.alpha.-ketoamide derivatives as inhibitors of thrombosis

a-Ketoamide derivatives, their pharmaceutically acceptable salts, compositions, diagnostic compositions and pharmaceutical compositions, which are useful for preventing or treating in a mammal a pathological condition characterized by thrombosis are described. a-Ketoamide derivatives, their pharmaceutically acceptable salts, compositions and diagnostic compositions, which are useful for in vivo imaging of thrombi in a mammal are also described. Methods of preventing or treating in a mammal a pathological condition characterized by thrombosis and methods of in vivo imaging of thrombi in a mammal are also disclosed.

FIELD OF INVENTION 
In one aspect, the present invention relates to novel compounds, their 
pharmaceutically acceptable salts, compositions and pharmaceutical 
compositions, which are useful for preventing or treating in a mammal a 
pathological condition characterized by thrombosis. Another aspect of the 
present invention is directed to novel compounds, their pharmaceutically 
acceptable salts, therapeutic compositions and diagnostic compositions 
which are useful for in vivo imaging of thrombi in a mammal. In yet 
another aspect, the present invention relates to methods of preventing, 
treating or diagnosing in a mammal a pathological condition characterized 
by thrombosis and methods of in vivo imaging of thrombin in a mammal. 
BACKGROUND OF INVENTION 
Normal hemostasis is the result of a complex balance between the processes 
of clot initiation and formation (blood coagulation) and clot dissolution 
(fibrinolysis). The complex interactions between blood cells, specific 
plasma proteins and the vascular surface, maintain the fluidity of blood 
unless injury and blood loss occur. 
Blood coagulation is the culmination of a series of amplified reactions in 
which several specific zymogens of serine proteases in plasma are 
activated by limited proteolysis. Nemerson, Y. and Nossel, H. L., Ann. 
Rev. Med., 33: 479 (1982). This series of reactions results in the 
formation of an insoluble fibrin matrix which is required for the 
stabilization of the primary hemostatic plug. The interaction and 
propagation of the activation reactions occurs through the extrinsic and 
intrinsic pathways of coagulation. 
The serine protease, thrombin, is the primary mediator of thrombus 
formation. Thrombin acts directly to cause formation of insoluble fibrin 
from circulating fibrinogen. In addition, thrombin activates the zymogen 
factor XIII to the active transglutaminase factor XIIIa which acts to 
covalently stabilize the growing thrombus by crosslinking the fibrin 
strands. Lorand, L. and Konishi, K., Arch. Biochem. Biophys., 105: 58 
(1964). Beyond its direct role in the formation and stabilization of 
fibrin rich clots, the enzyme has profound bioregulatory effects on a 
number of cellular components within the vasculature and blood. Shuman, M. 
A., Ann. NY Acad. Sci., 405: 349 (1986). 
It is believed that thrombin is the most potent agonist of platelet 
activation, and it has been demonstrated to be the primary 
pathophysiologic-mediator of platelet-dependent arterial thrombus 
formation. Edit, J. F. et al., J. Clin. Invest., 84: 18 (1989). 
Thrombin-mediated platelet activation leads to ligand-induced 
inter-platelet aggregation principally due to the bivalent interactions 
between adhesive ligands such as fibrinogen with the platelet integrin 
receptor glycoprotein IIb/IIIa which assume their active conformation 
following thrombin activation of the cell. Berndt, M. C. and Phillips, D. 
R., Platelets in Biology and Pathology, pp 43-74, Elsevier/North Holland 
Biomedical Press (Gordon, J. L. edit. 1981). Thrombin-activated platelets 
can more effectively support additional thrombin production through the 
assembly of new prothrombinase (factor Xa and Factor Va) and tenase 
(factor IXa and factor VIIIa) catalytic complexes on the membrane surface 
of intact activated platelets and platelet-derived microparticles, 
following thrombin-mediated activation of the non-enzymatic cofactors V 
and VIII, respectively. Tans, G. et al., Blood, 77: 2641 (1991). This 
positive feedback process results in the local generation of high 
concentrations of thrombin within the vicinity of the thrombus which 
supports further thrombus growth and extension. Mann, K. G. et al., Blood, 
75: 1 (1990). 
In contrast to its prothrombotic effects, thrombin has been shown to 
influence other aspects of hemostasis. These include its effect as an 
important physiological anticoagulant. The anticoagulant effect of 
thrombin is expressed following binding of thrombin to the endothelial 
cell membrane glycoprotein, thrombomodulin. This is thought to result in 
an alteration of the substrate specificity of thrombin thereby allowing it 
to recognize and protolytically activate the circulating zymogen, protein 
C, to give activated protein C (aPC). Musci, G. et al., Biochemistry, 27: 
769 (1988). The activation of protein C by thrombin in the absence of 
thrombomodulin is poor. 
Thrombin has also been shown to be a potent direct mitogen for a number of 
cell types, including cells of mesenchymal origin such as vascular smooth 
muscle cells. Chen, L. B. and Buchanan, J. M., Proc. Natl. Acad. Sci. USA, 
72: 131 (1975). The direct interaction of thrombin with vascular smooth 
muscle also results in vasoconstriction. Walz, D. A. et al., Proc. Soc. 
Expl. Biol. Med., 180: 518 (1985). Thrombin acts as a direct secretagogue 
inducing the release of a number of bioactive substances from vascular 
endothelial cells including tissue plasminogen activator. Levin, E. G. et 
al., Thromb. Haemost., 56: 115 (1986). In addition to these direct effects 
on vascular cells, the enzyme can indirectly elicit proliferation of 
vascular smooth muscle cells by the release of several potent growth 
factors (e.g. platelet-derived growth factor and epidermal growth factor) 
from platelet a-granules following thrombin-induced activation. Ross, R., 
N. Engl. J. Med., 314: 408 (1986). 
Many significant disease states are related to abnormal hemostasis. With 
respect to the coronary arterial vasculature, local thrombus formation due 
to the rupture of an established atherosclerotic plaque is the major cause 
of acute myocardial infarction and unstable angina. Moreover, treatment of 
an occlusive coronary thrombus by either thrombolytic therapy or 
percutaneous transluminal coronary angioplasty (PTCA) is often accompanied 
by an acute thrombotic reclosure of the affected vessel which requires 
immediate resolution. With respect to the venous vasculature, a high 
percentage of patients undergoing major surgery in the lower extremities 
or the abdominal area suffer from thrombus formation in this vascular bed 
which can result in reduced blood flow to the affected extremity and a 
predisposition to pulmonary embolism with high risk of mortality. 
Disseminated intravascular coagulopathy is commonly associated with septic 
shock, certain viral infections and cancer and is characterized by the 
rapid consumption of coagulation factors and disseminated vascular 
microthrombosis which may result in leukocyte activation, inflammation and 
organ failure. 
Arterial thrombosis is a major clinical cause of morbidity and mortality. 
It is the primary cause of acute myocardial infarction which is one of the 
leading causes of death in the Western world. Arterial rethrombosis also 
remains one of the primary causes of failure following enzymatic or 
mechanical recanalization of occluded coronary vessels using thrombolytic 
agents or percutaneous transluminal coronary angioplasty (PTCA), 
respectively. Ross, A. M., Thrombosis in Cardiovascular Disorder, p. 327, 
W. B. Saunders Co. (Fuster, V. and Verstraete, M. edit. 1991); Califf, R. 
M. and Willerson, J. T., Id. at p 389. In contrast to thrombotic events in 
the venous vasculature, arterial thrombosis is the result of a complex 
interaction between fibrin formation resulting from the blood coagulation 
cascade and cellular components, notably platelets, which make up a large 
percentage of arterial thrombi. There is currently no clinically approved 
effective therapy for the treatment or prevention of acute arterial 
thrombosis or rethrombosis since heparin, the most widely used clinical 
anticoagulant administered i.v., has not been shown to be universally 
effective in this setting. Prins, M. H. and Hirsh, J., J. Am. Coll. 
Cardiol., 67: 3A (1991). 
Besides the unpredictable, recurrent thrombotic reocclusion which 
frequently occurs following PTCA, a profound restenosis of the recanalized 
vessel occurs in 30 to 40% of patients 1 to 6 months following this 
procedure. Califf, R. M. et al., J. Am. Coll. Cardiol., 17: 2B (1991). 
Many of these patients require further treatment with either a repeat. 
PTCA or coronary artery bypass surgery to relieve the newly formed 
stenosis which results in restriction of blood supply to the myocardium. 
Restenosis of a mechanically damaged vessel is not the direct result of a 
thrombotic process but instead is the result of a proliferative response 
of the vascular smooth muscle cells constituting the wall of the artery. 
Over time this results in a decreased luminal diameter of the affected 
vessel and decreased blood flow due to increased cellular and pericellular 
mass. Id. As for arterial thrombosis, there is currently no effective 
pharmacologic treatment for the prevention of vascular restenosis 
following mechanical recanalization. 
The need for safe and effective therapeutic anticoagulants has in one 
aspect focused on the role of thrombin as the final enzyme in the process 
of blood coagulation. 
As previously mentioned, recurrent arterial thrombosis remains one of the 
leading causes of failure following enzymatic or mechanical recanalization 
of occluded Coronary vessels using thrombolytic agents or percutaneous 
transluminal coronary angioplasty (PTCA), respectively. After lysis of a 
clot by enzymatic means, residual thrombi may be responsible for 
reocclusion of the recanalized coronary artery via increased thrombus 
growth. Gash, A. K. et al., Am. J. Cardiol., 57: 175 (1986); Shaer, D. H. 
et al., Circulation, 76: 57 (1984). Mechanical recanalization by coronary 
angioplasty may not prevent reocclusion, and in the presence of a residual 
thrombus, may precipitate acute reocclusion, requiring bypass surgery. 
Sugrue, D. et al., Br. Heart J., 56: 62 (1986). The development of methods 
for direct thrombus imaging have been stimulated by these clinical 
problems. 
In vivo diagnostic imaging for intravascular thrombi has been reported. 
These imaging methods use compounds which are detectable by virtue of 
being labelled with radioactive or paramagnetic atoms. For example, 
platelets labelled with the gamma emitter, In-111, have been reported as 
an imaging agent for detecting thrombi. Thakur, M. L. et al., Thromb. 
Res., 9: 345 (1976); Powers et al., Neurology, 32: 938 (1982). A 
thrombolytic enzyme, such as streptokinase, labelled with the gamma 
emitter Tc-99m, has been proposed as an imaging agent. Wong, D. W., U.S. 
Pat. No. 4,418,052 (1983). The fibrin-binding domains of Staphylcoccus 
aureus derived protein A labelled with the gamma emitters, I-125 and 
I-131, have been proposed as imaging agents. Pang, R. H. L., U.S. Pat. No. 
5,011,686 (1991). Monoclonal antibodies having specificity for fibrin (in 
contrast to fibrinogen) and labelled with the gamma emitter, Tc-99m, have 
been proposed as imaging agents. U.S. Pat. Nos. 5,024,829 (1991) to 
Berger, H. J. et al.; and 4,980,148 (1990) to Dean, R. T. et al. The use 
of the paramagnetic contrasting agent, gadolinium 
diethylenetriaminepentaacetic acid, in magnetic resonance imaging of 
patentis treated by thrombolysis for acute myocardial infarction has been 
reported. De Roos, A. et al., Int. J. Card. Imaging, 7: 133 (1991). 
Most preferred natural substrates for thrombin are reported to contain an 
uncharged amino acid in the P3 recognition subsite. For example, the 
thrombin cleavage site on the Aa chain of fibrinogen, which is a 
physiological substrate for thrombin, is reported to contain a glycine 
residue in this position while the cleavage site on the Bb chain contains 
a serine, as shown below: 
##STR1## 
Peptidyl derivatives having an uncharged residue in the P3 position which 
are believed to bind to the active site of thrombin and thereby inhibit 
the conversion of fibrinogen to fibrin and cellular activation have been 
reported. Additionally, these derivatives have either an aldehyde, 
chloromethyl ketone or boronic acid functionality associated with the P1 
amino acid. For example, substrate-like peptidyl derivatives such as 
D-phenylalanyl-prolyl-argininal (D-Phe-Pro-Arg-al), 
D-phenylalanyl-prolyl-arginine-chloromethyl ketone (P-K) and 
acetyl-D-phenylalanyl-prolyl-boroarginine (Ac-(D-Phe)-Pro-boroArg) have 
been reported to inhibit thrombin by directly binding to the active site 
of the enzyme. Bajusz, S., Symposia Biologica Hungarica, 25: 277 (1984), 
Bajusz, S. et al, J. Med. Chem., 33: 1729 (1990) and Bajusz, S. et al., 
Int. J. Peptide Protein Res. 12: 217 (1970); Kettner, C. and Shaw, E., 
Methods Enzymol., 80: 826 (1987); Kettner, C. et al., EP 293,881 
(published Dec. 7, 1988); Kettner, C., et al., J. Biol. Chem., 265: 18209 
(1990). These molecules have been reported to be potent anticoagulants in 
the prevention of platelet-rich arterial thrombosis. Kelly, A. B. et al., 
Thromb. Haemostas., 65: 736 at abstract 257 (1991). 
Peptidyl compounds which are said to be active site inhibitors of thrombin 
but which are said to differ in structure from those containing a 
uncharged amino acid in the P3 recognition subsite have been reported. The 
compound, Argatroban (also called 
2R,4R-4-methyl-1-N-2-(3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L- 
argininal!-2-piperdinecarboxylic acid), is also reported to bind directly 
to the active site of thrombin and has been thought to be the most potent 
and selective compound in the class of non-peptidyl inhibitors of this 
enzyme. Okamoto, S. et al., Biochem. Biophys. Res. Commun., 101: 440 
(1981). Argatroban has been reported to be a potent antithrombotic agent 
in several experimental models of acute arterial thrombosis. Jang, I. K. 
et al., in both Circulation, 81: 219 (1990) and Circ. Res., 67: 1552 
(1990). 
Peptidyl compounds which are said to be inhibitors of thrombin and whose 
mode of action is thought to be by binding to the active site as well as 
an accessory or exo-site on the enzyme have been reported. For example, 
hirudin and its various peptidyl derivatives have been reported to inhibit 
both conversion of fibrinogen to fibrin and platelet activation by binding 
to either both the active site and exo-site, or to the exo-site only, of 
thrombin. Markwardt, F., Thromb. Haemostas., 66: 141 (1991). Hirudin is 
said to be one of the most potent inhibitors of thrombin known. Marki, W. 
E. and Wallis, R. B., Thromb. Haemostas., 64: 344 (1990). Hirudin is 
reported to inhibit thrombin by binding to both its anion-binding exo-site 
and to its catalytic active site, sites which are distinct and physically 
distant from each other. Rydel, T. J. et al., Science, 249: 277 (1990). 
Its potency as measured by the inhibitory constant ("Ki") was determined 
to be 22.times.10.sup.-15 M. Stone et al., Biochemistry, 25: 4622, 4624 
(1986). Hirudin has been reported to be a potent antithrombotic agent in 
vitro and in vivo. Markwardt, F. et al., Pharmazie, 43: 202 (1988); Kelly, 
A. B. et al., Blood, 77: 1 (1991). In addition to its antithrombotic 
effects, hirudin has been reported to also inhibit smooth muscle 
proliferation and the associated restenosis following mechanical damage to 
a atherosclerotic rabbit femoral artery. Sarembock, I. J. et al., 
Circulation, 84: 232 (1991). 
Hirudin has been reported to be a 65 amino acid polypeptide which was 
originally isolated from leech salivary gland extracts. The primary amino 
acid sequence, as shown below, has been reported. Krstenansky J. L. et 
al., Thromb. Hemostasis, 63: 208 (1990). 
##STR2## 
The primary amino acid sequence of various isoforms of hirudin has also 
been reported. Scharf M. et al., FEBS Lett., 255: 105 (1989). The 
C-terminal portion (comprised of amino acids 56 to 64) of hirudin has been 
reported to be the minimal domain required for the binding of hirudin to 
the exo-site of thrombin. Krstenansky, J. L., et al., Thromb. Hemostasis, 
63: 208 (1990); Mao, S. J. T., et al., Biochemistry, 27: 8170 (1988); 
Krstenansky, et al., FEBS Lett., 211: 10 (1987). Peptides similar to this 
C-terminal portion have been reported to inhibit thrombin-induced clot 
formation and/or thrombin-mediated platelet aggregation. 
Hirugen has been reported to be a peptide derived from the anionic 
carboxy-terminus of hirudin. It is reported to bind only to the anion 
binding exo-site of thrombin and thereby inhibit the formation of fibrin 
but not the catalytic turnover of small synthetic substrates which have 
access to the unblocked active site of the enzyme. Maraganore, J. M. et 
al., J. Biol. Chem., 264: 8692 (1989.). The region of hirudin represented 
by hirugen has been shown using x-ray crystallographic techniques to bind 
directly to the exo-site of thrombin. Skrzypczak-Jankun, E. et al., 
Thromb. Haemostas., 65: 830 at abstract 507 (1991). Moreover, the binding 
of hirugen has also been reported to enhance the catalytic turnover of 
certain small synthetic substrates by thrombin, indicating that a 
conformational change in the enzyme active site may accompany occupancy of 
the exo-site. Naski, M. C. et al., J. Biol. Chem., 265: 13484 (1990); Liu, 
L. W. et al., J. Biol. Chem., 266: 16977 (1991). Hirugen also is reported 
to block thrombin-mediated platelet aggregation. Jakubowski, J. A. and 
Maraganore, J. M., Blood, 75: 399 (1990). The inhibition of 
thrombin-induced fibrin clot formation resulting from substitution of the 
various amino acid residues on a C-terminal peptide of hirudin has also 
been reported. Krstenansky, J. L., et al., Thromb. Hemostasis, 63: 208 
(1990). 
A chimeric peptide has been reported to be comprised of a C-terminal 
peptide of hirudin (amino acids 53 to 64) coupled to a peptide containing 
an Arg-Gly-Asp (RGD) sequence. The C-terminal peptide, with or without the 
RGD-containing peptide, is said to inhibit both thrombin-induced clot 
formation and thrombin-mediated platelet aggregation with an IC.sub.50 of 
0.6 mM and 7 mM, respectively. Church, F. C. et al., J. Biol. Chem., 266: 
11975 (1991). 
Another chimeric peptide, Hirulog, has been reported to be a synthetic 
molecule comprised of a hirugen-like sequence (amino acids 53 to 64 of 
hirudin) linked by a glycine-spacer region to the peptide, 
D-phenylalanyl-prolyl-arginine. The latter portion of this peptide is said 
to be based on a preferred substrate recognition site for thrombin. The 
hirugen-like sequence is said to be located at the C-terminus of this 
peptide. Maraganone, J. M. et al., Biochemistry, 29: 7095 (1990); 
Maraganone, J. M. et al., International Application No. WO 91/02750 
(published Mar. 7, 1991); and Dimaio, J. et al., International Application 
No. WO 91/19734 (published Dec. 26, 1991). Hirulog is said to bind to 
thrombin in a bivalent manner and this binding is characterized by an Ki 
of 2.56.times.10.sup.-9 M. The D-phenylalanyl-prolyl-arginine peptide is 
said to bind to the catalytic site of thrombin, whereas the hirugen-like 
sequence binds to its anion-binding exo-site. Witting, J. I. et al., 
Biochem. J., 283: 737 (1992). Hirulog has been reported to be an effective 
antithrombotic agent in vivo, preventing both fibrin-rich and 
platelet-rich thrombosis. Maraganone, J. M. et al., Thromb. Haemostas., 
65: 651 at abstract 17 (1991). 
Hirulog has been reported to have the structure, 
H-(D-Phe)-Pro-Arg-Pro-(Gly).sub.4 
-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, and is said to be potent 
thrombin inhibitor. The substitution of various amino acids on the 
hirugen-like sequence of Hirulog and the effect thereof on binding 
constant has been reported. Bourdon, P. et al., FEBS, 294: 163 (1991). 
Substitution of the D-phenylalanine residue with a b-cyclohexyl-D-alanine 
residue is said to provide a more potent thrombin inhibitor, characterized 
by a Ki of 0.077.times.10.sup.-9 M. Witting, J. I. et al., Biochem. J., 
287: 663, 664 (1992). Addition of a methylene group between the arginine 
a-carbon and carbonyl of Hirulog is said to provide a non-cleavable 
thrombin inhibitor characterized by a Ki of 7.4.times.10.sup.-9 M, while 
substitution of a methylene group for this carbonyl alone is said to 
provide a poor thrombin inhibitor having a Ki of greater than 
2000.times.10.sup.-9 M. Kline, T. et al., Biochem. Biophys. Res. Commun., 
177: 1049, 1052-1054 (1991). N-acetyl-D-Phe-Pro Arg-yC(.dbd.O)--CH.sub.2 
!--CH.sub.2 --CH.sub.2 --CH.sub.2 
--(C.dbd.O)-Gln-Ser-His-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Le 
u-Gln is said to be potent thrombin non-cleavable inhibitor having a. Ki of 
0.14.times.10.sup.-9 M. Dimaio, J. et al., International Application, at 
page 44. 
Cyclotheonamide A and B, isolated from the marine sponge, Theonella, a 
genus of marine sponges, have been reported to be inhibitors of thrombin 
with an IC.sub.50 of 0.076 mg/mL (9.9.times.10.sup.-8 M). Structurally, 
they have been characterized as cyclic peptides containing an arginine 
a-keto amide moiety. Fusetani et al., J. Am. Chem. Soc. 112: 7053-7054 
(1991).and Hagihara et al., J. Am. Chem. Soc, 114: 6570-6571 (1992). It 
has been proposed that the a-keto group of the cyclotheonamides may 
function as an electrophilic mimic of the Arg-X scissile amide bond of the 
thrombin substrates. Hagihara et al., Id. at 6570. The partial synthesis 
of cyclotheonamide A and the total synthesis of cyclotheonamide B have 
been reported. Wipf et al., Tetrahedron Lett., 33: 4275-4278 (1992) and 
Hagihara et al., J. Am. Chem. Soc, 114: 6570-6571 (1992). 
a-Keto-amide derivatives of other amino acids and peptides have also been 
reported to be inhibitors of proteases. For example, 
L-valyl-L-valyl-3-amino-2-oxovaleryl-D-leucyl-L-valine had been reported 
to be an inhibitor of prolyl endopeptidase. Nagai et al., J. Antibiotics, 
44: 956-961 (1991). 3-Amino-2-oxo-4-phenylbutanoic acid amide has been 
reported to be an inhibitor of arginyl aminopeptidase (with a Ki of 1.5 
mM), cytosol aminopeptidase (with a Ki of 1.0 mM) and microsomal 
aminopeptidase (with a Ki of 2.5 mM). Ocain et al., J. Med. Chem., 35: 
451-456 (1992). 2-Oxo-2-(pyrrolidin-2-yl)acetyl derivatives have been 
reported to be inhibitors of prolyl endopeptidase. Someno et al., European 
Patent Application No. 468,339 (published Jan. 29, 1992). Certain 
a-keto-amide derivatives of peptides have been reported to inhibit various 
serine and cysteine proteases. Powers J. C., International Application No. 
WO 92/12140 (published Jul. 23, 1992). 
a-Keto ester derivatives of N-protected amino acids and peptides have also 
been reported as inhibitors of serine proteases, such as neutrophil 
elastase and cathepsin. G. Mehdi et al., Biochem. Biophys. Res. Commun., 
166: 595-600 (1990) and Angelastro et al., J. Med. Chem., 33: 11-13 
(1990). 
SUMMARY OF INVENTION 
The present invention includes novel compounds useful for preventing or 
treating in a mammal a pathological condition characterized by thrombus 
formation. 
Among other factors, the present invention is based on our discovery of a 
novel class of compounds which are surprisingly active as inhibitors of 
thrombin. According to a preferred aspect, provided are certain compounds 
which by virtue of their novel structures exhibit the ability to inhibit 
thrombin in a potent manner substantially exceeding that of thrombin 
inhibitors described in the art. Their high potency allows the preferred 
compounds of the present invention to be especially useful in the 
formulation of compositions, therapeutic compositions and diagnostic 
compositions which can be administered at comparatively lower doses for 
the various therapeutic or diagnostic procedures in which they are useful. 
