an alkyl group comprised of 3 to 5 carbons
attached to an amino, guanidino, or isothiouronium group. Preferrably, the R2 is -(CH2)z-
X. A more preferred value of R2 is -(CH2)z-X where z is 3 to 4. Examples of morepreferred values of R2 include 3-guanidino-propyl, 3-amino-propyl, and 4-amino-butyl.
Most preferred is 3-guanidino-propyl.
Abbreviations and terms prefixed by "boro-" indicate amino acids of Formula I
wherein the terminal carboxyl group-C02H has been replaced by a boronic functionality




Thus, "boroarginine" or "boroArg-" refers to boronic acid analogs of arginine;
"borolysine" or



- 10-

~A i 333~08
1 1

"boroLys-" refers to boronic acid analogs of Iysine; and "boroornithine" or "boroOrn-"
refers to boronic acid analog of ornithine. The prefix "homo", as in "homoboroarginine"
or "homoboroArg-", refers to boroarginine analogs in which the side chain has anadditional methylene group. "Irg" refers to the isothiouronium analog of arginine or
homoarginine in which the thiouronium group, -S-C(NH)NH2, replaces the guanidinogroup, -NH-C(NH)-NH2, and "borolrg-" or "borohomolrg-" is the abbreviation for the
corresponding boronic acid analog.
In naming compounds of the invention, Y' and y2 are simplified by the suffix "-F"
for the difluoroboranes (y1 = y2 = -F), "-OH" for the unprotected boronic acids (y1 = y2
= -OH), "-C6H12" for the pinacol esters (yl and y2, taken together, are -C6H12), and "-
C1oH16" for the pinanediol esters (Y' and y2, taken together, are -C1oH16).
The present invention also contemplates physiologically acceptable salts of
Formula 1. These salts include acid addition salts, for example, salts of benzene sulfonic
acid (BSA), hydrochloric acid (HCI), hydrobromic acid (HBr), acetic acid, trifluoroacetic
acid (TFA), succinic acid, citric acid, or other suitable acid addition salts. When
employed in naming compounds of the present invention, these salts shall be introduced
in the compound name by a " ".
Contemplated classes of compounds within the scope of the present invention
include the following amino acids of the D- or L-configuration. A first class includes
compounds wherein A1 is Ala, Pro, Gly, Val, Leu, lle or Met, that is, an amino acid having
a neutral side chain. A second class includes compounds wherein A1 is Phe, Trp or Tyr,
that is, an amino acid having an aromatic side chain. A third class includes compounds
wherein A' is Lys or Arg, that is, a basic

12 ~A i 333208

amino acid, and a fourth class includes compounds wherein A' is Ser or Thr, that is, an
amino acid with a hydroxy side chain. Finally, a fifth class includes compounds wherein
A1 is Asp, Glu, Asn or Gln, that is, an amino acid with an acidic or a carboxamido side
chain. Preferable values of A' substituents, include Lys, Phe, Pro, Ala, Leu, Gly, Glu, Val,
Thr, lle, Met, Tyr, Trp, Arg, Asp, Asn and Gln. One preferable class of such substituents
includes Lys, Phe, Pro, Ala, Leu, Gly, Glu, Val and Thr.
The foregoing principal classes include subclasses corresponding to preferred
values of R2, and these subclasses are further subtended into groups defined by preferred
values for A2 and for N-terminal protecting group R'.
Preferred values for A2 include all amino acids having a D-configuration, most
preferably (D)Phe. Other preferrable values for A2 are (D or L) Phe, (D or L) Ala, (D or L)
Leu, (D or L) Pro, (D or L) Glu and (D or L) Gly. Another class of A2 substituents includes
(L) Glu and (D) Val.
Preferrably, the Formula I compounds have a total of two to four amino acid
substituents, including the boro amino acid analog. A three amino acid compound which
has Pro in the A' position and boroArg as the boro amino acid analog, such as




are particularily suited as inhibitors of thrombin, having an IC 50 of significantly less than
5nM.
Obvious equivalents of the foregoing compounds include compounds comprising
less common or modified amino acids, for example, norleucine, hydroxyproline,

13 C~ l 333208

pyroglutamic acid or other derivatives, including residues with side chain protecting
groups, capable of incorporation into the alpha-aminoboronic acid peptides of the present
nventlon .
Specific compounds within the scope of the invention, named in accordance with
the conventions described above, inciude the following examples:
Ac-(D,L)Phe-boroArg-C,OH,6-BSA
Ac-Phe-boroOrn-C,OH,6-BSA
Ac-Phe-boroArg-ClOH16-HCI
H-(D)Phe-Pro-borolrg-C,OH,6HBr HCI
Boc-(D)Phe-Pro-borolrg-C10H16 HBr
Ac-Phe-borolrg-C10H16-HBr
Ac-Ala-Lys(Boc)-boroOrn-C10H16 BSA
Ac-Ala-Lys(Boc)-borolrg-C10H16 HBr
Boc-(D)Phe-Pro-boroArg-C10H16 BSA
Boc-(D)Phe-Phe-Borolrg-C10H16 HBr
H-(D)Phe-Pro-boroArg-C10H16 HCI
Boc-(D)Phe-Phe-boroOrn-C10H16BSA
Boc-(D)Phe-Phe-boroArg-C10H16 BSA
Ac-Ala-Lys(Boc)-boroArg-C10H16 BSA
Ac-(D)Phe-Pro-boroArg-C10H16 HCI
Ac-(D)Phe-Pro-boroArg-OH HCI
Boc-Leu-Gly-Leu-Ala-borolrg-C10H16 HBr
Boc-Leu-Gly-Leu-Ala-boroOrn-C10H16 BSA
Boc-Leu-Gly-Leu-Ala-boroArg-C10H16 BSA
Bz-Pro-Phe-boroOrn-C10H16 BSA
Bz-Pro-Phe-boroArg-C10H16 BSA
Boc-Ala-Phe-(D,L)borolrg-C6H12 HBr
Bz-Glu(OBu)-Gly-borolrg-C10H16 HBr
Bz-Glu-Gly-boroArg-C10H16 BSA
Bz-Glu(OBu)-Gly-boroOrg-C10H16 BSA
Bz-Glu(OBu)-Gly-boroArg-C10H16 BSA
Bz-Pro-Phe-borolrg-C10H16 HBr
Z-Phe-Gly-Gly-borolrg-C10H16 HBr
Boc-Ala-Phe-(D,L)borohomolrg-C6H12 HBr

14 ~A ~ 333208

Bz-Pro-Phe-boroArg-OH HCI
Bz-Pro-Phe-boroArg-F
H-(D)Phe-Pro-boroArg-C,OH,6 2HCI
H-(D)Phe-Phe-boroArg-C,OH,6 2HCI
Ac-Ala-Lys-boroArg-ClOH,6 2HCI
H-Leu-Gly-Leu-Ala-boroArg-C,OH,6-HCI BSA
Boc-Ala-Phe-(D,L)boroLys-C6H,2 HCI
H-Ala-Phe-(D,L)boroLys-C6H,2-2HCI
Boc-(D)Val-Leu-boroLys-C6H,2 HCI
Ac-Phe-boroLys-C6H,2 HCI
Bz-Glu-Gly-boroArg-C,OH,6 BSA
H-(D)Phe-Phe-borolrg-C,OH,6-2HBr
H-Leu-Gly-Leu-Ala-borolrg-C,OH,6-2HBr
H-Ala-Phe-(D,L)borolrg-C6H,2-2HBr
Bz-Glu-Gly-borolrg-C,OH,6 HBr
H-Ala-Phe-(D,L)boroHomolrg-C6H,2-2HBr
Ac-Ala-Lys-borolrg-C,OH,6-2HBr
Bz-boro I rg-C6H 1 2-H Br
Bz-boroOrn-C6Hl2 BSA
Bz-boroArg-C6Hl2-BSA
Ac-Leu-Thr(oBu)-boroorn-cloH16 BsA
Ac-Leu-Thr(OBu)boroArg-ClOHl6-BSA
Ac-Leu-Thr-boroArg-ClOH,6 BSA
Ac-Lys(Boc)-Pro-boroOrn-ClOH,6-BSA
Ac-Lys(Boc)-Pro-boroArg-ClOH,6-BSA
Ac-Lys-Pro-boroArg-C,OH,6 BSA
Ac-Ala-Glu(OBu)-boroOrn-C,OH,6 BSA
Ac-Als-Glu(OBu)-boroArg-C,OH,6-BSA
Ac-Ala-Glu-boroArg-C,OH,6 BSA
Boc-Val-Val-boroLys-C6H,2-BSA
H-Val-Val-boroLys-C6H,2 BSA TFA
Boc-(D)Phe-Phe-boroLys-C6H,2-BSA
H-(D) Phe-Phe-boroLys-C6H, 2 BSA TFA
Boc-Glu-Phe-boroLys-C6H,2 BSA
PyroGlu-Phe-boroLys-C6H,2 BSA

- 14-

- 15 .,A 1 333208

The invention also provides compositions and methods for inhibiting trypsin-likeserin proteases, including but not limited to thrombin, plasma kallikrein and plasmin, and
for treating aberrant physiological conditions, including but not limited to blood
coagulation and inflammation in mammals. The compositions of the present invention
comprise an effective amount of a compound of Formula I and a physiologically
acceptable carrier or diluent. In practicing the method of the invention, the compounds
or compositions can be used alone or in combination with one another, or in combination
with other therapeutic agents. They can be administered orally, parenterally,
intravenously, subcutaneously, intramuscularly, colonically, rectally, nasally or
intraperitoneally in a variety of dosage forms. The useful dosage to be administered and
the mode of administration will vary depending upon the age, weight and mammal
treated, and the particular compounds employed. Typically, therapy is initiated at lower
dosage levels with dosage being increased until the desired effect is achieved.
The present invention further contemplates two classes of critical intermediates to
compounds of Formula 1, the compounds of Formulas ll and lll. The Formula ll
intermediates includes compounds of the formula




[FORMULA ll]
wherein
Y3 is a moiety derived from a dihydroxy




- 15-

~ h i 333208
16

compound having at least two hydroxy groups separated by at least two connectingatoms in a chain or ring, said chain or ring comprising 1 to about 20 carbon atoms;
R3 is a substituted alkyl selected from the group consisting of -(CH2)z-W',
-CH(CH3)-(CH2)2,-W',-CH2-CH(CH3)-CH2-W', -(CH2)2-CH(CH3)-W', and-(CH2)2-CH(CH3)2-
W';
W and W', independently, are Cl or Br; and
z is 3 to 5.
A particularly preferred compound of Formula ll is one wherein R3 is -(CH2)z-W' and z is 3
to 4.
A second class of intermediates includes compounds of the formula




[FORMULA lll]

wherein
A', A2, A3, Y3, R', n, o, p and q are as previously defined;
R4 is a substituted alkyl selected from the group consisting of -(CH2)z-W2,
-CH(CH3)-(CH2)2-W2,-CH2-CH(CH3)-CH2-W2, -(CH2)2-CH(CH3)-W2, and-(CH2)2-CH(CH3)2-w2;
W2 is Cl, Br or N3 and
z is 3 to 5.
Contemplated classes of compounds within the scope of Formula lll are as described for
the analogous Formula I compounds. A particularly preferred compound of Formula lll is
one wherein R4 is -(CH2)z-W2 and z is 3 to 4.



- 16-

17 CA 1 333208

PreParation of Inhibitors
Temperatures are in C. The numbered compounds shown in the schematic
entitled "Synthesis Scheme", illustrated below, are referred to in the text according to
their respective numbers. "NMR", as used herein, signifies proton nuclear magnetic
resonance.

- SYNTHES ~S SCHEME
... . .
~--cn,-ctl-et~ 3 8r-CH~-CH~-CH~-8 /


P;n~nedlol o /

8r CH2 CH2 CH2 8~ 1 /
2 ~
CHCt~-LI- Br-CH2-CH~.CH~CHCI-80~-C,OH"

t(cH~)~ 5
(CH~)~SII~N LI-
Br~CH~CH2-CH~-C H~BO~.C~OH"

~ H~CI
3 ~qu Ha
Br-~CH~)~.CH-80~.C,OH"

mp 1~ 5-C
P~P Peptide-NH-CH-BO~-C~OH"
~CH~)~
~r
N~-N~- Pepllde-NH-CH-80~-C~OH"

(CH~)~
N~

~ f~` - 1333~8
18


H, P~C
P~ptldo Il:l CH-BO,~CIoH"
O~SO,H
~CH,~,
NH,' OSO,-

cy~n-mlde ~eptlde~N11-CH-BO~C~0H~
elh-nol 100'C ~CH~)~
~IH
C =NH
NH~- OSO~-




lon ~chon~ OH
Peptl~ Nll "H~8
or BCI~ ¦ OH
( C~ H2~,
NH
C=NH
NH~- Cl

queou~ HFPepl'do NH-CH~B

~CH~)~
NH
C=NH
NH~- Ct

thioure~
ô P~ptide-NH~CH~BO~C~OH~
(CH~)~
S
C=NH
NH~ Br



- lB -

19 ~,A i ~ 0~

Following the procedures set forth herein, the Formula I compounds of the present
invention are obtainable in a high useable purity, that is, an 80-100% pure form.
Starting materials are available in high purity from chemical suppliers or can be
readily synthesized by procedures known to those skilled in the art. The Synthesis
Scheme shows the general order in which the compounds of this invention were
synthesized. Compounds 1-4 are prepared as described by Matteson et al.,
Organometallics 3: 1284-1288 (1984), except that the procedure was modified to allow
large scale preparation.
Compound 1 is prepared by hydroboration of an alkene halide with catechol
borane. The components are heated in tetrahydrofuran or some other inert solvent and
the product is isolated by distillation. The halo-substituted alkyl boronic acid-catechol
ester is transesterified by allowing it to react with a suitable diol (alpha-pinanediol,
pinacol, 2,3-butandiol, etc) in tetrahydrofuran. (+)-Alpha-pinanediol is preferred in view
of the observations in Matteson et al., J. Am. Chem. Soc. 103: 5241 (1981) that steric
restraints in the molecule allow the stereo specific addition of the -CHCI- group in
formation of Compound 3 and the subsequent introduction of an amino group in the "L"
configuration. Structures 3-9 in the Synthesis Scheme are shown with the pinanediol
protecting 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 solubility. Compound 2 is then purified either by chromatography on
silica gel, by distillation, or is used without additonal purification. Compound 2, as the
pinanediol ester is obtained in close to analytical purity by the removal of solvent.
Additional purification can be achieved by silica gel




- 19-

~A 1 333208

chromotography. For the pinacol ester of Compound 2, final purification by distillation is
preferred .
Compound 3 is prepared by the homologation of 2 using CHCI2-Li+. This reagent ismade by treating methylene chloride with n-butyllithium in tetrahydrofuran at -100. To
Compound 2 is added 0.65 equivalents of zinc chloride at -100. The mixture is allowed
to slowly warm to room temperature and is stirred over night. Compound 3 is obtained
after evaporating solvent, then dissolving the residue in hexane, followed by washing the
organic phase with water, drying it with magnesium sulfate, and finally evaporating the
hexane. Compound 3 is used without further purification when it is protected as the
pinanediol ester and alternately, it can be distilled when it is protected as a pinacol ester.
Compound 4 is prepared by treating the alpha-chloro-substituted boronic acid
ester, Compound 3, with [(CH3)3Si]2N-Li+. Hexamethyldisilazane is dissolved in
tetrahydrofuran and an equivalent of n-butyllithium is added at -78. The mixture is
allowed to warm to room temperature and then, after recooling to -78, an equivalent of
3 is added in tetrahydrofuran. The mixture is allowed to slowly come to room
temperature and to stir over night. The alpha-bis[trimethylsilane]-protected amine is
isolated by evaporating solvent and adding hexane under anhydrous conditions. Insoluble
residue is removed by filtration under a nitrogen blanket yielding a hexane solution of
Compound 4.
Compound 5 is obtained by cooling the hexane solution of Compound 4 to -78
and adding three equivalents of hydrogen chloride. The solution is slowly allowed to
warm to room temperature and is stirred for 1.5-2 h. Compound 5 is then isolated by
filtration and is purified further by dissolving in




