Methods of increasing thrombomodulin expression

A method of increasing thrombomodulin expression comprising administering to a human in need of treatment an effective amount of a compound having the formula ##STR1## wherein R.sup.1 and R.sup.3 are independently hydrogen, --CH.sub.3, ##STR2## wherein Ar is optionally substituted phenyl; R.sup.2 is selected from the group consisting of pyrrolidino, hexamethyleneamino, and piperidino; or a pharmaceutically acceptable salt of solvate thereof. wherein R.sup.1 and R.sup.3 are independently hydrogen, --CH.sub.3, ##STR3## wherein Ar is optionally substituted phenyl; R.sup.2 is selected from the group consisting of pyrrolidino, hexamethyleneimino, and piperidino; and pharmaceutically acceptable salts and solvates thereof. Also encompassed by the invention is a method of inhibiting a thrombotic disorder or event which includes administering to a human in need thereof an effective amount of a compound of formula 1. Also encompassed by the invention is a method of increasing Protein C activation rate which includes the administration of a compound of formula 1.

BACKGROUND OF THE INVENTION 
The process of blood coagulation, thrombosis, is triggered by a complex 
proteolytic cascade leading to the formation of thrombin. Thrombin 
proteolytically removes activation peptides from the A.alpha. and 
B.beta.-chains of fibrinogen, which is soluble in blood plasma, initiating 
insoluble fibrin formation. 
Anti-coagulation is currently achieved by the administration of heparins 
and coumarins. Parenteral pharmacological control of coagulation and 
thrombosis is based on inhibition of thrombin through the use of heparins. 
Heparins act indirectly on thrombin by accelerating the inhibitory effect 
of endogenous antithrombin III (the main physiological inhibitor of 
thrombin). Because antithrombin III levels vary in plasma and because 
surface-bound thrombin seems resistant to this indirect mechanism, 
heparins can be an ineffective treatment. Because certain coagulation 
assays are believed to be associated with efficacy and safety, heparin 
levels are typically monitored with coagulation assays (particularly the 
activated partial thromboplastin time (APTT) assay). Coumarins impede the 
generation of thrombin by blocking the posttranslational 
gamma-carboxylation in the synthesis of prothrombin and other proteins of 
this type. Because of their mechanism of action, the effect of coumarins 
can only develop slowly, 6-24 hours after-administration. Further, they 
are non selective anticoagulants. Coumarins also require monitoring with 
coagulation assays (particularly the prothrombin time assay). 
To better understand the invention, the following brief description of the 
coagulation enzyme system is provided. The coagulation system, sometimes 
referred to as the "cascade", is best looked at as a chain reaction 
involving the sequential activation of zymogens into active serine 
proteases which eventually lead to the production of the enzyme, thrombin. 
Thrombin, through limited proteolysis, converts plasma fibrinogen into the 
insoluble gel, fibrin. Two key events in the coagulation cascade are the 
conversion of clotting Factor X to Xa by clotting factor IXa and the 
conversion of prothrombin into thrombin by clotting factor Xa. 
Both of these reactions occur on cell surfaces, most notably the platelet 
endothelial cell surfaces, and both reactions require cofactors. The major 
cofactors, factors V and VIII, circulate as relatively inactive 
precursors, but when the first few molecules of thrombin are formed, 
thrombin activates, by limited proteolysis, the cofactors. The activated 
cofactors, Va and VIIIa, accelerate, by about three orders of magnitude, 
both the conversion of prothrombin into thrombin and the conversion of 
factor X to factor Xa. 
Activated protein C overwhelmingly prefers two plasma protein substrates 
which it hydrolyzes and irreversibly destroys. These plasma protein 
substrates are the activated forms of clotting cofactors V and VIII 
(cofactors Va and VIIIa, respectively). Activated protein C only minimally 
degrades the inactive precursors, clotting factors V and VIII. In dogs, 
activated protein C has been shown to sharply increase circulating levels 
of the major physiological fibrinolytic enzyme, tissue plasminogen 
activator. 