According to one aspect, compounds of the present invention are provided 
which are represented by the formula: 
##STR3## 
wherein R.sub.1 is alkyl of 1 to about 12 carbon atoms, alkenyl of about 3 
to about 6 carbon atoms, aryl of about 6 to about 14 carbon atoms, aralkyl 
of about 6 to about 15 carbon atoms, aralkenyl of about 8 to 15 carbon 
atoms, alkoxy of 1 to about 12 carbon atoms, alkenyloxy of about 3 to 
about 8 carbon atoms, aryloxy of about 6 to about 14 carbon atoms, or 
aralkyloxy of about 6 to about 15 carbon atoms; 
A is selected from the group consisting of 
##STR4## 
wherein R' is H, alkyl of 1 to about 6 carbon atoms, or aralkyl of about 6 
to about 15 carbon atoms and R" is alkyl of 1 to 6 carbon atoms or aralkyl 
of about 6 to about 15 carbon atoms; 
m is 1, 2 or 3; 
B is a peptide represented by the formula, B.sub.1 -B.sub.2 -B.sub.3 
-B.sub.4 -B.sub.5, wherein B.sub.1 is peptide of 5 to 8 amino acids, 
B.sub.2 is Arg, Asn, Asp or Gln; B.sub.3 is Gly; B.sub.4 is Asp; and 
B.sub.5 is Nap, Phe, Tha, Trp or Tyr; 
C is a peptide represented by the formula: C.sub.1 -C.sub.2 -C.sub.3 
-C.sub.4 -C.sub.5 -C.sub.6 -C.sub.7 -Z, wherein C.sub.1 is Glu; C.sub.2 is 
Ala, Glu or Pro; C.sub.3 is Ile, Leu or Ser; C.sub.4 is Hyp, Leu or Pro; 
C.sub.5 is Asp, Glu, Ala-Asp, Ala-Glu, Asp-Asp, Asp-Glu, Glu-Asp or 
Glu-Glu; C.sub.6 is Ala, Ile, Tyr, Tyr(O-SO.sub.3 H), Tyr(3-iodo), 
Tyr(3,5-diiodo), Ala-Tyr, Ala-Tyr(O--SO.sub.3 H), Ala-Tyr(3-iodo) or 
Ala-Tyr(3,5-diiodo): C.sub.7 is Ala, Asp, Cha, Leu or Tyr; and Z is --OH 
or --NH.sub.2 ; or pharmaceutically acceptable salt thereof. 
In another aspect, the present invention includes pharmaceutical 
compositions useful for preventing or treating in a mammal a pathological 
condition characterized by thrombus formation, comprising a 
pharmaceutically acceptable carrier and a therapeutically effective amount 
of a compound of the present invention. 
In another aspect, the present invention includes methods of preventing or 
treating in a mammal a pathological condition characterized by thrombus 
formation. 
In another aspect, the present invention includes novel compounds which are 
useful for in vivo imaging of thrombi in a mammal. According to a 
preferred aspect, compounds of the present invention include those 
represented by the formula: 
##STR5## 
wherein R.sub.1 is alkyl of 1 to about 12 carbon atoms, alkenyl of about 3 
to about 6 carbon atoms, aryl of about 6 to about 14 carbon atoms, aralkyl 
of about 6 to about 15 carbon atoms, aralkenyl of about 8 to 15 carbon 
atoms, alkoxy of 1 to about 12 carbon atoms, alkenyloxy of about 3 to 
about 8 carbon atoms, aryloxy of about 6 to about 14 carbon atoms, or 
aralkyloxy of about 6 to about 15 carbon atoms; 
A is selected from the group consisting of 
##STR6## 
wherein R' is H, alkyl of 1 to about 6 carbon atoms, or aralkyl of about 6 
to about 15 carbon atoms and R" is alkyl of 1 to 6 carbon atoms or aralkyl 
of about 6 to about 15 carbon atoms; 
m is 1, 2 or 3; 
D is a peptide represented by the formula, D.sub.1 -D.sub.2 -D.sub.3 
-D.sub.4 -D.sub.5, wherein 
D.sub.1 is (Gly).sub.p -X-(Gly).sub.q when D.sub.2 is Arg, Asn, Asp or Gln, 
or D.sub.1 is --(Gly).sub.p+q -Gly-- when D.sub.2 is X, wherein p and q 
are independently selected integers from 1 to 7, such that their sum is 4 
to 7, and X has the formula: 
##STR7## 
wherein r is an integer selected from 2 to 6, L is a chelating means for 
chelating a radioactive or paramagnetic atom, and Y is an attaching means 
for attaching chelating means to the amino group; 
D.sub.3 is Gly; 
D.sub.4 is Asp; and 
D.sub.5 is Nap, Phe, Tha, Trp or Tyr; 
E is a peptide represented by the formula: E.sub.1 -E.sub.2 -E.sub.3 
-E.sub.4 -E.sub.5 -E.sub.6 -E.sub.7 -Z, wherein 
E.sub.1 is Glu; 
E.sub.2 is Ala, Glu or Pro; 
E.sub.3 is Ile, Leu or Ser;. 
E.sub.4 is Hyp, Leu or Pro; 
E.sub.5 is Asp, Glu, Ala-Asp, Ala-Glu, Asp-Asp, Asp-Glu, Glu-Asp or 
Glu-Glu; 
E.sub.6 is Ala, Ile, Tyr(3-iodo), Tyr(3,5-diiodo), Tyr(O--SO.sub.3 H) , 
Ala-Tyr(3-iodo) , Ala-Tyr(3,5-diiodo), or Ala-Tyr(O--SO.sub.3 H); 
E.sub.7 is Ala, Asp, Cha, Leu or Tyr; and 
Z is --OH or --NH.sub.2 ; or pharmaceutically acceptable salt thereof. 
In another aspect, the present invention includes compositions which are 
useful for in vivo imaging of thrombi in a mammal, comprising a compound 
of the present invention which is capable of being detected outside the 
body. Preferred are compositions comprising a compound of the present 
invention and a detectable label, preferably a radioactive or paramagnetic 
atom. 
In another aspect, the present invention provides diagnostic compositions 
which are useful for in vivo imaging of thrombi in a mammal, comprising a 
pharmaceutically acceptable carrier and a diagnostically effective amount 
of a compound or composition of the present invention. 
In another aspect, the present invention includes methods which are useful 
for in vivo imaging of thrombi in a mammal. 
Definitions 
In accordance with the present invention and as used herein, the following 
terms are defined with the following meanings, unless explicitly stated 
otherwise. 
The term "alkoxy" refers to the group --OR wherein R is alkyl. 
The term "alkyl" refers to saturated aliphatic groups including 
straight-chain, branched-chain and cyclic groups. 
The term "alkenyl" refers to unsaturated hydrocarbyl groups which contain 
at least one carbon-carbon double bond and includes straight-chain, 
branched-chain and cyclic groups. 
The term "alkenyloxy" refers to the group --OR wherein R is alkenyl. 
The term "amino acid" refers to and includes the L-isomers of the naturally 
occurring a-amino acids, as well as nonnatural a-amino acids such as those 
used in peptide synthesis of analogs of naturally occurring peptides. The 
naturally occurring amino acids include glycine (Gly), alanine (Ala), 
valine (Val), leucine (Leu), isoleucine (Ile), serine (Ser), methionine 
(Met), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), tryptophan 
(Trp), cysteine (Cys), proline (Pro), histidine (His), aspartic acid 
(Asp), asparagine (Asn), glutamic acid (Glu), glutamine (Gln), 
g-carboxyglutamic acid, arginine (Arg), ornithine (Orn) and lysine (Lys). 
Examples of nonnatural a-amino acids include alloisoleucine, 
2-aminobutyric acid (Abu), a-cyclohexylglycine b-cyclohexylalanine (Cha), 
homoarginine (HArg), hydroxyproline (Hyp), homoserine (HSer), norleucine 
(Nle), norvaline (Nva), phenylalanines substituted on its phenyl ring with 
one or more alkyl, alkenyl, aryl, aralkyl, alkoxy, alkenyloxy, aryloxy, 
aralkyloxy, alkylsulfonic, alkylphosphonic, sulfate, phosphate, halogen or 
nitro groups, b-(2-thienyl)-alanine (Tha), b-furanylalanine (Fua), 
b-pyridylalanine (Pya), b-benzothienylalanine (Btha), 
b-(2'naphthyl)-alanine (Nap), 0-alkylated derivatives of serine, threonine 
or tyrosine, S-alkylated cysteine, 4-phenylacetic acid, 3-iodotyrosine, 
3,5-diiodotyrosine, lysine and ornithine substituted with an alkyl group, 
and D-isomers of naturally occurring amino acids. 
The term "anionic amino acid" refers to Phe, Cha or Tyr which are either 
mono- or di-substituted with a carboxyl, phosphoryl, or sulfonyl group on 
their respective aromatic or cyclic alkyl rings, as well as Glu, Asp, 
phosphothreonine, phosphoserine, phosphotyrosine, 3-sulfotyrosine, 
4-sulfotyrosine, 5-sulfotyrosine, 3-methylphosphonyltyrosine, and 
3-methylsulphonyltyrosine. 
The term "aryl" refers to aromatic groups which have at least one ring 
having a conjugated pi electron system and includes carbocyclic aryl, 
heterocyclic aryl and biaryl groups, all of which may be optionally 
substituted. 
The term "aryloxy" refers to the group --OR wherein R is aryl. 
The term "aralkyl" refers to an alkyl group substituted with an aryl group. 
Suitable aralkyl groups include benzyl, picolyl, and the like, all of 
which may be optionally substituted. 
The term "aralkenyl" refers to an alkenyl group substituted with an aryl 
group. Suitable aralkenyl groups include styrenyl and the like, all of 
which may be optionally substituted. 
The term "aralkyloxy" refers to the group --OR wherein R is aralkyl. 
The term "lipophilic amino acid" refers to Tyr, Trp, Phe, Leu, Nle, Val, 
Cha, or Pro. 
The term "methylene" refers to --CH.sub.2 --. 
In addition, the following abbreviations stand for the following: 
"Bn" refers to benzyl. 
"Boc" refers to t-butoxycarbonyl. 
"Boc.sub.2 O" refers di-t-butyldicarbonate. 
"BocAsp.sup.Bn -OH" refers to N-Boc-L-aspartic acid-(b-benzyl ester). 
"BocPro-OH" refers to N-Boc-L-proline. 
"BOP" refers to 
benzotriazol-1-yloxy-tris-(dimethylamino)-phosphonium-hexafluorophosphate. 
"Brine" refers to an aqueous saturated solution of sodium chloride. 
"Cbz" refers to benzoyloxy carbonyl. 
"DCA" refers to dichloroacetic acid. 
"DEC" refers to dicyclohexylcarbodiimide. 
"3,4-dehydroPro" refers to 3,4-dehydroproline. 
"EDAC-HCl" refers to 1-ethyl-3=(3-dimethylaminopropyl)carbodiimide 
hydrochloride salt. 
"EDT" refers to ethanedithiol. 
"Fmoc" refers to 9-fluorenymethyloxycarbonyl. 
"HBTU" refers to 2-(1H-benzotriazol-1-yl)-1,1,3,3 
tetramethyluroniumhexafluorophosphate. 
"HOBT" refers to 1-hydroxybenzotriazole. 
"Nap" refers to b-(2'-naphthyl)-alanine. 
"TFA" refers to trifluoroacetic acid. 
"Tha" refers to b-(2-thienyl)-alanine. 
"Tyr(O-SO3H)" refers to tyrosine substituted on its aromatic ring hydroxyl 
with a sulfate group. 
"Tyr(3-iodo)" refers to 3'-iodotyrosine. 
"Tyr (3,5-diiodo)" refers to 3',5'-diiodotyrosine

DETAILED DESCRIPTION 
Compounds and Their Preparation 
A. Preferred Compounds 
The present invention provides novel compounds useful for preventing or 
treating in a mammal a pathological condition characterized by thrombosis. 
These compounds are represented by formula I. 
##STR8## 
These compounds of formula I include those wherein m is 1, 2 or 3. 
Preferred are those compounds wherein m is 2. 
The compounds of the present invention also include those wherein B is a 
peptide represented by the formula: B.sub.1 -B.sub.2 -B.sub.3 -B.sub.4 
-B.sub.5, wherein B.sub.1 is peptide of 5 to 8 amino acids, B.sub.2 is 
Arg, Asn, Asp or Gln; B.sub.3 is Gly; B.sub.4 is Asp; and B.sub.5 is Nap, 
Phe, Tha, Trp or Tyr. Preferred are those compounds, wherein B is selected 
from the group consisting of -Gly-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe- SEQ. 
ID. NO. 4! or -Gly-Gly-Gly-Gly-Gly-Arg-Gly-Asp-Phe-. SEQ. ID. NO. 5! 
The compounds of formula I also include those wherein C is a peptide 
represented by the formula: C.sub.1 -C.sub.2 -C.sub.3 -C.sub.4 -C.sub.5 
-C.sub.6 -C.sub.7 -Z, wherein C.sub.1 is Glu; C.sub.2 is Ala, Glu or Pro; 
C.sub.3 is Ile, Leu or Ser; C.sub.4 is Hyp, Leu or Pro; C.sub.5 is Asp, 
Glu, Ala-Asp, Ala-Glu, Asp-Asp, Asp-Glu, Glu-Asp or Glu-Glu; C.sub.6 is 
Ala, Ile, Tyr, Tyr(O--SO.sub.3 H), Tyr(3-iodo), Tyr(3,5-diiodo), Ala-Tyr, 
Ala-Tyr(O--SO.sub.3 H), Ala-Tyr(3-iodo) or Ala-Tyr(3,5-diiodo); C.sub.7 is 
Ala, Asp, Cha, Leu or Tyr; and Z is --OH or --NH.sub.2. Preferred are 
those compounds, wherein C is selected from the group consisting of 
-Glu-Glu-Ile-Pro-Glu-Tyr-Leu-OH, SEQ. ID. NO. 6! 
-Glu-Glu-Ile-Pro=Glu-Glu-Tyr-Leu-OH, SEQ. ID. NO. 7! 
-Glu-Glu-Ile-Pro-Glu-Tyr-Leu-NH.sub.2 SEQ. ID. NO. 8! or 
-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-NH.sub.2. SEQ. ID. NO. 9! Especially 
preferred are those compounds having B as 
-Gly-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe- SEQ. ID. NO. 10! and C as 
-Glu-Glu-Ile-Pro-Glu-Tyr-Leu-OH; SEQ. ID. NO. 11! B as 
-Gly-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe- SEQ. ID. NO. 12! and C as 
-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH; SEQ. ID. NO. 13! B as 
-Gly-Gly-Gly-Gly-Gly-Arg-Gly-Asp-Phe- SEQ. ID. NO. 14! and C as 
-Glu-Glu-Ile-Pro-Glu-Tyr-Leu-OH; SEQ. ID. NO. 15! or B as 
-Gly-Gly-Gly-Gly-Gly-Arg-Gly-Asp-Phe- SEQ. ID. NO. 16! and C as 
-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. SEQ. ID. NO.17! 
The compounds of formula I also include those wherein R.sub.1 which is an 
alkyl of 1 to about 12 carbon atoms, alkenyl of about 3 to about 6 carbon 
atoms, aryl of about 6 to about 14 carbon atoms, aralkyl of about 6 to 
about 15 carbon atoms, aralkenyl of about 8 to 15 carbon atoms, alkoxy of 
1 to about 12 carbon atoms, alkenyloxy of about 3 to about 8 carbon atoms, 
aryloxy of about 6 to about 14 carbon atoms, or aralkyloxy of about 6 to 
about 15 carbon atoms. Compounds of the present invention include those 
wherein R.sub.1 is cyclohexyl, 4-heptyl, 3-methylpentyl, 2-methytpropyl, 
3-octyl or 2-phenylethyl. Preferred are those compounds, wherein R.sub.1 
is 4-heptyl. 
The compounds of formula I also include those wherein A is selected from 
the group consisting of 
##STR9## 
wherein R' is H, alkyl of 1 to about 6 carbon atoms, or aralkyl of about 6 
to about 15 carbon atoms and R" is alkyl of 1 to 6 carbon atoms or aralkyl 
of about 6 to about 15 carbon atoms. The preferred compounds of the 
present invention include those which are potent inhibitors of human 
a-thrombin, which potency is characterized by an inhibitor constant, Ki, 
of less than 0.050 nM. 
As previously noted the present invention is based on our discovery of the 
novel compounds of formula I. Certain of the compounds of the present 
invention by virtue of their novel structures have imparted to them the 
ability to inhibit thrombin with a potency substantially exceeding that of 
thrombin inhibitors reported in the art. This substantial enhanced potency 
exhibited by these preferred compounds allows then to be used in the 
formulation of compositions, therapeutic compositions and diagnostic 
compositions which can then be administered at comparatively and 
advantageously lower doses in the various therapeutic or diagnostic 
procedures in which they are useful. 
The substantial difference in potency of the preferred compounds of the 
present invention over compounds described in the art is exemplified in 
Example A. Certain preferred compounds of the present invention have been 
found to have an inhibitor constant (Ki) against a-thrombin in the range 
of 0.0019 to 0.040 nM, while a compound of the art, Hirulog-1, was found 
to a have Ki of 0.437 nM under the same assay conditions. The improvement 
in potency provided by the preferred compounds of the present invention is 
therefore at least ten-fold, and as demonstrated, can exceed a 
hundred-fold. 
Compounds illustrative of the present invention include: 
##STR10## 
Preferred compounds of the present invention include: 
##STR11## 
In another aspect, the present invention also provides a class of novel 
compounds useful for imaging of thrombi in a mammal. Preferred compounds 
that class include those of formula I, wherein 
m is 2; 
B is -Gly-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe- SEQ. ID. NO. 62! or 
-Gly-Gly-Gly-Gly-Gly-Arg-Gly-Asp-Phe-; SEQ. ID. NO. 63! 
C is -Gly-Gly-Ile-Pro-Glu-Tyr(3-iodo)-Leu-OH, SEQ. ID. NO. 64! 
-Glu-Glu-Ile-Pro-Glu-Tyr(3,5-diiodo)-Leu-OH, SEQ. ID. NO. 65! 
-Glu-Glu-Ile-Pro-Glu-Tyr(3-iodo)-Leu-NH.sub.2, SEQ. ID. NO. 66! 
-Glu-Glu-Ile-Pro-Glu-Tyr(3,5-diiodo)-Leu-NH.sub.2, SEQ. ID. NO. 67! 
-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(3-iodo)-Leu-OH, SEQ. ID. NO. 68! 
-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(3,5-diiodo)-Leu-OH, SEQ. ID. NO. 69! 
-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(3-iodo)-Leu-NH.sub.2. SEQ. ID. NO. 70! or 
-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(3,5-diiodo)-Leu-NH.sub.2, SEQ. ID. NO. 71! 
wherein at least one of the iodine atoms therein is either I-123, I-125 or 
I-131; 
R.sub.1 is cyclohexyl, 4-heptyl, 3-methylpentyl, 2-methylpropyl, 3-octyl or 
2-phenylethyl; and 
A is --CO.sub.2 H and --CO.sub.2 CH.sub.3. Especially preferred are those 
compounds wherein B is -Gly-Gly-Gly-Gly-Gly-Asn-Gly-Asp-Phe- SEQ. ID. NO. 
72! or -Gly-Gly-Gly-Gly-Gly-Arg-Gly-Asp-Phe-, SEQ. ID. NO. 73! and C is 
-Glu-Glu-Ile-Pro-Glu-Tyr(3-iodo)-Leu-OH, SEQ. ID. NO. 74! 
-Glu-Glu-Ile-Pro-Glu-Tyr(3,5-diiodo)-Leu-OH, SEQ. ID. NO. 75! 
-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(3-iodo)-Leu-OH, SEQ. ID. NO. 76! or 
-Glu-Glu-Ile-Pro-Glu-Glu-Tyr(3,5-diiodo)-Leu-OH; R.sup.1 is 4-heptyl; 
SEQ. ID. NO. 77! and A is --CO.sub.2 H. 
In another aspect, the present invention provides another class of novel 
compounds which are useful for in vivo imaging of thrombi in a mammal. 
These compounds include those having formula II. 
##STR12## 
The compounds of formula II include those wherein m is 1, 2, or 3. The 
preferred compounds will have m equal to 1. 
The compounds of formula II also include those wherein E is a peptide 
represented by the formula: E.sub.1 -E.sub.2 -E.sub.3 -E.sub.4 -E.sub.5 
-E.sub.6 -E.sub.7 -Z, wherein E.sub.1 is Glu; E.sub.2 is Ala, Glu or Pro; 
E.sub.3 is Ile, Leu or Ser; E.sub.4 is Hyp, Leu or Pro; E.sub.5 is Asp, 
Glu, Ala-Asp, Ala-Glu, Asp-Asp, Asp-. Glu, Glu-Asp or Glu-Glu; E.sub.6 is 
Ala, Ile, Tyr(3-iodo), Tyr(3,5-diiodo), Tyr(O--SO.sub.3 H), 
Ala-Tyr(3-iodo), Ala-Tyr(3,5-diiodo), or Ala-Tyr(O--SO.sub.3 H); E.sub.7 
is Ala, Asp, Cha, Leu or Tyr; and Z is --OH or --NH.sub.2. The preferred 
compounds will have E which is -Glu-Glu-Ile-Pro-Glu-Tyr-Leu-OH, SEQ. ID. 
NO. 78! -Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH, SEQ. ID. NO. 79! 
-Glu-Glu-Ile-Pro-Glu-Tyr-Leu-NH.sub.2 SEQ. ID. NO. 80! or 
-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-NH.sub.2 SEQ. ID. NO. 81!. Especially 
preferred compounds will have E which is -Glu-Glu-Ile-Pro-Glu-Tyr-Leu-OH 
SEQ. ID. NO. 82! or -Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. SEQ. ID. NO. 83 
! 
The compounds of formula II include those wherein D is a peptide 
represented by D.sub.1 -D.sub.2 -D.sub.3 -D.sub.4 -D.sub.5, wherein 
D.sub.1 is (Gly).sub.p -X-(Gly).sub.q when D.sub.2 is Arg, Asn, Asp or 
Gln, or D.sub.1 is -(Gly).sub.p+q -Gly- when D.sub.2 is X, wherein p and q 
are independently selected from the integers, 1 to 7, such that their sum 
is 4 to 7; and X has the formula: 
##STR13## 
wherein r is an integer selected from 2 to 6, L is a chelating means for 
binding of a radioactive or paramagnetic atom, and Y is an attaching means 
for attaching chelating means; D.sub.3 is Gly; D.sub.4 is Asp; and D.sub.5 
is Nap, Phe, Tha, Trp or Tyr. The preferred compounds of the present 
invention will have D which is -Gly-Gly-X-Gly-Gly-Asn-Gly-Asp-Phe-, SEQ. 
ID. NO.84! -Gly-Gly-X-Gly-Gly-Arg-Gly-Asp-Phe-, SEQ. ID. NO. 85! or 
-Gly-Gly-Gly-Gly-Gly-X-Gly-Asp-Phe-, SEQ. ID. NO. 86! Especially 
preferred are the compounds which have r equal to 4, or L-lysine. 
In these compounds represented by formula II, the attaching means, Y, 
includes groups which are capable of covalently bonding with both the 
e-amino group of L-lysine and the chelating means. For example, Y may be 
--C(.dbd.S)--, --C(.dbd.O)--, --C(.dbd.NH)--(CH.sub.2).sub.6 
--C(.dbd.NH)--, --C(.dbd.O)--(CH.sub.2).sub.6 --C(.dbd.O)--, 
##STR14## 
and the like. Especially preferred compounds will have a Y which is 
--C(.dbd.S)-- or 
##STR15## 
Also, in the compounds represented by formula II, the chelating means, L, 
includes groups capable of covalently bonding to Y and covalently or 
noncovalently binding to either a radioactive or paramagnetic atom. The 
chelating means include those which are customarily used for complexing 
radioactive or paramagnetic atoms. These include chelating means 
containing 3 to 12, preferably 3 to 8, methylene phosphonic acid groups, 
methylene carbohydroxamic acid groups, carboxyethylidene groups, or 
especially carboxymethylene groups, which are bonded to a nitrogen atom. 
If only one or two of the acid groups are bonded to a nitrogen atom, then 
that nitrogen is bonded to another nitrogen atom having such groups by an 
optionally substituted ethylene group or by up to four separated ethylene 
units separated by a nitrogen or oxygen or sulfur atom. Preferred as a 
complexing means is diethylenetriamine-N,N,N',N",N"-pentaacetic acid 
(DTPA). DTPA is well known in the art as a chelating means for the 
radioactive atom indium-111 (In-111), technetium-99m (Tc-99m), and the 
paramagnetic atom gadolinium (Gd). Khaw, et al., Science, 209: 295 (1980); 
Paik C. H. et al., U.S. Pat. Nos. 4,652,440 (1987); Gries, H. et al., 
4,957,939 (1990). Especially preferred for chelating means, L, is 
1-(p-aminobenzyl)diethylenetriaminepentaacetic acid. Also included as 
chelating means are compounds which contain sulfhydryl or amine moieties, 
the total of which in any combination is at least four. These sulfhydryl 
or amine moieties are separated from each other by at least two atoms 
which can be either carbon, nitrogen, oxygen, or sulfur. Especially 
preferred for chelating means, L, is metallothionein which is well known 
in the art as a chelating means for Tc-99m. 