- 20 -

~- A i 3 3 3 ~ 0 8
21

chloroform and removing insoluble material. Compound 5 is obtained as a white
crystalline solid by removing the chloroform by evaporation and crystallizing the residue
for ethyl acetate.
The above process of converting Compound 3 to Compound 5 surprisingly results
in analytically pure preparations of Compound 5 which then allows Compound 6 to be
obtained without the difficulty normally encountered in coupling heterogenous material.
The art teaches or strongly suggests that Compound 4 has to be purified prior toconversion to Compound 5 in order to obtain pure samples. The only known procedure
for the preparation of pure alpha-aminoboronic acids is that disclosed in Shenvi U.S.
4,537,773 and used in Shenvi et al., U.S. 4,499,082. In the Shenvi et al. disclosure,
compounds analgous to Compound 4, except that they have aromatic and alkyl side
chains, are purified by distillation. Compound 4 is unstable to the Shenvi et al. distillation
and an altered product is obtained.
Compound 6, the N-acyl or N-peptidyl form of Compound 5, can be prepared by
two different routes. The first is a modification of the procedure described by Matteson
et al., Organometallics 3: 1284-1288 (1984) in which Compound 4, prepared in situ
(without evaporation of solvent and removal of salts by filtration), is treated with an
equivalent of acetic acid and an excess of acetic anhydride to yield N-Acetyl-NH-
CH[(CH2)3Br]BOz-pinanediol. This method is applicable to the coupling of highly reactive
acid chloride of N-Acetyl-phenylalanine (Ac-Phe-CI) with the modification that prior
treatment with acetic acid is omitted. When acetic acid is added in conjunction with Ac-
Phe-CI, 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-


22 CA ~ 333208

CH[(CH2)3Br]B02-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 HCI 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 oneequivalent of triethylamine (or other stericly hindered base) are added to a solution of
Compound 5

~iA ~ 333208
23

dissolved in either cold chloroform or tetrahydrofuran. The reaction mixture is routinely
stirred one hour at -20 followed by 1-2 h of stirring at room temperature. Insoluble
material is removed by filtration, the solvent removed by evaporation, and the residue
dissolved in ethyl acetate. The organic solution is washed with 0.20 N hydrochloric acid,
5% aqueous sodium bicarbonate, and saturated aqueous sodium chloride. The organic
phase is then dried over anhydrous sodium sulfate, filtered, and subjected to evaporaton
to yield a partial solid in most cases. For a number of compounds, further purification of
Compound 6 was deemed unnecessary. Methods which are applicable for the
purification of Compound 6 are silical gel chromatography, crystallization in some cases,
and gel permeation chromatography using SephadexTM LH-20 and methanol as a solvent.
The latter method is preferred. Typically, NMR spectra indicated the -CH2-Br band at
delta 3.45 and a sharp singlet band at delta 0.80-0.95 for one of the methyl group in the
pinanediol protecting group or singlet at delta 1.3 for the pinacol group.
The peptide alkyl halide, Compound 6, is then converted to the alkyl azide,
Compound 7, by treatment with two equivalents of sodium azide in dimethylformamide at
100 for 3 h. In all cases, this reaction appeared to go smoothly without altering
reaction conditions. The NMR spectrum of Compound 7 in CDCI3, typically indicated a
delta 0.1-0.2 ppm upfield shift of the -CH2-Br on conversion to the azide. Further
purification can be obtained by LH-20 chromatography, but it is not necessary for a many
of the peptides.
The boroOrnithine peptides, Compound 8, are prepared routinely by catalytic
hydrogenation of the alkyl azides, Compound 7, in the presence of 10% Pd/C and one
equivalent of benzene sulfonic acid in alcohol.




- 23 -

~A~1 3332i~
24

Hydrogenations are run on a Parr apparatus. Alternately, the hydrogenations can be run
at atmospheric pressure and mineral acids can be substituted for benzene sulfonic acid.
It should be noted that it is necessary to use peptide protecting groups which are stable
to catalytic hydrogenation. Such peptide protecting groups are known to those skilled in
the art and are discussed in The Peptides (E. Gross and J. Meienhofer eds.) vol 3,
Academic Press, New York, (1981). The preferred protecting groups are the t-
butyloxycarbonyl group for amino groups, and t-butyl ethers and esters for hydroxy and
carboxylic acid side chains. Other suitable protecting groups include
diisopropylmethyloxycarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl,
biphenylisopropyloxycarbonyl and tosyl. It is expected that conversion of the azide to the
amine by reduction with other reducing agents can be achieved using reagents such as
stannous chloride and trialkyl phosphites as described by Maiti et al., Tetrahedron Lett.,
27: 1423-1424 (1986) and Koziara et al., Synthesis, 202-204 (1985). These reagents
are expected to be compatible with peptide protecting groups which are labile to catalytic
hydrogenation. The boroOrnithine peptides are routinely chromatogramed on SephadexTM
LH-20 and are white amorphous solids after trituration with ether.
BoroArginine peptides, Compound 9, are prepared by allowing the corresponding
boroOrnithine peptide, Compound 8, to react with a least a 4-fold excess of cyanamide
(50 mg/mL) in absolute ethanol at 100. Initially the components are allowed to react 2-
3 days under a blanket of nitrogen with a water cooled condenser in place. Watercooling is discontinued and the reaction mixture is allowed to concentrate slowly over a
period of several days. The completion of the reaction is determined by the progressive
ncrease in




- 24 -

CA 1 333208

the intensity of material staining with Sakaguchi stain for the guanidino group of the
boroArginine moiety and the disappearance of material staining positive with ninhydrin
stain for the amino group of the boroOrnithine moiety on reverse phase thin layer plates
run in methaol:water (85:15). Typically, the boroArginine peptides streaked from the
origin of the plate, the boroOrnithine peptides traveled as discrete spots in the middle of
the plate, and cyanamide traveled with the solvent front allowing each component to be
identified. Specific stains for the guanidino group and the amino group are commonly
used in peptide synthesis. Compound 9 was purified by gel permeation chromatography
using SephadexTM LH-20 and methanol as a solvent. This chromatographic step readily
separates the boroArginine peptides from low molecular weight byproducts and unreacted
cyanamide. In most cases, no further purification is needed. However, it is essential that
the guanidation reaction of Compound 8 be permitted to run to completion since it is
difficult, if not impossible to separate a mixture of Compounds 8 and 9. Final products
are obtained as amorphous white solids by trituration with ether and, in most case, are of
analytical purity as determined by NMR, mass spectral, and combustion analyses.
It should be noted that guanidation of Compound 8 with cyanamide has been
found to be very dependent upon reaction conditions. First, as discussed 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 solvents are oftenrequired. In an initial survey of reactions to guanidate Compound 8, Compound 8 as the
hydrogen chloride salt, was refluxed with cyanamide in ethanol




- 25 -

~ A ~ ~33208
26

for several hours. The desired product, Compound 9, was not detectable. Attempts to
guanidate Compound 8 using the successful conditions noted above except that
tetrahydrofuran was substituted for absolute ethanol failed to yield detectable product.
Similarly, when an attempt was made to guanidate Compound 8 using these conditions,
except that the benzene sulfonic acid salt of the amino group of the boroOrnithine
peptide was neutralized prior to guanidation, Compound 9 was present only at a barely
detectable level. The preferred conditions involve reactions with the benzene sulfonic
acid salt of Compound 8 (unneutralized). Successful reactions have also been run with
the corresponding hydrogen chloride salt.
Usual methods of guanidation of ornithine peptides, to yield the corresponding
arginine peptides, used by those skilled in the art of peptide synthesis are theneutralization of the amine of the ornithine peptide and coupling with either S-alkyl or 0-
alkyl isoureas or guanyl-3,5-dimethylpyrazole nitrate, as described by Barany et al., in The
Peptides (E. Gross and J. Meienhofer eds) vol 2, pp. 169-175, Academic Press, New
York,(1980). Bannard et al., Can. J. Chem. 36: 1541-1549 (1958) have surveyed
different methods of guanidation of amines and found that guanyl-3,5-dimethyl pyrazole
is superior to the use of S-methyl isourea and concludes that guanidation with cyanamide
is unacceptable although it is described in the early literature. Reactions run with S-
methyl isourea hydrogen iodide in ethanol and guanyl-3,5-dimethyl pyrazole under a
variety of conditions failed to guanidate the boroOrnithine peptide. The lack of reactivity
in this case is probably due to the formation of an internal Lewis acid base complex
between the amino group of the ornithine side chain and the boronic acid ester.
Synthesis of Compound 9 by the treatment of




- 26 -

CA I 333208
27

Compound 6 with guanidine in ethanol was also an unacceptable method of synthesis.
Compound 6, approximately 50% pure, was isolated from the reaction of guanidine with
6 in less than 1% yield.
The guanidino group of boroArginine-pinanediol behaves in a fashion similar to the
guanidino group of the natural amino acid arginine once it is incorporated into the
molecule. For example, the alpha-amino groups can be selectively acylated with an
anhydride without effecting the guanidino group of boroArginine. Thus, it is ourexpectation that Compound 9 can be prepared by the synthesis of H-boroArginine-
pinanediol and subsequentially adding the N-protected form of the peptide portion of the
molecule using the mixed anhydride procedure and similarily, di-, tri-, etc. peptide analogs
containing boroArginine can be extended in length by coupling additional amino acids or
peptides .
Additional purification of the protected boroArginine peptides can be achieved by
ion exchange chromatography on SP SephadexTM. The peptides are dissolved in 20%
acetic acid and applied to the column in its H+ form. After washing the column with
20% acetic acid, product is eluted by running a gradient from 0-0.3 N hydrochloric acid
in 20% acetic acid. The product is eluted as a mixture of pinanediol ester and free
peptide boronic acid. A homogenous preparation is obtained by treating the mixture with
pinanediol 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

vA i ~33208
28

compounds are readily separated by chromatography on LH-20. The second procedure is
a modification of the method of Kinder et al., J. Med. Chem. 28: 1917-1925 (1985).
The boronic acid ester is treated with a 2-3 fold excess of boron trichloride in methylene
chloride for 5 min at -78 and the mixture is allowed to stir 15 min in a 0 ice bath.
Water is slowly added to hydrolyze excess boron trichloride to boric acid and hydrochloric
acid. The reaction is further diluted with 20% acetic acid to yield a final concentration of
hydrochloric acid of 0.05 M. The concentration of hydrochloric acid is based on the
initial quantity of boron trichloride used in the reaction. The aqueous phase is applied to
a SP-SephadexTM column and product is eluted as the hydrochloride salt as described
above. The free boronic acid peptides were obtained as white amorphous solids.
Compound 10 can be converted to the difluoroborane, Compound 11, using a
modification of the procedure of Kinder et al., J. Med. Chem. 28: 1917-1925 (1985).
The peptide boronic acid is treated with a 5-fold molar excess of 0.50 N aqueoushydrofluoric acid at room temperature. Excess hydrofluoric acid and water are removed
by Iyophilization and the resulting solid is triturated with ether to yield desired product as
a white amorphous solid.
In the foregoing description, the preparation of the free boronic acid, Compound10, is from boroArginine-pinanediol ester and the preparation of the difluoroborane acid,
Compound 11, is from Compound 10. The procedure for the removal of the ester
protecting group should applicable to acyl peptides of boroOrnithine, boroLysine, and
boroHomoarginine protected as either pinanediol, pinacol, or other ester protecting group.
Similarly, the corresponding free boronic acids can be converted to difluoroboranes.




- 28 -

~A I 333208
29

The preferred side chain protecting groups and N-terminal protecting groups of the
peptide portion of molecules are those stable to catalytic hydrogenation and liable to
anhydrous hydrogen chloride or trifluoroacetic acid. These criteria are readily met by the
t-butyloxycarbonyl amino protecting group and t-butyl ethers and esters for hydroxy and
acidic side chains. To remove these groups, the peptides are treated with 4 N hydrogen
chloride in dioxane at room temperature. The deprotected peptide is isolated by either
evaporating solvent or by precipitation with ether. Particular care should be taken with
peptides containing an acidic side chain to remove all hydrogen chloride by evaporation.
This insures that the boroArginine peptide is maintained as benzene sulfonic acid salt.
Other peptide can be isolated as either a mixed hydrogen chloride-benzene sulfonic acid
salt or most can be converted to the hydrogen chloride salt by passage through a anion
exchange column in the C1- ion form.
Isothiouronium derivatives of Compound 6 are prepared by treatment of Compound
6 with thiourea in absolute ethanol to yield Compound 12, analogs of the peptideboroArginine esters, Compound 10. Routinely, the alkyl halides were allowed to stir with
a 4-5 fold excess of thiourea for several days at room temperature. The product is
separated, when necessary, for unreacted Compound 6 by trituration with ether.
Compound 6 is readily soluble in ether for most peptides while the product is insoluble.
Final purification, removal of excess thiourea, is achieved by chromatography onSephadexTM LH-20 in methonol and trituration with ether to yield final products as
hydrogen bromide salts. Side chain and N-terminal protecting groups are removed by
treatment with anhydrous hydrogen bromide or other anhydrous acid.