The activation of protein C, however, involves thrombin, the final serine 
protease in the coagulation cascade, and an endothelial cell 
membrane-associated glycoprotein, thrombomodulin. Thrombomodulin forms a 
tight 1:1 stoichiometric complex with thrombin. Thrombomodulin, when 
complexed with thrombin, modifies substantially the functional properties 
of thrombin. Thrombin, in the coagulation pathway, normally clots 
fibrinogen, activates platelets, and converts clotting cofactors V and 
VIII to their activated forms, Va and VIIIa. Thrombin, alone, acts to 
activate Protein C, but only very slowly and inefficiently. In contrast, 
thrombin, when in the a 1:1 complex with thrombomodulin, fails to clot 
fibrinogen, does not activate platelets, and does not convert clotting 
factors V and VIII to their activated forms. The thrombin:thrombomodulin 
complex promotes the activation of protein C with the rate constant of 
protein C activation being as great as 20,000-fold higher for the 
thrombin:thrombomodulin complex than the rate constant for thrombin alone. 
Activated protein C, therefore, is an antithrombotic agent with a wider 
therapeutic index than other anticoagulants, such as heparin and the oral 
hydroxycoumarin-type anticoagulants, such as warfarin. Neither protein C 
nor activated protein C is effective until thrombin is generated at some 
local site. Activated protein C is virtually ineffective without thrombin, 
because thrombin is needed to convert clotting factors V to Va and VIII to 
VIIIa. As noted, the activated forms of these two cofactors are the 
preferred substrate for activated protein C. The protein C zymogen, when 
infused into patients, will remain inactive until thrombin is generated. 
Without the thrombomodulin:thrombin complex, the protein C zymogen is 
converted into activated Protein C at a very slow rate. 
The present invention is directed to the discovery that the compounds of 
the present invention, as defined below, increase thrombomodulin 
expressions and have oral bioavailability. 
SUMMARY OF THE INVENTION 
This invention provides methods of increasing thrombomodulin expression 
comprising administering to a human in need thereof an effective amount of 
a compound of formula I --CH.sub.3, 
##STR4## 
wherein R.sup.1 and R.sup.3 are independently hydrogen, 
##STR5## 
wherein Ar is optionally substituted phenyl; 
R.sup.2 is selected from the group consisting of pyrrolidino, 
hexamethyleneimino, and piperidino; and pharmaceutically acceptable salts 
and solvates thereof. 
Also encompassed by the invention is a method of inhibiting a thrombotic 
disorder or event which includes administering to a human in need thereof 
an effective amount of a compound of formula 1. 
Also encompassed by the invention is a method of increasing Protein C 
activation rate which includes the administration of a compound of formula 
1.

DETAILED DESCRIPTION OF THE INVENTION 
The current invention concerns the discovery that a select group of 
2-phenyl-3-aroylbenzothiophenes (benzothiophenes), those of formula I, are 
useful for increasing thrombomodulin expression. The methods of use 
provided by this invention are practiced by administering to a human or 
mammal in need thereof a dose of a compound of formula I or a 
pharmaceutically acceptable salt or solvate thereof, that is effective to 
increase thrombomodulin expression. The present method includes both 
medical therapeutic and/or prophylactic treatment, as appropriate. 
Inhibit is defined to include its generally accepted meaning which includes 
retarding, preventing, restraining, slowing or reversing. 
Raloxifene, a compound of this invention is the hydrochloride salt of a 
compound of formula 1, wherein R.sup.1 and R.sup.3 are hydrogen and 
R.sup.2 is 1-piperidinyl. 
Generally, the compound is formulated with common excipients, diluents or 
carriers, and compressed into tablets, or formulated as elixirs or 
solutions for convenient oral administration, or administered by the 
intramuscular or intravenous routes. The compounds can be administered 
transdermally, and may be formulated as sustained release dosage forms and 
the like. 
The compounds used in the methods of the current invention can be made 
according to established procedures, such as those detailed in U.S. Pat. 
Nos. 4,133,814, 4,418,068, and 4,380,635 all of which are incorporated by 
reference herein. In general, the process starts with a benzo[b]thiophene 
having a 6-hydroxyl group and a 2-(4-hydroxyphenyl) group. The starting 
compound is protected, alkylated or acylated, and deprotected to form the 
formula I compounds. Examples of the preparation of such compounds are 
provided in the U.S. patents discussed above. Optionally substituted 
phenyl includes phenyl and phenyl substituted once or twice with C.sub.1 
-C.sub.6 alkyl, C.sub.1 -C.sub.4 alkoxy, hdroxy, nitro, chloro, fluoro, or 
tri(chloro or fluoro)methyl. 