The compounds of the present invention also include those wherein R.sub.1 
is an alkyl of about 1 to about 12 carbon atoms, alkenyl of about 3 to 
about 6 carbon atoms, aryl of about 6 to about 14 carbon atoms, aralkyl of 
about 6 to about 15 carbon atoms, aralkenyl of about 8 to 15 carbon atoms, 
alkoxy of about 1 to about 12 carbon atoms, alkenyloxy of about 3 to about 
8 carbon atoms, aryloxy of about 6 to about 14 carbon atoms, or aralkyloxy 
of about 6 to about 15 carbon atoms. Compounds of formula II include those 
having an R.sub.1 which is cyclohexyl, 4-heptyl, 3-methylpentyl, 
2-methylpropyl, 3-octyl or 2-phenylethyl. 
Preferred compounds will have an R.sub.1 which is 4-heptyl. 
The compounds of formula IT also include those, wherein A is selected from 
the group consisting of 
##STR16## 
wherein R' is H, alkyl of 1 to about 6 carbon atoms, or aralkyl of about 6 
to about 15 carbon atoms and R" is alkyl of 1 to 6 carbon atoms or aralkyl 
of about 6 to about 15 carbon atoms. The preferred compounds will have an 
A which is 
##STR17## 
Especially preferred compounds will have an A which is --CO.sub.2 H. 
The preferred compounds of formula I and II also include their 
pharmaceutically acceptable salts. The term "pharmaceutically acceptable 
salts" includes salts of compounds derived from the combination of a 
compound of formula I or II and an organic or inorganic acid. These 
compounds are useful in both free base and salt form. These salts include 
acid addition salts, for example, salts of hydrochloric, hydrobromic, 
acetic acid, and benzene sulfonic acid and the like. In practice, the use 
of the salt form amounts to use of the base form; both forms are within 
the scope of the present invention. 
B. Preparation 
The preferred compounds of the present invention can be synthesized using 
conventional preparative and recovery methods known to those skilled in 
the art of peptide synthesis. Solid phase or liquid phase methods, or both 
can be utilized. 
A preferred synthesis route for the straight-chain peptide intermediates, 
especially the smaller peptides (of shorter chain length, that is, having 
from about 3 to about 50 amino acid residues) of the invention is the 
solid phase method. This method is well known in the art and is described 
in references such as Merrifield, J., Am. Chem. Soc. 85: 2149-2154 (1963); 
Science 150: 178-185 (1965); and Science 232: 341-347 (1986); Vale et al., 
Science 213: 1394-1397 (1981); and Marke et al., J. Am. Chem. Soc. 103: 
3178 (1981). Other preparative methods which may be employed include the 
processes of Houghten, Proc. Natl. Acad. Sci(USA) 82: 5132 (1985). Further 
background information on established solid phase synthesis procedures 
which can be used for the preparation of the compounds described herein is 
set forth in the treatise by Stewart and Young, Solid Phase Peptide 
Synthesis, W. H. Freeman & Co., San Francisco, 1969; in the review chapter 
by Merrifield, J., Advances in Enzymology, Vol. 32, pp 221-296, 
Interscience Publishers, New York, (F. F. Nold, E., 1969); and in Erickson 
and Merrifield, The Proteins, Vol. 2, p 255 et seq., Academic Press, New 
York, ((Neurath and Hill ed. 1976). 
Solid phase peptide synthesis is generally commenced from the C-terminus of 
the peptide by coupling a protected a-amino acid to a suitable resin, such 
as Fmoc-amino acid-4-(hydroxymethyl)phenoxymethylcopoly (styrene-1% 
divinylbenzene) resin (Wang resin), Boc-amino 
acid-4-(oxymethyl)-phenylacetamidiomethyl copoly (styrene-1% 
divinylbenzene) resin (PAM resin), hydroxymethylphenoxymethyl polystyrene 
resin (HMP resin) or a RINK (dimethoxyphenyl-Fmoc aminomethyl!phenoxy 
resin) resin. The RINK resin is a modified benzhydrylamine resin that 
contains ortho and para electron-donating methoxy groups. 
In the solid phase method, the compounds of the present invention can be 
synthesized by sequential coupling of protected amino acid derivatives 
onto a solid phase using the reagents known in the art. Such reagents are 
readily available from chemical vendors as Aldrich, Sigma, Nova 
Biochemicals, Advanced ChemTech, Bachem and the like. 
During the solid phase synthesis of the compounds of the present invention, 
the functional groups of the requisite amino acid derivatives or analogs 
used are protected by blocking groups to prevent cross reaction during the 
coupling procedure. As such, they are referred to herein as protected 
amino acids or amino acid analogs. Examples of suitable blocking groups 
and their use are described in The Peptides: Analysis, Synthesis, Biology, 
Academic Press, Vol. 3 (E. Gross & Meienhofer edit. 1981) and Vol. 9 (S. 
Udenfriend & J. Meienhofer edit. 1987). 
A suitably protected amino acid or amino acid analog will have blocking 
groups on its a-amino group and, if necessary, on its side chain 
functionality. Examples of suitable blocking groups for the a-amino group 
include acyl protecting groups, for example, formyl, acetyl, benzoyl, 
trifluoroacetyl, succinyl and methoxysuccinyl aromatic urethane protecting 
groups, for example, benzyloxycarbonyl and aliphatic urethane protecting 
groups, for example, tert-butyloxycarbonyl (Boc), adamantyloxycarbonyl or 
fluorenylmethyloxycarbonyl (Fmoc) groups. Numerous suitable amino terminal 
protecting groups are known. See, for example, The Peptides, Vol. 3, pp 
3-88. Other suitable protecting groups are known to those skilled in the 
art. The preferred amino terminal protecting groups include 
t-butyloxycarbonyl (Boc) and 9-fluorenymethyloxycarbonyl (Fmoc). 
The sequential coupling of protected amino acids or amino acid analogs to 
the solid phase or growing peptide chain on the solid phase comprises 
converting the free carboxyl group of the protected amino acid or amino 
acid analog to an "activated" derivative wherein its carboxyl group is 
rendered more susceptible to reaction with the free N-terminal a-amino 
group of the target amino acid or peptide having an associated a-keto 
amide functionality. For example, the free carboxyl of the a-amino 
protected (N-protected) amino acid can be converted to a mixed anhydride 
by reaction of a N-protected amino acid with ethyl choloroformate, 
pivaloyl chloride or like acid chlorides. Alternatively, the carboxyl of 
the a-amino protected amino acid can be converted to an active ester such 
as a 2,4,5-trichloropheyl ester, a pentachlorophenol ester, a 
pentafluorophenyl ester, a p-nitrophenyl ester, a N-hydroxysuccinimide 
ester, or an ester formed from 1-hydroxybenzotriazole. Another coupling 
method involves use of a suitable coupling agent such as 
N,N'-dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide. Other 
appropriate coupling agents are disclosed in The Peptides: Analysis, 
Structure, Biology, Vol. I: "Major Methods of Peptide Bond Formation", 
Academic Press, New York, E. Gross & J. Meinenhofer edits, 1979). 
The preferred method of solid phase coupling uses either t-Boc-protected 
amino acids or amino acid analogs or Fmoc protected amino acids or amino 
acid analogs which are coupled to the N-terminus free a-amino of the 
growing peptide chain attached to the solid phase resin. In this method, 
the coupling reagents include 1-hydroxybenzotriazole (HOBT) and 
2-(1H-benzotriazol-1-yl)-1,1,3,3 tetramethyluronium hexafluorophosphate 
(HBTU), dicyclohexylcarbodiimide (DCC) or BOP, either alone or in 
combination with 1-hydroxybenzotriazole (HOBT). Preferred methods as 
discussed above are described in Example 1. 
A preferred method of preparation of the compounds Of the present invention 
involves the use of the amino acid analog, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy) methanamido-2-hydroxyhexanoic 
acid. This intermediate of the present invention is depicted in formula 
III below: 
##STR18## 
FIG. 1 illustrates the preferred method of synthesis of this intermediate, 
which is explained in detail in Examples 2 to 7. In FIG. 1, "i" represents 
potassium cyanide, potassium bicarbonate, water; "ii" represents 
HCl/water/dioxane; "iii" represents dry HCl/methanol; "iv" represents 
Boc.sub.2 O/THF/NaHCO.sub.3 /H.sub.2 O/; "v" represents 
lithiumhydroxide/methanol/water; and "vi" represents Dowex-50 acid form. 
The coupling of requisite amino acids, amino acid analogs and other groups 
to the solid phase gives an intermediate of the present invention, the 
peptide-solid phase as shown in formula IV below: 
##STR19## 
wherein R.sub.1 and m are defined as for formula T above; A' is selected 
from the group consisting of 
##STR20## 
wherein R" is alkyl of 1 to about 6 carbons, or aralkyl of about 6 to 
about 15 carbon atoms; 
B' is a peptide represented by the formula: B.sub.1 '-B.sub.2 '-B.sub.3 
'-B.sub.4 '-B.sub.5 ', wherein 
B.sub.1 ' is peptide of 5 to 8 amino acids whose side chain group is 
protected, or B.sub.1 ' is (Gly).sub.p -X-(Gly).sub.q when B.sub.2 ' is 
Arg, Asn, Asp or Gln whose side chain group is protected, or B.sub.1 ' is 
-(Gly).sub.p+q -Gly- when B.sub.2 ' is X, wherein p and q are 
independently selected from the integers, 1 to 7, such that their sum is 4 
to 7, and X has the formula: 
##STR21## 
wherein r is an integer selected from 2 to 6, L is a chelating means for 
binding of a radioactive or paramagnetic atom, and Y is an attaching means 
for attaching chelating means; 
B.sub.2 ' is Arg, Asn, Asp or Gln whose side chain group is protected; 
B.sub.3 ' is Gly; 
B.sub.4 ' is Asp whose side chain group is protected; and 
B.sub.5 ' is Nap, Phe, Tha, Trp or Tyr whose side chain group is protected; 
C.sub.1 ' is Glu whose side chain group is protected; 
C.sub.2 ' is Ala, Glu or Pro whose side chain group is protected; 
C.sub.3 ' is Ile, Leu or Ser whose side chain group is protected; 
C.sub.4 ' is Hyp, Leu or Pro whose side chain group is protected; 
C.sub.5 ' is Asp, Glu, Ala-Asp, Ala-Glu, Asp-Asp, Asp-Glu, Glu-Asp or 
Glu-Glu whose side chain groups are protected; 
C.sub.6 ' is Ala, Ile, Tyr, Tyr(O-SO.sub.3 H), Tyr(3-iodo), 
Tyr(3,5-diiodo), Ala-Tyr, Ala-Tyr(O-SO.sub.3 H), Ala-Tyr(3-iodo) or 
Ala-Tyr(3,5-diiodo) whose side chain group or groups are protected; 
C.sub.7 ' is Ala, Asp, Cha, Leu or Tyr whose side chain group is protected; 
Z' is --O--or --NH--; and 
SP is a solid phase insoluble in solvents and solutions employed in solid 
phase peptide synthesis. 
A "side chain group" of an amino acid refers to its substituent on the 
a-carbon which characterizes the amino acid. Among these side chain 
groups, some must be protected to prevent side reactions involving their 
substituent groups during peptide synthesis, for example, when they are 
used in the tBoc or Fmoc coupling chemistries. Side chain groups having 
substituent groups that must be protected to be useful in these peptide 
synthesis methods include those associated with Arg, Asn, Asp, Cys, Gln, 
Glu, His, Lys, Orn, Ser, Thr, Trp and Tyr. Typical protecting groups are 
shown in the Examples provided hereinunder. Other protecting groups are 
known in the art. 
The hydroxy group incorporated into the intermediate represented by formula 
IV by coupling of the compound of formula III may be oxidized to a keto 
group by treatment with oxidant to provide yet another intermediate of the 
present invention represented by formula V below. 
##STR22## 
wherein, R.sub.1, m, A', B', C.sub.1 ', C.sub.2 ', C.sub.3 ', C.sub.4 ', 
C.sub.5 ', C.sub.6 ', C.sub.7 ', Z' and SP are as defined for the 
intermediates represented by formula IV. 
Upon completion of the coupling of requisite amino acids, amino acid 
analogs and other groups to give an intermediate of the present invention, 
a peptide-solid phase, the hydroxy group incorporated therein by coupling 
of the compound of formula III may be oxidized to a keto group by 
treatment with oxidant to provide yet another intermediate of the present 
invention. In the preferred method of oxidation, a peptide-solid phase is 
treated with EDAC-HCl and DCA in dry dichloromethane and dry 
dimethylsulfoxide, the details of which are given in Example 8. The 
oxidation of a-hydroxy acids using these reagents in a liquid phase system 
has been described in Edwards et al., J. Am. Chem. Soc., 114: 1854 at 1861 
(1992). 
After the desired peptide sequence is completed, the intermediate peptide 
is cleaved from the resin and the protecting groups are removed. 
Cleavage/deprotection methods would include the treatment of the 
resin-bound peptide with reagents such as hydrofluoric acid containing 
anisole or trifluoroacetic acid containing phenol, EDT and thioanisole. 
The preferred method of cleavage from the resin and deprotection is 
described in Example 8. 
The desired peptide is isolated from the cleavage/deprotection solution by 
techniques such as filtration, centrifugation or extraction with diethyl 
ether. The peptide can then be purified by high performance liquid 
chromatography (HPLC) or other such methods of protein purification. 
Exemplars of the preparation of the some of compounds of the present 
invention are found in Examples 8, 10 through 14, 19 through 22, and 34 
through 40. 
The compounds of the present invention are distinguished by their ability 
to inhibit the catalytic activity of thrombin. The compounds of the 
present invention may be prepared for assay by dissolving them in buffer 
to give solutions containing concentrations such that assay concentrations 
range from 0 and 100 mM in one assay. In the assay to determine the 
inhibitor constant, Ki, for a compound of the present invention, 
chromogenic synthetic substrate for thrombin is added to a solution 
containing test compound and thrombin and the catalytic activity of the 
enzyme is determined spectrophometrically. A preferred method of 
determining Ki is shown in Example A. 
Preferred compounds of the present invention have a Ki of less than about 
0.050 nM, particularly in this thrombin assay or an equivalent assay. 
Another aspect of the present invention provides compounds which are which 
are useful for in vivo imaging of thrombi in a mammal, wherein the 
compound represented by formulas I or II is covalently labelled with a 
radioactive atom. 
A radioactive iodine isotope such as I-123, I-125, or I-131 may be 
covalently attached to the tyrosine group of the compounds of formula I 
using radioactive sodium or potassium iodide and a chemical oxidizing 
agent, such as sodium hypochlorite, chloramine T, or the like, or by an 
enzymatic oxidizing system, such as lactoperoxidase, glucose oxidate and 
glucose, or using Boulton Hunter reagent. An embodiment of these compounds 
of the present invention and their preparation is disclosed in Example 24. 
As described above, the compounds of the present invention as depicted in 
formula II include those wherein D.sub.1 is (Gly).sub.p -X-(Gly).sub.q 
when D.sub.2 is Arg, Ash, Asp or Gln, or D.sub.1 is -(Gly).sub.p+q -Gly- 
when D.sub.2 is X, wherein p and q are independently selected from the 
integers, 1 to 7, such that their sum is 4 to 7, and X has the formula: 
##STR23## 
wherein r is an integer selected from 2 to 6, L is a chelating means for 
binding of a radioactive or paramagnetic atom, and Y is an attaching means 
for attaching chelating means. These compounds may be prepared using the 
methods disclosed above and those disclosed in Examples 26 thorugh 31. 
Compositions and Their Preparation 
A. Pharmaceutical Compositions 
The compositions and pharmaceutical compositions comprising the compounds 
of formula I of the present invention are functional inhibitors of 
thrombin, and can be used to prevent or treat a pathological condition 
characterized by thrombus formation. 
Pathological conditions characterized by thrombus formation include those 
involving the arterial and venous vasculature. With respect to the 
coronary. arterial vasculature, thrombus formation may result from the 
rupture of an established atherosclerotic plaque. Such thrombosis is the 
major cause of acute myocardial infarction and unstable angina, as well as 
also characterizing the reocclusive coronary thrombus formation following 
either thrombolytic therapy or percutaneous transluminal coronary 
angioplasty (PTCA). 
With respect to the venous vasculature, thrombus formation characterizes 
the condition observed in patients undergoing major surgery in the lower 
extremities or the abdominal area who often suffer from thrombus formation 
in the venous vasculature resulting in reduced blood flow to the affected 
extremity and a predisposition to pulmonary embolism. A systemic form of 
abnormal activation of coagulation is designated disseminated 
intravascular coagulopathy and commonly occurs within both vascular 
systems during septic shock, certain viral infections and cancer; it is a 
condition wherein there is rapid consumption of coagulation factors and 
systemic coagulation which results in the formation of life-threatening 
microvascular thrombi occurring throughout the vasculature leading to 
widespread organ failure. 
Accordingly, the present invention provides pharmaceutical compositions for 
preventing or treating a pathological condition in a mammal characterized 
by thrombus formation. These pharmaceutical compositions are comprised of 
a therapeutically effective amount of compound or compounds of the present 
invention and a pharmaceutically acceptable carrier. The "therapeutically 
effective amount" of the composition required as a dose will depend on the 
route of administration, the type of mammal being treated, and the 
physical characteristics of the specific mammal under consideration. These 
factors and their relationship to determining this dose are well known to 
skilled practitioners in the medical arts. Also, the therapeutically 
effective amount and method of administration can be tailored to achieve 
optimal efficacy but will depend on such factors as weight, diet, 
concurrent medication and other factors which those skilled in the medical 
arts will recognize. Preferred as a "therapeutically effective amount" for 
a daily dose of the pharmaceutical composition is between about 1 .mu.g/kg 
body weight of a mammal to be treated to about 5 mg/kg body weight of a 
compound or compounds of the present invention. 
The pharmaceutical compositions of the present invention containing a 
therapeutically effective amount of the compounds of the present invention 
may be formulated with a pharmaceutically acceptable carrier to provide 
sterile solutions, suspensions for injectable administration; and the 
like. In particular, injectables can be prepared in conventional forms, 
either as liquid solutions or suspensions, solid forms suitable for 
solution or suspension in liquid prior to injection, or as emulsions. 
Suitable excipients are, for example, water, saline, dextrose, mannitol, 
lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, or 
the like. In addition, if desired, the injectable pharmaceutical 
compositions may contain minor amounts of nontoxic auxilliary substances, 
such as wetting agents, pH buffering agents, and the like. If desired, 
absorption enhancing preparations (e.g., liposomes) may be utilized. 
Pharmaceutically acceptable carriers or diluents for therapeutic use are 
well known in the pharmaceutical art, and are described, for example, in 
Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro 
edit. 1985). 
The present invention also encompasses pharmaceutical compositions prepared 
for storage or administration. These would additionally contain 
preservatives, stabilizers, and dyes. For example, sodium benzoate, sorbic 
acid and esters of p-hydroxybenzoic acid may be added as preservatives. 
Id. at 1449. In addition, antioxidants and suspending agents may be used. 
Id. 
B. Compositions Containing Radioactive Atoms 
The present invention also includes compositions which are useful for in 
vivo imaging of thrombi in a mammal, wherein the compositions are 
comprised of a compound of formula I or II complexed with a radioactive 
atom. 
Compounds of formula I can be labelled with radioactive iodine as described 
above. 
For the compounds of formula II, suitable radioactive atoms include Co-57, 
Cu-67, Ga-67, Ga-68, Ru-97, Tc-99m, In-111, In-113m, Hg-197, Au-198, and 
Pb-203. Some radioactive atoms have superior properties for use in 
radiochemical imaging techniques. In particular, technetium-99m (Tc-99m) 
is an ideal radioactive atom for imaging because of its nuclear 
properties. It is a gamma emitter, and has a single photon energy of 140 
keV, a half-life of about 6 hours, and it is readily available from a 
Mo-99/Tc-99 generator. Rhenium-186 and -188 also have gamma emission which 
allows it to be imaged. Preferred compositions contain the radioactive 
atom, Tc-99m. 
Compositions of the present invention are conveniently prepared by 
complexing a compound of formula II with radioisotopes which are suitable 
for detection externally. The gamma emitters, indium-111 and 
technetium-99m, are preferred as radioactive atoms because they are 
detectable with a gamma camera and have favorable half-lives in vivo. 
The compounds FIG. II can be labelled by any of the many techniques known 
in the art to provide a composition of the present invention. For example, 
these compounds can be labelled through a chelating agent such as 
diethylenetriaminepentaacetic acid (DTPA) or metallothionein, both of 
which can be covalently attached to the compound of formula II. 
In general, the compositions of the present invention containing 
technetium-99m are prepared by forming an aqueous mixture of 
technetium-99m and a reducing agent and a water-soluble ligand, and then 
contacting the mixture with a compound of the present invention 
represented by formula II. For example, the imaging compounds of this 
invention are made by reacting technetium-99m (in an oxidized state) with 
the compounds of the present invention having a chelating means in the 
presence of a reducing agent to form a stable complex between 
technetium-99m in a reduced state (IV or V valence state). 
One embodiment of the composition of the present invention is prepared by 
labeling a compound of formula II having a DTPA chelating means with 
technetium-99m. This may be accomplished behind a lead shield by combining 
a predetermined amount (as 5mg to 0.5 mg) of compound of the present 
invention with an aqueous solution containing citrate buffer and stannous 
reducing agent, then adding freshly eluted sodium pertechnetate containing 
a predetermined level of radioactivity (as 15 mCi). After allowing an 
incubation of the mixture at room temperature, the reaction mixture is 
loaded into a shielded syringe through a sterile filter (0.2-0.22 micron), 
then is dispensed into 0.9% saline for injection, if desired. 
Another embodiment of the compositions of the present invention is prepared 
by labeling a compound of formula II having a metallothionein chelating 
means with technetium-99m. This may be accomplished by combining aqueous 
sodium pertechnetate-99m with aqueous stannous glucoheptonate to form a 
soluble complex of technetium-99m (in reduced state) with two 
glucoheptonate molecules, then combining this solution with a compound of 
the formula II having a metallothionein attached thereto. After incubating 
the mixture for a period of time and under conditions which allow for an 
exchange of the technetium-99m from the glucoheptonate complex to the 
metallothionein of the compound of formula II, the technetium-labeled 
composition of the present invention is formed. In particular, an exemplar 
of this composition and its preparation is disclosed in Example 32. 
The source of technetium-99m should preferably be water soluble. Preferred 
sources are alkali and alkaline earth metal pertechnetate 
(TcO.sub.4.sup.-). Technetium-99m is most preferably obtained in the form 
of fresh sodium pertechnetate from a sterile technetium-99m generator (as 
from a conventional Mo-99/Tc-99m generator). However, any other source of 
physiologically acceptable technetium-99m may be used. 
Reducing agents for use in the method are physiologically acceptable for 
reducing technetium-99m from its oxidized state to the IV or V valence 
state or for reducing rhenium from its oxidized state. Reducing agents 
which can be used are stannous chloride, stannous fluoride, stannous 
glucoheptonate, stannous tartarate, and sodium dithionite. The preferred 
agents are stannous reducing agents, especially stannous chloride or 
stannous glucoheptonate. The amount of reducing agent is that amount 
necessary to reduce the technetium-99m to provide for the binding to the 
chelating means of a compound of formula II in this radioisotope's reduced 
state. For example, stannous chloride (SnCl.sub.2) is the reducing agent 
and can used in range from 1-1,000 mg/mL. Especially preferred 
concentrations are about 30-500 mg/mL. 
Citric acid complexes with technetium-99m quickly to form a stable 
technetium-99m-citrate complex. Upon contact with a compound of formula 
II, substantially quantitative transfer of technetium-99m from its citrate 
complex to the chelating means of the compound of formula II is achieved 
rapidly and under mild conditions. The amount of citric acid (as sodium 
citrate) can range from about 0.5 mg/ml up to the amount maximally soluble 
in the medium. Preferred amounts of citric acid range from 15 to 30 mg/ml. 
The amount of compound of formula II having a chelating means can range 
from 0.001 to about 3 mg/mL, preferably about 0.017 to about 0.15 mg/mL. 
Finally, technetium-99m in the form of pertechnetate can be used in 
amounts of preferably about 1-50 mCi. The amount of mCi per mg of compound 
of the present invention is preferably about 30-150. 
The reaction between the compound of formula II and the metal ion-transfer 
ligand complex is preferably carried out in an aqueous solution at a pH at 
which the compound of formula II is stable. By "stable", it is meant that 
the compound remains soluble and retains its inhibitory activity against 
a-thrombin. Normally, the pH for the reaction will be from about 5 to 9, 
the preferred pH being about 6-8. The technetium-99m-citrate complex and a 
compound of formula II are incubated, preferably at a temperature from 
about 20.degree. C. to about 60.degree. C., most preferably from about 
20.degree. C. to about 37.degree. C., for a sufficient amount of time to 
allow transfer of the metal ion from the citrate complex to the chelating 
means of the compound of formula II. Generally, less than one hour is 
sufficient to complete the transfer reaction under these conditions. 