- 29 -

CAl 333208

Biological Activity
The biological activity of compounds of the present invention is demonstrated byboth in vitro and in 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 measuredin both the presence and absence of inhibitor and the precent 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 thecomponents 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 Kj'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 Kj can
be obtained from the percent activity (inhibition) data and the concentration of



- 30 -

C~ 1 333208
31

inhibitor. A level of inhibitor of about 20-fold higher than Kj is required to inhibit a
reaction 95% and a level of inhibitor of about 50-fold higher than Kj is required for 98%
inhibition .
Plasma kallikrein preferentially hydrolyses and liberates bradykinin. BoroArginine
peptides containing Phe adjacent to the boroArginine are the most effective inhibitors of
this enzyme. For example, 10 nM H-(D)Phe-Phe-boroArg-C1OH16 inhibits kallikrein greater
than 95%. No significant differences are observed between the effectiveness of the
boroArginine pinanediol esters and the corresponding isothiouronium analogs (borolrg-).
In addition, no differences are observed in the effectiveness of the unprotected boronic
acid and corresponding difluoroborane.
Results similar to those with kallikrein are obtained for thrombin in assays with
synthetic substrates, except that thrombin has a much higher affinity for inhibitors with
proline in the site adjacent to the boroArginine. The most effective inhibitor is Ac-(D)Phe-
Pro-boroArg-C,OH16 which inhibits thrombin 99% at a concentration of 5 nM. The most
potent inhibitor reported in the literature is N-alpha-(2-naphthylsulfonyl-glycyl)-4-
amidinophenyl-alanine piperidide, which has a Kj of 6 nM. It was reported by B. Kaiser et
al., Thromb. Res. 43: 613-620 (1986) and Sturzebecher et al., Thromb. Res. 29: 635-
642 (1983). The relationship between inhibitor concentration, Kj, and percent inhibition,
as previously described, suggests that the Kj of Ac-(D)Phe-Pro-boroArg-C10H16 is in the
picomolar range. Furthermore, the effectiveness of inhibitors having a (D)Phe-Pro-
boroArg- sequence appear relatively insensitive to the presence 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

CA i 333208
32

group inhibit thrombin similarily, each showing an l.C. 50 of less than 5 nM.
The effectiveness of inhibitors in reactions in which they compete with natural
substrates for target enzymes is measured in vitro in blood coagulation assays. Two
different assays are used, the APTT (activated partial thromboplastin times) and PT
(prothrombin times) assays. These assays mimic the blood clotting process in vivo.
Blood coagulation occurs through either of two pathways, each consisting of a cascades
of zymogen activation steps. The pathways are termed the intrinsic and the extrinsic
pathways (see L. Lorand, Methods in Enzymology 45: 31-37 (1976). The intrinsic
pathway is initiated by negatively charged surfaces in which plasma kallikrein, factor Xll
and factor IX are activated and then factors IX and X and prothrombin are activated in
calcium dependent steps. Thrombin, the last protease in the cascade, hydrolyses
fibrinogen to fibrin which results in clot formation. In the APTT assay, plasma
components are activated by exposure to negatively charged surfaces and then clotting
times are measured after calcium is added to the system. In the extrinsic pathway,
tissue thromboplastin activates factor Vll which then activates factor X leading to the
activation of thrombin. These events are measured in the prothrombin times assay.
Peptides of boroArginine and the corresponding isothiouronium analogs effectively
inhibit blood clotting in both of these assays. The most effective inhibitors of the present
invention for thrombin are the most effective for both assays. On the other hand,
inhibitors of kallikrein, while less potent clotting inhibitors, inhibit the APTT assay
(kallikrein is involved in the initiation of this assay) more effectively than the PT assay.
This is clearly shown in Figure 1 by the effect of H-(D)Phe-Pro-boroArg-C10H16




- 32 -

CA I 333208
33

(thrombin inhibitor) on the relative clotting times of plasma. It demonstrates the
selectivity which can be achieved by varying a single amino acid in the tripeptide inhibitor
in a rather complex biological system. The effective levels of thrombin inhibitors are in
the same molar range as heparin. Usually, 0.2-0.4 units of heparin per mL of plasma
increases clotting times 2-2.5 fold. If one assumes an average molecular weight of
15,000 for heparin and specific activity of 150 units/mg, its molar concentration is 86-
170 nM. The concentration of the boroArginine peptides required to increase clotting
times in the APTT assay are in the range of 170-230 nM. It should be noted that heparin
is a cofactor for the high molecular weight protease inhibitor, anti-thrombin lll.
The stability of the boroArginine peptides in human plasma is shown by incubating
them with plasma at a concentration effective to delay the clotting process. Samples of
the inhibitors are removed at increasing time intervals and their ability to delay clotting is
measured at each interval. No change in the clotting time indicates no change in the
inhibitory activity of the inhibitors during incubation in plasma. No significant change in
inhibitor activity was observed except for H-(D)Phe-Pro-boroArg-C,OH,6, which lost
activity after 24 h. The inhibitors of this invention are also stable for 24 h in phosphate
buffer at pH 7.5 except for H-(D)Phe-Pro-boroArg- C,OH,6, which lost inhibitory activity
within one hour. The greater instability of this inhibitor in buffer suggests that phosphate
buffer plays a role in destabilizing the compound.
The in vivo data supplied clearly indicates the efficiacy of the subject compounds
as inhibititors of blood coagulation in mammalian systems.
Compounds of the present invention are also

34 ~ A, 1 ;)3~)2 38

effective anti-inflammatory agents as shown by the inhibition of rat ear edema when the
compounds are applied topically along with with an irritant. The molecular basis for this
pharmacological activity is unknown, since multiple events occur during inflammation.
However, proteases which increases vascular permeability, such as plasma kallikrein
which liberates kinins and enzymes of the complement system which liberate the
anaphylatoxin peptides, are thought to be implicated in the inflammatory process.
Finally, pepitides of boroLysine were shown to effectively inhibit plasmin, an
enzyme which plays a key role in hemostasis.
Utility
N-Acyl and N-peptide alpha-aminoboronic acids which are analogs of ornithine,
arginine, Iysine and homoarginine of the present invention represent a novel class of
potent, reversible inhibitors of trypsin-like enzymes. Trypsin-like enzymes are a group of
proteases which hydrolyze peptide bonds at basic residues liberating either a C-terminal
arginyl or Iysyl residue. Among these enzymes are the proteases of the blood coagulation
system (factors, Xlla, Xla, IXa, Vlla, Xa, and thrombin), the fibrinolytic system
(plasminogen activators and plasmin), the complement system (C1s, C1r, C3 convertase,
factor D, etc.), pancreatic trypsin (which as a digestive function), and acrosin, (which is a
protease associated with sperm and required for fertilization).
The ability of the compounds of this invention to inhibit trypsin-like proteases has
been determined by inhibiting two different trypsin-like enzymes, human thrombin and
plasma kallikrein. Compounds of the present invention are much more potent inhibitors
of both of these enzymes than other known reversible inhibitors. For example, the most
effective inhibitor




- 34 -

~A I 333208

of thrombin reported to date is N-alpha-(2-naphthyl-sulfonyl-glycyl)-4-
amidinophenylalanine piperidine which a Kj of 6 nM. Compounds of the present invention
almost completely inhibit thrombin at a concentration of 5 nM indicating a Kj of ~ 1 nM,
and thus provide excellant candidates for the control of thrombin mediated processes
such as blood coagulation. The most effective boroArginine peptide inhibits blood
clotting as demonstrated by the increase in the APT times and PT times. Its level of
effectiveness is similar to that of heparin on a molecular basis. In addition, the
compounds are stable in human plasma. The compounds can be used as anticoagulants
in the preparation of plasma for protein isolation as well as for clinical testing.
An additional example is the protease, plasmin, which has a pivotal role in the Iysis
of blood clots. Peptides containing borolysine were prepared and tested and found to be
active inhibitors of plasmin.
Compounds of the present invention are effective in controlling proteolysis in vivo
and should be pharmaceutically effective in the treatment of diseases in mammals arising
from uncontrolled protease activity. Notable among these are conditions associated with
thrombosis and consumptive coagulopathy. Coronary thrombosis plays an important
contributing role in myocardial infarction. Consumptive coagulopathy, a condition marked
by decreases in blood coagulation factors and plasma protease inhibitor, is observed in
patients with acute pancreatitis and disseminated intravascular coagulation (DIC). It is
expected that compounds of the present invention can be used in place of heparin with
the advantage that heparin's plasma cofactor, anti-thrombin 111, is not consumed in the
reaction. Also, thrombocytopenia, a side effect of heparin treatment, should not be
observed. Furthermore, compounds of the present invention are expected to be




- 35 -

CA ~ 333208
36

valuable in the treatment of diseases in which there is a deficiency of natural inhibitors of
trypsin-like enzymes such as heritary edema. This disorder arises from a deficiency of C1
inhibitor, the major inhibitor of plasma kallikrein.
Finally, compounds of the present invention have demonstrated effective anti-
inflammatories activity in vivo.
Synthesis Examples
The examples which follow illustrate particular embodiments of the invention. All
melting points reported are uncorrected. All parts are by weight and all temperatures are
reported in degrees Celsius. Proton nuclear magnetic resonance (NMR or 1 H NMR)
reports chemical shifts in delta units, parts per million downfield from the internal
tetramethylsilane standard. Various abbreviations employed throughout include: TFA =
trifluoracetic acid; DMF = N,N-dimethylformamide; MS = mass spectrometry; TLC =
thin layer chromatography; RP-TCL = reverse phase thin layer chromatography. Theester protecting groups for the boronic acids are abbreviated: -C6H,2 = the pinacol group
and -C,oH16 = the pinanediol group. "Irg" is the abbreviation for the isothiouronium
analog of arginine (Arg) and the prefix "homo" indicates structures in which the side
chain contains an additional methylene group. All amino acid residues are in the "L"
configuration unless specified.
TLC and RP-TLC were conducted on E. Merk Silica Gel 60 Plates (Catalog # 5534,
E. M. Sciences, Gibbstown, NJ) and Whatman KC1 8F Reverse Phase Plates (Catalog #
4803-600, Whatman Co., Clifton, NJ), respecively. Neutral compounds were visualized
under UV light and after exposure to iodine vapors. Compounds with free amino groups
were stained with ninhydrin and compounds with guanidino groups were stained with the




- 36 -

37 ~ s2Q~

Sakaguchi stain. The Sakaguchi stain exhibits a considerable specificity for themonosubstituted guanidines such as those present in the boroArginine peptides (see
Chemistry of the Amino Acids, 3: (1984) Greenstein and Winitz, eds., Robert E. Krieger
Publishing Co., Malabar, FL).

Example 1a
1-Amino-4-bromo-butyl boronate pinanediol-hydrogen chloride, NH2-CH[(CH2)3Br]BO2-

CloH,6 HCI

4-Bromo-1-chlorobutyl boronate pinanediol was prepared by the method in
Matteson et al ., Organometallics 3: 1284- 1288 (1984), except conditions were modified
for large scale preparations. In a typical experiment, allyl bromide ~173 mL, 2.00 moles)
was hydroborated with catechol borane (240 mL, 2.00 moles) by the addition of the
borane to allyl bromide and then heating the reaction for 4 h at 100 under a nitrogen
atmosphere. The product, 3-bromopropyl boronate catechol (bp 95-102, 0.25 mm) was
isolated in a yield of 49% by distillation. The catechol ester (124 9, 0.52 moles) was
transesterified with (+)alpha-pinanediol (88 9, 0.52 moles) by mixing the component in
50 mL of tetrahydrofuran (THF) and allowing them to stir for 0.5 h at 0 and 0.5 h at
room temperature. Solvent was removed by evaporation and 250 mL of hexane was
added. Catechol was removed as a crystalline solid. Quantitative removal was achieved
by successive dilution to 500 mL and to 1000 mL with hexane and removing crystals at
each dilution. Product (147 9) was obtained as an oil by evaporating solvent.
Analysis for C,3H2202BrB:
Calculated: C = 51.85%, H = 7.38%, and Br = 26.54.
Found: C = 52.85%, H = 7.30%, and Br = 26.58%.

C~ 1 333208
38

4-Bromo-1-chlorobutyl boronate pinanediol was prepared by homologation of the
corresponding propyl boronate. Methylene chloride (34.8 mL, 0.540 moles) was
dissolved in 500 mL of THF, 1.54 N n-butyllithium in hexane (350 mL, 0.540 moles) and
was slowly added at-100. 3-Bromopropyl boronate pinanediol (148 9, 0.490 moles)was dissolved in 500 mL of THF, cooled to the freezing point of the solution, and added
to the reaction mixture. Zinc chloride (33.5 9, 0.246 moles) was dissolved in 250 mL of
THF, cooled to 0, and added to the reaction mixture in several portions. The reaction
mixture, while stirring, was allowed to warm slowly overnight to room temperature.
Solvent was evaporated and the residue was dissolved in hexane and washed with
water. After drying over anhydrous magnesium sulfate and filtering, solvent was
removed to yield the desired product (140 9).
1-Amino-4-bromobutyl boronate pinanediol was prepared first by dissolving
hexamethyldisilizane (28.0 9, 80.0 mmoles) in 30 mL of THF, cooling the solution to -
78, and adding 1.62 N n-butyllithium in hexane (49.4 mL, 80.0 mmoles). The solution
was allowed to slowly warm to room temperature and was then recooled to -78 and 4-
bromo-1-chlorobutyl boronate pinanediol (28.0 9, 80.0 mmoles) in 20 mL of THF was
added. The mixture was allowed to slowly warm to room temperature and to stir
overnight. Solvent was removed by evaporation and 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 mmoles)
was added. The reaction was allowed to warm slowly to room temperature, at whichtemperature it was stirred for 2 h. The resulting product (20 9) was isolated as a solid by
filtration. After drying in vacuo, the crude product was dissolved in chloroform and
insoluble material was




- 38 -

~A I 333208
39

removed by filtration. The filtrate was evaporated and the residue dissolved in ethyl
acetate. The product crystallized from ethyl acetate to yield 15.1 g (mp 142-144.5).
[a]D25 = + 16.7 + 0.80, C = 1.0 in absolute ethanol.
Analysis for C14H26N02BrClB:
Calculated: C=45.87%, H=7.16%, N=3.82%, and B=2.95%.
Found: C =45.76%, H = 7.21 %, N = 3.79%, and B = 3.04%.

Example 1 b
(D,L)1-Amino-4-bromobutyl boronate pinacol-HCI
(DlL)NH2-cH[(cH2)3Br]Bo2-c6Hl2-Hcl

4-bromo-1-chlorobutyl boronate pinacol was prepared by the method described for
the corresponding pinanediol (Example 1 a) except pinacol was substituted for pinanediol
and 3-bromopropyl boronate pinacol (bp 60-64, 0.35 mm) and 4-bromo-1-chlorobutyl
boronate pinacol (bp 110-112, 0.20 mm) were distilled.
Analysis for C1OH1902BrClB:
Calculated: C=40.38% and H=6.45%.
Found: C=40.70% and H=6.37%.

1-Amino-4-bromobutyl boronate pinacol-hydrogen chloride was also prepared by
the procedure in Example 1 a. The final product was crystallized for ethyl acetate:hexane
in yield of 52%.
Analysis for C1OH22N02BrClB:
Calculated: C = 38.19%, H = 7.05%, N = 4.45%, Cl = 11.27% and Br = 25.41 %.
Found: C = 38.28%, H = 7.39%, N = 4.25%, Cl = 11.68% and Br = 26.00%

Example 1c
1-Amino-4-chlorobutyl boronate pinacol-hydrogen chloride (D,L)NH2-CH[(CH2)3CI]B02-
C6H12 HCl


- 39 -

C A i ~332~8

3-Chloropropyl boronate catechol (bp 80-850, 0.30 mm) and 3-chloropropyl
boronate pinacol(bp 63, 0.20 mm) were prepared by the method in Example 1 a except
allyl chloride was substituted for allyl bromide and pinacol was substituted for pinanediol.

Analysis for CgH1802ClB:
Calculated: C = 52.85%, H = 8.89%, and Cl = 17.33%.
Found: C = 53.41 %, H = 8.15%, and Cl = 16.81 %.

Homologation was also conducted by the procedure in Example 1 a and the product
was isolated by distillation (bp 95, 0.25 mm) in a yield of 65%.
Analysis for C,oH,902CI2B:
Calculated: C=47.47%, H=7.58%, and Cl=28.02%.
Found: C = 47.17%, H = 7.45%, and Cl = 27.75.

1-Amino-4-chlorobutyl boronate pinacol HCI was prepared by a procedure identicalto Example 1 a. The product crystallized from ethyl acetate to yield 8.8 g (mp 132-
135.5) and 2.2 g (mp 145- 147). The product melting 145-147 was used for
analyses.
Analysis for C,OH22N02CI2B:
Calculated: C = 44.47%, H = 8.23%, N = 5.19%, and B = 4.00%.
Found: C = 44.01 %, H = 8.23%, N = 4.77%, and B = 3.80% .

ExamPle 1d
(D, L) 1 -Amino-5-bromopentyl boronate-pinacol HCI
(DlL)NH2-cH[(cH2)4Br]Bo2c6H12-Hcl
4-bromobutyl boronate pinacol was prepared by the method described for 3-
bromopropyl boronate pinanediol (Example 1a) except 4-bromo-1-butene was substituted
for allyl bromide and pinacol was substituted for pinanediol. The product was isolated as
an oil (bp 77, 0.3 mm). Homologation yielded 5-bromo-1-chloropentyl boronate pinacol.


- 40 -

41 CAl J32~8

MS(CI) for C"H2102BrClB:
Calculated - H: 310.47.
Found: 310.