The compounds used in the methods of this invention form pharmaceutically 
acceptable acid and base addition salts with a wide variety of organic and 
inorganic acids and bases and include the physiologically acceptable salts 
which are often used in pharmaceutical chemistry. Such salts are also part 
of this invention. Typical inorganic acids used to form such salts include 
hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, 
hypophosphoric and the like. Salts derived from organic acids, such as 
aliphatic mono and dicarboxylic acids, phenyl substituted alkanoic acids, 
hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and 
aromatic sulfonic acids, may also be used. Such pharmaceutically 
acceptable salts thus include acetate, phenylacetate, trifluoroacetate, 
acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, 
hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, 
naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, 
.beta.-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caprate, 
caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate, 
heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, 
mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, 
phthalate, teraphthalate, phosphate, monohydrogenphosphate, 
dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, 
phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, 
bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzene-sulfonate, 
p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 
2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1sulfonate, 
naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartarate, 
and the like. A preferred salt is the hydrochloride salt. 
The pharmaceutically acceptable acid addition salts are typically formed by 
reacting a compound of formula I with an equimolar or excess amount of 
acid. The reactants are generally combined in a mutual solvent such as 
diethyl ether or benzene. The salt normally precipitates out of solution 
within about one hour to 10 days and can be isolated by filtration or the 
solvent can be stripped off by conventional means. 
Bases commonly used for formation of salts include ammonium hydroxide and 
alkali and alkaline earth metal hydroxides, carbonates, as well as 
aliphatic and primary, secondary and tertiary amines, aliphatic diamines. 
Bases especially useful in the preparation of addition salts include 
ammonium hydroxide, potassium carbonate, methylamine, diethylamine, 
ethylene diamine and cyclohexylamine. 
The pharmaceutically acceptable salts generally have enhanced solubility 
characteristics compared to the compound from which they are derived, and 
thus are often more amenable to formulation as liquids or emulsions. 
Pharmaceutical formulations can be prepared by procedures known in the art. 
For example, the compounds can be formulated with common excipients, 
diluents, or carriers, and formed into tablets, capsules, suspensions, 
powders, and the like. Examples of excipients, diluents, and carriers that 
are suitable for such formulations include the following: fillers and 
extenders such as starch, sugars, mannitol, and silicic derivatives; 
binding agents such as carboxymethyl cellulose and other cellulose 
derivatives, alginates, gelatin, and polyvinyl pyrrolidone; moisturizing 
agents such as glycerol; disintegrating agents such as calcium carbonate 
and sodium bicarbonate; agents for retarding dissolution such as paraffin; 
resorption accelerators such as quaternary ammonium compounds; surface 
active agents such as cetyl alcohol, glycerol monostearate; adsorptive 
carriers such as kaolin and bentonite; and lubricants such as talc, 
calcium and magnesium stearate, and solid polyethyl glycols. 
The compounds can also be formulated as elixirs or solutions for convenient 
oral administration or as solutions appropriate for parenteral 
administration, for instance by intramuscular, subcutaneous or intravenous 
routes. Additionally, the compounds are well suited to formulation as 
sustained release dosage forms and the like. The formulations can be so 
constituted that they release the active ingredient only or preferably in 
a particular part of the intestinal tract, possibly over a period of time. 
The coatings, envelopes, and protective matrices may be made, for example, 
from polymeric substances or waxes. 
The particular dosage of a compound of formula I required to increase 
thrombomodulin expression, inhibit a thrombotic disorder or event, or 
increase the Protein C activation rate, according to this invention, will 
depend upon the severity and nature of the condition, the route of 
administration, and related factors that will be decided by the attending 
physician. Generally, accepted and effective daily doses will be from 
about 0.1 to about 1000 mg/day, and more typically from about 50 to about 
200 mg/day. Such dosages will be administered to a subject in need of 
treatment from once to about three times each day, or more often as needed 
to effectively increase thrombomodulin expression, increase Protein C 
activation, or inhibit thrombotic disorders. 
It is usually preferred to administer a compound of formula I in the form 
of an acid addition salt, as is customary in the administration of 
pharmaceuticals bearing a basic group, such as the piperidino ring. It is 
also advantageous to administer such a compound by the oral route. For 
such purposes the following oral dosage forms are available. 
Formulations 
In the formulations which follow, "active ingredient" means a compound of 
formula I. 