Alternative compositions of the present invention include a In-111 labeled 
compound of the present invention. An embodiment of these compositions and 
its preparation is disclosed in Example 33. This exemplar teaches 
conditions for preparation of In-111 complex with a compound of formula II 
which has thereon a DTPA chelating means. 
C. Compositions Containing Paramagnetic Atoms 
The present invention also includes compositions of the compounds of the 
present invention which are useful for in vivo imaging of thrombi in a 
mammal, comprised of a compound represented by formula II complexed to a 
paramagnetic atom. 
Preferred paramagnetic atoms are divalent or trivalent ions of elements 
with an atomic number of 21 to 29, 42, 44 and 58 to 70. Suitable ions 
include chromium(III), manganese(II), iron(III), iron(II), cobalt(II), 
nickel(II), copper(II), praseodymium(III), neodymium(III), samarium(III) 
and ytterbium(III). Because of their very strong magnetic moments, 
gadolinium(III), terbium(III), dysprosium(III), holmium(III), and 
erbium(III) are preferred. Especially preferred for the paramagnetic atom 
is gadolinium(III). 
The compositions of the present invention may be prepared by combining a 
compound of formula II with a paramagnetic atom. For example the metal 
oxide or a metal salt (for example, nitrate, chloride or sulfate) of a 
suitable paramagnetic atom is dissolved or suspended in a medium comprised 
of water and an alcohol, such as methyl, ethyl or isopropyl alcohol. This 
mixture is added to a solution of an equimolar amount of the compound of 
formula II in a similar aqueous medium and stirred. The reaction mixture 
may be heated moderately until the reaction is completed insoluble 
compositions formed may be isolated by filtering, while soluble 
compositions may be isolated by evaporation of the solvent. If acid groups 
on the chelating means are still present in the composition of the present 
invention, inorganic or organic bases, and even amino acids, may be added 
to convert the acidic complex into a neutral complex to facilitate 
isolation or purification of homogenous composition. Organic bases or 
basic amino acids may be used as neutralizing agents, as well as inorganic 
bases such as hydroxides, carbonates or bicarbonates of sodium, potassium 
or lithium. 
The compositions of the present invention may be isolated by 
crystallization by adding solvents soluble in water as lower alcohols 
(methyl, ethyl, isopropyl alcohol), lower ketones (acetone), polar ethers 
(tetrahydrofuran, dioxane, 1,2-dimethoxyethane) to an aqueous solution 
containing the compositions of the present invention. 
D. Diagnostic Compositions 
The present invention also includes diagnostic compositions which are 
useful for in vivo imaging of thrombi in a mammal, comprising a 
pharmaceutically acceptable carrier and a diagnostically effective amount 
of compositions derived from the compounds of formula I or II. 
Compositions such as those described in paragraphs B and C hereinabove may 
be conveniently used in these diagnostic compositions. 
The "diagnostically effective amount" of the composition required as a dose 
will depend on the route of administration, the type of mammal being 
treated, and the physical characteristics of the specific mammal under 
consideration. These factors and their relationship to determining this 
dose are well known to skilled practitioners in the medical diagnostic 
arts. Also, the diagnostically effective amount and method of 
administration can be tailored to achieve optimal efficacy but will depend 
on such factors as weight, diet, concurrent medication and other factors 
which those skilled in the medical arts will recognize. In any regard, the 
dose for imaging should be sufficient for detecting the presence of the 
imaging agent at the site of a thrombus in question. Typically, radiologic 
imaging will require that the dose provided by the pharmaceutical 
composition of the present invention be about 5 to 20 mCi, preferably 
about 10 mCi. Magnetic resonance imaging will require that the dose 
provided be about 0.001 to 5 mmole/kg, preferably about 0.005 to 0.5 
mmole/kg of a compound of formula II complexed with paramagnetic atom. In 
either case, it is known in the art that the actual dose will depend on 
the location of the thrombus. 
"Pharmaceutically acceptable carriers" for in vivo use are well known in 
the pharmaceutical art, and are described, for example, in Remington's 
Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). 
The pharmaceutical compositions of the present invention may be formulated 
with a pharmaceutically acceptable carrier to provide sterile solutions or 
suspensions for injectable administration. In particular, injectables can 
be prepared in conventional forms, either as liquid solutions or 
suspensions, solid forms suitable for solution or suspension in liquid 
prior to injection, or as emulsions. Suitable excipients are, for example, 
water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium 
glutamate, cysteine hydrochloride, or the like. In addition, if desired, 
the injectable pharmaceutical compositions may contain minor amounts of 
nontoxic auxilliary substances, such as wetting agents, pH buffering 
agents, and the like. If desired, absorption enhancing preparations (e.g., 
liposomes) may be utilized. 
The present invention also encompasses diagnostic compositions prepared for 
storage or administration. These would additionally contain preservatives, 
stabilizers and dyes. For example, sodium benzoate, sorbic acid and esters 
of p-hydroxybenzoic acid may be added as preservatives. Id. at 1449. In 
addition, antioxidants and suspending agents may be used. Id. 
Methods of Use 
A. Therapeutic Methods 
The methods of the present invention offer a significant advantage over the 
previous methods of preventing and arresting in vivo thrombus formation in 
mammals known in the art. This advantage is based on the fact that the 
compounds, composition and pharmaceutical compositions of the present 
invention are extremely potent inhibitors of thrombin which are not 
proteolytically degraded by thrombin. Because this provides a long-lasting 
inhibitory effect on abnormal thrombus formation in vivo, the present 
invention thereby provides novel methods useful for preventing or treating 
in a mammal a pathological condition characterized by thrombus formation. 
According to one embodiment of the present invention, a method is provided 
for treating or preventing in a mammal a pathological condition 
characterized by thrombus formation comprising administering to said 
mammal a therapeutically acceptable amount of the compound or 
pharmaceutical composition of the present invention. 
In employing the compounds, compositions or pharmaceutical compositions in 
vivo by this method, administering can be accomplished in a variety of 
ways, including parenterally, employing a variety of dosage forms. As will 
be apparent to one skilled in the art, the useful in vivo dosage to be 
administered and the particular mode of administration will wary depending 
upon the age, weight and mammalian species treated, the particular 
compounds employed, and the specific use for which these compounds are 
employed. Furthermore, the compounds, compositions or pharmaceutical 
compositions can be administered alone or in combination with one another, 
or in combination with other therapeutic or diagnostic agents. 
The compounds, compositions or pharmaceutical compositions can be 
administered in vivo, ordinarily in a mammal, preferably in a human, or in 
vitro. 
The determination of the "therapeutically effective amount" or effective 
dose of the compound, composition or pharmaceutical composition, that is, 
the dosage levels necessary to achieve the desired result, will be within 
the ability of one skilled in the medical arts. This dose will depend on 
the route of administration, the type of mammal being treated, and the 
physical characteristics of the specific mammal under consideration. These 
factors and their relationship to determining this dose are well known to 
skilled practitioners in the medical arts. Also, the therapeutically 
effect amount and method of administration can be tailored to achieve 
optimal efficacy but will depend on such factors as weight, diet, 
concurrent medication and other factors which those skilled in the medical 
arts will recognize. Preferred as a "therapeutically effective amount" for 
a daily dose of the pharmaceutical composition is between about 1 mg/kg 
body weight of a mammal to be treated to about 5 mg/kg body weight of the 
compound or compounds of the present invention. Typically, administration 
is commenced at lower dosage levels, with dosage levels being increased 
until the desired effect is achieved. Once dosage ranges are established, 
these compositions may be given as a bolus, followed by intraveneous 
administration at a predetermined rate. 
B. Diagnostic Methods. 
The in vivo imaging methods of the present invention also offer several 
advantages over previous imaging techniques for the detection or 
monitoring of the presence, size, regression or increase of a thrombus. In 
particular, the present invention provides compounds, compositions and 
diagnostic compositions have been designed to bind extremely tightly to 
the thrombin associated with a thrombus and thereby reduce "background" 
due to circulating radioactivity or paramagnetism arising from unbound 
imaging agent. Furthermore, in vivo imaging by intracoronary injection of 
the compounds, compositions or diagnostic compositions of the present 
invention, is expected to be almost instantaneous since these imaging 
agents would saturate the thrombin bound to the thrombus immediately. 
Accordingly, the present invention also includes methods for in vivo 
imaging of a thrombus in a mammal, comprising the steps of: (1) 
administering to a mammal a diagnostically acceptable amount of a 
compound, composition, or diagnostic composition of the present invention 
and (2) detecting a thrombus in a blood vessel. 
The term "in vivo imaging" as used herein relates to methods of the 
detection of a thrombus in a mammal, as well as the monitoring of the 
size, location and number of thrombi in a mammal, as well as dissolution 
or growth of the thrombus. 
In employing the compounds, compositions or diagnostic compositions in vivo 
by this method, "administering" is accomplished parenterally, in either a 
systemic or local targeted manner. Systemic administration is accomplished 
by injecting the compounds, compositions or diagnostic compositions of the 
present invention into a convenient and accessible vein or artery. This 
includes but is not limited to administration by the antecubutal vein. 
Local targeted administration is accomplished by injecting the compounds, 
compositions or diagnostic compositions of the present invention proximal 
in flow to a vein or artery suspected to contain thrombi distal to the 
injection site. This includes but is not limited to direct injection into 
the coronary arterial vasculature to image coronary thrombi, into the 
carotid artery to image thrombi in the cerebral vasculature, or into a 
pedal vein to image deep vein thrombosis of the leg. 
Also, the manner of delivery of a composition of the present invention to 
the site of a thrombus is considered within the scope of the term 
"administering". For example, a compound represented by formula II having 
a chelating means attached thereto may be injected into the mammal, 
followed at a later time by the radioactive atom thereby forming in vivo 
at the site of the thrombus the composition comprising the compound of 
formula II complexed to radioactive atom. Alternatively, a composition 
comprising the compound of formula II complexed to radioactive atom may be 
injected into the mammal. 
The "diagnostically effective amount" of the compounds, compositions or 
diagnostic compositions used in the methods of the present invention will, 
as previously mentioned, depend on the route of administration, the type 
of mammal being treated, and the physical characteristics of the specific 
mammal under treatment. These factors and their relationship to 
determining this dose are well known to skilled practitioners in the 
medical diagnostic arts. In any regard, the dose for in vivo imaging 
should be sufficient for detecting the presence of the imaging agent at 
the site of a thrombus in question. Typically, radiologic imaging will 
require that the dose provided by the diagnostic composition of the 
present invention be about 5 to 20 mCi, preferably about 10 mCi. Magnetic 
resonance imaging will require that the dose provided by the diagnostic 
composition be about 0.001 to 5 mmole/kg, preferably about 0.005 to 0.5 
mmole/kg of a compound of formula II complexed with paramagnetic atom. In 
either case, it is known in the art that the actual dose will depend on 
the location of the thrombus. 
The detecting of a thrombus by imaging is made possible by the presence of 
radioactive or paramagnetic atoms localized at such thrombus. 
The radioactive atoms associated with the compositions and diagnostic 
compositions of the present invention are preferably imaged using a 
radiation detection means capable of detecting gamma radiation, such as a 
gamma camera or the like. Typically, radiation imaging cameras employ a 
conversion medium (wherein the high energy gamma ray is absorbed, 
displacing an electron which emits a photon upon its return to the orbital 
state), photoelectric detectors arranged in a spatial detection chamber 
(to determine the position of the emitted photons), and circuitry to 
analyze the photons detected in the chamber and produce an image. 
The paramagnetic atoms associated with the compositions and diagnostic 
compositions of the present invention detected in magnetic resonance 
imaging (MRI) systems. In such systems, a strong magnetic field is used to 
align the nuclear spin vectors of the atoms in a patient's body. The field 
is disturbed by the presence of paramagnetic atoms localized at a thrombus 
and an image of the patient is read as the nuclei return to their 
equilibrium alignments. 
To assist in understanding the present invention, the following examples 
are included which describe the results of a series of experiments. The 
following examples relating to this invention should not, of course, be 
construed as specifically limiting the invention and such variations of 
the invention, now known or later developed, which would be within the 
purview of one skilled in the art are considered to fall within the scope 
of the invention as described herein and hereinafter claimed. 
EXAMPLES 
Example 1 
General Solid-Phase Synthesis Methods 
The solid phase syntheses of the compounds of the present invention were 
performed using an Applied Biosystems Model 430A peptide synthesizer. 
Either t-Boc or Fmoc chemistry was used to implement coupling of suitably 
protected amino acids to the resin or growing peptide chain thereon. The 
resin wash step as used herein involved placing the resin in 5 to 7 mL of 
a specified solvent, followed by agitation of the mixture for about 1 
minute. All steps are conducted at room temperature unless stated 
otherwise. 
t-BOC Coupling Protocol: 
1. Starting resin (having thereon 0.5 mmole of covalently attached amino 
acid) was transferred to a 40 mL reaction vessel. 
2. The resin was washed once with dichloromethane. 
3. The wash was drained, then 5 to 7 mL 25% trifluoroacetic acid (in 
dichloromethane) was added and the mixture was agitated for about 3 
minutes. 
4. The liquid was drained, then 5-7 mL of 50% trifluoroacetic acid (in 
dichloromethane) was added to the resin and the mixture was agitated for 
about 16 minutes. 
5. The liquid was drained, then the resin was washed five times with 
dichloromethane. 
6. The resin was washed twice with 5% diisopropylethylamine (in 
N-methylpyrrolidone). 
7. The resin was washed six times with N-methylpyrrolidone. 
8. 3.3 mL N-methylpyrrolidone was added to 2 mmole of the N-a-t-Boc amino 
acid, followed by 2 mL of 1M HOBT (in N-methylpyrrolidone). The mixture 
was intermittently mixed for about 3 minutes, then was transferred to 2 mL 
of 1M dicyclohexylcarbodiimide (in N-methylpyrrolidone). This mixture was 
intermittently mixed for about 25 minutes, then resulting dicyclohexylurea 
was filtered out by transferring the mixture containing the activated 
N-a-t-Boc amino acid into the reaction vessel containing the resin. The 
resin-containing solution was then agitated for about 60 minutes. 
9. The liquid was drained, then the resin was washed five times with 
dichloromethane. 
10. The coupling cycle (steps 2 to 8) was repeated starting at step 2 until 
the desired peptide was completed. 
Fmoc Coupling Protocol: 
1. Starting resin (having thereon 0.25 mmole of covalently attached amino 
acid) was transferred to a 40 mL reaction vessel. 
2. The resin was washed once with N-methylpyrrolidone. 
3. The liquid was drained, then 5 to 7 mL 20% piperidine (in 
N-methylpyrrolidine) was added and the mixture was agitated for about 6 
minutes. 
4. The liquid was drained, then the resin was washed five times with 
N-methylpyrrolidone. 
5. 2.4 mL N-methylpyrrolidone was added to 1 mmole of the N-a-Fmoc amino 
acid, followed by 2.2 mL of 0.45M HBTU-HOBT (in dimethylformamide) and 
then the mixture was intermittently mixed for about 6 minutes. The mixture 
was transferred to the reaction vessel containing the resin. 0.34 mL of 
diisopropylethylamine was then added and the solution was agitated for 
about 30 minutes. 
6. The liquid was drained, then the resin was washed five times with 
N-methylpyrrolidone. 
7. The coupling cycle (steps 2 to 6) was repeated starting at step 2 until 
the desired peptide was completed. 
Example 2 
Preparation of alpha-N-t-butoxycarbonyl-N.sup.g -nitroargininal 
##STR24## 
A. Procedure 1: 
The following procedure for the synthesis of a-t-butoxycarbonyl-N.sup.g 
-nitro-argininal 2 is an example of a general procedure for the 
preparation of Boc-amino acid aldehydes, see Patel et al., Biochim. 
Biophys. Acta, 748, 321-330 (1983). 
In 200 mL dry THF, 12.7 g Boc-N.sup.g -nitro-arginine (40 mmoles) and 7.0 g 
carbonyldiimidazole (CDI; 43 mmoles) were added at room temperature and 
allowed to stir for 30 minutes. The reaction mixture was cooled to 
-78.degree. C. and 35 mL of a solution of LiAlH.sub.4 (1M in THF) were 
added dropwise over thirty minutes. The reaction was allowed to stir for 
an additional hour at -78.degree. C. Next, 18 mL of acetone were added and 
this mixture was quickly added to 400 mL of 1N HCl. The mixture was 
extracted twice with 100 mL of ethyl acetate. The ethyl acetate washes 
were combined and then washed two times each with 100 mL water, 100 mL 
saturated NaHCO.sub.3 and 100 mL saturated NaCl. The solution was dried 
(MgSO.sub.4) and concentrated to a foam. The crude weight of the 
a-t-butoxycarbonyl-N.sup.g -nitro-argininal was 6.36 g (21 mmole; yield 
52%). 
B. Procedure 2: 
Alternatively, 2 was synthesized by a modification of the procedure of 
Fehrentz, J. A. and Castro, B., Synthesis, 676 (1983). 
11.4 mL of N-methyl piperidine was slowly added to a stirred suspension of 
8.42 g (94 mmole) of N,O-dimethylhydroxylamine in 75 mL dichloromethane 
which had been cooled to about 0.degree. C. The solution was allowed to 
stir for 20 minutes which gave the free hydroxylamine, then was kept cold 
for use in the next step. 
In a separate flask, 30.0 g (94 mmole) of Boc-N.sup.g -nitroarginine was 
dissolved by heating in about 1400 mL of tetrahydrofuran, then the mixture 
was cooled under nitrogen to 0.degree. C. 11.4 mL of N-methylpiperidine 
and 12.14 mL (94 mmole) of isobutylchloroformate was added and the mixture 
stirred for 10 minutes. The free hydroxylamine prepared above was added 
all at once and the reaction mixture was allowed to warm to room 
temperature, then stirred overnight. 
The resulting precipitate was filtered off, then washed with 200 mL of 
tetrahydrofuran. After concentrating the filtrates to about 150 mL under 
vacuum, 200 mL of ethyl acetate was added, followed by ice to cool the 
solution. The cooled ethyl acetate phase was washed with two 75 mL 
portions of 0.2N hydrochloric acid, two 75 mL portions of 0.5N sodium 
hydroxide, one portion of 75 mL of brine, then the organic phase was dried 
over anhydrous magnesium sulfate. Upon concentration in vacuum, 22.7 g 
(70% yield) of solid Boc-N.sup.g -nitroarginine 
N-methyl-O-methylcarboxamide was recovered. Thin layer chromatographic 
analysis in 9:1 dichloromethane/methanol (silica gel) showed one spot. 
A flask was placed under a nitrogen atmosphere and cooled to -50.degree. 
C., then charged with 70 mL (70 mmole) of 1N lithium aluminum hydride (in 
tetrahydrofuran) and 500 mL of dry tetrahydrofuran. 50 mL of a solution 
containing 66 mmole of Boc-N.sup.g -nitroarginine 
N-methyl-O-methylcarboxamide in dry tetrahydrofuran was slowly added while 
the temperature of the reaction mixture was maintained at -50.degree. C. 
After allowing the reaction mixture to warm to 0.degree. C. by removal of 
the cooling, it was recooled to -30.degree. C., at which temperature, 100 
mL (0.2 mole) of 2N potassium bisulfate was added with stirring over about 
a 10 to 15 minute period. The reaction mixture was then allowed to stir at 
room temperature for 2 hours. After filtering off the precipitate, the 
filtrate was concentrated to 100 mL under vacuum. The concentrate was 
poured into 800 mL ethyl acetate, then was successively washed with two 50 
mL portions of 1N hydrochloric acid, two 50 mL portions of saturated 
sodium bicarbonate, one 50 mL portion of brine. The combined aqueous 
extracts were extracted with 3-100 mL portions of ethyl acetate. All of 
the ethyl acetate washes were combined, then was dried over anhydrous 
magnesium sulfate. The mixture was concentrated under vacuum to yield 18.5 
g (95%) of the title compound. 
Example 3 
Preparation of 
N-(nitroguanidino-1-(S)-(cyanohydroxymethyl)butyl)-1-(1,1-dimethylethoxy)m 
ethanamide 
##STR25## 
A solution of 25.2 g (83.1 mmoles) of alpha-Boc-N.sup.g -nitro-argininal 2 
in 680 mL tetrahydrofuran was added to a solution of 136 g (1.36 moles) of 
potassium bicarbonate and 27.6 g (423 mmoles) of potassium cyanide in 680 
mL of water. This two phase mixture was allowed to stir vigorously for 
thirty minutes. The stirring was discontinued and the phases were 
separated. The aqueous phase was extracted three times with 500 mL ethyl 
acetate. The tetrahydrofuran phase was diluted with 1000 mL of ethyl 
acetate. The organic phases were combined and extracted successively with 
water and brine. This solution was dried over anhydrous magnesium sulfate 
and concentrated under vacuum to give 28.1 g of the above-identified 
product as a white foam. This material can be purified by flash 
chromatography (0 to 6% methanol in dichloromethane) or carried through 
the next steps directly. .sup.1 H NMR (CD.sub.3 OD) d 1.37 (s, 9H), 1.53 
(m, 2H), 1.7 (m, 2H), 3.19 (m, 2H), 3.65 (m, 1H), 4.29 (d, J=7 Hz, 0.35H), 
4.48 (d, J=4 Hz, 0.65H). 
Example 4 
Preparation of 6-nitroguanidino-3-(S)-amino-2-hydroxyhexanoic Acid 
Hydrochloride Salt 
##STR26## 
The 26.0 g (.about.83 mmole) crude cyanohydrin 3 was dissolved in 450 mL 
dioxane, and 450 mL concentrated aqueous hydrochloric acid was slowly 
added with stirring. This addition was accompanied by vigorous gas 
evolution. This solution was heated to reflux and stirred for 15 hours. 
After this period of time, the reaction was allowed to cool to room 
temperature and then concentrated under vacuum to a thick brown syrup of 
6-nitroguanidino-3-(S)-amino-2-hydroxyhexanoic acid hydrochloride salt. 
This was used directly in the next step. 
Example 5 
6-Nitroguanidino-3-(S)-amino-2-hydroxyhexanoic Acid Methyl Ester 
##STR27## 
This crude acid 4 was concentrated several times from methanol under vacuum 
and then dissolved in 750 mL of saturated anhydrous hydrochloric acid in 
methanol. This suspension was refluxed for three hours, allowed to cool to 
room temperature and concentrated under vacuum. This gave crude 
6-nitroguanidino-3-(S)-amino-2-hydroxyhexanoic acid methyl ester 
hydrochloride salt as a thick brown syrup. This was used directly in the 
next step. 
Example 6 
6-Nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
Acid Methyl Ester 
##STR28## 
The amino ester 5 from above was dissolved in a mixture of 300 mL of 
saturated sodium bicarbonate and 300 mL tetrahydrofuran. This mixture was 
treated with di-t-butyldicarbonate (30 g, 137 mmoles) and allowed to stir 
vigorously for 16 hours. The resulting mixture was extracted with ethyl 
acetate (1000 mL). The organic layer was washed successively with water 
then brine, dried over anhydrous magnesium sulfate and concentrated to a 
small volume under vacuum. The product was purified by flash 
chromatography (0 to 10% methanol/dichloromethane) to give 13.5 g (49% 
yield) of the above-identified product as an off-white foam. .sup.1 H NMR 
(CDCl.sub.3) d 1.41 and 1.45 (s, 9H), 1.7 (m, 4H), 3.2 (m, 2H); 3.82 and 
3.84 (s, 3H), 4.10 (m, 1H), 4.19 (bs, 0.65H), 4.33 (bs, 0.35H), 5.02 (d, 
J=10 Hz; 1H), 5.17 (d, J=10 Hz, 1H). 
Example 7 
Preparation of 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
Acid 
##STR29## 
A solution of the compound 6 (5.0 g, 13.8 mmole) in 100 mL of methanol was 
treated with 17.mL of 1M lithium hydroxide. This solution was allowed to 
stir overnight and then treated with 20 mL of Dowex-50 resin X8 400 
(H.sup.+ form) in 50 mL of deionized water. This solution was swirled for 
15 minutes then passed through a 4.times.4 cm. column of the same resin, 
the column was washed with 1:1 methanol:water and the combined filtrates 
were concentrated to dryness under vacuum. The residue was dissolved in 
100 mL acetonitrile and concentrated to dryness, this process was repeated 
two more times to give 4.2 g (87 % yield) of the above-identified compound 
as an off-white foam. .sup.1 H NMR (CD.sub.3 OD) d 1.42 and 1.42 (s, 9H), 
1.7 (m, 4H), 3.3 (m, 2H), 3.95 (m, 1H), 4.19 (bs, 0.65H), 4.33 (bs, 
0.35H), 4.15 (d, J=l Hz, 0.65H), 4.38 (d, J=4 Hz). 