The final product, 1-amino-5-bromopentyl boronate pinacol HCI, was prepared by
the procedure in Example 1 a in a yield of 35%.
Analysis for C1'H24NO2BrBCI:
Calculated: C=40.22%, H=7.36%, N=4.26%, Cl=10.79%, Br=24.32%, and
B=3.29%.
Found: C = 39.23%, H = 7.18%, N = 4.04%, Cl = 15.21 % and Br = 25.66%, and
B = 3.75%

Example 2
Boc-(D)Phe-Pro-NH-CH[(CH2)3Br]BO2-C,OH,6

Boc-(D)Phe-Pro-OH was produced by first preparing the dipeptide benzyl ester andthen removing the ester by catalytic hydrogenation. Boc-(D)Phe-OH (10.0 9, 37.7
mmoles) was dissolved in 50 mL of THF and N-methylmorpholine (4.14 mL, 37.7
mmoles) was added. The solution was cooled to -20 and isobutyl chloroformate (4.90
mL, 37.7 mmoles) was added. After 5 min, H-Pro-OBzl.HCI (9.11 9, 37.7 mmoles),
dissolved in 50 mL of chloroform and cooled to -20, was added. Triethylamine (5.25
mL, 37.7 mmoles) was added and the mixture was stirred for 1 h at -20 and 2 h at
room temperature. The reaction mixture was filtered and the filtrate 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 organic phase
was dried over anhydrous sodium sulfate, filtered, and evaporated to yield 15.2 9 of Boc-
(D)Phe-Pro-OBzl as an oil. The benzyl ester (15.2 g) was dissolved in 100 mL of
methanol and it was hydrogenated at an initial pressure of 40 psi on a Parr


- 41 -

~, A l 333208
42

apparatus in the presence of 0.5 9 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 9 of the desired product (mp
176.5-177).
Analysis for C1gH26N205:
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]BO2-C1OH16 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 wascooled 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 1 a, NH2-
CH[(CH2)3Br]BO2-C1OH16 HCI, (5.0 9, 13.6 mmoles) dissolved in 10 mL of cold
chloroform. Cold THF (10 mL) and triethylamine (1.90 mL, 13.6 mmoles) were addedand 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.




- 42 -

43 Cl~ 333208

MS(FAB) for C33H49N306BBr:
Calculated + H: 674.30
Found: 674.30

Example 3
Boc-(D)phe-pro-NH-cH[(cH2)3N3]Bo2-cloHl6

Boc-(D)Phe-Pro-NH-CH[(CH2)3Br]B02-C1OH16, the product of Example 2, (4.4 9,
6.54 mmoles) was dissolved in 7 mL of DMF and sodium azide (0.919 9, 14.1 mmoles)
was added. The mixture was heated at 100 for 3 h. Ethyl acetate (100 mL) was added
to the reaction mixture and it was washed with water and with saturated aqueous
sodium chloride. The organic phase was dried over anhydrous sodium sulfate, filtered,
and subjected to evaporation. A yield of 4.1 9 of a solid resulted. This material was
chromatographed on a 2.5 X 50 cm column of LH-20 in methanol. Fractions containing
the desired product were pooled, liquid was evaporated to yield 2.3 q of the azide. TLC
in methanol:chloroform (1:9) indicated a single spot, Rf 0.76.
Analysis for C33H48N606B:
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 C33H48N606B
Calculated + H: 637.39.
Found: 637.49.

ExamPle 4
Boc-(D)Phe-Pro-NH-CH[(CH2)3NH2]B02-C~OH16benzene sulfonic 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 9
of 10% Pd/C and benzene sulfonic acid (2.19 g, 13.8


- 43 -

~A ~ s33208
44

mmoles). After 1 h, catalyst was removed and the solution was evaporated to yield a
solid which was triturated with hexane to yield 9.9 9 of the desired product. RP-TLC in
methanol:water (85:15) indicated a UV spot, RF 0.91, and a ninhydrin positive spot, RF
0.52.

Example 5
Boc-(D)Phe-Pro-NH-CH[(CH2)3-NH-C(NH)NH2]B02-C10H,6 benzene sulfonic acid.
Boc-(D)Phe-Pro-boroArg-C1OH16benzene sulfonic acid

Boc-(D)Phe-Pro-boroOrn-C1OH16benzene sulfonic acid, Example 4, (4.6 9, 6.11
mmoles) was refluxed at 100 in 20 mL of absolute ethanol containing cyanamide (50
mg/mL). The progress of the reaction was monitored by RP-TLC in methanol:water
(85:15) in which the disappearance of the ninhydrin spot for the amine starting material
(Rf 0.54) and the appearance of the Sakaguchi streak of the product (Rf 0-0.13) was
observed. Product could be detected after refluxing 18 h and its level progressively
increased with time. After 7 days, amine could not be detected and the reaction solution
was concentrated to an approximate 50% solution through passive evaporation. Thereaction solution was filtered, concentrated, and chromatographed on a 2.5 X 100 cm
column of LH-20 in methanol. Fractions containing the desired product were pooled and
subjected to evaporation to yield 3.7 9 of the desired product. A portion (2.3 9) was
crystallized for ethyl acetate:hexane to yield 0.89 9 and the residue (1.2 9) was obtained
as a solid by triturating with ether. In separate experiments.
MS(FAB) for C34H53N606SB:
Calculated + H: 653.42
Found: 653.38




- 44 -

`~ A i 333~08

Analysis for C4oH59N6o9sB-H2o
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-C,OH16-2HCI

Boc-(D)Phe-Pro-boroArg-C1OH,6-benzene sulfonic acid, the product of Example 5,
(1.17 9, 1.54 mmoles) was reacted with 5 mL of % 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(O.80 9).
MS~FAB) for C29H45N604B
Calculated + H: 553.37.
Found: 553.40 and 538.40 (unidentified).
Analysis of H-( D) Phe-Pro-boroArg-C, oH 16-1 BSA TFA:
Found: 553.4
Examples 7 - 8
Ac-(D)Phe-Pro-boroArg-C,OH,6-HCI (Example 7)
Ac-(D)Phe-Pro-boroArg-OH-HCI (Example 8)
Boc-(D)Phe-Pro-boroArg-C,OH,6-benzene sulfonic acid, the product of Example 5,
(0.86 9, 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 (0.70 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)


- 45 -

CA ~ 333208
46

and sodium bicarbonate (0.42 g, 5.0 mmoles) were added. The mixture was stirred for
20 min at room temperature. Ethyl acetate (50 mL) and water (5 mL) were added. The
phases were separated and the organic phase was dried over anhydrous sodium sulfate,
filtered, and solvent removed by evaporation to yield 0.56 g of a partial solid.
The sample was dissolved in 4 mL of glacial acetic acid and diluted with 16mL ofwater. It was immediately applied to a column containing 15 mL of SP-SephedexTM (H+
form) and equilibrated with 20% acetic acid. The column was washed with 300 mL of
20% acetic acid and then a linear gradient from 100 mL of 20% acetic acid to 100 mL of
20% acetic acid adjusted to 0.30 N hydrochloric acid was run. Fractions collected from
0.08 to 0.17 N hydrochloric acid contained the N-acetyl peptide (0.29 g) as a mixture of
the free boronic acid and pinanediol ester.
The pinanediol ester and the free boronic acid were separated by chromatography
on a 2.5 X 100 cm column of LH-20 in methanol. The fraction size was 8.2 mL. Thepinanediol ester (102 mg) eluted in fraction 41-43 while free boronic acid (131 mg)
slowly eluted in fractions 45-129.
MS(FAB) (Example 7):
Ac-(D)Phe-Pro-boroArg-C,OH16) for C31H47N605B:
Calculated + H: 595.33.
Found: 595.33.
MS (FAB) (Example 8):
Ac-(D)Phe-Pro-boroArg-OHHCI) for C21H33N605:
Calculated + H: 449.60.
Found: 579.24-581.24.
The latter result could not be interpreted. However, NMR was consistent with thestructure of the free boronic acid since definitive bands for pinanediol group such as the
methyl groups singlets observed at



- 46 -

~ Q 1 333208
47

delta 0.85(3H), 1.30(3H), and 1.36(3H) were absent. As added proof of structure, a
sample of the free boronic was re-esterified to give the product in Example 7. An
analytical sample (20 mg) was treated with a 2-fold excess of pinanediol (14 mg) in 3 mL
of methanol for 5 min. Solvent was evaporated and excess pinanediol was removed by
trituration of the sample with ether to yield the product (26 mg).
MS(FAB) (Found: 595.38) and NMR were consistent with that expected for the esterified
product and were almost identical to the pinanediol product of Example 7.

Example 9
Ac-Phe-boroArg-C,OH,6-HCI

Ac-Phe-NH-CH-[(CH2)3Br]BO2-C10H16 was prepared by the procedure described in
Example 2. The mixed anhydride of Ac-Phe-OH (0.565 9, 2.73 mmoles) was prepared in
10 mL of THF and coupled to NH2-CH[(CH2)3Br]BO2-C,OH16-HCI (the product of Example
1 a, 1.00 9, 2.73 mmoles) dissolved in 10 ml of cold THF to yield 1.47 9 of a white
foam. This material was stirred with hexane overnight to yield a solid, 1.01 9 (mp
106.5-109).
Analysis for C25H36N204BrB:
Calculated: C=57.81, H=7.00%, N=5.40%, Br=15.40%,
B = 2.08% . Found: C = 58.33 %, H = 7.33%, N = 4.76%, Br = 14.18%,
B = 1.80% .
MS(FAB) for C25H36N204BrB:
Calculated + H: 519.20.
Found: 519.23.

Ac-Phe-NH-CH[(CH2)3N3]BO2-C~OH~6 was prepared by treating Ac-Phe-NH-
CH[(CH2)3Br]BO2-C,OH,6 (3.22 9, 6.20 mmoles) with sodium azide by the procedure
described in Example 3. Product from the reaction (3.03


- 47 -

C A 1 333208
48

g) was chromatographed on LH-20. Fractions containing the desired product were
pooled and evaporated. The residue was triturated with hexane to yield 2.21 9 of the
azide.
Ac-Phe-boroOrn-C,OH,6benzene sulfonic acid was prepared from Ac-Phe-NH-
CH[(CH2)3N3]B02-C10H,6 (2.21 9, 4.59 mmoles) by the procedure in Example 4 except
hydrogenation was performed at atmospheric pressure. After filtration, and the
evaporation of solvent, the desired product (2.22 9) was obtained by triturating with
ether.
Ac-Phe-boroArg-ClOH,6benzene sulfonic acid was prepared by treating Ac-Phe-
boroOrn-C,OH,6-benzene sulfonic acid (2.0 9, 3.26 mmoles) with a 10 mL solution of
cyanamide (100 mg/mL) in ethanol. The guanidation procedure in Example 5 was used
except the reaction time was 3 days and the reaction mixture contained a mixture of
starting material and product. This required an additional purification step which most
probably could have been eliminated by a longer reaction time. The solution was
concentrated and chromatogramed on 2.5 X 100 cm column of LH-20 in methanol. Thefractions containing the desired product, detected by Sakaguchi stain, were pooled and
subjected to evaporation to yield 1.4 9. The resulting material (1.2 g) was dissolved in 6
mL of acetic acid and diluted with 30 mL of water to yield a milky solution. It was
applied to a 30 mL column of SP-SephedexTM C-25 (H +form) equilibrated in 20%
aqueous acetic acid. The column was 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. Fractions eluted from the column from
0.12 N to 0.16 N hydrochloric acid were pooled to yield 0.42 q of the desired peptide as
a mixture of the free boronic acid and pinanediol ester. The mixture was dissolved in
methanol




- 48 -

CA 1 333208
49

(10 ml) and 80 mg of pinanediol was added to esterify the free boronic acid. After
stirring for 30 min, solvent was evaporated and the residue was triturated with ether to
yield 0.28 9 of the desired product.
Analysis for C26H40N504B HCI 2H20:
Calculated: C = 54.78%, H = 8.15%, N = 12.30%, and B = 1.90.
Found: C=55.34, H=7.83, N=11.66, and B=1.99.
MS(FAB) for C26H40Nso4B
Calculated + H: 498.32.
Found: 498.31.

Example 10
Ac-(D,L)Phe-(D,L)boroArg-C6H,2

The intermediate, Ac-(D,L)Phe-(D,L)-NH-CH[CH2)3Br]BO2-C6H,2, was prepared by a
modification of the procedures of Examples 1 b and 2. The acid chloride of Ac-Phe-OH
was prepared by reacting Ac-Phe-OH (30 g, 0.145 moles) with phosphorous
pentachloride (30 g, 0.144 moles) in 175 mL of THF at -10. The reaction was stirred at
0 for approximately 1 h, then diluted to a volume of 350 mL with cold ether. The
product was isolated as a solid, washed with cold ether, and dried in vacuo to yield 21 9.
The activated Ac-Phe derivative (14.8 9, 65.6 mmoles) was dissolved in 40 mL of THF
and added to the product of the reaction 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 as a



- 49 -

CA i ~33208

crystalline solid (1.37 9, mp 146.5-148). In a separate experiment, the following
analysis was obtained.
Analysis for C2,H32N204BrB:
Calculated: 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%, Br = 16.46%, and B = 3.40%.

The alkyl bromide was converted to the corresponding azide by the procedure in
Example 3. The product crystallized from ethyl acetate (mp 143-144).
Analysis for C21H32N5O4B:
Calculated: C=58.74%, H=7.53%, N=16.31%, and B=2.53%.
Found: C = 58.85%, H = 7.48%, N = 16.53%, and B = 2.93%.

The azide was converted to Ac-(D,L)Phe-(D,L)boroOrn-C6H12-benzene sulfonic acid
by the procedure in Example 4 except hydrogenation was conducted at atmospheric
pressure .
Ac-(D,L)Phe-(D,L)boroOrn-C6H12-benzene sulfonic acid (0.243 9, 0.433 mmoles)
was reacted with cyanamide (0.020 9, 0.476 mmoles) at 100 in 2 mL of absolute
ethanol overnight. The solution was concentrated and triturated with ether to yield 0.21
g of a white solid. RP-TLC of the material indicated the characteristic streak staining
positive with the Sakaguchi stain for the boroArginine peptides, Rf 0-0.55, and a discrete
spot, Rf 0.68, corresponding to unreacted starting 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 with ether.
MS(FAB) for C22H37NsO4B:
Calculated + H: 446.30.
Found: 446.23 and 404.19 (correspondingto the




- 50 -

Cb 1 333208
51

unreacted boroOrn peptide).
Note that the method of Example 5 is a superior method for preparing the
boroArginine peptides and differs in that a larger excess of cyanamide and longer reaction
times are used.

Example 11
Boc-(D)Phe-Phe-boroArg-C,OH16benzene sulfonic acid

Boc-(D)Phe-Phe-OH was prepared by the method described for Boc-(D)Phe-Pro-OH
in Example 2. Following hydrogenation of the benzyl ester, material crystallized from
chloroform: hexane yielding the desired peptide (mp 133- 133.5 ) .
Analysis for C23H28N205:
Calculated: C = 66.96%, H = 6.86%, and N = 6.79%.
Found: C = 66.75%, H = 6.79%, and N = 6.56%.