Formulation 1: Gelatin Capsules Hard gelatin capsules are prepared using 
the following: 
______________________________________ 
Quantity 
Ingredient (mg/capsule) 
______________________________________ 
Active ingredient 0.1-1000 
Starch, NF 0-650 
Starch flowable powder 0-650 
Silicone fluid 350 centistokes 
0-15 
______________________________________ 
The ingredients are blended, passed through a No. 45 mesh U.S. sieve, and 
filled into hard gelatin capsules. 
Examples of specific capsule formulations of raloxifene that have been made 
include those shown below: 
Formulation 2: Raloxifene capsule 
______________________________________ 
Ingredient Quantity (mg/capsule) 
______________________________________ 
Raloxifene 1 
Starch, NF 112 
Starch flowable powder 
225.3 
Silicone fluid 350 centistokes 
1.7 
______________________________________ 
Formulation 3: Raloxifene capsule 
______________________________________ 
Ingredient Quantity (mg/capsule) 
______________________________________ 
Raloxifene 5 
Starch, NF 108 
Starch flowable powder 
225.3 
Silicone fluid 350 centistokes 
1.7 
______________________________________ 
Formulation 4: Raloxifene capsule 
______________________________________ 
Ingredient Quantity (mg/capsule) 
______________________________________ 
Raloxifene 10 
Starch, NF 103 
Starch flowable powder 
225.3 
Silicone fluid 350 centistokes 
1.7 
______________________________________ 
Formulation 5: Raloxifene capsule 
______________________________________ 
Ingredient Quantity (mg/capsule) 
______________________________________ 
Raloxifene 50 
Starch, NF 150 
Starch flowable powder 
397 
Silicone fluid 350 centistokes 
3.0 
______________________________________ 
The specific formulations above may be changed in compliance with the 
reasonable variations provided. 
A tablet formulation is prepared using the ingredients below: 
Formulation 6: Tablets 
______________________________________ 
Quantity 
Ingredient (mg/tablet) 
______________________________________ 
Active ingredient 0.1-1000 
Cellulose, microcrystalline 
0-650 
Silicon dioxide, fumed 0-650 
Stearate acid 0-15 
______________________________________ 
The components are blended and compressed to form tablets. 
Alternatively, tablets each containing 0.1-1000 mg of active ingredient are 
made up as follows: 
Formulation 7: Tablets 
______________________________________ 
Quantity 
Ingredient (mg/tablet) 
______________________________________ 
Active ingredient 0.1-1000 
Starch 45 
Cellulose, microcrystalline 
35 
Polyvinylpyrrolidone 4 
(as 10% solution in water) 
Sodium carboxymethyl cellulose 
4.5 
Magnesium stearate 0.5 
Talc 1 
______________________________________ 
The active ingredient, starch, and cellulose are passed through a No. 45 
mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone 
is mixed with the resultant powders which are then passed through a No. 14 
mesh U.S. sieve. The granules so produced are dried at 
50.degree.-60.degree. C. and passed through a No. 18 mesh U.S. sieve. The 
sodium carboxymethyl starch, magnesium stearate, and talc, previously 
passed through a No. 60 U.S. sieve, are then added to the granules which, 
after mixing, are compressed on a tablet machine to yield tablets. 
Suspensions each containing 0.1-1000 mg of medicament per 5 mL dose are 
made as follows: 
Formulation 8: Suspensions 
______________________________________ 
Quantity 
Ingredient (mg/5 ml) 
______________________________________ 
Active ingredient 0.1-1000 mg 
Sodium carboxymethyl cellulose 
50 mg 
Syrup 1.25 mg 
Benzoic acid solution 0.10 mL 
Flavor q.v. 
Color q.v. 
Purified water to 5 mL 
______________________________________ 
The medicament is passed through a No. 45 mesh U.S. sieve and mixed with 
the sodium carboxymethyl cellulose and syrup to form a smooth paste. The 
benzoic acid solution, flavor, and color are diluted with some of the 
water and added, with stirring. Sufficient water is then added to produce 
the required volume. 