Example 8 
Preparation of 
##STR30## 
This compound was prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling 
Boc-L-leucine-Pam Resin, the starting resin, was purchased from Advanced 
ChemTech (Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine was first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-proline, N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl 
ester, N-Boc-L-phenylalanine, N-Boc-L-aspartic acid-b-cyclohexyl ester, 
N-Boc-glycine, N-Boc-asparagine, N-Boc-glycine, N-Boc-glycine, 
N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
acid 7, N-Boc-L-proline, and N-Boc-L-aspartic acid-b-cyclohexyl ester. In 
the final coupling cycle, 2 mmole of 2-propylpentoic acid was coupled in 
the same manner as described for the N-Boc amino acids. 
(b) Oxidation 
The peptide resin was transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide was oxidized to a keto group 
by treating the resin to two oxidation cycles. Each oxidation cycle was 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and.2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 4 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. 
(c) Deprotection and Removal 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin were transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture was cooled 
to -20.degree. C. and 10 mL of hydrofluoric acid (HF) was distilled into 
the reaction vessel. The mixture was first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether was added, then 
decanted. The resin was then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 0.1M ammonium bicarbonate. The extracts were combined and then 
extracted with 2-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase was frozen and lyophilized to yield crude 
product. The resin was further extracted with 50 mL of 40% 
acetonitrile+0.1% trifluoroacetic acid (in water), the extract stripped of 
acetonitrile in vacuo, frozen, and then lyophilized to yield a more crude 
product. 
(d) HPLC Purification 
The crude product was dissolved in 20% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and was put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent was monitored at 210 nm. A 20 minute 
gradient of 20% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) was run at a flowrate of 1 mL/minute. Title compound 
was collected at a retention time of 19.0 minutes. Fast atom bombardment 
mass spectrometry gave observed molecular weight of 2133.8 a.m.u.; 
calculated molecular weight was 2133.3 a.m.u. 
Example 9 
Preparation of 
##STR31## 
This compound was prepared using the Fmoc Coupling Protocol as described in 
Example 1, followed by deprotection and removal from the resin, and HPLC 
purification. 
(a) Preparation on Resin 
Fmoc-L-leucine-Pam Resin, the starting resin, was purchased from Advanced 
ChemTech (Louisville, Ky.). 
N-Fmoc-O-t-butyl-L-tyrosine was first coupled to the resin, followed by 
N-Fmoc-L-glutamic acid-g-t-butyl ester, N-Fmoc-L-proline, 
N-Fmoc-L-isoleucine, N-Fmoc-L-glutamic acid-g-t-butyl ester, 
N-Fmoc-L-glutamic acid-g-t-butyl ester, N-Fmoc-L-phenylalanine, 
N-Fmoc-L-aspartic acid-b-t-butyl ester, N-Fmoc-glycine, 
N-a-Fmoc-N-b-(trityl)-L-asparagine, N-Fmoc-glycine, N-Fmoc-glycine, 
N-Fmoc-glycine, N-Fmoc-glycine, N-Fmoc-L-proline, N-a-Fmoc-N.sup.g 
-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginine, N-Fmoc-L-proline, 
and N-Boc-L-aspartic acid-b-t-butyl ester. In the final coupling cycle, 2 
mmole of 2-propylpentoic acid was coupled in the same manner as described 
for the N-Fmoc amino acids. 
(b) Deprotection and Removal from Resin 
Ten mL of trifluoroacetic acid, 0.75 g of phenol, 0.25 mL of ethanedithiol 
(EDT), 0.5 mL of water, and 0.5 mL of thioanisole were combined to give 
cleavage mixture. The cleavage mixture was cooled on an ice bath, 
transferred to 0.30 g of the peptide resin. After stirring the reaction 
mixture for 2.5 hours at room temperature, the resin was filtered off, 
washed with 3-15 mL portions of trifluoroacetic acid, and 3-15 mL portions 
of dichloromethane. The combined filtrates were then concentrated in vacuo 
to an oil and titurated with 5 mL of diethyl ether to yield a participate. 
The participate was filtered off, then redissolved in 50 mL of water. The 
solution was extracted with 3-25 mL portions diethyl ether, then frozen 
lyophilized to yield crude product. 
(c) HPLC Purification 
The crude product was dissolved in 15% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and was put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent was monitored at.210 nm. A 20 minute 
gradient of 15% to 40% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) was run at a flowrate of 1 mL/minute. Title compound 
was collected at a retention time of 18.5 minutes. Fast atom bombardment 
mass spectrometry gave observed molecular weight of 2144.4 a.m.u.; 
calculated molecular weight was 2145.3 a.m.u. 
Example 10 
Preparation of 
##STR32## 
This compound was prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling 
Boc-L-leucine-Pam Resin, the starting resin, was purchased from Advanced 
Chemtech (Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine was first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-proline, 
N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-Boc-asparagine, 
N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
acid 7, N-Boc-L-proline, and N-Boc-L-aspartic acid-b-cyclohexyl ester. In 
the final coupling cycle, 2 mmole of 2-propylpentoic acid was coupled in 
the same manner as described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin was transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide was oxidized to a keto group 
by treating the resin to three oxidation cycles. Each oxidation cycle was 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 4 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. In the last two oxidation cycles, the oxidation time was 
3 hours for each cycle. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin were transferred to a plastic reaction vessel. After purging-the 
vessel and associated lines with nitrogen, the reaction mixture was cooled 
to -20.degree. C. and 10 mL of hydrofluoric acid (HF) was distilled into 
the reaction vessel. The mixture was first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether was added, then 
decanted. The resin was then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts were combined and 
then extracted with 3-25 mL portions of diethyl ether, saving the aqueous 
phase each time. The aqueous phase was frozen and lyophilized to yield 
crude product. 
(d) HPLC purification. 
The crude product was dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and was put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent was monitored at 210 nm. A 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) was run at a flowrate of 1 mL/minute. Title compound 
was collected at a retention time of 12.0 minutes. Fast atom bombardment 
mass spectrometry gave observed molecular weight of 2262.0 a.m.u.; 
calculated molecular weight was 2262.4 a.m.u. 
Example 11 
Preparation of 
##STR33## 
This compound was prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, was purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine was first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-proline, 
N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-a-Boc-N.sup.g 
-tosyl-L-arginine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
N-Boc-glycine, N-Boc-glycine, 6-nitroguanidino-3-(S)-(1,1-dimethylethoxy) 
methanamido-2-hydroxyhexanoic acid 7, N-Boc-L-proline, and 
N-Boc-L-aspartic acid-b-cyclohexyl ester. In the final coupling cycle, 2 
mmole of 2-propylpentoic acid was coupled in the same manner as described 
for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin was transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide was oxidized to a keto group 
by treating the resin to three oxidation cycles. Each oxidation cycle was 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 2 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. In the last two oxidation cycle, the oxidation time was 2 
hours for each cycle. After the oxidation was complete, the resin was 
washed three times 5 to 7 mL each with dimethylformamide, dichloromethane, 
methanol and diethylether. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin were transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture was cooled 
to -20.degree. C. and 15 mL of hydrofluoric acid (HF) was distilled into 
the reaction vessel. The mixture was first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether was added, then 
decanted. The resin was then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts were combined and 
then extracted with 3-25 mL portions of diethyl ether, saving the aqueous 
phase each time. The aqueous phase was frozen and lyophilized to yield 
crude product. 
(d) HPLC purification. 
The crude product was dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and was put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent was monitored at 210 nm. A 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) was run at a flowrate of 1 mL/minute. Title compound 
was collected at a retention time of 12.0 minutes. Fast atom bombardment 
mass spectrometry gave observed molecular weight of 2304.6 a.m.u.; 
calculated molecular weight was 2304.5 a.m.u. 
Example 12 
Preparation of 
##STR34## 
This compound is prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, is purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine is first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-proline, N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl 
ester, N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-Boc-asparagine, 
N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2- hydroxyhexanoic 
acid 7, N-Boc-L-proline, and N-Boc-L-aspartic acid-b-methyl ester. In the 
final coupling cycle, 2 mmole of 2-propylpentoic acid is coupled in the 
same manner as described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin is transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide is oxidized to a keto group 
by treating the resin to three oxidation cycles. Each oxidation cycle is 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 2 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. In the last two oxidation cycle, the oxidation time is 2 
hours for each cycle. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin are transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture is cooled 
to -20.degree. C. and 10 mL of hydrofluoric acid (HF) was distilled into 
the reaction vessel. The mixture is first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether is added, then 
decanted. The resin is then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts are combined and then 
extracted with 3-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase is frozen and lyophilized to yield crude 
product. 
(d) HPLC purification. 
The crude product is dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent is monitored at 210nm. A 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. The title 
compound was collected at a retention time of 19.2 minutes. Fast atom 
bombardment mass spectrometry gave an observed molecular weight of 2275.4 
a.m.u.; the calculated molecular wieght was 2275.0. 
Example 13 
Preparation of 
##STR35## 
This compound was prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, was purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine was first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-proline, 
N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-Boc-asparagine, 
N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
acid 7, N-Boc-L-proline, and N-Boc-L-aspartic acid-b-methyl ester. In the 
final coupling cycle, 2 mmole of 2-propylpentoic acid was coupled in the 
same manner as described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin was transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide was oxidized to a keto group 
by treating the resin to three oxidation cycles. Each oxidation cycle was 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide;. deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 2 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. In the last two oxidation cycles, the oxidation time was 
2 hours for each cycle. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin were transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture was cooled 
to -20.degree. C. and 10 mL of hydrofluoric acid (HF) was distilled into 
the reaction vessel. The mixture was first stirred for 30 minutes at 
-20.degree. C., then for 90 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether was added, then was 
decanted. The resin was then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts were combined and 
then extracted with 3-25 mL portions of diethyl ether, saving the aqueous 
phase each time. The aqueous phase was frozen and lyophilized to yield 
crude product. 
(d) HPLC purification. 
The crude product was dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent was monitored at 210 nm. A 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. 
The title compound was collected at a retention time of 19.2 minutes. Fast 
atom bombardment mass spectrometry gave an observed molecular weight of 
2275.4 a.m.u.; the calculated molecular weight was 2275.0. 
Example 14 
Preparation of 
##STR36## 
This compound is prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, is purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine is first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-proline, 
N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-a-Boc-N.sup.g 
-tosyl-L-arginine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
N-Boc-glycine, N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
acid 7, N-Boc-L-proline, and N-Boc-L-aspartic acid-b-methyl ester. In the 
final coupling cycle, 2 mmole of 2-propylpentoic acid is coupled in the 
same manner as described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin is transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide is oxidized to a keto group 
by treating the resin to three oxidation cycles. Each oxidation cycle is 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 2 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. In the last two oxidation cycle, the oxidation time is 2 
hours for each cycle. After the oxidation was complete, the resin was 
washed three times 5 to 7 mL each with dimethylformamide, dichloromethane, 
methanol and diethylether. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin are transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture is cooled 
to -20.degree. C. and 15 mL of hydrofluoric acid (HF) is distilled into 
the reaction vessel. The mixture is first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether is added, then 
decanted. The resin is then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts are combined and then 
extracted with 3-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase is frozen and lyophilized to yield crude 
product. 
(d) HPLC purification. 
The crude product is dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent is monitored at 210nm. A 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. 
Example 15 
Preparation of 3-cyano-2-(1,1-dimethylethoxy) methanamido propionic acid 
##STR37## 
20.0 g (86 mmol, 1 equiv.) of Boc-L-asparagine (from Bachem or Sigma) was 
dissolved in 120 ml of dry pyridine and 20.0 g (97 mmol, 1.3 equiv.) of 
dicyclohexylcarbodiimide dissolved in 60 ml of dry pyridine was added 
dropwise over a period of 30 minutes. The reaction was stirred for 3 hours 
at 23.degree. C. and filtered through a 2 .mu.m nylon filter. The filtrate 
was concentrated in vacuo on a rotovap and 100 ml of water was added. The 
pH was adjusted to 10 with 40% sodium hydroxide (aq.) and the solution 
filtered through a 2 .mu.m nylon filter once again. The filtrate was 
passed through a 120 ml bed of Dowex 50.times.8-400 ion exchange resin and 
the resin washed with four column volumes of 1:1 methanol:water. The 
filtrate was concentrated in vacuo to yield 17.5g (95% yield) of product 
as a white solid. .sup.1 H-NMR (CD.sub.3 OD): 4.40 p.p.m (m, 1H); 2.95 
p.p.m. (m, 2H); 1.40 p.p.m. (s, 9H). 
Example 16 
Preparation of 3-tetrazolyl-2-(1,1-dimethylethoxy)methanamido propionic 
acid 
##STR38## 
17.5 g (82 mmol, 1 equiv.) of 3-cyano-2-(1,1-dimethylethoxy) 
methanamido-propionic acid 15 was dissolved in 125 mL of tetrahydrofuran 
and 40.5 g (129 mmol, 1.5 equiv.) was added. The reaction mixture was 
brought to reflux and held there for 3 days. The reaction mixture was 
cooled and the volatiles removed in vacuo on the rotovap. The residue was 
dissolved in 300 mL of 0.5M sodium hydroxide and this aqueous solution was 
washed with ethyl acetate (4.times.100 mL). The aqueous layer was passed 
through a 125 mL bed of Dowex 50.times.8-400 ion exchange resin and the 
resin washed with four column volumes of 1:1 methanol:water. The volatiles 
were removed in vacuo on the rotovap to yield 17.9 g of the product as a 
white solid (85% yield). .sup.1 H-NMR (CD.sub.3 OD): 4.55 p.p.m (m, 1H); 
3.40 p.p.m. (m, 2H); 1.40 p.p.m. (s, 9H). This material is suitable for 
use in solid-phase peptide synthesis. 
Example 17 
Preparation of 
3-(N-2-methyl)tetrazolyl-2-(1,1-dimethylethoxy)methanamidopropionic acid, 
methyl ester and 
3-(N-3-methyl)tetrazolyl-2-(1,1-dimethylethoxy)methan-amidopropionic acid, 
methyl ester 
##STR39## 
1.5 g (5.8 mmol, 1.0 equiv.) of 
3-tetrazolyl-2-(1,1-dimethylethoxy)methan-amidopropionic acid 16 was 
dissolved in 13 mL of dry dimethylformamide and 3.9 g (12.0 mmol, 2.1 
equiv.) of cesium carbonate was added. This was followed by the addition 
of 930 .mu.L (14.5 mmol, 2.5 equiv.) of methyl iodide via syringe. The 
reaction mixture was stirred at 23.degree. C. for 3 hours and poured into 
50 mL of 0.5M hydrochloric acid. The resulting mixture was extracted with 
ethyl acetate (3.times.50 mL). The combined organics were washed with 50 
mL 0.5M hydrochloric acid, 50 mL saturated sodium bicarbonate, and 50 mL 
brine. After drying over sodium sulfate, the organics were decanted and 
the volatiles removed in vacuo on the rotary evaporator to yield a mixture 
of the title compounds as a yellow oil. The isomers were separated by 
chromatography on silica gel (50% ethyl acetate/hexane) with one isomer 
eluting first (Rf=0.3 vs. Rf=0.15 of the other isomer on silica gel 
developing in 50% ethyl acetate/hexane). Fractions containing pure product 
were combined and the volatiles removed on the rotovap to yield 0.60 g of 
pure product for each of the title compounds. .sup.1 H-NMR (CDCl.sub.3): 
The second-eluting isomer gave 5.8 p.p.m (d, 1H); 4.75 p.p.m (m, 1H); 4.05 
p.p.m (s, 3H); 3.75 p.p.m. (s, 3H); 3.4 p.p.m (m, 2H); 1.5 p.p.m. (s, 9H). 
The first-eluting isomer gave: 5.75 p.p.m (d, 1H); 4.75 p.p.m (m, 1H); 
4.30 p.p.m (s, 3H); 3.75 p.p.m. (s, 3H); 3.65 p.p.m (m, 2H); 1.7 p.p.m. 
(s, 9H). 
Example 18 
Preparation of 3-(N-2-methyl)tetrazolyl-2-(1,1-dimethylethoxy) 
methanamidopropionic acid or 
3-(N-3-methyl)tetrazolyl-2(1,1-dimethylethoxy)methanamidopropionic 
##STR40## 
0.5 g (1.75 mmol, 1.0 equiv.) of 
3-(N-2-methyl)tetrazolyl-2-(1,1-dimethylethoxy)methanamidopropionic acid 
methyl ester (or 
3-(N-3-methyl)tetrazolyl-2-(1,1-dimethylethoxy)methanamidopropionic acid 
methyl ester) 17 is dissolved in 12 mL of methanol and 2.3 mL (1.3 equiv.) 
of 1.0M lithium hydroxide (aq.) is added. The reaction is stirred for 2 
hours at 23.degree. C. when starting material can no longer be seen by TLC 
analysis (1:1 ethyl acetate/hexane). The reaction mixture is passed 
through a 10 mL bed of Dowex 50.times.8-400 ion exchange resin and the 
resin is washed with four column volumes of 1:1 methanol:water. The 
solvents are removed in vacuo to yield the title product. 
Example 19 
Preparation of 
##STR41## 
This compound is prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, is purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine is first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-proline, N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl 
ester, N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-Boc-asparagine, 
N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
acid 7, N-Boc-L-proline, and 3-tetrazolyl-2- 
(1,1-dimethylethoxy)methanamido propionic acid 16. In the final coupling 
cycle, 2 mmole of 2-propylpentoic acid is coupled in the same manner as 
described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin is transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide is oxidized to a keto group 
by treating the resin to two oxidation cycles. Each oxidation cycle is 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 4 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin are transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture is cooled 
to -20.degree. C. and 10 mL of hydrofluoric acid (HF) is distilled into 
the reaction vessel. The mixture is first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether is added, then 
decanted. The resin is then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts are combined and then 
extracted with 3-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase is frozen and lyophilized to yield crude 
product. 
(d) HPLC purification. 
The crude product is dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent is monitored at 210 nm. A. 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. 
Example 20 
Preparation of 
##STR42## 
This compound is prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, is purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine is first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-proline, 
N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-a-Boc-N.sup.g 
-tosyl-L-arginine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
N-Boc-glycine, N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
acid 7, N-Boc-L-proline, and 
3-tetrazolyl-2-(1,1-dimethylethoxy)methanamido propionic acid 16. In the 
final coupling cycle, 2 mmole of 2-propylpentoic acid is coupled in the 
same manner as described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin is transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide is oxidized to a keto group 
by treating the resin to three oxidation cycles. Each oxidation cycle is 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 2 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. In the last two oxidation cycle, the oxidation time is 2 
hours for each cycle. After the oxidation was complete, the resin was 
washed three times 5 to 7 mL each with dimethylformamide, dichloromethane, 
methanol and diethylether. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin are transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture is cooled 
to -20.degree. C. and 15 mL of hydrofluoric acid (HF) is distilled into 
the reaction vessel. The mixture is first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether is added, then 
decanted. The resin is then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts are combined and then 
extracted with 3-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase is frozen and lyophilized to yield crude 
product. 
(d) HPLC purification. 
The crude product is dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent is monitored at 210 nm. A 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. 
Example 21 
Preparation of 
##STR43## 
This compound is prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, is purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine is first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-proline, N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl 
ester, N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-Boc-asparagine, 
N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2- hydroxyhexanoic 
acid 7, N-Boc-L-proline, and 
3-(N-2-methyl)tetrazolyl-2-(1,1-dimethylethoxy) methanamidopropionic acid 
18. In the final coupling cycle, 2 mmole of 2-propylpentoic acid is 
coupled in the same manner as described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin is transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide is oxidized to a keto group 
by treating the resin to two oxidation cycles. Each oxidation cycle is 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 4 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin are transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture is cooled 
to -20.degree. C. and 10 mL of hydrofluoric acid (HF) is distilled into 
the reaction vessel. The mixture is first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether is added, then 
decanted. The resin is then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts are combined and then 
extracted with 3-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase is frozen and lyophilized to yield crude 
product. 
(d) HPLC purification. 
The crude product is dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and was put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent was monitored at 210nm. A 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) was run at a flowrate of 1 mL/minute. 
Example 22 
Preparation of 
##STR44## 
This compound is prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, is purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine is first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-proline, 
N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc -glycine, N-a-Boc-N.sup.g 
-tosyl-L-arginine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
N-Boc-glycine, N-Boc-glycine, 6-nitroguanidino-3-(S)-(1,1-dimethylethoxy) 
methanamido-2-hydroxyhexanoic acid 7, N-Boc-L-proline, and 3-(N-2-methyl) 
tetrazolyl-2-(1,1-dimethylethoxy) methanamidopropionic acid 18. In the 
final coupling cycle, 2 mmole of 2-propylpentoic acid is coupled in the 
same manner as described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin is transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide is oxidized to a keto group 
by treating the resin to three oxidation cycles. Each oxidation cycle is 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 2 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. In the last two oxidation cycle, the oxidation time is 2 
hours for each cycle. After the oxidation was complete, the resin was 
washed three times 5 to 7 mL each with dimethylformamide, dichloromethane, 
methanol and diethylether. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin are transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture is cooled 
to -20.degree. C. and 15 mL of hydrofluoric acid (HF) is distilled into 
the reaction vessel. The mixture is first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether is added, then 
decanted. The resin is then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts are combined and then 
extracted with 3-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase is frozen and lyophilized to yield crude 
product. 
(d) HPLC purification. 
The crude product is dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent is monitored at 210 nm. A 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. 
Example 33 
Preparation of 
##STR45## 
The compound of Example 8 is sulfated at its tyrosine residue using the 
procedure of Nakahara et al., Anal. Biochem., 154: 194-199 (1986). 
1.5 mg (7.times.10.sup.- 7 mole) of the compound of Example 8 is dissolved 
in 0.050 mL of dimethylformamide and then was dried under a flow of 
nitrogen. The compound is redissolved in 0.040 mL of dimethylformamide 
containing 2.times.10.sup.-5 mole of sulfuric acid, 0.012 mg 
(0.6.times.10.sup.-7 mole) of N,N'-dicyclohexylcarbodiimide in 0.010 mL of 
dimethylformamide is added, and the reaction mixture is mixed by swirling. 
The reaction is allowed sit for about 5 to 10 minutes, then 0.75 mL of 
deionized water is added. Insoluble reaction products are removed by 
centrifugation in a microfuge apparatus. The solvent is removed under a 
flow of nitrogen. 
The crude product is dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent is monitored at 210 nm. A 20 minute 
gradient of 0% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. Fractions are 
collected, dried in a speed-vac apparatus and redissoved in deionized 
water. The column fractions are assayed as disclosed in Example A and 
selected for their ability to inhibit a-thrombin. 
Example 24 
Preparation of I-123 labelled 
##STR46## 
The compound of Example 8 is covalently at its tyrosine with iodine-123. 
The compound of Example 8 is reacted with I-123 Bolton Hunter Reagent (New 
England Nuclear, Boston, Mass.) in 0.1M sodium borate buffer, pH 9.0 so 
that the title compound would have a specific activity greater than 5 
mCi/mg. After the labelling, the I-123 labelled title compound is isolated 
by desalting the reaction mixture by passage through a Biogel P2 column, 
which is equilibrated with 0.01M sodium phosphate, pH 7.2, containing 0.15 
M sodium chloride. 
Example 25 
Preparation of Hirulog-1 
(D-Phe)-Pro-Arg-Pro-(Gly).sub.4 
-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH 48! 
25 
This compound was prepared using the Fmoc Coupling Protocol as described in 
Example 1, followed by deprotection and removal from the resin, and HPLC 
purification. 
(a) Preparation on resin. 
Fmoc-L-leucine-Pam Resin, the starting resin, was purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Fmoc-O-t-butyl-L-tyrosine was first coupled to the resin, followed by 
N-Fmoc-L-glutamic acid-g-t-butyl ester, N-Fmoc-L-proline, 
N-Fmoc-L-isoleucine, N-Fmoc-L-glutamic acid-g-t-butyl ester, 
N-Fmoc-L-glutamic acid-g-t-butyl ester, N-Fmoc-L-phenylalanine, 
N-Fmoc-L-aspartic ac id-b- t-butyl ester, N-Fmoc-glycine, 
N-a-Fmoc-N-b-(trityl)-L-asparagine, N-Fmoc-glycine, N-Fmoc-glycine, 
N-Fmoc-glycine, N-Fmoc-glycine, N-Fmoc-L-proline, N-a-Fmoc-N.sup.g 
-(2,2,5,7,8-pentamethylchroman-6-sulfonyl)-L-arginine, N-Fmoc-L-proline, 
and N-Fmoc -D-phenylalanine. 
(b) Deprotection and removal from resin. 