Boc-(D)Phe-Phe-NH-CH[(CH2)3Br]BO2-C1OH,6 was prepared by coupling Boc-(D)Phe-
Phe-OH (6.00 9, 14.5 mmoles) to NH2-CH[(CH2)3Br]BO2-C,OH,6 HCI (Example 1a, 5.33 9,
14.5 mmoles) using the procedure described in Example 2 except that the LH-20
chromatography step was eliminated. The product crystallized from ethyl acetate to yield
2.47 9 (mp 132-134) in the first crop and 5.05 9 (mp 133-135) in a second crop. RP-
TLC in methanol:water (85:15) indicated a single spot, Rf 0.29.
Analysis for C37H5,N306BrB:
Calculated: C = 61.32%, H = 7.11 %, N = 5.80%, Br = 11.03%.
Found: C = 61.21 %, H = 7.02%, N = 5.59%, Br = 10.22%.

Boc-(D)Phe-Phe-NH-CH[(CH2)3N3]BO2-C,OH,6 was prepared by treating the
corresponding alkyl bromide (7.15 9, 9.87 mmoles) with sodium azide using the
procedure in Example 3, except the LH-20 chromatography step was not needed for
purification. The product


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~ b i j33208
52
_

emerged from an ethyl acetate:hexane solution as a gel and was isolated and washed
with hexane to yield 3.0 9 in the first crop and 2.9 9 in a second crop.
Boc-(D)Phe-Phe-boroOrn-C1OH,6benzene sulfonic acid was prepared from the azide
(5.37 g, 7.82 mmoles) by the procedure in Example 4 to yield 5.33 9. RP-TLC
methanol:water (85:15) indicated an intense ninhydrin positive spot, Rf 0.42, and a weak
ultraviolet (UV) light spot, 0.92. (The UV spot at Rf 0.92 is typical of amines or
guanidino compounds which are benzene sulfonic acid salts.)
MS(FAB) for C37H53N406B:
Calculated + H: 661.76.
Found: 661.14.

Boc-(D)Phe-Phe-boroArg-C,OH16 was obtained by the procedure in Example 5. The
boroOrnithine peptide (4.83 9, 5.90 mmoles) was treated with cyanamide (50 mg/mL) in
20 mL of absolute ethanol for 7 days. A portion of the reaction mixture corresponding to
1.0 9 of starting material was removed and heated separately in the absence of a reflux
condenser overnight to obtain complete conversion of the amine to the guanidino
compound. Following chromatography on LH-20 and trituration of the product with
ether, 0.52 9 of the desired product were obtained.
Analysis for C44H61N609SB:
Calculated: C = 61.38%, H = 7.16%, N = 9.76%, B = 1.25%.
Found: C=59.69%, H=7.41%, N=9.82%, B=1.26%.
MS(FAB) for C38H55N606B
Calculated + H: 703.43.
Found: 703.49.

Example 12
H-(D)Phe-Phe-boroArg-C,OH,6-2HCI



- 52 -

53 CA 1~33208

Boc(D)Phe-Phe-boroArg-C,OH16benzene sufonic acid (Example 11, 0.59 9, 1.25
mmoles) was deblocked by the procedure in Example 6 except that the sample was
applied to the ion exchange column in 20% ethanol and the column was eluted with 20%
ethanol. The product (0.424 9) was obtained as a white solid.
MS(FAB) for C33H47N604B:
Calculated + H: 603.38.
Found: 603.41.

Example 13
Ac-Ala-Lys(Boc)-boroArg-C,OH,6-benzene sulfonic acid

Ac-Ala-Lys(Boc)-OH was prepared by coupling the
N-hydroxysuccinimide ester of Ac-Ala-OH, prepared by the method of Anderson et al., J.
Am. Chem. Soc. 86: 1839, (1964), to H-Lys( Boc)-OH . The N-hydroxy-succinimide of
Ac-Ala-OH (6.25 9, 27.4 mmoles) was dissolved in 30 mL of dioxane and was added to a
solution of H-Lys(Boc)-OH (7.50 9, 30.4 mmoles) dissolved in a solution consisting of 30
mL of 1.0 N sodium hydroxide and triethylamine (2.12 mL, 15.0 mmoles). The reaction
mixture was stirred overnight, then acidified with hydrochloric acid. Sufficient dry
sodium chloride to nearly saturate the solution was added. The product was extracted
into ethyl acetate and it was washed with 0.2 N hydrochloric acid prepared in saturated
aqueous sodium chloride. The organic phase was dried over anhydrous sodium sulfate
and filtered. Solvent was removed by evaporation. The product was crystallized from
ethyl acetate:hexane to yield 7.3 9 (mp 86-89).
Ac-Ala-Lys(Boc)-NH-CH[(CH2)3Br]BO2-C,OH16 was prepared by the procedure of
Example 2 except that the product was purified by fractional crystallization from ethyl
acetate. The product (1.13 g) obtained in the




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CA 1 333208
54

second and third crops exhibited a single spot on RP-TLC in methanol:water (85:15), with
an Rf 0.51. The TLC plate was exposed to hydrochloric acid fumes wherein the resulting
amine was detected after the addition of ninhydrin stain.
Ac-Ala-Lys(Boc)-NH-CH[CH2)3N3]BO2-C1OH,6 was prepared from the corresponding
alkyl bromide (1.95 9, 2.90 mmoles) by the procedure in Example 3 except that the
product was purified by crystallizing it from ethyl acetate rather than LH-20
chromatography. Crude product (1.60 9) crystallized to yield 0.55 9 (mp 79-84) and
0.96 9 of residue. The analysis of the crystalline product follows.
Analysis for C30H52N77B:
Calculated: C = 56.86%, H = 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,OH,6benzene sulfonic acid was prepared from the
corresponding alkyl azide (0.433 9, 0.683 mmoles) using the method described in
Example 4. The catalyst and solvent were removed, then the product (0.45 9) was
obtained by trituration with ether.
MS(FAB) for C30H54N507B:
Calculated + H: 608.42.
Found: 608.49.

Ac-Ala-Lys(Boc)-boroArg-C,OH,6benzene sulfonic acid was prepared by reacting
the corresponding boroOrnithine peptide with cyanamide using the method described in
Example 5. The chromatography fractions containing the desired product were triturated
with ether to yield 0.83 9 as a white solid.
Analysis for C37H62N70,0BS:
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%.



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C~ i 333208

Example 14
Ac-Ala-Lys-boroArg-ClOH,6 2HCI

Ac-Ala-Lys~Boc)-boroArg-C10H16benzene sulfonic acid ~0.200 9, 0.248 mmoles)
was deblocked by the procedure in Example 6. Following ion exchange, evaporation of
solvent, drying in vacuo, and triturating with ether, 0.14 9 of material were obtained.
MS(FAB) for C26H48N705B:
Calculated + H: 550.39.
Found: 550.42.

Example 15
Boc-Leu-Gly-Leu-Ala-boroArg-C,OH16benzene sulfonic acid

Boc-Leu-Ala-OBzl was prepared by the procedure for dipeptide synthesis in
Example 2. Boc-Leu-Ala-OBzl (23.7 g, 57.7 mmoles) was dissolved in 40 mL of
anhydrous trifluoroacetic acid. After 15 min, excess trifluoroacetic acid was removed by
evaporation and the residue was treated with ether to yield H-Leu-Ala-OBzl.trifluoroacetic
acid as a crystalline product (22.8 9).
Analysis for C18H25N205E3:
Calculated: C = 53.19%, H = 6.21 %, and N = 6.89%.
Found: C = 53.37%, H = 5.68%, and N = 6.84%.

Boc-Gly-Leu-Ala-OBzl was prepared by coupling Boc-Gly-OH (5.70 9, 32.6 mmoles)
to H-Leu-Ala-OBzl using the mixed anhydride procedure described in Example 2. The
product (13.8 9) 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


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CA i 333208
56

ethyl acetate and treated with anhydrous hydrogen chloride. The resulting product was
precipitated by the addition of ether to yield 7.7 9 of H-Gly-Leu-Ala-OBzl-HCI in a first
crop .
Boc-Leu-Gly-Leu-Ala-OBzl was prepared by coupling Boc-Leu-OH (2.62 g, 10.5
mmoles) to H-Gly-Leu-Ala-OBzl using the mixed anhydride procedure described in
Example 2. The resulting product was crystallized from ethyl acetate:hexane to yield 2.7
g (mp 95-96) in the first crop.
Analysis for C29H46N407:
Calculated: C=61.89%, H=8.26%, and N=9.96.
Found: C = 62.00%, H = 8.40%, and N = 9.83%.

Boc-Leu-Gly-Leu-Ala-OH was prepared by the catalytic hydrogenation of the benzylester (2.6 g, 4.62 mmoles) by the procedure described in Example 2 to yield 2.1 g. The
resulting product was crystallized from hot ethyl acetate to yield 1.4 g.
Analysis for C22H40N407:
Calculated: C = 55.90%, H = 8.55%, and N = 11.86%.
Found: C = 55.42%, H = 8.47%, and N = 11.73%.

Boc-Leu-Gly-Leu-Ala-NH-CH[(CH2)3Br]BO2-C~OH~6 was prepared by coupling Boc-
Leu-Gly-Leu-Ala-OH (1.40 g, 2.96 mmoles) to the amine from Example 1a. This was
done using the procedure in Example 2 except that the chromatographic step was
eliminated. The product crystallized from ethyl acetate:hexane to yield 1.17 9. TLC in
methanol:chloroform (1 :9) indicated a single spot Rf 0.68.
Analysis for C36H63N508BrB:
Calculated: C = 55.10%, H = 8.11 %, N = 8.93%, and B = 1.38%.
Found: C=55.96%, H=8.30%, N=8.74%, and B=1.33%.

The corresponding azide was prepared by the


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CA 1 333208
57

procedure described in Example 3 in a yield of 97% and it was converted to Boc-Leu-Gly-
Leu-Ala-boroOrn-C,OH,6 by the method described in Example 4. An analytical sample was
prepared by precipitating the product with ether and then chromatographing it on LH-20,
and reprecipitating it from chloroform with hexane.
MS(FAB) for C36H65N608B:
Calculated + H: 721.50.
Found: 721.55.

Boc-Leu-Gly-Leu-Ala-boroArg-C,OH16-benzene sulfonic acid was prepared by the
method described in Example 5. The corresponding boroOrnithine peptide (0.695 9,0.791 mmoles) was reacted with 5 mL of a cyanamide solution (50 mg/mL) in absolute
ethanol. The above mixture was chromatographed and triturated with ether, wherein
0.41 9 of the desired product was obtained.
MS(FAB) for C37H67N808B:
Calculated + H: 763.53.
Found: 763.8.

Example 16
H-Leu-Gly-Leu-Ala-boroArg-C,OH,6benzene sulfonic acid

Boc-Leu-Gly-Leu-Ala-boroArg-C,OH,6benzene sulfonic acid (Example 15, 0.050 9,
0.0543 mmoles) was reacted with 2 mL of 4 N hydrogen chloride in dioxane for 5 min at
room temperature. Solvent and excess hydrogen chloride were removed by evaporation.
The sample 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(FAB) for C32H59N806B:
Calculated + H: 663.47.



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~A I 333208
58

Found: 663.50.

Example 17
Bz-Glu(OBu)-Gly-boroArg-C1OH16benzene sulfonic acid

Bz-Glu(OBu)-Gly-NH-CH[(CH2)3Br]BO2-C1OH16 was prepared by coupling Bz-
Glu(OBu)-Gly-OH to the amine according to the method described in Example 2. Thecorresponding azide was prepared by the method described in Example 3 and
boroOrnithine peptide were prepared by the method described in Example 4.
MS(FAB) for C32H49N407B:
Calculated + H: 613.38.
Found: 613.60.

The final product was obtained by the method described in Example 5.
MS(FAB) for C33H51N607B:
Calculated + H: 655.40.
Found: 655.37.
Analysis for C39H57N6010SB:
Calculated: C = 57.62%, H = 7.08%, N = 10.34%, and B = 1.33%.
Found: C = 57.43%, H = 7.25%, N = 9.91 %, and B = 1.23%.

Example 18
Bz-Glu-Gly-boroArg-C1OH16benzene sulfonic acid

Bz-Glu (OBu)-Gly-boroArg-C1OH 16 benzene sulfonic acid (0.13 9, 0.16 mmoles) wasdissolved in 5 mL of dioxane, benzene sulfonic acid (0.10 9, 0.66 mmoles) was added,
and the solution 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 9). The material was chromatographed on a 2.5 X 50 cm column of
LH-20 in methanol. Fractions containing the

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C~ 1 333208
59

desired product were subject to evaporation and the residue was triturated with ether to
yield 53 mg of the desired product.
MS(FAB) for C29H43N607B:
Calculated + H: 599.34.
Found: 599.35 + 613.36 (unidentified).

Example 18a
Bz-Glu-Gly-boroArg-C10H,6benzene sulfonic acid

Bz-Glu(OBu)-Gly-boroArg-C,0H16-benzene sulfonic acid (Example 17, 0.20 g, 0.246
mmoles) was treated with anhydrous hydrogen chloride by the procedure described in
Example 6 for 45 min. After the material was triturated with ether, NMR indicated that
approximately 30% of the t-butyl protecting group was still present. The product was
then reacted with anhydrous TFA for 45 min at room temperature. TFA was removed by
evaporation and the residue was triturated with ether to yield 143 mg.
MS(FAB) for C29H43N6O7B:
Calculated + H: 599.34.
Found: 599.35.

ExamPle 1 9
Bz-Pro-Phe-boroArg-C,0H16benzene sulfonic acid

Bz-Pro-Phe-OH (mp 200-201 ) was prepared by the method described in Example
2 for dipeptide synthesis.
Analysis for C21H22N204:
Calculated: C = 68.82%, H = 6.06%, and N = 7.65%.
Found: C = 68.91 %, H = 6.09%, and N = 7.47%.

Bz-Pro-Phe-NH-CH[(CH2)3Br]BO2-C,0H16 was prepared by coupling
Bz-Pro-Phe-OH to the amine using

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CA 1 333208

the general method described in Example 2 except the chromatography step was
eliminated. TLC in methanol:chloroform (1 :9) indicated a major spot at Rf 0.72 and a
trace at Rf 0.86.
MS(FAB) for C35H45N305BBr:
Calculated + H: 678.27.
Found: 677.95.

The alkyl halide was converted to the azide and to the boroOrnithine peptide by the
procedures described in Examples 3 and 4.
MS(FAB) for (Bz-Pro-Phe-boroOrn-C,OH,6) C35H47N405B:
Calculated + H: 615.37.
Found: 615.42.
Bz-Pro-Phe-boroArg-C,OH,6benzene sulfonic acid was prepared by the method
described in Example 5.
MS(FAB) for C36H49N605B:
Calculated + H: 657.39.
Found: 657.13.
Analysis for C42H55N60gSB:
Calculated: C = 61.90%, H = 6.82%, N = 10.31 %, and B = 1.33%.
Found: C=60.16%, H=7.27%, N= 9.79%, and B=1.44%.

Example 20
Bz-Pro-Phe-boroArg-OH HCI
Bz-Pro-Phe-boroArg-C~OH~6benzene sulfonic acid (Compound of Example 19, 0.64
9, 0.79 mmoles) was dissolved in 4 mL of methylene chloride and cooled to -78. It was
added to flask containing 4 mL of 0.50 N boron trichloride, which has been prepared by
diluting 1.0 N boron trichloride (Aldrich Chemical Co., Milwaukee, Wl) 50% with dry
methylene chloride, in a dry ice bath. The solution was stirred for 5 min at -78, then
the flask was transferred to a 0 ice bath where the solution was stirred for 15 min.
Cold water (5 mL)

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CA i 333208
61

was added slowly then the solution was diluted to 120 mL with 20% acetic acid. The
organic phase which separated was removed and discarded. The aqueous phase was
applied to a 20 mL column of SP-SephedexTM which was equilibrated with 20% acetic
acid. The column was washed with approximately 150 mL of 20% acetic acid then
subjected to a linear gradient from 200 mL of 20% acetic acid to 200 mL of 20% acetic
acid containing 0.30 N hydrochloric acid. The product eluted when the concentration of
hydrochloric acid was between 0.08 and 0.15 N. The desired product (0.19 9) was
obtained after evaporating the solvent, drying the residue in vacuo, and triturating it with
ether.
MS(FAB) for C26H35N60sB
Calculated + H: 523.29.
Found: 579.34 (unidentified).
Analysis for C26H36N605ClB
Calculated: C = 53.29%, H = 6.55%, N = 14.34%, and B = 1.84%.
Found: C = 53.27%, H = 6.58%, N = 13.25%, and B = 1.89%.