In a further embodiment the invention relates to treatment, in a human or 
animal, of conditions where inhibition of a thrombotic disorder is 
required. Thrombotic disorders or events, or thromboembolic diseases, 
include a wide variety of acquired disease states involving intravascular 
coagulation, including deep vein thrombosis, pulmonary embolism, 
myocardial ischemia, myocardial infarction, cerebral thrombosis, general 
states, local hypercoaguable states and generalized tissue injury as it 
relates to the inflammatory process, microvascular arterial thrombosis, 
peripheral arterial thrombosis, emboli originating from the heart or 
peripheral arteries, acute myocardial infarction, thrombotic strokes, and 
disseminated intravascular coagulation. Disseminated intravascular 
coagulation occurs as a complication to numerous disease states including 
major trauma, major surgery, heat stroke, septicemia, acute and chronic 
liver disease, malignancies including solid tumors, leukemias and 
lymphomas, a wide variety of bacterial, fungal, parasitic and viral 
infections, obstetrical complications, hemolytic processes, cardiogenic 
shock, circulatory collapse from any cause, severe progressive strokes, 
snake bites, collagen vascular disorders, purpura fulminans, acute 
pancreatitis, allergic vasculitis, polycythemia vera, thrombocythemia, and 
ulcerative colitis among others. Although not yet discovered, congenital 
deficiencies in the expression of the thrombomodulin may be demonstrated 
in patients with thromboembolic problems. Acute episodes in such patients 
will be amenable to treatment with the compounds of Formula 1. 
Compounds of formula 1 are also believed to be useful in the treatment of 
thrombotic strokes. Thrombomodulin levels are low in vessels in the brain, 
thus upregulation will be important in thrombotic stroke. Today, strokes 
are not usually treated with conventional anticoagulants. Treatment of 
strokes with either heparin or oral anticoagulants, although occasionally 
beneficial, carries a high risk for bleeding into the infarcted brain 
area, thereby aggravating the neurological deficit accompanying the 
stroke. 
Further, the present compounds are believed to be useful in the treatment 
of acute myocardial infarction because there is evidence that the major 
mechanism of action of thrombomodulin and derivatives provided, that is, 
the activation of the protein C anticoagulant pathway, constitutes a 
highly effective means of achieving antithrombotic effects on the arterial 
side of the circulation, as well as reducing reperfusion injury. In 
current trials with thrombolytic agents in acute myocardial infarction and 
from animal experiments, it would appear that heparin, as adjunct therapy, 
is relatively ineffective, as an antithrombotic agent on the arterial side 
of the circulation. 
Also, the present compounds are believed to be useful in the treatment of 
disseminated intravascular coagulation "DIC"). There is experimental 
evidence that inflammatory mediators such as IL-1, TNF, and LPS endotoxin 
sharply down regulate the expression of thrombomodulin on endothelial 
cells leading in turn to defective activation of the protein C 
anticoagulant pathways. Heparin and the oral anticoagulants have been 
given to patients with disseminated intravascular coagulation in extensive 
clinical trials, but the results of these trials have been disappointing. 
Characteristically, patient with disseminated intravascular coagulation 
have widespread thrombi involving the microcirculation with concomitant 
and often severe bleeding problems, which result from "consumption" of 
essential clotting factors, which have been first activated and then 
inactivated during the formation of widespread microcirculatory fibrin 
thrombi. 
Because thrombomodulin is down regulated by a variety of inflammatory 
mediators, a preferred use of the compounds is in correcting inflammatory 
conditions associated with microvascular thrombosis. Broadly this would 
include any condition marked by dysfunctional vascular endothelium as 
occurs in sepsis, injuries involving major tissue damage and trauma, 
systemic inflammatory response syndrome, sepsis syndrome, septic shock, 
and multiple organ dysfunction syndrome, including DIC. 
Evidence exists that conventional anticoagulant drugs, particularly 
warfarin, are useful in the treatment of invasive malignant tumors. Many 
tumor cells produce substances which trigger the activation of the 
coagulation system resulting in local fibrin deposits. These fibrin 
deposits function as "nests" in which cancer cells can divide to form 
metastatic lesions. In one clinical study, it was shown that patients 
receiving warfarin in addition to cancer chemotherapy for treatment of 
small cell carcinoma of the lung live longer and have less extensive 
metastatic lesions than patients receiving chemotherapy alone. However, 
the cancer chemotherapy utilized in this study was less intensive than 
that considered optimal in clinical oncology today. The more intensive 
forms of cancer chemotherapy almost always produce a sharp decline in the 
platelet count, and thrombocytopenia combined with warfarin therapy puts 
the patient in an unacceptably high risk for serious bleeding 
complications. 
The compounds are expected to have utility in other diseases where blood 
coagulation could be a fundamental contributing process or source of 
secondary pathology, such as cancer, including matastisis, inflammatory 
diseases, including arthritis, and diabetes. The anti-coagulant compound 
is administered orally, parenterally e.g. by intravenous infusion (iv), 
intramuscular injection (im) or subcutaneously (sc). 