Ten mL of trifluoroacetic acid, 0.75 g of phenol, 0.25 mL of ethanedithiol 
(EDT), 0.5 mL of water, and 0.5 mL of thioanisole were combined to give 
cleavage mixture. The cleavage mixture was cooled on an ice bath, 
transferred to 0.30 g of the peptide resin. After stirring the reaction 
mixture for 2.5 hours at room temperature, the resin was filtered off, 
washed with 3-15 mL portions of trifluoroacetic acid, and 3-15 mL portions 
of dichloromethane. The combined filtrates were then concentrated in vacuo 
to an oil and then titurated with 5 mL of diethyl ether to yield a 
participate. The participate was filtered off, then redissolved in 50 mL 
of 0.1M ammonium bicarbonate. The solution was extracted with 3-25 mL 
portions diethyl ether, then frozen lyophilized to yield crude product. 
(c) HPLC Purification. 
The crude product was dissolved in 15% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and was put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent was monitored at 210 nm. A 20 minute 
gradient of 15% to 30% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) was run at a flowrate of 1 mL/minute. Title compound 
was collected at a retention time of 17.0 minutes. Fast atom bombardment 
mass spectrometry gave observed molecular weight of 2178.6 a.m.u.; 
calculated molecular weight was 2179.0 a.m.u. 
Example 26 
Preparation of 
##STR47## 
This compound is prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, is purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxcarbonyl)-L-tyrosine is first coupled to the resin, 
followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-proline, 
N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-Boc-asparagine, 
N-Boc-glycine, N-Boc-glycine, N-a-Boc-N-e-Cbz-lysine, N-Boc-glycine, 
N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
acid 7, N-Boc-L-proline, and N-Boc-L-aspartic acid-b-cyclohexyl ester. In 
the final coupling cycle, 2 mole of 2-propylpentoic acid is coupled in the 
same manner as described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin is transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide is oxidized to a keto group 
by treating the resin to two oxidation cycles. Each oxidation cycle is 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 4 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin are transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture is cooled 
to -20.degree. C. and 10 mL of hydrofluoric acid (HF) was distilled into 
the reaction vessel. The mixture is first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether is added, then 
decanted. The resin is then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 0.1M ammonium bicarbonate. The extracts are combined and then 
extracted with 2-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase is frozen and lyophilized to yield crude 
product. The resin is further extracted with 50 mL of 40% acetonitrile 
+0.1% trifluoroacetic acid (in water), the extract stripped of 
acetonitrile in vacuo, frozen, and then lyophilized to yield a more crude 
product. 
(d) HPLC purification. 
The crude product is dissolved in 20% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent is monitored at 210 nm. A 20 minute 
gradient of 20% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. 
Example 27 
Crosslinking of Peptide from Example 8 to Metallothionein 
##STR48## 
The following procedure used corresponds to that outlined by Brown, et. 
al., Analytical Biochemistry, 172: 22 (1988). Succinimidyl 
4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, purchased from 
Pierce Chemical Co. and recrystallized from acetone) is first dissolved in 
methylsulfoxide (DMSO, 1 mg/ml) and diluted to 50% with water immediately 
prior to use. The peptide prepared in Example 26 is placed in buffer (1 
mg/ml, 10 mM sodium phosphate, 150 mM sodium chloride, pH=7.5) and 
incubated with a freshly prepared SMCC solution at 4.degree. C. for 16 
hours such that the molar ratio of SMCC to peptide is 5:1. The unreacted 
SMCC is removed on a Sephadex G-15 column and the fractions containing 
peptide are pooled and lyophilized. A solution of 10 molar equivalents of 
metallothionein Zn.sub.7 MT, pure apo-MT is isolated from rabbit liver as 
described in Pande, et. al., Biochemistry, 24: 6717 (1985) and converted 
to the fully metallated Zn.sub.7 MT form as described by Morelock & Tolman 
in Metallothionein (Kagi & Nordberg, eds.), pp. 247-253. Birkhauser, 
Basel! in 50 mM Tris-HCl, pH=9.0 was added to an equal volume of 
SMCC-activated peptide at a concentration of 1.5 mg/ml. The mixture was 
incubated at 4.degree. C. for 16 hours. and the peptide-metallothionein 
conjugate purified on a Sephadex G-50 column. 
Example 28 
Preparation of 1-(p-nitrobenzyl)diethylene triaminepentaacetic acid, 
penta-t-butyl ester 
##STR49## 
1-(p-nitrobenzyl)diethylenetriamine trihydrochloride is prepared as 
described by Brechbiel, et. al., Inorg. Chem., 25: 2772 (1986). This 
material is suspended in dry THF (0.3M) along with 20 equivalents of 
potassium carbonate. 10 equivalents of t-butyl bromoacetate (available 
from Aldrich Chemical Co.) was added and the reaction mixture is sonicated 
under nitrogen at 60.degree. C. for 24 hours or until the reaction is 
complete as judged by TLC. The salts are filtered off and the volatiles 
removed in vacuo. Pure product is obtained by chromatography on silica 
(ethyl acetate/hexane). 
Example 29 
Preparation of 1-(p-aminobenzyl)diethylene triaminepentaacetic acid, 
penta-t-butyl ester 
##STR50## 
1- (p-nitrobenzyl) diethylenetriaminepentaacetic acid, penta-t-butyl ester 
is taken up in ethanol (0.1M) and placed in an atmospheric hydrogenation 
apparatus. The solution is purged with nitrogen and 10% Pd/C is added. The 
reaction mixture is then purged with hydrogen and stirred rapidly at 
ambient temperature until a hydrogen atmosphere until starting material is 
consumed by TLC analysis. The catalyst is filtered off and the volatiles 
removed in vacuo to yield crude product. This material is used as is for 
the subsequent reaction. An analytical sample can be obtained by 
chromatography on silica (ethyl acetate/hexane). 
Example 30 
Preparation of 1-(p-isothiocyanatobenzyl)diethylene triaminepentaacetic 
acid, penta-t-butyl ester 
##STR51## 
1-(p-aminobenzyl)diethylenetriaminepentaacetic acid, penta-t-butyl ester is 
taken up in chloroform (0.2M) and placed in a round bottom flask equipped 
with magnetic stirring. 3 equivalents of diisopropylethylamine is added 
followed by the addition of 1.2 equivalents of a 0.1M solution of 
thiophosgene in chloroform. Stirring is continued until the starting 
material is consumed by TLC analysis. The volatiles are removed in vacuo 
and pure product obtained by chromatography on silica (ethyl 
acetate/hexane). 
Example 31 
Preparation of thrombin-binding peptide which is attached to a 
diethylenetriaminepentaacetic acid chelator during solid-phase synthesis 
##STR52## 
This compound is prepared using the tBOC Coupling Protocol as described in 
Example 1. The single lysine residue incorporated in the peptide is 
protected at the epsilon nitrogen as a 9-fluorenylmethyloxycarbamate and 
is selectively deprotected followed by reaction with 
1-(p-isothiocyanatobenzyl) ethylenediaminetetraacetic acid, penta-t-butyl 
ester. This is followed by oxidation, deprotection of the other protecting 
groups, removal of the peptide from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, is purchased from Advanced 
ChemTech; Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine is first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-proline, 
N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-Boc-asparagine, 
N-Boc-glycine, N-Boc-glycine, N-a-Boc-N-e-fmoc-lysine, N-Boc-glycine, 
N-Boc-glycine, 6-nitroguanidino-3-(S)-(1,1-dimethylethoxy) 
methanamido-2-hydroxyhexanoic acid 7, N-Boc-L-proline, and 
N-Boc-L-aspartic acid-b-cyclohexyl ester. In the final coupling cycle, 2 
mmole of 2-propylpentoic acid is coupled in the same manner as described 
for the N-Boc amino acids. 
(b) Lysine Fmoc-deprotection 
1. The resin is washed once with N-dimethylformamide. 
2. The liquid is drained, then 5 to 7 mL 20% piperidine (in 
N-dimethyformamide) is added and the mixture was agitated for 3-5 minutes. 
3. The liquid is drained, then the resin is washed five times with 
N-dimethyformamide. 
(c) Coupling to chelating agent 
1. 5 equivalents of 1-(p-isothiocyanatobenzyl)ethylenediaminetetraacetic 
acid, penta-t-butyl ester in DMF is added and the mixture agitated for 2 
hours. 
2. The liquid is drained, then the resin is washed five times with 
N-dimethyformamide. 
(d) Oxidation. 
The peptide resin is transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide is oxidized to a keto group 
by treating the resin to three oxidation cycles. Each oxidation cycle is 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 4 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. In the last two oxidation cycle, the oxidation time is 3 
hours. 
(e) Deprotection and Removal. 
It is important to realize that after removal of the protecting groups on 
the chelator, metal free conditions must be observed. That is, all 
glassware used must be rinsed prior to use with dilute metal-free HCl 
followed by rinsing with metal-free water to neutrality. All aqueous 
solutions must be prepared using metal-free water. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin are transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture is cooled 
to -20.degree. C. and 10 mL of hydrofluoric acid (HF) is distilled into 
the reaction vessel. The mixture is first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether is added, then 
decanted. The resin is then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts are combined and then 
extracted with 3-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase is frozen and lyophilized to yield crude 
product. 
(f) HPLC purification. 
The crude product is dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent was monitored at 210 nm. A 20 minute 
gradient of 10% to 30% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. If possible, 
the column should be packed in a metal free container and non-metallic 
lines used. 
Example 32 
.sup.99m Tc Labelling of a thrombin-binding peptide which is crosslinked to 
metallothionein 
The labelling protocol is similar to that used by Brown, et. al., 
Analytical Biochemistry, 172: 22 (1988). A .sup.99 Mo/.sup.99m Tc 
generator (DuPont) is the source of .sup.99m TcO.sub.4 --. When reacted 
with stannous glucoheptonate (1 mL of .sup.99m TcO.sub.4 -- added to a 
Glucoscan kit) (DuPont), the .sup.99m TcO(GH).sub.2 transchelates to 
metallothionein and metallothionein-peptide conjugates Morelock & Tolman 
in Metallothionein (Kagi & Nordberg, eds.), pp. 247-253. Birkhauser, 
Basel!. Specifically, 1 volume of the compound of Example 27 is mixed with 
1 volume of 0.3M sodium phosphate, pH=6.2, followed by 1 volume of 
.sup.99m TcO(glucoheptonate).sub.2. After 2 hours at ambient temperature, 
the percentage of incorporation of the .sup.99m Tc into peptide-chelator 
conjugate was quantitated by silica TLC chromatography in saline. 
Peptide-bound .sup.99m Tc remained at the origin while the .sup.99m 
TcO(GH).sub.2 moved to the solvent front. 
Example 33 
.sup.111 In Labelling of a Thrombin-Binding Peptide which is Attached to a 
Diethylenetriaminepentaacetic Acid Chelator 
The labelling protocol is similar to that used by Westerberg, et. al., J. 
Med. Chem. 32: 236 (1989). Carrier-free indium-111 chloride is added to an 
aliquot of the compound of Example 31 (100 .mu.L at a concentration of 10 
mg/mL in 0.05M citrate buffer, pH 6). After a 30 minute incubation at room 
temperature, the radiochemical yield of indium-111-labeled peptide is 
determined by incubating an aliquot (50 .mu.L) of the solution with 0.05M 
DTPA, pH 6 (25 .mu.L) for 10 minutes and then diluting this solution 
50-fold with normal saline and spotting 3 .mu.L of the resulting solution 
onto a TLC plate. Meares, et. al., Anal. Biochem., 142: 68 (1984). 
Subsequent TLC analysis indicates the amount of indium-111 bound to 
peptide. 
Example 34 
Preparation of 
##STR53## 
This compound is prepared using the tBOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, is purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine is first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-proline, N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl 
ester, N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-a-Boc-N.sup.g 
-tosyl-L-arginine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
N-Boc-glycine, N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
acid 7, N-Boc-L-proline, and N-Boc-L-methionine sulfone. In the final 
coupling cycle, 2 mmole of 2-propylpentanoic acid is coupled in the same 
manner as described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin is transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry. dichloromethane. 
The a-hydroxy group of the resin-bound peptide is oxidized to a keto group 
by treating the resin to three oxidation cycles. Each oxidation cycle is 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 2 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. The oxidation time is 2 hours for each cycle. After the 
oxidation was complete, the resin was washed three times 5 to 7 mL each 
with dimethylformamide, dichloromethane, methanol and diethylether. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin are transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture is cooled 
to -20.degree. C. and 15 mL of hydrofluoric acid (HF) is distilled into 
the reaction vessel. The mixture is first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether is added, then 
decanted. The resin is then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts are combined and then 
extracted with 3-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase is frozen and lyophilized to yield crude 
product. 
(d) HPLC purification. 
The crude product is dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent is monitored at 210nm. A 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. 
Example 35 
Preparation of a-Boc-L-serine-b-lactone 
##STR54## 
5.44 g (24.4 mmol, 1.0 equiv.) of Boc-L-serine hydrate (crushed) and 6.4 g 
(24.4 mmol, 1.0 equiv.) of triphenylphosphine (crushed) were dried in 
Vacuo at room temperature for 48 hours over phosphorous. pentoxide. The 
triphenylphosphine was taken up in 100 mL of 8:2 anhydrous 
acetonitrile/dry THF and cooled to -45.degree. C. (acetone/dry ice bath) 
under nitrogen. 3.84 mL (24.4 mmol, 1.0 equiv.) of diethylazodicarboxylate 
(DEAD) was added dropwise via syringe over about 15 minutes and the 
reaction mixture was stirred for an additional 10 minutes at -55.degree. 
C. A thick slurry of the DEAD-triphenylphosphine adduct resulted. The 
previously dried Boc-L-serine was taken up in 100 mL of anhydrous 
acetonitrile and added dropwise via canula to the activated 
DEAD/triphenylphosphine reagent. The reaction mixture was stirred at 
-5.degree. C. for 1 hour and warmed to ambient temperature and stirred for 
an additional 1.5 hours. The volatiles were removed on the rotary 
evaporator and the resulting crude product was immediately taken up in a 
minimum amount of methylene chloride and flash chromatographed (30% ethyl 
acetate/hexane) to yield a white solid (2.53 g, 55 % yield). .sup.1 H-NMR 
(CDCl.sub.3); 1.47 ppm (s, 9H), 4.42 ppm (t, 1H), 4.46 ppm (t, 1H), 5.11 
ppm (quart., 1H), 5.26 (hr. s., 1H). .sup.13 C-NMR (CDCl.sub.3); 28.0 ppm, 
59.3 ppm, 66.5 ppm, 81.2 ppm, 154.5 ppm, 169.4 ppm. 
Example 36 
Preparation of a-N-Boc-b-amino-L-alanine 
##STR55## 
Anhydrous ammonia was bubbled through 400 mL of anhydrous acetonitrile for 
25 minutes. To this saturated solution was added dropwise 2.0 g of 
N-Boc-L-serine-b-lactone 35 in 200 mL of anhydrous acetonitrile over 1 
hour. After addition was complete, the reaction mixture was stirred at 
ambient temperature for an additional 16 hours. The volatiles were removed 
in vacuo (a liquid nitrogen trap was used to trap the ammonia) to yield a 
white solid (2.1 g, 96 % yield). It was one spot by TLC (Rf=0.8; 70% 
propanol/water). .sup.1 H-NMR (CDCl.sub.3); 1.47 ppm (s, 1H), 3.20 ppm 
(dd, 1H), 3.39 ppm (dd, 1H), 4.16 ppm (br. s, 1H). .sup.13 C-NMR; 28.3 
ppm, 41.9 ppm, 53.7 ppm, 82.3 ppm, 158.2 ppm, 175.7 ppm. 
Example 37 
Preparation of a-N-Boc-b-(methylsulfonylamino)-L-alanine 
##STR56## 
To a solution of a-N-Boc-b-amino-L-alanine 36 (1.92 g, 10 mmol) in 50 mL 
dry dichloromethane at 0.degree. C., is added triethylamine (2.79 mL, 20 
mmol) followed by mesyl chloride (1.55 mL, 20 mmol) dropwise. After the 
addition, the reaction mixture is warmed to room temperature and allowed 
to stir for two hours. After this time, the reaction mixture is poured 
into 50 mL of ethyl acetate and 50 mL of 1M aqueous hydrochloric acid, and 
the title compound is allowed to partition into the organic phase. The 
organic phase is separated, dried over anhydrous magnesium sulfate, then 
reduced to dryness in vacuo to provide the title compound. 
Example 38 
Preparation of 
##STR57## 
This compound is prepared using the t-BOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, is purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine is first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-proline, N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl 
ester, N-Boc-L-glutamic acid-g-cyclohexyl ester; N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-a-Boc-N.sup.g 
-tosyl-L-arginine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
N-Boc-glycine, N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
acid 7, N-Boc-L-proline, and a-N-Boc-b-(methylsulfonylamino)-L-alanine 37. 
In the final coupling cycle, 2 mmole of 2-propylpentoic acid is coupled in 
the same manner as described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin is transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide is oxidized to a keto group 
by treating the resin to three oxidation cycles. Each oxidation cycle is 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 2 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. In the last two oxidation cycle, the oxidation time is 2 
hours for each cycle. After the oxidation was complete, the resin was 
washed three times 5 to 7 mL each with dimethylformamide, dichloromethane, 
methanol and diethylether. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin are transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture is cooled 
to -20.degree. C. and 15 mL of hydrofluoric acid (HF) is distilled into 
the reaction vessel. The mixture is first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether is added, then 
decanted. The resin is then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts are combined and then 
extracted with 3-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase is frozen and lyophilized to yield crude 
product. 
(d) HPLC purification. 
The crude product is dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent is monitored at 210 nm. A 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. 
Example 39 
Preparation of a-N-Boc-b-(methoxycarbonylamino)-L-alanine 
##STR58## 
To a suspension of a-N-Boc-b-amino-L-alanine 36 (1.92 g, 10 mmol) and solid 
potassium carbonate (2.76 g, 20 mmol) in 50 mL dry tetrahydrofuran at room 
temperature, is added methylchloroformate (f.55 mL, 20 mmol) dropwise. The 
reaction mixture is allowed to stir for twelve hours, then 100 mL of ethyl 
acetate is added. The reaction mixture is then decanted away from the 
potassium carbonate, poured into 50 mL of ethyl acetate and 50 mL of 1M 
aqueous hydrochloric acid, and the title compound is allowed to partition 
into the organic phase. The organic phase is separated, dried over 
anhydrous magnesium sulfate, and then reduced to dryness in vacuo to 
provide the title compound. 
Example 40 
Preparation of 
##STR59## 
This compound is prepared using the t-BOC Coupling Protocol as described in 
Example 1, followed by oxidation, deprotection and removal of the peptide 
from the resin, and HPLC purification. 
(a) Coupling. 
Boc-L-leucine-Pam Resin, the starting resin, is purchased from Advanced 
ChemTech, Louisville, Ky.). 
N-Boc-O-(2-bromobenzyloxycarbonyl)-L-tyrosine is first coupled to the 
resin, followed by N-Boc-L-glutamic acid-g-cyclohexyl ester, 
N-Boc-L-proline, N-Boc-L-isoleucine, N-Boc-L-glutamic acid-g-cyclohexyl 
ester, N-Boc-L-glutamic acid-g-cyclohexyl ester, N-Boc-L-phenylalanine, 
N-Boc-L-aspartic acid-b-cyclohexyl ester, N-Boc-glycine, N-a-Boc-N.sup.g 
-tosyl-L-arginine, N-Boc-glycine, N-Boc-glycine, N-Boc-glycine, 
N-Boc-glycine, N-Boc-glycine, 
6-nitroguanidino-3-(S)-(1,1-dimethylethoxy)methanamido-2-hydroxyhexanoic 
acid 7, N-Boc-L-proline, and a-N-Boc-b-(methoxycarbonylamino)-L-alanine 
39. In the final coupling cycle, 2 mmole of 2-propylpentoic acid is 
coupled in the same manner as described for the N-Boc amino acids. 
(b) Oxidation. 
The peptide resin is transferred to another reaction vessel and washed 
twice with 5 to 7 mL of dry dichloromethane. 
The a-hydroxy group of the resin-bound peptide is oxidized to a keto group 
by treating the resin to three oxidation cycles. Each oxidation cycle is 
performed by suspending the resin in a mixture of 5 mL of dry 
dichloromethane and 5 mL of dry dimethylsulfoxide; deoxygenating the 
mixture with nitrogen; adding 5 mmole 
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride salt 
(EDAC-HCl), 2 mmole dichloroacetic acid (DCA), 2 mL of dry dichloromethane 
and 2 mL of dry dimethylsulfoxide; stirring the reaction mixture for 2 
hours; then finally washing the resin three times with 5 to 7 mL of dry 
dichloromethane. In the last two oxidation cycle, the oxidation time is 2 
hours for each cycle. After the oxidation was complete, the resin was 
washed three times 5 to 7 mL each with dimethylformamide, dichloromethane, 
methanol and diethylether. 
(c) Deprotection and Removal. 
The peptide resin and a volume of anisole numerically equal to the weight 
of resin are transferred to a plastic reaction vessel. After purging the 
vessel and associated lines with nitrogen, the reaction mixture is cooled 
to -20.degree. C. and 15 mL of hydrofluoric acid (HF) is distilled into 
the reaction vessel. The mixture is first stirred for 30 minutes at 
-20.degree. C., then for 120 minutes at 0.degree. to 10.degree. C. After 
removing the HF by evaporation, 20 mL diethyl ether is added, then 
decanted. The resin is then transferred to an extraction funnel, washed 
with 3-20 mL portions of diethyl ether, then extracted with 3-50 mL 
portions of 20% acetic acid (in water). The extracts are combined and then 
extracted with 3-25 mL portions of diethyl ether, saving the aqueous phase 
each time. The aqueous phase is frozen and lyophilized to yield crude 
product. (d) HPLC purification. 
The crude product is dissolved in 10% acetonitrile (in water containing 
0.1% trifluoroacetic acid) and is put onto a 2.5.times.300 mm C18 reverse 
phase column (VYDAC) and the effluent is monitored at 210 nm. A 20 minute 
gradient of 10% to 35% acetonitrile (in water containing 0.1% 
trifluoroacetic acid) is run at a flowrate of 1 mL/minute. 
Example A 
Amidolytic Thrombin Assay 
The ability of the compounds of the present invention to act as inhibitors 
of thrombin catalytic activity in comparison to Hirulog-1 (compound 25) 
was assessed by determining their inhibition constant, Ki. 
Enzyme activity was determined using the chromogenic substrate Pefachrome 
t-PA (CH.sub.3 SO.sub.2 
-D-hexahydrotyrosine-glycyl-L-arginine-p-nitroaniline, obtained from 
Pentapharm Ltd.). The substrate was reconstituted in deionized water prior 
to use. Purified human a-thrombin was obtained from Enzyme Research 
Laboratories, Inc. The buffer used for all assays was HBSA (10 mM HEPES, 
pH 7.5, 150 mM sodium chloride, 0.1% bovine serum albumin) 
Ki values were determined for test compounds using the following 
methodologies: 
1) For test compounds exhibiting slow binding or slow-tight binding 
kinetics (as compounds 8, 10 and 11), Ki values were determined using the 
relationships developed by Williams and Morrison, Methods in Enzymology, 
63: 437 (1979) by determining the apparent first-order rate constant 
(k.sub.obs) which describes the rate of equilibration from the initial to 
the steady state velocity (Vs). The assay was conducted by combining in 
appropriate wells of a Corning microtiter plate, 50 mL of HBSA, 50 mL of 
the test compound at a specified concentration diluted in HBSA (or HBSA 
alone for V.sub.o(uninhibited velocity) measurement), and 50 mL of the 
chromogenic substrate diluted in HBSA. At time zero, 50 mL of a-thrombin 
diluted in HBSA, was added to the wells yielding a final concentration of 
0.25 nM in a total volume of 200 mL. Velocities of Pefachrome-tPA 
substrate hydrolysis which occurred over a 40 minute time period was 
measured by the change in absorbance at 405 nm using a Thermo Max.RTM. 
Kinetic Microplate Reader. The concentration of substrate in this assay 
was 400 mM (.about.10-times Km) and the extent of substrate hydrolysis was 
less than 5% over the course of this assay. The linear relationship 
between k.sub.obs and inhibitor concentration is indicative of a 
competitive, one step mechanism and was used to calculate k.sub.on and 
k.sub.off which yielded a value for Ki after taking into consideration the 
concentration and Km (38.5 mM) of the substrate in the assay. 
2) A second method was used to measure the intrinsic dissociation constant 
(Ki*) for compound 8 which is independent of the inhibitory mechanism. In 
this assay, HBSA (50 mL), a-thrombin (50 .mu.L, 0.25 nM) and inhibitor (50 
.mu.L, covering a broad concentration range, 10-1000 pM), were combined in 
appropriate wells and incubated for 30 minutes at room temperature prior 
to the addition of substrate Pefachrome-t-PA (50 .mu.L, 260 mM, 
.about.7-times Km). The initial velocity of Pefachrome t-PA hydrolysis was 
measured by the change in absorbance at 405 nm using a Thermo Max.RTM. 