Esterification of the product with pinanediol as described in Example 8a gave a
product whose NMR and MS properties were consistent with the starting ester of
Example 19.
MS(FAB) for C36H49N605B:
Calculated + H: 657.40.
Found: 657.39.




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CA 1 333208
62

Example 21
Bz-Pro-Phe-boroArg-F-hydrogen chloride
Bz-Pro-Phe-NH-CH[(CH2)3NH-C(NH)NH2]BF2 HCI

Bz-Pro-Phe-boroArg-F was prepared by a modification of the procedure described
by Kinder et al., J. Med. Chem., 28: 1917-1925, (1985). Free boronic acid (Compound
of Example 20, 0.100 9., 0.179 mmoles) was dissolved in 2 mL of water. To it, 0.040
mL of 48% hydrofluoric acid was added at room temperature. A gummy precipitant
formed almost instantly. The reaction was stirred for 10 min, then the mixture was
frozen and excess hydrofluric acid and water were removed in vacuo. The residue was
dissolved in methanol, concentrated, and triturated with ether. A yield of 0.093 9 was
obtained .
MS(FAB) for C26H33N603BF2:
Calculated + H: 527.29.
Found: 527.31 and additional masses characteristic of the free boronic acid.
Analysis for C26H34N603BF2cl-H20
Calculated: C=53.47%, H=6.25%, N=14.47%, B=1.86%, and F=6.54%.
Found: C=54.00%, H=6.40%, N=13.48%, B=1.95%, and F=7.06%.

Example 22
Boc-(D)Phe-Pro-borolrg-C,OH,6-HBr

Borolrg- is the abbreviation for -NH-CH[(CH2)3S-C(NH)NH2]B02- in which the
isothiouronium group replaces the guanidino of boroArginine. Boc-(D)Phe-Pro-NH-
CH[(CH2)3Br]B02-C,OH,6 (Compound of Example 2, 1.00 9, 1.61 mmoles) was dissolved
in 4 mL of absolute ethanol and thiourea (0.37 g, 4.82 mmoles) was added. The mixture
was stirred overnight at room temperature. The solution was concentrated and the



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33208
63

residue was triturated with ether to yield 0.58 9 of solid. The resulting solid was
chromatographed on a 2.5 X 50 cm column of LH-20 in methanol. Pooled fractions
containing the desired product were subjected to evaporation to yield 0.26 9 of product.
The sample was triturated with ether to yield 0.150 9 of an amorphous solid.
MS(FAB) for C34H53N506BS:
Calculated + H: 670.38.
Found: 670.39.
Analysis for C34H53N506SBrB.
Calculated: C = 54.40%, H = 7.13%, N = 9.33%, and B = 1.44%.
Found: C = 54.10%, H = 7.39%, N = 9.27%, and B = 1.47%.

The ether soluble residue obtained from this reaction consisted mainly of starting
material which was converted to isothiouronium salt by longer reaction periods.
This general procedure was used to prepare other isothiouronium salts except in
some cases a 4-fold excess of thiourea and 3-4 day reaction times were used.

Example 23
H-(D)-Phe-Pro-borolrg-C,OH,6-HBr,HCI

Boc-(D)Phe-Pro-borolrg-C10H16-HBr (Compound of Example 22, 0.050 9, 0.067
mmoles) was reacted with 1 mL of 4 N hydrogen chloride in dioxane for 15 min at room
temperature. Solvent was evaporated and the residue was triturated with ether to yield
0.040 9 of a white solid.
MS(FAB) for C29H45N504SB:
Calculated + H: 571.29.
Found: 570.47.




- 63 -

64 ~A 1333208

Example 24
Ac-Ala-Lys(Boc)-borolrg-C,OH,6 HBr

Ac-Ala-Lys(Boc)-NH-CH[(CH2)3Br]B02-C,OH,6 (from Example 13, 0.700 9, 1.04
mmoles) was reacted with thiourea (0.320 9, 4.00 mmoles) for 4 days in 4 mL of
absolute ethanol. The product was purified by the procedure described in Example 22.
Following chromatography, 0.28 9 of the desired product were obtained. Trituration with
ether yielded 0.173 9 of the product as an amorphous white solid.
MS(FAB) for C3,H55N607SB:
Calculated + H: 667.8.
Found: 667.
Analysis for C31H56N607SBrB:
Calculated: C=49.79%, H =7.56%, N = 11.24%, and B= 1.44%.
Found: C=49.20%, H =7.62%, N = 11.31 %, and B= 1.36%.

Example 25
Ac-Ala-Lys-borolrg-C10H,6-2HBr

Ac-Ala-Lys(Boc)-borolrg-C,OH,6HBr (the compound of Example 24, 0.050 9,
0.067 mmoles) was dissolved in 1 mL of methanol and hydrogen bromide gas was
bubbled though the solution of 10 min. Solvent was removed by evaporation and the
residue was triturated with ether to yield the desired product as a solid (49 mg).
MS(FAB) for C36H47N605SB:
Calculated + H: 567.35.
Found: 567.41.
ExamPle 26
Ac-Phe-borolrg-C,OH,6 HBr
Ac-Phe-NH-CH[(CH2)3Br]B02-C,OH,6 (From Example


- 64 -

CA 1 333208

9, 1.00 g, 2.41 mmoles) was reacted with a 3-fold excess of thiourea in 5 mL of
absolute ethanol following the procedure described in Example 22. The product (0.284
g) was obtained as a white amorphous solid. Additional product was obtained by again
reacting any remaining ether soluble material with thiourea and repeating the purification
procedure .
MS(FAB) for C26H39N404SB:
Calculated + H : 5 15 . 29 .
Found: 51 5.29.
Analysis for C26H39N404SB.
Calculated: C = 52.44%, H = 6.79%, N = 9.41%, and B = 1.82%.
Found: C = 52.83%, H = 6.89%, N = 8.47%, and B = 1 .85%.

Example 27
Bz-Pro-Phe-borolrg-C,OH~6-HBr

Bz-Pro-Phe-NH-CH[(CH2)3Br]B02C,OH,6 (product from Example 19, 0.500 g, 0.737
mmoles) was used to prepare the product of this example by following the procedure
described in Example 22. Product (0.358 g) was obtained as a white solid.
MS(FAB) for C36H48N505SB:
Calculated + H: 674.35.
Found: 674. 27.
Analysis for C36H49N505SBBr.
Calculated: C = 57.29%, H = 6.56%, N = 9.28%, and B = 1 .43%.
Found: C=57.46%, H=6.45%, N=8.78%, and B=1.38%

Example 28:
Boc-Leu-Gly-Leu-Ala-borolrg-C,OH,6 HBr
Boc-Leu-Gly-Leu-Ala-NH-CH[(CH2)3Br]B02-C,OH,6 (product from Example 15, 0.770
g, 0.980 mmoles) was used to prepared the isothiouronium analog of this example using
the procedure described in Example 22.

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CA 1 333208
66

Following chromatography of the reaction products, the final product (0.400 9) was
obtained as a white solid by trituration with hexane.
MS(FAB) for C37H66N708SB:
Calculated: + H: 780.48.
Found: 780.52.
Analysis for C37H67N508SBrB.
Calculated: C=51.62%, H=7.86%, 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-borolrg-C,OH16 2HBr

Boc-Leu-Gly-Leu-Ala-borolrg-C,OH16HBr (compound of Example 28, 0.100 9, 0.12
mmoles) was dissolved in 1 mL of methanol and 1 mL of 0.7 N hydrogen bromide in
methylene chloride was added. The mixture was stirred for 15 min at room temperature.
Solvent and excess hydrogen bromide were removed by evaporation and the residue was
triturated with ether to yield the desired product in almost quantitative yield.MS(FAB) for C32H58N706SB:
Calculated + H: 680.43.
Found: 680.50.

Example 30
Bz-Glu(OBu)-Gly-borolrg-C10Hl6 HBr
Bz-Glu(OBu)-Gly-NH[(CH2)3Br]B02-C,OH,6 (product from Example 17, 0.293 9,
0.433 mmoles) was used to prepare the isothiouronium analog (0.220 9) using the
procedure described in Example 22.
MS(FAB) for C33H50N507SB:
Calculated + H: 672.36.
Found: 672.3.
Analysis for C33H5,N507SBBr:

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Ch i 333208
67

Calculated: C=52.66%, H=6.84%, N=9.31%, and B=1.44%.
Found: C=52.38%, H=6.76%, N=8.81%, and B=1.46%.
Example 31
Bz-Glu-Gly-borolrg-C,OH,6 HBr

Bz-Glu(OBu)-Gly-borolrg-C,OH,6 HBr (the product of Example 30, 0.050 g, 0.066
mmoles) was dissolved in 1 mL of TFA and stirred for 1 h at room temperature.
Hydrogen bromide in methylene chloride (0.35 mmoles) was added and the liquid of the
resulting solution was evaporated. The residue was triturated with ether to yield 47 mg.
MS(FAB) for C29H42N507SB:
Calculated + H: 616.30.
Found: 616.34.

Example 32
Boc-(D)Phe-Phe-borolrg-C,OH,6 HBr

Boc-(D)Phe-Phe-NH-CH[(CH2)3Br]B02-C,OH,6 (compound from Example 11, 1.50 g,
2.07 mmoles) was used to prepare the isothiouronium analog (0.90 9) using the
procedure described in Example 22.
MS(FAB) for C38H54N506SB:
Calculated + H: 719.84.
Found: 720.
Analysis for C38H55N506SBBr:
Calculated: C = 56.99%, H = 6.94%, N = 8.75%, and B = 1.35%.
Found: C = 55.89%, H = 6.87%, N = 8.59%, and B = 1.18%.

Example 33
H-(D)Phe-Phe-borolrg-C,OH,6 2HBr
Boc-(D)Phe-Phe-borolrg-C~OH,6-HBr (compound of Example 20, 0.20 9, 0.25
mmoles) was reacted with

- 67 -

CA 1 333208
68

hydrogen bromide by the procedure described in Example 29 to yield 188 mg of the
desired product.
MS(FAB) for C33H46N504SB:
Calculated + H: 620.34.
Found: 620.40.

Example 34
Z-Phe-Gly-Gly-borolrg-C,OH,6 HBr
Z-Phe-Gly-Gly-NH-CH[(CH2)3Br]BO2-C,OH,2 was prepared by coupling Z-Phe-Gly-Gly-
OH to the amine (Example 1 a) using the procedure described in Example 2.
Analysis for C35H46N407BBr:
Calculated: C = 57.93%, H = 6.40%, N = 7.72%, and B = 1.49%.
Found: C = 58.42%, H = 6.83%, N = 7.74%, and B = 1.96%.

The alkyl halide (1.00 g, 1.38 mmoles) was converted to the isothiouronium analog
by the method in Example 22 to yield product (0.87 g) as a white amorphous solid.
MS(FAB) for C36H49N607SB:
Calculated + H: 721.36.
Found: 721.32.
Analysis for C36H50N607SBBr:
Calculated: C = 54.00%, H = 6.31 %, N = 10.50%, and B = 1.35%.
Found: C = 53.17%, H = 6.50%, N = 10.03%, and B = 1.25%.

Example 35
Boc-Ala-Phe-(D,L)borolrg-C6H,2 HBr
Boc-Ala-Phe-OMe was prepared using the mixed anhydride procedure described in
Example 2.
Analysis for C18H26N205:
Calculated: C = 61.70%, H = 7.48%, N = 7.99%.
Found: C = 61.51 %, H = 7.56%, N = 7.92%.

- 68 -

C A 1 333208
69

The methyl ester was hydrolyzed with base to yield Boc-Ala-Phe-OH in a yield of
56%. Boc-Ala-Phe-NH-CH[(CH2)3Br]BO2-C6H,2 was prepared by coupling Boc-Ala-Phe-OH
to NH2-CH[(CH2)3Br]BO2-C6H12 HCI (Example 1 b) using the method described in Example
2, except LH-20 chromatography was not used.
Boc-Ala-Phe-NH-CH[(CH2)3Br]BO2-C6H12 (1.00 9, 1.72 mmoles) was reacted with
thiourea using the procedure described in Example 22 to yield the isothiouronium analog
(0.485 9) as a white solid.
MS(FAB) for C28H46N506SB:
Calculated + H: 592.33.
Found: 592.60.
Analysis for C28H47N506SBBr:
Calculated: C = 50.00%, H = 7.06%, N = 10.41 %, and B = 1.61 %.
Found: C = 39.50%, H = 7.24%, N = 10.22%, and B = 1.41 %.

Example 36
H-Ala-Phe-(D,L)borolrg-C6H12-2HBr

Boc-Ala-Phe-borolrg-C6H12-HBr (Example 35, 0.10 9, 0.149 mmoles) was reacted
with hydrogen bromide by the procedure described in Example 29 to yield the desired
product in almost quantitative yield.
MS(FAB) for C23H38N5O6SB:
Calculated + H: 492.28.
Found: 492.26.

Example 37
Boc-Ala-Phe-(D,L)boroHomolrg-C6H12-HBr
Boc-Ala-phe-NH-cH[(cH2)4-s-c(NH)NH2]Bo2-c6H12 HBr
Boc-Ala-PHe-OH (from Example 35) was coupled to the amine (Example 1d) to
yield Boc-Ala-Phe-NH-CH[(CH2)4Br]BO2-C6H12. The procedure in Example 2 was used
except the LH-20 chromatography step was not needed

- 69 -

CA 1 333208

for purification. An analytical sample was obtained by chromatography on silica gel using
ethyl acetate as an eluent.
MS(FAB) for C28H45N306BrB:
Calculated + H: 610.27.
Found: 610.24.
Analysis for C28H45N306BrB.
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 9, 0.883 mmoles) was reacted with thiourea using the
precedure in Example 22. The product (0.23 g) was obtained as an amorphous white
solid after trituration with ether.
MS(FAB) for C29H48N506S:
Calculated + H: 606.35.
Found: 606.38.
Analysis for C29H49N506SBBr.
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)boroHomolrg-C6H,2-2HBr

Boc-Ala-Phe-(D,L)boroHomolrg-C6H,2HBr (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 C24H40N504SB:
Calculated + H: 506.30.
Found: 506.39.

Example 39

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~A I 333208
71

Boc-Ala-phe-(D~L)boroLys-c6Hl2 Hcl
Boc-Ala-Phe-NH-CH[(CH2)4NH2] BO2-C6H,2 benzene sulfonic acid

Boc-Ala-Phe-NH-CH[(CH2)4Br]BO2-C6H,2 (from Example 35) was converted to
the alkyl azide using the procedure in Example 3 except the LH-20 chromatography step
was not needed for purification. The azide was hydrogenated using the method
described in Example 4 except 2 equivalents of benzene sulfonic acid were used and the
hydrogenation time was 2 h to yield the final product in a yield of 40% (mp 154-160,
dec) .
MS(FAB) for C28H46N406B:
Calculated + H: 547.38.
Found: 547.43.