The method of this invention also is practiced in conjunction with a clot 
lysing agent e.g. tissue plasminogen activator (tPA), modified tPA, 
streptokinase or urokinase to reduce reperfusion injury. In cases when 
clot formation has occurred and an artery or vein is blocked, either 
partially or totally, a clot lysing agent is usually employed. A compound 
of the invention can be administered prior to or along with the lysing 
agent or subsequent to its use alone or along with the lysing agent and 
preferably further is administered along with aspirin to prevent the 
reoccurrence of clot formation. 
The method of this invention is also practiced in conjunction with a 
platelet glycoprotein IIb-IIIa receptor antagonist, that inhibits platelet 
aggregation. A compound of the invention can be administered prior to or 
along with the IIb-IIIa receptor antagonist or subsequent to its use to 
prevent the occurrence or reoccurrence of clot formation. 
The method of this invention is also practiced in conjunction with aspirin. 
A compound of the invention can be administered prior to or along with 
aspirin or subsequent to its use to prevent the occurrence or reoccurrence 
of clot formation. As stated above, preferably a compound of the present 
invention is administered in conjunction with a clot lysing agent and 
aspirin. 
ASSAYS 
Assay 1 
To further understand the action(s) of the compounds of formula 1, intimal 
smooth muscle cells and its role in enhancing anticoagulation of blood, it 
is necessary to investigate changes in thrombomodulin (TM) activity on the 
surface of these cell types. The compounds of formula 1 may also be used 
to reverse/correct any effects of mediators that tend to down regulate TM 
activity on the surface of these cells. 
Approximately 40,000-80,000 early passaged endothelial (arterial, venous, 
or microvascular), or intimal smooth muscle cells are seeded and grown to 
confluency in 24-well cell culture plates. The cell monolayer is 
subsequently washed 2-3 times with either Hank's buffered saline solution 
(HBSS), or serum-free medium (SFM). For a period of 24 hours, varying 
concentrations of a compound of formula 1 (ranging from micromolar to 
subpicomolar) are added to the cells in triplicate. The cells residing in 
negative control wells are maintained on serum-free medium with an amount 
of vehicle equivalent among all wells. 
The existing method to measure cell surface TM activity is performed by 
using a two-phase amidolytic assay. During the first phase of the assay, 
following rinsing of the cells with HBSS or SFM, 0.4 ml SFM containing 
human protein C (final concentration 11.2 ug/ml) and human alpha-thrombin 
(final concentration 0.1 NIHU/ml) are added to the monolayer and incubated 
at 37.degree. C. and 5% CO.sub.2. At 15, 30, and 45 minute time points, 
100 ul of medium is removed from each well and added to 50 ul of excess 
hirudin (20 anti-thrombin U/ml) in microtiter wells for 5 minutes at 
37.degree. C. in order to arrest further thrombin activity. In the absence 
of cells, SFM plus protein C and alpha-thrombin, as described above, is 
used as a negative control and treated similarly. 
In the second phase of the assay, 50 ul of 3mM 2366, a chromogenic 
substrate of protein C, is added to the conditioned media/hirudin mixture 
and the OD.sub.405 is measured by an automatic plate reader to monitor the 
kinetics of TM activity over a 4 minute period. Upon completion of this 
kinetic assay, measurement of total protein is performed using the BCA 
method. The final TM activity is expressed as % increase. Compound A is a 
compound of formula 1 wherein R.sup.1 and R.sup.3 are hydrogen, and 
R.sup.2 is 1-pyrrolidino. 
______________________________________ 
Compound A TM Activity 
conc. (.mu.M) (% increase) 
______________________________________ 
1 .times. 10.sup.-4 
14 
1 .times. 10.sup.-2 
15 
1 .times. 10.sup.-1 
15 
1.00 17 
10.00 17 
______________________________________ 
Assay 2 
The baboon model of E. Coli-induced sepsis as described in U.S. Pat. No. 
5,009,889 (incorporated herein by reference) is used to illustrate the 
compounds of formula 1 effects as antithrombotics and their ability to 
correct inflammation-induced endothelial dysfunction. 
Utility of the compounds of the invention is illustrated by the positive 
impact on thrombomodulin expression, thrombotic disorder, or Protein C 
activation rate characteristics displayed by any of the above assays.