Kinetic Microplate Reader over a 2 minute period in which less than 5% of 
the added substrate was utilized. The relationship between the ratio of 
the inhibited steady-state velocity (Vs) and the uninhibited velocity (Vo) 
to the concentration of inhibitor I.sub.t !, was analyzed using Equation 
1 developed by Morrison , J. F., Biochim. Biophys. Acta, 185: 269 (1969) 
for inhibitors which deplete a significant amount of the total enzyme 
(E.sub.t) over the course of the assay (designated tight binding 
inhibitors): 
EQU Vs/Vo={(E.sub.t !-I.sub.t !-Ki*)+(I.sub.t !+Ki*=E.sub.t !).sup.2 
+4Ki*E.sub.t !!.sup.1/2 }/2E.sub.t ! Equation 1 
Ki* was determined by fitting Vs/Vo verses I! using Equation 1 by 
non-linear regression analysis. 
3) The final method for Ki determination was used for test compounds 
showing rapid, reversible kinetics of inhibition (compound 9 and compound 
25 (Hirulog-1)),using the assay protocol described above for slow-binding 
inhibitors (Method 1). Ki values were determined by non-linear regression 
analysis of the initial velocities of substrate hydrolysis taken over a 2 
minute period following the addition of a-thrombin, at several substrate 
(15-300 mM) and inhibitor concentrations (Compound 9, 0-300 mM; Hirulog-1 
(Compound 25), 0-5 nM) using the relationships developed by Dixon, M., 
Biochem. J., 129: 197 (1972). The best fit of the data for both compounds 
was to the equation describing competitive inhibition. 
Table I below gives the Ki values for test compounds 8 to 11 in comparison 
with Hirulog-1 (compound 25). 
TABLE I 
______________________________________ 
Inhibitor Constants (Ki).sup.a 
Compound Ki (nM) Ki* (nM) 
______________________________________ 
Compound 8 0.0019 0.0014 
Compound 9 46.06 NA 
Compound 10 0.040 ND 
Compound 11 0.0078 ND 
Compound 25 0.437 NA 
(Hirulog-1) 
______________________________________ 
.sup.a The data is representative of at least two independent experiments 
run in triplicate. 
NAnot applicable 
NDnot performed 
The good agreement between the values of Ki and Ki* seen for compound 8 
indicate that this compound inhibits thrombin in a predominately one-step 
competitive, tight-binding mechanism. This data also demonstrates the 
remarkable increase in inhibitory potency that can be achieved singlely 
through the incorporation of the a-keto-amide transition-state 
functionality as evidenced by the almost 23,000-fold difference in Ki 
between compound 8 and 9. In addition this data also demonstrates that 
compound 8 is 230-fold more potent than the prototypical bifunctional, 
clearable, thrombin inhibitor, Hirulog-1 (compound 25), as determined by 
direct comparison under the assay conditions described above even though 
the calculated Ki for Hirulog-1 (compound 23) is lower than previously 
reported by Witting, J. I. et al., Biochem. J., 283:737 (1992). Compound 
10 differs from compound 8 in having an additional glutamic acid residue 
at position C.sub.5 in group C as described in Formula I within the 
Detailed Description. Although the sequence of this peptide in compound 10 
is identical to that described for compound 25 (Hirulog-1), it resulted in 
an over 20-fold decrease in the inhibitory potency compared to the parent, 
compound 8 having only a single glutamic acid at this position. Changing 
the asparagine residue in compound 10 at position B.sub.2 in group B shown 
in Formula I to arginine, results in an approximately 5-fold increase in 
inhibitory potency compared to compound 10, indicating that the overall 
charge density in the region of this compound encompassing groups B and C 
in Formula I may be important in determining the overall potency of this 
inhibitor. 
Example B 
Thrombin-Induced Clotting of Purified Fibrinogen 
Compound 8 and Hirulog-1 (compound 25) were compared in an assay designed 
to measure the inhibition of thrombin using purified fibrinogen as the 
substrate using a modification of the method described by Witting, J. I. 
et al., Biochem. J. 283:737 (1992). Compound 8 or Hirulog-1 (compound 25) 
were pre-incubated with a-thrombin over a broad concentration range at 
25.degree. C. in 300 mL of Buffer A (150 mM NaCl, 30 mM CaCl.sub.2, 10 mM 
imidazole, 8.8 mg/mL polyethylene glycol (PEG 6000), pH 7.4) for 0, 30, 
and 60 minutes prior to the addition 100 mL of purified human fibrinogen 
(American Diagnostica, Greenwich, Conn.) which had been reconstituted in 
Buffer B (30 mM NaCl, 10 mM imidazole, pH 7.4) to a concentration of 8 
ng/mL. The clotting time of fibrinogen following the addition of the 
thrombin/inhibitor complex was measured optically using the Coag-A-Mate XC 
automated coagulometer (General Diagnostics, Organon Technica, Oklahoma 
City, Okla.). 
The results of this assay are shown in FIG. 2. In this figure, Compound 8 
(solid symbols) and Hirulog-1 (compound 25; open symbols) were 
preincubated with a-thrombin for 0 (m,1) 30 (.DELTA.,s), and 60 (o,n) 
minutes prior to the addition of fibrinogen. 
The data in FIG. 2 demonstrate that Compound 8 can dose-dependently inhibit 
the generation of fibrin catalyzed by thrombin as measured by an increase 
in clotting time relative to the uninhibited control. This inhibition is 
reflected as an increase in the relative clotting time which is the ratio 
of the control (uninhibited) clotting time/experimental clotting time. The 
control clotting time in this assay is 16.2.+-.0.78 sec. 
In addition this data demonstrates that compound 8 is significantly more 
potent than Hirulog-1 (compound 25) in this assay and is not 
proteolytically inactivated by thrombin upon prolonged incubation (up to 
60 minutes) with the enzyme prior to the addition of the fibrinogen 
substrate. This is in contrast to Hirulog-1 (compound 25) which appears to 
be sensitive to proteolytic degradation with re-emergence of thrombin 
activity consistent with the results presented by Witting, J. I. et al., 
Biochem. J., 283:737 (1992) and Rubens, F. D., et al., Thromb. Haemostas., 
69:130-134 (1993). 
Example C 
Ex Vivo coagulation assay 
The ex vivo anticoagulant effects of compound 8 in comparison with 
Hirulog-1 (compound 25) were determined by measuring the prolongation of 
the activated partial thromboplastin time (APTT) over a broad 
concentration range of each added inhibitor, using pooled normal human 
plasma. Fresh frozen pooled normal human plasma was obtained from George 
King Biomedical, Overland Park, Kans.. Measurements APTT was made using 
the Coag-A-Mate RA4 automated coagulometer (General Diagnostics, Organon 
Technica, Oklahoma City, Okla.) using the Automated APTT reagent (Organon 
Technica, Durham, N.C.) as the initiator of clotting according to the 
manufacturers instructions. The assay was conducted by making a series of 
dilutions of the test compounds in rapidly thawed plasma followed by 
adding 200 mL to the wells of the assay carousel. 
FIG. 3 depicts the effect of compound 8 (open symbols) and Hirulog-1 
(compound 25) (closed symbols) on the activated partial thromboplastin 
time (APTT) of normal citrated human plasma. 
As shown in this figure, both compounds prolonged the APTT in a dose 
dependent manner. 
Example D 
EX Vivo Platelet Aggregation Assay Thrombin-Induced Aggregation in Washed 
Human Platelets 
The ability of compound 8 and Hirulog-1 (compound 25) to inhibit 
thrombin-induced platelet aggregation were determined using washed human 
platelets. Washed human platelets were prepared from freshly isolated 
venous blood obtained from normal, healthy volunteers who had not taken 
any medication which might affect platelet function, according to the 
procedure of Connolly, T. M. et al., J. Biol. Chem., 267: 6893. (1992). 
Inhibitors, were added in a volume of 20 mL to 350 mL of prewarmed washed 
platelets in a siliconized glass cuvette (Chronolog Corp., Hayertown, Pa.) 
followed by 10 mL of 0.25M CaCl.sub.2. The rate and extent of aggregation 
were measured for 5-15 minutes by the change in light transmission in a 
stirred cuvette, following the addition of 20 mL of a-thrombin (final 
concentration 2 nM), using a Chronolog Whole Blood Aggregometer equipped 
with the Aggro/Link data acquisition system (Chronolog Corp., Hayertown, 
Pa.). The extent of inhibition was determined by measuring the change in 
the amplitude (extent) of the aggregation response compared to the control 
(uninhibited) response over the 1 minute period following the addition of 
thrombin. The inhibitory effect of the test compounds was measured over a 
broad concentration range and is reported as the concentration required to 
inhibit aggregation by 50% (IC.sub.50). 
TABLE II 
______________________________________ 
Effect of Compounds 8 and Hirulog-1 (compound 25) 
on thrombin-induced platelet aggregation using 
washed human platelets. 
Compound IC.sub.50 (nM)* 
______________________________________ 
Compound 8 25 .+-. 8 
Hirulog-1 (compound 25) 
16 .+-. 3 
______________________________________ 
*represents the mean .+-. standard deviation from three independent 
determinations. 
This data indicates that compound 8 and Hirulog-1 (compound 25) equally 
inhibit thrombin-induced platelet aggregation in vitro when the extent of 
aggregation is measured for the initial 1 minute following thrombin 
addition. However, as shown in FIG. 4A, the maximal inhibition of 
aggregation with compound 8 obtained at a concentration of 20 nM was 
stable with no recovery of aggregation over the 15 minute course of the 
assay. This was in contrast to Hirulog-1 (compound 25) which did not cause 
sustained inhibition of the aggregation response at the same or higher 
concentrations of inhibitor (FIG. 4B). The functional differences between 
compound 8 and Hirulog-1 (compound 25) in the platelet aggregation assay 
is similar to the effects of these two inhibitors in the purified fibrin 
formation assay shown in FIG. 2 and can be attributed to the 
susceptibility of Hirulog-1 (compound 25) to proteolytic inactivation by 
thrombin which does not occur with compound 8 due to the presence of the 
proteolytically stable transition state functionality. 
Example E 
Experimental Models of Thrombosis in Rats 
The antithrombotic properties of compound 8 and Hirulog-1 (compound 25) 
were evaluated using the following established in vivo experimental models 
of acute thrombosis. 
1. Venous Stasis in Rats. 
This is one of the most commonly used models in the evaluation of 
antithrombotic compounds. Hladovec, J. Thromb. Res., 43:539-544 (1986) In 
this model a localized clot made up of primarily fibrin is formed in a 
segment of the inferior vena cava (IVC) in which an artificial stasis is 
induced by ligature following the systemic infusion of thromboplastin used 
as the thrombogenic stimulus. Talbot, M. D., et. al., Thromb. Haemostas., 
61: 77-80 (1989) The antithrombotic effect of the compound 8 and Hirulog-1 
(compound 25), was determined by measuring the final clot weight recovered 
from the isolated segment of the IVC as the primary end point in the model 
following systemic, intravenous administration of each compound at several 
dosing regimens. 
Male Harlan Sprague Dawley rats (420-450 g) were acclimated at least 72 
hours prior to use. The animals were fasted for 12 hours prior to surgery 
with free access to water. The animals were anesthetized with a sodium 
pentobarbital (Nembutal) given intraperitoneally at a dose of 50 mg/kg 
body weight and placed on a isothermal pad to maintain body temperature. 
The level of anesthesia was monitored every 15 minutes by: neuro-response 
to a tail pinch, respiration and body core temperature. The desired depth 
of surgical anesthesia was maintained by administering subsequent doses (5 
mg/kg) intravenously. The left femoral artery was catheterized with 
polyethylene tubing (PE50) using standard procedures for blood pressure 
monitoring and blood sampling. The left and right femoral veins were 
catheterized with PE50 tubing for delivery of anesthetic and test 
compounds respectively. 
Following anesthesia the animals were randomized in either a control 
(saline infusion) or treatment group (Compound 8 or Hirulog-1 (compound 
25)) with at least 4 animals per group per dose. The compounds or saline 
were administered via the femoral catheter as a bolus infusion of 100 
mg/kg followed by a continuous intravenous infusion for a period of 30 
minutes of 10, 20 or 40 mg/kg/min for compound 8 and 40, 60 or 80 
mg/kg/min for Hirulog-1 (compound 25). Blood pressure, heart rate, core 
temperature and respiration were monitored continuously. 
The abdomen of the animals is opened by making a vertical midline incision 
followed by isolation of the IVC using dissection. The segment extends 
from below the renal to above the iliac vessels (about 2 cm in length). 
The peripheral blood vessels are tied off and a ligature is loosely placed 
at the distal and proximal ends of the isolated segment. Rabbit brain 
thromboplastin (RBT)(Sigma Chemical Co., St. Louis Mo.) is prepared by 
resuspending the contents of the vial with 2 mL of sterile saline 
pre-warmed to 37.degree. C. At the end of the 30 minute infusion period, 
RBT is systemically administered as a bolus injection (1.5 mL/kg) via the 
femoral catheter. Stasis is induced within the isolated IVC segment by 
securing the proximal and distal ligature 10 sec following the 
administration of the RBT. Following a 30 minute period of stasis the tied 
off IVC segment is removed and the contents weighed. Clot formation was 
defined as %clot: Weight of the isolated clot/(Weight of the intact 
segment-the weight of the empty segment)! .times.100. This method was used 
to correct for slight differences in segment size and fluid content. 
Following termination of the experiment the animal was euthanized with a 
120 mg/kg dose of Nembutal. 
The efficacy of the compound 8 compared to Hirulog-1 (compound 25) in this 
in vivo model is shown in Table III below. 
TABLE III 
______________________________________ 
Efficacy of the Compound 8 and Hirulog-1 
(compound 25) in the Rat Venous Stasis Model. 
Treatment Group % Clot.sup.a 
______________________________________ 
Control 25.18 .+-. 0.86 (n = 6) 
Compound 8 
Group 1 22.03 .+-. 3.6 (n = 4) 
Group 2 8.25 .+-. 5.1* (n = 4) 
Group 3 0** (n = 4) 
Hirulog-1 (compound 25) 
Group 1 25.75 .+-. 3.71 (n = 4) 
Group 2 26.50 .+-. 3.59 (n = 4) 
Group 3 25.75 .+-. 0.41 (n = 4) 
______________________________________ 
Control-no treatment 
Group 10.1 mg/kg i.v. bolus + 10 (compound 8) or 40 (Hirulog1 (compound 
25)) mg/kg/min i.v. infusion 
Group 20.1 mg/kg i.v. bolus + 20 (compound 8) or 60 (Hirulog1 (compound 
25)) mg/kg/min i.v. infusion 
Group 30.1 mg/kg i.v. bolus + 40 (compound 8) or 80 (Hirulog1 (compound 
25)) mg/kg/min i.v. infusion 
.sup.a % Clot is defined as: Isolated clot/(Intact segment - Empty 
segment)! .times. 100. Numbers represent the mean .+-. S.E.M. 
*p .ltoreq. 0.05 vs Control by oneway ANOVA followed by NewmanKuels Test. 
**p .ltoreq. 0.01 vs Control by oneway ANOVA followed by NewmanKuels Test 
 
This data demonstrates that compound 8 is very efficacious in 
dose-dependently preventing venous thrombus formation induced by stasis 
and thromboplastin in this rat model of venous thrombosis compared to 
Hirulog-1 (compound 25) which did not have any effect of thrombus 
formation in this setting. The lack of efficacy observed for Hirulog-1 
(compound 25) in this model is presumably a result of the proteolytic 
inactivation of this inhibitor by thrombin generated within the isolated 
IVC segment during the 30 minutes of induced stasis. This is in contrast 
to compound 8 which is not proteolytically inactivated by thrombin under 
these conditions and thus serves as an effective antithrombotic agent in 
this model. This in vivo data correlates well with the results obtained 
with these two compounds in vitro in the purified fibrin formation and in 
ex vivo thrombin-induced platelet aggregation assays described above. 
2. Rat model of FeCl.sub.3 -induced platelet-dependent arterial thrombosis. 
This is a well characterized model of platelet dependent, arterial 
thrombosis which has been used in the evaluation potential antithrombotic 
compounds such as direct thrombin inhibitors. Kurz, K. D., Main, B. W., 
and Sandusky, G. E., Thromb. Res., 60: 269-280. (1990). In this model a 
platelet-rich, occlusive thrombus is formed in a segment of the rat 
carotid artery treated with a fresh solution of FeCl.sub.3 absorbed to a 
piece of filter paper. The FeCl.sub.3 is thought to diffuse into the 
treated segment of artery and causes de-endothelialization resulting in 
thrombus formation. The effect of a test compound on the incidence of 
occlusive thrombus formation following the application of the FeCl.sub.3 
is monitored by ultrasonic flowtometry and is used as the primary end 
point. The use of flowtometry is a modification of the original procedure 
in which thermal detection of clot formation was employed. Kurz, K. D., 
Main, B. W., and Sandusky, G. E., Thromb. Res., 60: 269-280 (1990). 
Male Harlan Sprague Dawley rats (420-450 g) were acclimated at least 72 
hours prior to use and fasted for 12 hours prior to surgery with free 
access to water. The animals were prepared, anesthetized with Nembutal 
with catheters for blood pressure monitoring, drug and anesthesia delivery 
being implanted as described above. The left carotid artery was exposed 
and isolated by making a midline cervical incision followed by blunt 
dissection and spreading techniques to separate a 2 cm segment of the 
vessel from the carotid sheath. A silk suture is inserted under the 
proximal and distal ends of the isolated vessel to provide clearance for 
the placement of a ultrasonic flow probe (Transonic) around the proximal 
end of the vessel. The probe is then secured with a stationary arm. 
Following surgery the animals were randomized in either a control (saline 
infusion) or treatment group with test compounds (compound 8 or Hirulog-1 
(compound 25)) with at least 3 animals per group per dose. The test 
compounds were administered as described above after placement of the flow 
probe and stabilization of the preparation for a period of 60 minutes 
prior to the thrombogenic stimulus. At t=0, a 3 mm diameter piece of 
filter paper (Whatman #3) soaked with 10 mL of a 35% solution of fresh 
FeCl.sub.3 (in water) was applied the segment of isolated carotid artery 
distal to the flow probe. Blood pressure, blood flow, heart rate, and 
respiration were monitored for 60 minutes. 
The incidence of occlusion (defined as the attainment of zero blood flow) 
was recorded as the primary end point. Following the 60 minute observation 
period the flow probe was removed and the area cleared of all excess 
fluid. The distal and proximal sutures were tied off and arterial clamps 
placed on the far proximal and distal ends of the segment. The isolated 
segment was cut out, blotted dry on filter paper and weighed. The segment 
was re-weighed following removal of the clot and the difference recorded 
as total % clot (see above). Weights were recorded on only those segments 
which had detectable thrombus. Following the procedure the animals were 
euthanized as described above. 
The efficacy of the compound 8 and Hirulog-1 (compound 25) in this in vivo 
model is shown in Table IV below. 
TABLE IV 
______________________________________ 
Efficacy of Compound 8 and Hirulog-1 (compound 25) 
in the FeCl.sub.3 Model of Thrombosis in Rats. 
Treatment Group.sup.a 
Incidence of Occlusion.sup.b 
% Clot.sup.c 
______________________________________ 
Control 6/6 70.9 .+-. 1.21 (n = 6) 
Compound 8 
Group 1 5/6 66.14 .+-. 3.83 (n = 5) 
Group 2 4/6 42.28 .+-. 12.6 (n = 4) 
Group 3 3/6 46.13 .+-. 6.75 (n = 3) 
Group 4 0/5* 0 
Hirulog-1 (compound 25) 
Group 1 3/3 70.1 .+-. 5.5 (n = 3) 
Group 2 5/6 59.42 .+-. 4.34 (n = 5) 
Group 3 5/6 45.3 .+-. 11.63 (n = 5) 
Group 4 0/6** 0 
______________________________________ 
.sup.a Controlno treatment (saline infusion) 
Group 10.1 mg/kg i.v. bolus + 5 mg/kg/min i.v. infusion 
Group 20.1 mg/kg i.v. bolus + 10 mg/kg/min i.v. infusion 
Group 30.1 mg/kg i.v. bolus + 20 mg/kg/min i.v. infusion 
Group 40.1 mg/kg i.v. bolus + 40 mg/kg/min i.v. infusion 
.sup.b occlusion is defined as the establishment of zero blood flow 
through the treated segment of the carotid artery. 
.sup.c % Clot is defined as: Isolated clot/(Intact segment - Empty 
segment)! .times. 100. Numbers represent the mean .+-. S.E.M. in the 
designated number of animals. 
*p .ltoreq. 0.01 vs Control by ChiSquare Analysis 
**p .ltoreq. 0.005 vs Control by ChiSquare Analysis 
These in vivo data demonstrated the antithrombotic efficacy of the Compound 
8 compared to Hirulog-1 (compound 25) in a rodent model of 
platelet-dependent arterial thrombosis. 
Example F 
Inhibition of Clot-Associated Thrombin 
The association of catalytically active thrombin with fibrin rich clot is 
believed to render this pool of thrombin activity resistant to the actions 
of certain anticoagulants such as heparin which require an interaction 
with the serpin antithrombin III for activity. Weitz, J. I. et.al., J. 
Clin. Invest., 86: 385-391 (1990). This resistance to inhibition is 
believed to be among the reasons why heparin anticoagulants are not 
particularly effective either in the prevention or treatment of arterial 
thrombosis (Hirsh, J. N. Engl. J. Med. 324: 1565-1574 (1991). 
Recombinant Hirudin (rHIR) (CGP 39393).was supplied by Ciba-Geigy, Horsham, 
UK. D-Phe-Pro-ArgCH.sub.2 Cl was obtained Calbiochem. 
The relative inhibition of fluid-phase and clot-associated thrombin 
activity by compound 8 was compared to Hirulog-1 (compound 25) and rHIR, 
using the methodologies of Weitz, J. I. et.al. J. Clin. Invest., 86: 
385-391 (1990). 
1. Inhibition of Fluid-phase Thrombin. 
Human a-thrombin (2 U/ml) was incubated with citrated plasma for 60 minutes 
at 37.degree. C. in the absence or presence of varying concentrations of 
each of the inhibitors. At timed intervals, 100 .mu.l aliquots were 
removed and the reaction was terminated by the addition of 
D-Phe-Pro-ArgCH.sub.2 Cl (5 .mu.M, final concentration). Unreacted 
fibrinogen was then precipitated with ethanol, and the ethanol 
supernatants were assayed for fibrinopeptide A (FPA). Thrombin addition to 
plasma results in rapid FPA generation which reaches a plateau in minutes 
as the thrombin is complexed and inactivated by fluid-phase 
antiproteases.(e.g. antithrombin III). 
FIG. 5A shows the effect of compound 8, Hirulog-1 and rHIR on the 
fluid-phase a-thrombin-mediated FPA generation in citrated human plasma. 
In this figure, the control (no inhibitor) group (o) was compared to 
compound 8 at a final concentration of 18.75 nM (1), Hirulog-1 at a final 
concentration of 500 nM (-) and rHIR at a final concentration of 4 nM(t). 
As indicated in FIG. 5A, all three inhibitors block thrombin-mediated FPA 
release. However, compound 8 and rHIR produce stable inhibition of FPA 
generation throughout the incubation period. In contrast, Hirulog-1 
(compound 25) produces only transient inhibition of thrombin-induced FPA 
generation. These findings are consistent with the data shown above in 
Example B for in vitro fibrin formation and further supports the concept 
that once Hirulog-1 undergoes proteolytic degradation within the 
Hirulog-1-thrombin complex, it can no longer fully inhibit thrombin 
activity. 
2. Inhibition of Clot-Bound Thrombin. 
Plasma clots were formed around wire hooks by the addition of CaCl.sub.2 
(final concentration, 25 mM) to 500 .mu.l aliquots of citrated plasma. 
After aging the clots for 60 minutes at 37.degree. C. with constant 
agitation, the clots were washed 10 times with 2 ml aliquots of 0.1M NaCl 
buffered with 0.05M Tris-HCL, pH 7.4 over an 18 hour period. The washed 
clots were then incubated in 1 ml aliquots of citrated plasma for 60 
minutes at 37.degree. C. in the absence or presence of the various 
inhibitors. At timed intervals, 100 .mu.l aliquots were removed and the 
reaction was terminated by the addition of the irreversible serine 
protease inhibitor, D-phenylalanyl-prolinyl-argininyl-CH.sub.2 Cl. 
Unreacted fibrinogen was precipitated with ethanol, and the ethanol 
supernatants were then assayed for FPA. 