Example 40
H-Ala-Phe-(D, L) boroLys-C6H,2 TFA benzene sulfonic acid

Boc-Ala-Phe-(D,L)boroLys-C6H,2benzene sulfonic acid (compound of Example 39)
was reacted with trifluoroacetic acid for 1 hr at room temperature. Solvent was
evaporated and the residue was triturated with ether to yield a solid.
MS(FAB) for C23H39N404B:
Calculated + H: 447.31.
Found: 447.31.
Anal. for C31H46N4OgSF3B-2H20
Calculated: C=49.34%, H=6.68%, N=7.42%, and B=1.43%.
Found: C=49.26%, H=5.94%, N=7.12%, and B=1.34%.

Example 41
Boc-(D)Val-Leu-boroLys-C6H,2benzene sulfonic acid
Boc-(D)Val-Leu-OH was prepared by the method described in Example 2. The
benzyl ester was obtained

72 CA 1 333208

in a yield of 76%.
MS(FAB) for C23H36N205:
Calculated + H: 421.27.
Found: 421.38.

Follwoing hydrogenation, the free acid was obtained in a yield of 100% as a white
crystalline solid.
Analysis for C,6H29N205:
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 was coupled to the amine (Example 1 d) using the method
described in Example 37 for the coupling of Boc-Ala-Phe-OH to yield Boc-(D)Val-Leu-NH-
CH[(CH2)4Br]BO2-C6H,2 in a yield of 97%.
MS(FAB) for C27H5,N306BBr:
Calculated + H: 604.31.
Fou nd: 604.31.

The alkyl bromide was converted to the corresponding azide in a yield of 85% by
the method described in Example 3, and the azide was hydrogenated by the method
described in Example 39 to yield the final product as a white solid in a yield of 62%.
MS(FAB) for C27H53N406B:
Calculated + H: 541.41.
Found: 541.46.
Analysis C33H59N409SB-1.5 H20:
Calculated: C = 54.62%, H = 8.61 %, N = 7.73%, and B = 1.49%.
Found: C = 54.58%, H = 8.59%, N = 7.92%, and B = 1.98%

ExamPle 42
Ac-Phe-boroLys-C6H12-benzene sulfonic acid

CA 1 333208


Example 42 was prepared according to the procedure described in Example 39.
Ac-Phe-NH-CH[(CH2)4Br]B02-C6H,2 was prepared in a yield of 72%.
MS(FAB) for C22H34N204BBr:
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 C22H37N304B:
Calculated: + H: 418.29.
Found: 418.31.
Analysis for C29H42N3C7SB-H20:
Calculated: C=56.66%, H=7.47%, N=7.08%, and B=1.82%.
Found: C = 56.88%, H = 7.43%, N = 7.22%, and B = 1.53%.

Example 43
Bz-~D,L)borolrg-C6H~2 HBr

Bz-(D,L)NH-CH[(CH2)3Br]B02C-6H12 was prepared by reacting the amine (Example
1 b, 5.0 9, 15.9 mmoles) with an equivalent of benzoyl chloride and two equivalents of
sodium bicarbonate in a mixture consisting of 4 mL of dioxane and 4 mL of water at 0.
After initially mixing the reagents, the reaction was diluted with 6 mL of 50%
dioxane:water and it was allowed to warm to room temperature. The reaction mixture
was stirred approximately 30 min at room temperature and then the product was
extracted into ethyl acetate and washed with water, 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 evaporated to yield a crystalline
product. After isolation and washing with ethyl acetate, 3.26 9 of compound (mp

CA i 333208
74

176-177) were obtained.
Analysis for C'7H25N03BrB:
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%.

The alkyl halide (1.00 9, 2.62 mmoles) was converted to corresponding
isothiouronium salt by the procedure described in Example 22. The product, 0.84 9, was
obtained as a white solid.
MS(FAB) for C18H28N303SB:
Calculated + H: 378.20.
Found: 378.21.
Analysis for C'8H29N303SBBr:
Calculated: C = 47.18%, H = 6.38%, N = 9.17%, and B = 2.36%.
Found: C=46.11%, H=6.71%, N=8.97%, and B=2.22%.

Example 44
Bz-(D,L)boroArg-C6H12benzene sulfonic acid
The alkyl halide (Example 43, 2.0 9, 5.25 mmoles) was converted to 0.97 9 of theazide (mp 138-139) using the procedure in Example 3. The azide was converted to Bz-
boroOrn-C6H12 benzene sulfonic acid in almost quantitative yield using the procedure in
Example 4.
MS(FAB) for C18H27N203B:
Calculated + H: 319.22.
Found: 319.26.
Bz-boroOrn-C6H12 benzene sulfonic acid (0.90 9, 1.84mmoles) was allowed to
react with cyanamide using the procedure in Example 5 to yield 0.65 9 of crystalline
product (mp 242-244).
FAB(MS) for C18H29N403B:
Calculated + H: 361.24.
Found: 361.24.

- 74 -

~ A 1 333208

Analysis for C24H35N406SB:
Calculated: C = 55.59%, H = 6.82%, N = 10.81 %, and B = 2.08%.
Found: C=54.60%, H=6.70%, N=11.24%, and B=1.87%.

Example 45
Ac-Leu-Thr(OBu)-boroArg-C,OH,6-benzene sulfonic acid

Ac-Leu-Thr(OBu)-OH was prepared by coupling Ac-Leu-OSu to H-Thr(OBu)-OH
using the procedure in Example 13 for dipeptide synthesis except the final product was
obtained as an amorphous white solid after chromatography on LH-20. Ac-Leu-Thr(OBu)-
OH (3.29 9, 9.90 mmoles) was coupled to the amine (Example 1 a) using the mixed
anhydride procedure in Example 2 except the LH-20 chromatography step was not
needed .
Ac-Leu-Thr(OBu)-NH-CH[(CH2)3Br]BO2-C10H16 was obtained as an amorphous white solid,
5.39 9. The alkyl halide was converted to the corresponding azide in a yield of 82%
using the procedure in Example 3 except a chromatography step was needed for further
purification. The azide (3.88 9, 6.42 mmoles) was hydrogenated by the procedure in
Example 4. The product,
Ac-Leu-Thr(OBu)-boroOrn-C,OH,6benzene sulfonic acid, was obtained in a yield of 74%
after chromatography of the product on LH-20 and trituration with ether.
MS(FAB) for C30H55N406B:
Calculated + H: 579.43.
Found: 579.48.

The boroOrnithine peptide was converted to the final product in a yield of 86% by
the procedure in Example 5.
MS(FAB) for C3,H57N606B:
Calculated + H: 621.45.
Found: 621.50.
Analysis for C37H63N6SOgB

- 75 -

CAl 333208
76

Calculated: C = 57.05%, H = 8.17%, N = 10.79%, B = 1.39%.
Found: C = 56.47%, H = 8.01 %, N = 10.93%, and B = 1.34%.

Example 46
Ac-Leu-Thr-boroArg-C1OH~6benzene sulfonic acid

Ac-Leu-Thr(OBu)-boroArg-C1OH,6benzene sulfonic acid (Example 45, 0.200 9,
0.257 mmoles) was dissolved in a mixture of 2 mL of methylene chloride and 2 mL of 4
N HCl:dioxane and was allowed to stir for 30 min at room temperature. Solvent was
evaporated and the residue was dried under high vacuum. The desired product was
obtained as a white solid in a yield of 97% by triturating with ether.
MS(FAB) for C27H49N606B:
Calculated + H: 565.39.
Found: 565.48.

Example 47:
Ac-Lys(Boc)-Pro-boroArg-C,OH,6benzene sulfonic acid

Ac-Lys(Boc)-Pro-OH was prepared by the methods described in Example 13. It
was obtained as a white solid (mp 160-161.5) after crystallization from ethyl acetate.
Ac-Lys(Boc)-Pro-OH (3.15 g, 8.18 mmoles) was coupled to the amine (Example 1 a) using
the procedure in Example 2. The product, 5.8 g, was used without further purification.
It was converted to the azide in a yield of 73% by the method in Example 3 afterchromatography on LH-20. Hydrogenation by the method in Example 4, chromatography
on LH-20, and trituration of the sample with ether gave Ac-Lys(Boc)-Pro-boroOrn-C,OH,6 benzene sulfonic acid in a yield of 81 %.
MS(FAB) for C32H55N507B:
Calculated + H: 634.43.
Found: 634.46.

CAl ~33208


The boroOrnithine peptide (2.0 9, 2.53 mmoles) reacted with cyanamide by the
procedure in Example 5 to yield 1.8 9 of the desired product as a white solid.
MS(FAB) for C33H57N707B:
Calculated + H: 676.46.
Found: 676.41.
Analysis for C39H63N70,0BS:
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-C,OH16 2HCI

Ac-Lys~Boc)-Pro-boroArg-C,OH,6benzene sulfonic acid ~Example 47, 0.30 9, 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 C28H49H705B:
Calculated + H: 576.40.
Found: 576.45.

Example 49
Ac-Ala-Glu~OBu)-boroArg-C,OH,6-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).
Analysis for C,4H24N206:
Calculated: C = 53.14%, H = 7.66%, and N = 8.85%.

CA I 333208

78

Found: C = 53.28%, H = 7.53%, and N = 9.08%.
Ac-Ala-Glu(OBu)-NH-CH[(CH2)3Br]BO2-C,OH,6 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 C28H47N607B:
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 C28H49N407B:
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 C29H51N607B
Calculated + H: 607.40.
Found: 607.41.
Analysis for C35H57N6010BS:
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-C1OH,6benzene sulfonic acid


- 78 -

Cb, 33320~
79

Ac-Ala-Glu(Bu)-boroArg-C1OH,6-benzene sulfonic acid (Example 49, 0.10 g, 0.131
mmoles) was dissolved in 10 mL of acetic acid and anhydrous HCI was bubbled through
the solution for 20 min. The solution was stirred at room temperature for 1.5 h and
solvent was evaporated to yield an oil. The desired product was obtained as a white
solid (82 mg) after drying in vacuo and trituration with ether.
MS(FAB) for C25H43N607B:
Calculated + H: 551.34.
Found: 551.41.

The following compounds were also prepared using substantially the same
procedures as in Examples 39 and 40 above:
Boc-Val-Val-boro Lys-C6H 12-BSA;
H-Val-Val-boro Lys-C6H 12-BSA TFA;
Boc-(D)Phe-Phe-boroLys-C6Hl2-BSA;
H-(D)Phe-Phe-boroLys-C6H12 BSATFA
Boc-Glu-Phe-boroLys-C6Hl2 BSA
PyroGlu-Phe-boroLys-C6Hl2 BSA

Biological Examples
In the following examples, ,u denotes micro.

Examples 51 - 71
Inhibition of Human Plasma Kallikrein

Human plasma kallikrein was obtained from Protogen AG (Switzerland). The
specific activity as described by the supplier is 15 units per mg. A unit is defined as the
quantity of enzyme required to hydrolyze 1 ,umole 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


- 79 -

CA I 333208


A stock solution of enzyme (1 unit/mL) was prepared in 50% glycerol-0.10M
sodium phosphate buffer, pH 7.5, containing 0.20 M sodium chloride and 0.1% PEG
6000 (polyethylene glycol). In standard assays, 10 ,uL of the stock kallikrein solution
were added to 990,uL of a solution consisting of 0.20 mM S2302 in 0.10 mM sodiumphosphate buffer, pH 7.5, containing 0.20 M sodium chloride and 0.1% PEG at 25.The effect of inhibitors were evaluated by monitoring enzymatic activity determined by
measuring the increase in absorbance at 405 nm with time both in the presence and
absence of inhibitors. Table 1 shows inhibitor levels and the activity remaining measured
in the time interval from 10 to 20 min following initiation of the reaction. Activity of the
controls were 0.0092 + 0.0095 min~'.

Table 1
Inhibition of Human Plasma Kallikrein

Conc. Percent
Ex Inhibitor (nM) Activity

51 Boc-(D)Phe-Phe-borolrg-C10H,6HBr 10 2
52 H-(D)Phe-Phe-boroArg-C,0H~62HCI 10 2.6
53 Boc-(D)Phe-Phe-boroArg-C,0H,6-BSA 10 5.2
54 Boc-Ala-Phe-(D,L)borolrg-C6H,2-HBr 10 15
Bz-Pro-Phe-boroArg-C,0H,6 BSA 10 15
56 Bz-Pro-Phe-boroArg-OH HCI 10 16
57 Bz-Pro-Phe-boroArg-F 10 18
58 Boc-Leu-Gly-Leu-Ala-boroArg-C~0H~6 BSA 10 30
59 Ac-Ala-Lys(Boc)-boroArg-C,0H,6 BSA 10 34
Ac-Phe-boroArg-C,0H~6 HCI 10 48
61 Ac-(D)-Phe-Pro-boroArg-C,0H,6-HCI 10 56


- 80 -

C~ ~ ~33208
81

(Table 1 Continued)
Conc. Percent
Ex Inhibitor (nM) Activity

62 H-(D)-Phe-Pro-boroArg-C10H,62 HCI 10 61
63 Ac-Ala-Lys(Boc)-borolrg-C10H16 HBr 50 1.4
64 Bz-Glu(OBu)-Gly-boroArg-C10H,6 BSA 50 1 1
Ac-Phe-borolrg-C10H16 HBr 50 17
66 Bz-Glu-Gly-boroArg-C,OH16 BSA 50 39
67 Ac-Ala-Lys-boroArg-C,OH16-2HCI 50 39
68 Boc-Ala-Phe-(D,L)borohomolrg-C6H,2-HBr 100 38
69 Boc-Ala-Phe-(D,L)boroLys-C6H12 HCI 1000 17
Boc-(D)Phe-Phe-boroOrn-C,OH16 BSA 10000 39
71 Boc-(D)Phe-Pro-boroOrn-C10H16BSA 10000 100

Examples 72 - 1 10
Inhibition of Thrombin (Esterase Activity)

Human thrombin (specific activity 2345 NIH units/mg) was obtained from R.Q.P.
Laboratories, South Bend, IN) (Lot HT102). A stock solution of thrombin was prepared in
0.010M PIPES Buffer, pH 6.0, containing 0.75 M sodium chloride. Assays of thrombin
were run according to the procedure of Green and Shaw, Anal. Biochem., 93: 223
(1979), in sodium phosphate buffer, pH 7.5, containing 0.20 M sodium chloride and
0.1% PEG 6000. The initial concentration of substrate was 0.10 mM and the
concentration 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~'.

C~ i 333208
82

Table 2
Inhibition of Thrombin
Conc. Percent
Ex. Inhibitor (nM) Activity

72 Ac-(D)Phe-Pro-boroArg-C,OH,6 HCI 5
73 Boc-(D)Phe-Pro-borolrg-C,OH,6-HBr 5 3
74 Boc-(D)Phe-Pro-boroArg-C,OH16 BSA 5 3
Ac-(D)Phe-Pro-boroArg-OH-HCI 5 3
76 H-(D)Phe-Pro-borolrg-C,OH,6 HBr HCI 5 4
77 H-(D)Phe-Pro-boroArg-C10H16 2HCI 5 7
78 Boc-(D)Phe-Phe-Borolrg-C10H16Br 5 48
79 H-(D)Phe-Phe-boroArg-C,OH,6 2HCI 1 0 1 0
H-Leu-Gly-Leu-Ala-boroArg-C10H16HCl BSA 10 25
81 Boc-(D)Phe-Phe-boroArg-C,OH16-BSA 1 0 32
82 H-Leu-Gly-Leu-Ala-borolrg-C10H16 2HBr 1 0 37
83 Boc-Leu-Gly-Leu-Ala-boroArg-C10H16 BSA 1 0 38
84 H-(D)Phe-Phe-borolrg-C10H,62HBr 10 49
Bz-Glu(OBu)-Gly-boroArg-C,OH,6-BSA 1 0 52
86 Bz-Glu(OBu-Gly-borolrg-C10H16 HBr 1 0 59
87 Boc-Leu-Gly-Leu-Ala-borolrg-C,OH,6 HBr 1 0 66
88 Boc-(D)Phe-Pro-boroOrn-C,OH16 BSA 1 00 1 8
89 Ac-Ala-Lys(Boc)-boroArg-C,OH,6-BSA 1 00 1 8
Z-Phe-Gly-Gly-borolrg-C,OH,6 HBr 1 00 46
91 Bz-Glu-Gly-boroArg-C~OH16-BSA 1 00 46
92 Ac-Ala-Lys(Boc)-borolrg-C,OH16 HBr 1 00 55
93 Bz-Pro-Phe-boroArg-OH HCI 1 000 1 8
94 Bz-Pro-Phe-boroArg-F 1 000 1 8
Bz-Pro-Phe-borolrg-C10H16 HBr 1 000 21
96 Boc-(D)Val-Leu-boroLys-C6H,2 HCI 1 000 21
97 Bz-Pro-Phe-boroArg-C,OH,6 BSA 1 000 24
98 Boc-Leu-Gly-Leu-Ala-boroOrn-C10H16 BSA 1 000 24
99 Boc-Ala-Phe-(D,L)borolrg-C6H12-HBr 1 000 28
1 00 Bz-Glu-Gly-borolrg-C10H16 HBr 1 000 39
1 01 Ac-Ala-Lys-boroArg-c1oH16 2HCI 1 000 45

- 82 -

., A 1 s33208
83

(Table 2 Continued)
Conc. Percent
Ex. Inhibitor (nM) Activity

102 Ac-Phe-boroArg-C,OH,6 HCI 1000 53
103 Ac-Phe-borolrg-C,OH,6 HBr 1000 64
104 Ac-Ala-Lys-borolrg-C1OH,6 2HBr 1000 68
105 H-Ala-Phe-(D,L)borolrg-C6H,2 2HBr 10000 23
106 Boc-Ala-Phe-(D,L)borohomolrg-C6H,2-HBr 10000 32
107 Boc-Ala-Phe-(D,L)boroLys-C6H,2 HCI 10000 46
108 H-Ala-Phe-(D,L)boroHomolrg-C6H,2-2HBr 10000 47
109 H-Ala-Phe-D,L)boroLys-C6H~2 2HCI 10000 89
1 10 Ac-Phe-boroLys-C6H12-HCI 10000 97

Examples 1 1 1- 1 24
Inhibition of Blood Coagulation As Shown by APTT and PT Determination

The effect of protease inhibitors on blood coagulation In vitro was determined by
measuring their effects on two different clinical parameters, the activated partial
thromboplastin times (APTT) and prothrombin times (PT). Reagents for each of these
assays were supplied by General Diagnostics, Jessup MD. Stock solutions of inhibitors
were prepared in 25 mM HEPES buffer, pH 7.5, containing 0.10 M sodium chloride. For
the APTT assay, the inhibitor solution (0.100 mL) was incubated with normal human
plasma (0.100 mL) and automated APTT reagent (0.100 mL). After incubation for 5.0
min at 37, calcium chloride (0.100 mL) was added and the clotting times, measured in
seconds, was determined on a fibrameter. The effects of the varying concentrations of
inhibitor on blood clotting times compared with the clotting times of controls run in the
absence of inhibitor, are shown in Table 3.
For PT assays, inhibitor solutions (0.100 mL)


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84 CA 1333208

were incubated with normal human plasma (0.100 mL) for 2 min at 37. Simplastin
reagent (0.200 mL) was then added and clotting times measured as shown in Table 4.
Table 5 provides a summary of the results in Tables 3 and 4, showing the
approximate concentrations of inhibitor required to increase the Activated Partial
Thromboplastin Times (2X APTT), and the Prothrombin Times (2XPT) two fold.

Compound
Designation In
Tables 3-5, 7-10 Name of Inhibitor
A Boc-(D)Phe-Pro-boroArg-C,OH,6
B H-(D)Phe-Pro-boroArg-C,OH,6
C Boc-(D)Phe-Pro-borolrg-ClOH16
D Boc-(D)Phe-Phe-Borolrg-C10H16
E Boc-(D)Phe-Phe-boroArg-C,OH16
F Ac-Phe-boroArg-C10H16
G Ac-Phe-borolrg-C,OH16

Table 3
Activated Partial Thromboplastin Times
(measured in seconds)

Example Number
111 112113 114 115 116 117
Conc. Compound
(nM) A B C D E F G
0 35.3 32 32.8 34.7 34.7 33.7 33.8
35.8 36.8 35 35.2
62.5 36.5 39.2
125 36.6 44.2 46.9 45.2 40.5 35.2 36.2
200 40.2 71.5 67 46.2 43.6 36.7 39.3
250 81.2 158.7 160
275 60.7 52.8 44.8 58.2
300 113.3 169.7 197.8
350 128.7 249.7 301.8 75.7 54.8 58.2 66.7
550 94.8 61.3 144.2 98.4

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Table 4
Prothrombin Times
(measured in seconds)
Example Number
118 119120 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
Inhibition of Blood Coagulation

Calc. Concentration
Compound 2X APTT(nM) 2XPT(nM)
A 230 800
B 170 460
C 200 650
D 370 1200
E 375 2600
F 325 2500
G 625 1200
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86

ExamPles 125- 127

Inhibition of Blood Coagulation as Shown by TT Determinations

The effect of the protease inhibitor Ac-(D)Phe-Pro-boroArg-OH (Example 8) on
blood coagulation in vitro was determined by measuring its effect on thrombin times (TT).
A mixture of 0.2 ml of normal rabbit plasma and 0.05 ml of buffer containing the inhibitor
at 6 times the desired final concentration was warmed to 37C. Clotting was initiated
by addition of thrombin (0.05 ml at 6 times the final concentration). The thrombin used
was purchased from Sigma Chemical Company (No. T-6634, activity 1190 NIH units per
mg protein) and prepared in buffer. The buffer employed for both the inhibitor and the
thrombin was 0.1 M Tris buffer (12.10 g/L), containing 0.154 M NaCI (8.84 g/L) and 2.5
mg/ml bovine serum albumin, pH 7.4. The clotting times, measured in seconds, were
determined using a fibrometer. The effects of the inhibitor on blood clotting times
compared with the blood clotting times of controls run in the absence of inhibitors, are
shown in Table 6. Values represent the average of at least three determinations. If
clotting did not occur within 300 seconds, the reaction was terminated.

Table 6
Thrombin Times

Ex. Thrombin Conc.Inhibitor Conc. Thrombin Times
(Il/ml) (nM)
--- 0.75 0 > 300
--- 0.83 0 226.9 + 14.8




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87

--- 1 0 147.2 + 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 50 32.7 + 0.8
the mean time needed for clotting, measured in seconds, + the standard deviation Examples 128 - 132
Stability of Inhibitors in Human Plasma As Measured by APTT
The stability of inhibitors in plasma was determined by their ability to inhibit blood
coagulation. First, a stock solutions (1.0,uM) 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 mixtures were made at 0, then aliquots (0.200 mL) were removed
and 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 times
(shown in hours) and clotting time (measured in seconds) for individual inhibitors are
shown in Table 7. Values for compounds E and F were determined simultaneously with
the Control. Values for compounds A, B and C were obtained on a different day.

88 CA 1 333208

Table 7
Stability of Inhibitors in Human Plasma

Example Number
128 129 130 131 132
Compound
Control F E A B C
Incubation
Time (h) Clotting Time (sec)

0 41.5 76.3 63.2 81.2 152.2 203.2
0.5 42.7 76.4 73.2 84.7 157.7 207.2
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 57.3
24 35.2 73.9 64.7 92.2 109.3 248.7
48 47.2 49.3 58.7

Examples 133-136
Stability of Inhibitors in Buffer
Inhibitors, each at a concentration of 1.0,uM, were incubated at room temperature
in 0.20 M sodium phosphate buffer, pH 7.5, containing 0.20 M sodium chloride and
0.10% PEG. Aliquots (4.0,uL) were removed and assayed in the thrombin assay as
described in Examples 72-110. The percent of thrombin activity remaining 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 loss of inhibitor activity.
Inhibitor B loses its biological activity over a period of an hour.

CA I 333208
89

Table 8
Stability of Inhibitors in Buffer

Percent Thrombin activity
Example No. Compound 0 hr 6.5 hr 24 hr
133 A 3.3 1.6 0.8
134 C 3.0 0.9
135 C 65 77

0 hr 0.5 hr 1 hr
136 B 2.0 15 100

Examples 137- 142
Inhibition of Blood Coagulation Following In Vivo Oral Dosing
Male rats (Sprague Dawley CD Rats, 130-140 g, supplied by Charles River Labs,
Inc., Wilmington, MA) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). A
midline incision was made on the ventral surface of the neck, and a polyethylene catheter
was inserted in one of the carotid arteries and exteriorized at the back of the neck. After
recovery from anesthesia, control blood samples were taken from the carotid artery
catheter, anticoagulated with sodium citrate, and centrifuged (2000 x 9, 10 minutes).
Plasma was transferred to plastic tubes and kept on ice until it was assayed. Thrombin
times were measured using a fibrometer, as described in Examples 125-127.
Rats were given either the protease inhibitor Ac-(D)Phe-Pro-boroArg-OH in a
vehicle, or the vehicle alone, by oral gavage in a volume of less than 4 ml. The vehicle
employed was 5% dimethylsulfoxide in saline.




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Blood samples were taken at various times after oral dosing and assayed as described
above. The results, shown in clotting times in seconds, are given in Table 9, below.
When clotting times exceeded 300 seconds, it is reported below as > 300. The
remaining data show the mean time needed for clotting, measured in seconds, + the
standard deviation.

Table 9
Inhibition of Blood Coagulation
Following In Vivo Oral Dosing
Ex. Time Control Inhibitor Concentration
(hr) 1 mg 2 mg 10 mg
137 .5 68 + 18 > 300 > 300 > 300
138 1 52 + 26 > 300 ND > 300
139 2 55 + 11 > 300 ND > 300
140 3 34 + 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 Blood Coagulation Following Oral Dosing
To further demonstrate the ability of this compound to inhibit blood coagulation in
vivo, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), a jugular vein
catheter was inserted, and the incision was closed. 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 units/kg




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thrombin over a period of one minute. All fourteen rats given only water died within ten
minutes of the thrombin infusion. In contrast only 8 out of 17 rats treated with the
inhibitor-containing water died within ten minutes, and the remainder survived one hour,
at which time they were euthanized.

Examples 144- 162
In Vivo Inhibition of Blood Coagulation Following Oral, Colonic and Rectal Administration

General Procedures:
Male Lewis rats weighing between 300-350 9 were anesthetized with sodium
pentobarbitol (50 mg/kg, i.p.) and the jugular vein was cannulated using a silastic tubing
attached to a polyethylene 50 tubing. The tubing was exteriorized at the back of the
neck and attached to a syringe through a stop cock. Blood samples (0.5 ml) were
withdrawn before and at different time intervals after dosing with the protease inhibitor
Ac-(D)Phe-Pro-boroArg-OH, into the syringe that was flushed with citrate buffer prior to
each collection. The blood samples were then transferred into vacutainers containing
citrate buffer. Also, after each collection the cannula was flushed with saline. The blood
samples were then centrifuged immediately (2500 rpm for 15 min) and 0.2 ml of the
plasma samples were used for clotting time measurements. The clotting time
measurements were carried out using a fibrometer as follows. First, plasma (0.2 ml) was
placed in a fibro cup, and pH 7.4 Tris buffer (50 microliters) was added. The plasma
buffer solution was incubated at 37C for 1 min, 50 microliters of a 24,u/ml thrombin
solution in Tris buffer was then added, and clotting time in seconds was measured.
When clotting time exceeded 300 seconds, it is reported below as >300.




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92

Oral dosing:
The jugular vein-cannulated rats were allowed to recover from anesthesia before
they were dosed orally. The protease inhibitor Ac-(D)Phe-Pro-boroArg-OH aqueous
solution, consisting of 3 mg of inhibitor per kg weight of rat (approximately 1 mg/rat) in a
volume of 0.75 ml of water per kg of rat, was administered by gavage. The results are
reported in Table 10, below.

Colonic administration:
A 3 cm incision was made in the abdomen of the jugular vein-cannulated rats while
they were still under anesthesia. The colon was located and was tied off at both the
beginning and the end. The protease inhibitor Ac-(D)Phe-Pro-boroArg-OH aqueous
solution, consisting of 3 mg of inhibitor per kg weight of rat (approximately 1 mg/rat) in a
volume of 1 ml of water per kg weight of rat, was injected at the beginning into the
colon cavity. The incision was closed using wound clips. The results are reported in
Table 11, below.

Rectal administration:
The procedure for rectal administration in the jugular vein-cannulated rats was as
described in Kamiya et al., J. Pharm. Sci., 71: 621 (1982). In brief, a device was made
consisting of a 0.89 cm and a 0.71 cm silicon rubber septa connected by a 2 cm length
of wire. This 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 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.




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93

Table 10
In Vivo Inhibition of
Blood Coagulation Following Oral Administration
Ex. Time(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
149 3.00 58.4 > 300
150 4.00 42.7 > 300
data represents the average for 2 rats
data represents the mean for 3 rats

Table 11
In Vivo Inhibition of
Blood Coagulation Following Colonic Administration
Ex. Time(hr) Control Inhibitor
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 represents the mean for 3 rats




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94

Table 12
In Vivo Inhibition of
Blood Coagulation Following Rectal Administration
Ex. Time(hr) Control Inhibitor
157 0 53.9 66.4
158 0.25 52.3 > 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 represents 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, a known
inflammatory agent, in an acetone carrier (Croton Solution) and the second consisting of
5% croton oil in an acetone carrier to which 10 mg/mL of a compound of the invention
was added (Compound Solution). The Croton Solution (10,uL), or alternatively theCompound Solution (10,~rL), was applied to the right ear of each animal (Sprague Dawley
CD Rats, 130-140 g, supplied by Charles River Labs, Inc., Wilmington, MA). The
acetone carrier alone (Acetone Solution) (10,uL) was applied to the left ear of each
animal. At 1 h following treatment, the animals were sacrificed, their ears removed and
1 /4 inch diameter disks 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




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known non-steroid anti-inflammatory (Indomethacin Solution), which was prepared and
applied in a manner substantially identical to the Compound Solution. Mean data are
shown in Table 13 for Compound F, Ac-Phe-boroArg-C,OH16. The term "dose" as used
below, indicates the amount of active anti-inflammatory ingredient in ,ug (Compounds A,
C, D, E, F or G, or Indomethacin, as the case may be) in the solution applied to each right
ear, and "n" indicates the number of rats used in each test. "SE" denotes standard error.
Examples 164-168 in Table 14 show the anti-inflammatory activity for Compounds A, C,
D, E, F and G which were run under essentially the same conditions (dose = 100,ug).
Table 13
Inhibition of Croton Oil Induced Inflammation
Example 163

Dose Mean Mean Mean
Right ear Right ear Left 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 8
Indometh. 100 20.6 15.6 5.0 + 2.8 55 8
Cmpd. F 100 18.9 16.6 2.3 + 0.7 79 8

Table 14
Inhibition of Croton Oil Induced Inflammation
Example No. Compound Percent Inhibition
164 G 69
165 E 82
166 D 93
167 A 59
168 C 76
This patent application is a division of Canadian Application No. 568224 filed 1988-05-
31.

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