FIG. 5B shows the effect of compound 8, Hirulog-1 and rHIR on the 
clot-bound a-thrombin-mediated FPA generation in citrated human plasma. In 
this figure, the control (no inhibitor) group (o) was compared to compound 
8 at a final concentration of 18.75 nM (1), Hirulog-1 at a final 
concentration of 500nM (-) and rHIR at a final concentration of 4 nM(t). 
As previously demonstrated by Weitz, J. I. et.al. J. Clin. Invest., 86: 
385-391 (1990) and shown in FIG. 5B, the incubation of a washed plasma 
clot in citrated plasma results in progressive, time-dependent FPA 
generation. Both compound 8 and rHIR were shown to stably inhibit 
clot-induced FPA generation. In contrast, Hirulog-1 only transiently 
inhibited FPA generation produced by the clot even at a .about.27-fold 
molar excess over the concentration of compound 8 (FIG. 5, panels A and 
B). 
Based on the data illustrated in FIGS. 5A and 5B, the inhibitory effects of 
the thrombin inhibitors against fluid-phase and clot-bound thrombin were 
compared based on the percent inhibition of free thrombin and clot-induced 
FPA generation produced by each inhibitor at 60 minutes incubation. FIG. 6 
depicts the comparative inhibitory effects of compound 8, Hirulog-1 and 
rHIR on fluid-phase (.rect-solid.) and clot-bound () a-thrombin-mediated 
FPA generation in citrated human plasma following a 60 minute incubation 
period. As illustrated in this figure, compound 8 and rHIR produce similar 
inhibition of both fluid-phase and clot-bound thrombin activity. In 
contrast, Hirulog-1 at a .about.27-fold molar excess over compound 8 had 
less inhibitory activity against clot-bound thrombin than it does against 
the free, fluid-phase enzyme following this incubation period. 
The results presented above clearly demonstrate that compound 8 can access 
and inhibit thrombin activity both free in plasma and more importantly 
bound to a fibrin-rich clot. The inhibitory profile of compound 8 is 
similar to that of rHIR in that the inhibition of these two pools of 
thrombin activity were stable for the 60 minute incubation period. This 
was in sharp contrast to Hirulog-1 which only showed transient inhibitory 
activity even at significantly higher molar concentrations. 
Example G 
Experimental Models of Thrombosis in Rabbits 
The antithrombotic properties of compound 8 and Hirulog-1 (compound 25) 
were evaluated using the following established in vivo experimental rabbit 
models of acute thrombosis. 
1. Rabbit Study of Venous Thrombus Growth. 
To determine the duration of antithrombotic efficacy with Compound 8 
following the termination of therapy, this inhibitor was compared to 
several other well-known antithrombotic agents in a rabbit model of venous 
thrombus growth described by Levi, M.et al., J. Thrombosis Haemostas., 66: 
218-221 (1991). In this model, a thrombin-induced clot is formed in the 
jugular vein of an anesthetized rabbit. Following the restoration of flow, 
the test compound is administered by intravenously bolus followed by 
infusion along with radiolabeled (.sup.125 I) autologous fibrinogen. The 
infusion of test compound is continued for 120 minutes. Following this 
time, the extent of fibrin accretion is determined by comparing the amount 
of radiolabel incorporated into the clot versus circulating in the 
peripheral blood. At this time, the infusion of the test compound is 
stopped and the experiment continued for another 60 minutes. At 180 
minutes, the extent of fibrin accretion is determined for the 
contralateral vessel. The data is expressed as "%Thrombus Growth" which is 
defined as (radioactivity in thrombus/radioactivity in blood).times.100. 
In this protocol, TAP(recombinant tick anticoagulant protein, a selective 
inhibitor of factor Xa described by Neeper, M. P. et. al., J. Biol. Chem. 
265:17746-17752 (1990)); recombinant Hirudin (rHIR) (CGP 39393) supplied 
by Ciba-Geigy, Horsham, UK.; and Hirulog-1. were separately administered 
intravenously as an initial bolus (0.5 mg/kg) followed by a continuous 
infusion (0.5 .mu.g/kg/min) for 120 minutes. Compound 8 was administered 
in two dosing regimens referred to as "Cmp8H" (1.0 mg/kg bolus +0.5 
.mu.g/kg/minute) and as "Cmp8M" (0.5mg/kg bolus +0.5.mu.g/kg/minute). LMWH 
(low molecular weight heparin, Fraxiparin, Sanofi, Paris, France) was 
administered as an initial bolus of 40 anti-Xa U/mg followed by a 0.33 
anti-Xa U/kg/min infusion. 
FIG. 7 shows the comparative inhibitory effects of compound 8 and other 
antithrombotic agents in this rabbit model of venous fibrin growth. In 
this figure, thrombus growth was assessed following the 120 minute 
infusion period (.rect-solid.) and 60 minutes after the termination of the 
infusion at 180 minutes (). "*" indicates a p.ltoreq.0.05 vs. time matched 
saline group, "**" indicates a p.ltoreq.0.001 versus time matched Compound 
8-M group. Results are presented as the Mean.+-.SEM. 
The data in this figure clearly demonstrate a clear reduction in thrombus 
size following termination of the compound 8 infusion period compared to 
the other antithrombotic agents. The data suggest that compound 8 is 
stably inhibiting preformed, fibrin-bound thrombin associated with the 
clot which results in reduced fibrin accretion. In the face of ongoing 
fibrinolysis, a reduction in clot size was observed. 
2. Rabbit jugular vein thrombosis/fibrinolysis model. 
In this series of experiments, the effects of compound 8 on enhancing the 
extent of endogenous fibrinolysis was compared to that of rHIR. 
New Zealand white rabbits of approximately 2.5 kg were anesthetized with 9 
mg Ketamin (Aescoket, Boxtel, the Netherlands) and 0.5 ml Rompun 2% 
(Bayer, Leverkusen, Germany) I.M. To maintain anesthesia, repeated 
intramuscular injections of Ketamin were given when appropriate. The 
carotid artery and the jugular veins were exposed through a medial 
incision in the neck. The carotid artery was cleared and a cannula (Baby 
Feeding Tube, 1.6 mm f) was introduced for the administration of the study 
medication and blood sampling. The jugular veins were cleared over a 
distance of 2 cm and all side branches were ligated. The veins were 
clamped both proximally and distally. 
Radiolabeled thrombi were produced in the jugular veins of the rabbit and 
the extent of fibrinolysis was determined by measuring the decrease in 
initial radioactivity of the preformed thrombi. Homologous rabbit blood 
was mixed with .sup.125 I-labeled fibrinogen (final radioactivity 
approximately 25 .mu.Ci per ml). An aliquot of 150 .mu.l l of this mixture 
was then aspirated in a 1 ml syringe containing 25 .mu.l thrombin (Human 
Thrombin T7009, Sigma Chemical Company, St. Louis, USA, 150 IU/ml) and 45 
.mu.l CaCl.sub.2 (0.25M) and quickly injected into the isolated venous 
segment. After 30 minutes of aging, the clamps were removed and the 
infusion of study medication started. The extent of endogenous 
fibrinolysis was assessed by measuring the remaining radioactivity of the 
thrombus at the end of the study as compared with the initial 
radioactivity and was expressed as a percentage of the initial thrombus 
volume. Both compound 8 and rHIR were administered as described in the 
previous study as an initial bolus (0.5mg/kg bolus) followed by a 
continuous infusion for 120 min (0.5 .mu.g/kg/min). 
FIG. 8 shows the effect of Compound 8 compared to rHIR on endogenous 
fibrinolysis of a preformed venous clot in a rabbit model of venous 
thrombosis. The extent of endogenous fibrinolysis was assessed following 
the 120 minutes infusion period (.rect-solid.) and 60 minutes after the 
termination of the infusion at 180 min (). "*" indicates a p.ltoreq.0.05 
versus time matched saline group, "**" indicates a p.ltoreq.0.001 versus 
time matched saline group, "t" indicates a p.ltoreq.0.001 versus time 
matched rHIR group. Results are presented as the Mean.+-.SEM. 
The data illustrated in FIG. 8 show that compound 8 can induce a greater 
degree of endogenous fibrinolysis resulting in a reduced clot size when 
compared to rHIR at the doses used. These results may explain the 
difference between these two agents in the thrombus growth experiments 
presented above in the rabbit model with respect to the size of the clot 
at the 180 minute time point. The stable inhibition of venous clot 
extension resulting in a reduction in clot size seen in this model may 
have important clinical implications for the treatment of established deep 
vein thrombosis (DVT). It is known that in most DVT patients there is an 
overall reduction in clot size which occurs over the course of continuous 
anticoagulant therapy with heparin or Coumadin. Hirsh, J., N. Engl. J. 
Med., 324:1565-1574 (1991). 
The potential of treating these patients for a short time with an agent 
such as compound 8 and obtaining long term antithrombotic efficacy 
resulting in a reduction in clot size could be a major advantage in 
reducing the period in which these patients are exposed to anticoagulant 
therapy. 
Example H 
Experimental Models of Thrombosis in Baboons 
1. Evaluation of Compound 8 in a baboon model of arterial thrombosis. 
The similarity of the coagulation responses between baboons and humans 
makes them an ideal experimental subject to study the effects of new 
antithrombotic agents on the thrombotic process. Harker, L. A. et al., 
Circulation, 83: Suppl IV IV-41-IV-55 (1991)). We therefore determined the 
the antithrombotic dose-duration response and corresponding hemostatic and 
hemorrhagic effects of compound 8 in a baboon model of heparin-resistant 
arterial thrombosis in a system which measures the formation of a 
platelet-rich thrombus on an exteriorized arterio-venous shunt as 
described by Harker, L. A. et al., Id. 
The antithrombotic doses of compound 8 were initially established using 
small amounts of reagent by infusing the drug into the boundary layer of 
rapidly flowing blood immediately proximal to a highly thrombogenic 
segment of Dacron vascular graft incorporated into an exteriorized chronic 
femoral arteriovenous (AV) shunt using a fusion device. The antithrombotic 
dose-response of compound 8 was then evaluated for drug administered by 
continuous intravenous infusion while concurrently assessing the 
corresponding antihemostatic effects. Subsequently, the duration of 
compound 8 therapy required to produce lasting antithrombotic effects were 
determined for vascular graft thrombosis by infusing a large fully 
antithrombotic dose of compound 8 for progressively longer periods of 
time, and following the accumulation of thrombus thereafter. 
a. Dose-response studies using the boundary layer infusion device. 
Initially, the dose-response between blood levels of compound 8 and the 
interruption of platelet-rich arterial type thrombus formation on highly 
thrombogenic segments of Dacron vascular graft interposed in exteriorized 
chronic femoral AV shunts was determined in a group of 4-5 animals using a 
proximal boundary layer infusion device. Drug was infused 
circumferentially through the pores of Goretex graft into the boundary 
layer interfacing blood flowing at 100 ml/min and the thrombogenic surface 
of 2 cm-long segments of Dacron vascular graft placed immediately distal 
to the infusion device (see below). The studies were begun by infusing 0.1 
ml/min of 500 nM compound 8 to achieve boundary layer concentrations of 
100 nM at the thrombogenic segment interface. Appropriate iteration of 
dosing established the concentration of drug required to reduce thrombus 
formation by half (IC.sub.50). With this approach the concentration of 
drug required to produce antithrombotic effects in vivo were established 
using less than 1/100 the amount of drug needed if systemic administration 
of the drug was employed. This approach minimizes the amount of drug 
needed for preliminary dose response and obviates possible toxicities. 
b. Systemic antithrombotic and antihemostatic comparison for Compound 8. 
A high-flow device which simulates arterial-type blood flow that generally 
exceeds 200 ml/min (shear rates of about 600-700 sec.sup.-1) was used to 
evaluate the antithrombotic effects of compound 8. Platelet-rich thrombus 
formation was induced by three different thrombogenic segments inserted as 
extension pieces into chronic arteriovenous (AV) access shunts in baboons 
as described by Kelly A. B. et al. in Blood, 77: 1006-1012 (1991) and 
Proc. Natl. Acad. Sci. USA, 89:6040-6044 (1992). Systemic administration 
is needed in order to coincidentally evaluate both antithrombotic benefits 
and hemostatic safety. Hemostatic function was also evaluated, including 
the template bleeding time to assess platelet hemostatic function, and the 
standard coagulation tests. 
The thrombogenic surface used in this study was a Dacron vascular graft as 
described by Kelly A. B. et al.,Id., incorporated into the exteriorized 
shunt. This thrombogenic surface was selected for study because it induces 
thrombus that is thrombin-mediated and platelet-dependent as well as 
highly reproducible and resistant to both aspirin and heparin in a 
clinically relevant manner. Based on the results in the boundary layer 
device (used to determine appropriate doses), compound 8 was continuously 
infused intravenously at rates required to produce high, intermediate and 
low antithrombotic effects. 
FIG. 9 shows the effect of compound 8 following systemic administration 
(infusion) over a 60 minute period to a conscious baboon on platelet 
deposition in the dacron vascular graft segment. The results are presented 
as the Mean.+-.SD with the corresponding replicates shown next to the 
treatment group in the figure. The control group(m) had 6 replicates, the 
group administered with 37 nmoles/minute/kg (-) had 3 replicates, the 
group administered with 75 nmoles/minute/kg (o) had 2 replicates and the 
group administered with 150 nmoles/minute/kg (.DELTA.) had 6 replicates. 
The various hemostatic parameters were also determined as shown in Table 
V. 
TABLE V 
______________________________________ 
Effects of Compound 8 on Blood markers of Thrombosis. 
Treatment Group 
37 75 150 
Parameter 
Control nmol/kg/min 
nmol/kg/min 
nmol/kg/min 
______________________________________ 
Platelet 4.65 .+-. 0.85 
1.57 .+-. 0.03 
1.17 .+-. 0.03 
0.39 .+-. 0.14 
deposition 
(.times. 10.sup.9) 
Compound 8 
0 13.5 .+-. 1.2 
23.5 .+-. 7.8 
62.8 .+-. 14.2 
levels (.mu.g/mL) 
Forearm 4.5 .+-. 0 
4 .+-. 0 9.8 .+-. 3.9 
10.9 .+-. 3.1 
Bleeding 
Time (min) 
Fibrinogen 
2.62 .+-. 0.35 
2.41 .+-. 0.45 
2.45 .+-. 0.45 
3.16 .+-. 0.78 
(mg/mL) 
APTT (sec) 
33 .+-. 1 &gt;300 &gt;300 &gt;300 
Platelet Count 
432 .+-. 28 
428 .+-. 35 
420 .+-. 0 
415 .+-. 56 
(.times. 10.sup.3) 
PF-4.sup.a (ng/mL) 
Pre-dosing 
2.7 .+-. 1.5 
2.6 2.8 1.3 
60 min 58.4 .+-. 13.3 
1.5 1.9 1.0 
bTG.sup.a (ng/mL) 
Pre-dosing 
2.5 .+-. 2.9 
2.1 2.6 2.1 
60 min 45.7 .+-. 19.3 
1.7 1.3 0.5 
FPA.sup.a (nM) 
Pre-dosing 
3.5 .+-. 1.1 
3.0 .+-. 0.9 
3.3 .+-. 1.7 
1.7 
60 min 26.8 .+-. 17.1 
1.45 .+-. 0.6 
0.75 .+-. 0.07 
0.7 .+-. 0.4 
TAT.sup.a (ng/mL) 
Pre-dosing 
7.5 .+-. 2.9 
5.9 .+-. 2.2 
6.5 .+-. 2.2 
8.4 .+-. 4.2 
60 min 53.3 .+-. 26 
2.6 .+-. 0.5 
2.2 .+-. 1.3 
2.0 .+-. 0.8 
______________________________________ 
.sup.a PF4 refers to platelet factor 4; bTG refers to bthromboglobulin; 
FPA refers to fibrinopeptide A; and TAT refers to thrombinantithrombin 
complexes. 
Interestingly as seen in Table V, there was no increase in forearm template 
bleeding time associated with the ED.sub.50 dose of compound 8 compared to 
a control time of 4 minutes. ED.sub.50 refers to the dose which gives a 
50% inhibition of platelet deposition. 
The observed data is in stark contrast to several other antithrombin-III 
independent antithrombin agents which have been evaluated in this model, 
including recombinant hirudin described by Kelly A B et al., Blood, 77: 
1006-1012 (1991)); the irreversible antithrombin peptide, 
D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone (D-FPR-CMK) described 
by Hanson S. R. and Harker L. A., Proc Natl Acad Sci., 85:3184-3188, 
(1988); the competitive antithrombin peptide,. 
D-phenyalanyl-prolinyl-boroarginine, (D-FPboroR); the bifunctional 
antithrombin peptide, Hirulog-1; the carboxy-terminal dodecapeptide of 
hirudin as described by Kelly AB et al., Proc. Natl. Acad. Sci. USA, 89: 
6040-6044 (1992); and arginine-based (argatroban) synthetic direct 
antithrombins as described by Harker L. A. et al., "Novel antithrombotic 
agents", Hemostasis and Thrombosis: Basic Principles and Clinical 
Practice, 3rd Ed. Philadelphia, J. P. Lippincott (Edits. Colman R. W. et 
al. 1992). All of these direct antithrombins interrupt platelet and fibrin 
deposition and thrombotic occlusion in a dose-dependent manner that is 
complete at the highest doses for all thrombogenic surfaces tested as 
shown in Table VI. 
TABLE VI 
______________________________________ 
Comparative Antithrombotic Effects of 
Compound 8 to Other Direct Antithrombins. 
Antithrombotic 
Antihemostatic 
Effects Effects 
ED.sub.50 Bleeding Time 
APTT 
Agent (nmol/kg/min) 
(min) (sec) 
______________________________________ 
Control -- 4 32 
Compound 8 35 4 &gt;300 
Hirulog-1 125 21 &gt;300 
r-Hirudin 5 12 130 
Argatroban &gt;800 &gt;30 &gt;300 
dF-P-R-al 250 14 145 
dF-P-boroR 25 12 95 
dF-P-R-CMK 25 12 95 
______________________________________ 
ED.sub.50Dose which gives a 50% inhibition of platelet deposition 
c. Duration of Compound 8 therapy for lasting antithrombotic effects. 
To assess the duration of the antithrombotic effect of compound 8 following 
a limited infusion period, compound 8 was infused intravenously at the 
highest, fully antithrombotic systemic dose (as determined above) for 3 
hours in animals bearing 4 segments of Dacron vascular graft in 
exteriorized chronic AV femoral shunts. Thrombus formation was assessed by 
gamma camera imaging. After discontinuing the infusion, subsequent 
accumulation of thrombus on the segments was measured for 2 of the 
segments throughout the following 3 hours, and again at 24 hours. The 
other 2 segments were transferred to the shunts of labeled shunt-bearing 
recipient animals to exclude the possibility that the thrombogenic 
response of blood was altered by the prior experimental procedures. 
FIG. 10 illustrates the results from a limited duration, high dose systemic 
infusion of compound 8 with the corresponding plasma levels shown in FIG. 
11. The infusion was over 3 hour period at a rate of 150 nmole/min/kg. In 
FIG. 10, the effect of this high-dose infusion of compound 8 (m) and of 
saline (l) on 24 hour platelet deposition and patency is shown. The 
results are presented as the Mean.+-.SD (n=6). FIG. 11 illustrates the 
plasma levels of compound 8 corresponding to the treatment groups shown in 
FIG. 10 for the 3 hour infusion period. The results are presented as the 
Mean.+-.SD (n=6). 
These data demonstrate that a limited (3 hours) high dose infusion of 
compound 8 results in an inhibition of platelet deposition for the 
duration of the experiment which was 24 hours. In addition, the graft 
segments remained patent with no reduction in blood flow. 
The results from this animal model demonstrate a dose-dependent reduction 
in platelet thrombus formation with a favorable antihemostatic profile 
compared to other antithrombin agents investigated in the same setting. 
The high degree of similarity between the baboon and human hemostatic 
systems suggests that similar antithrombotic efficacy in humans would be 
expected with this compound as well. 
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(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: 
GlyGlyGlyGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: 
GluGluIleProGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: 
GlyGlyGlyGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 38: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 39: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 40: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 41: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 42: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 43: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 44: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 45: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 46: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 47: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 48: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 49: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 50: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 51: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 52: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 53: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 54: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 55: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 56: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 57: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 58: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 59: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 60: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 61: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
GluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 62: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62: 
GlyGlyGlyGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 63: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: 
GlyGlyGlyGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 64: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 65: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 66: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 67: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 68: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 69: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 70: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 71: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 72: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: 
GlyGlyGlyGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 73: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: 
GlyGlyGlyGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 74: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 75: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 76: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 77: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 78: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78: 
GluGluIleProGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 79: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 80: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80: 
GluGluIleProGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 81: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 82: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: 
GluGluIleProGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 83: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 84: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 3 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84: 
GlyGlyXaaGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 85: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: 
GlyGlyXaaGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 86: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86: 
GlyGlyGlyGlyGlyXaaGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 87: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87: 
ProGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 88: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 15 is Tyr(O-SO3H). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
XaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 89: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 15 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: 
GlyGlyGlyGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
XaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 90: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90: 
GlyGlyLysGlyGlyAsnGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 91: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91: 
GlyGlyAsnGlyAspPheGluGluIleProGluTyrLeu 
1510 
(2) INFORMATION FOR SEQ ID NO: 92: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 13 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92: 
GlyGlyAsnGlyAspPheGluGluIleProGluTyrLeu 
1510 
(2) INFORMATION FOR SEQ ID NO: 93: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 94: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO:95: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95: 
GlyGlyGlyGlyGlyArgGlyAspPheGluGluIleProGlu 
1510 
TyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 96: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96: 
GlyGlyGlyGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 97: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: 
GlyGlyGlyGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 98: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98: 
GluGluIleProGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 99: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 100: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 101: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 102: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 103: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 104: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104: 
GluGluIleProGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 105: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 106: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 107: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 108: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 109: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 110: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110: 
GlyGlyGlyGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 111: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111: 
GluGluIleProGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 112: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112: 
GlyGlyGlyGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 113: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 114: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114: 
GlyGlyGlyGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 115: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115: 
GluGluIleProGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 116: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116: 
GlyGlyGlyGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 117: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 118: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 119: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119: 
GlugluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 120: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 121: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 122: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 123: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 123: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 124: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 125: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa at location 7 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125: 
GluGluIleProGluGluXaaLeu 
(2) INFORMATION FOR SEQ ID NO: 126: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126: 
GlyGlyGlyGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 127: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 6 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 127: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 128: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 128: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 129: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 129: 
GlyGlyGlyGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 130: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 7 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 130: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 131: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 7 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 131: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 132: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 132: 
GlyGlyGlyGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 133: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 6 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 133: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 134: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 134: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 135: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 135: 
GlyGlyGlyGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 136: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 7 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 136: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 137: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 7 is Tyr(3,5-diiodo) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 137: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 138: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 1 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 138: 
XaaGlyGlyGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 139: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 2 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 139: 
GlyXaaGlyGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 140: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 3 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 140: 
GlyGlyXaaGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 141: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 4 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 141: 
GlyGlyGlyXaaGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 142: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 5 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 142: 
GlyGlyGlyGlyXaaAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 143: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 1 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 143: 
XaaGlyGlyGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 144: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 2 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 144: 
GlyXaaGlyGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 145: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 3 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 145: 
GlyGlyXaaGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 146: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in position 4 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 146: 
GlyGlyGlyXaaGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 147: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 5 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 147: 
GlyGlyGlyGlyXaaArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 148: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 6 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 148: 
GlyGlyGlyGlyGlyXaaGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 149: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 149: 
GluGluIleProGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 150: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 150: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 151: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 6 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 151: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 152: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 152: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 153: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 7 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 153: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 154: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 7 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 154: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 155: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 155: 
GluGluIleProGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 156: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 156: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 157: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 6 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 157: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 158: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 6 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 158: 
GluGluIleProGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 159: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 7 is Tyr(3-iodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 159: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 160: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 7 is Tyr(3,5-diiodo). 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 160: 
GluGluIleProGluGluXaaLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 161: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 161: 
GluGluIleProGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 162: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 8 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 162: 
GluGluIleProGluGluTyrLeu 
15 
(2) INFORMATION FOR SEQ ID NO: 163: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 3 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 163: 
GlyGlyXaaGlyGlyAsnGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 164: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 3 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 164: 
GlyGlyXaaGlyGlyArgGlyAspPhe 
15 
(2) INFORMATION FOR SEQ ID NO: 165: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 9 AMINO ACIDS 
(B) TYPE: AMINO ACID 
(D) TOPOLOGY: LINEAR 
(ii) MOLECULE TYPE: PEPTIDE 
(ix) FEATURE: 
(D) OTHER INFORMATION: 
Xaa in location 6 is X. 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 165: 
GlyGlyGlyGlyGlyXaaGlyAspPhe 
15 
__________________________________________________________________________