1. Field of the Invention
The present invention relates to novel compounds that function as proteolytic enzyme inhibitors, and particularly to a new class of thrombin inhibitors.
2. Related Art
Proteases are enzymes that cleave proteins at single, specific peptide bonds. Proteases can be classified into four generic classes: serine, thiol or cysteinyl, acid or aspartyl, and metalloproteases (Cuypers et al., J. Biol. Chem. 257:7086 (1982)). Proteases are essential to a variety of biological activities, such as digestion, formation and dissolution of blood clots, reproduction and the immune reaction to foreign cells and organisms. Aberrant proteolysis is associated with a number of disease states in man and other mammals. The human neutrophil proteases, elastase and cathepsin G, have been implicated as contributing to disease states marked by tissue destruction. These disease states include emphysema, rheumatoid arthritis, corneal ulcers and glomerular nephritis. (Barret, in Enzyme Inhibitors as Drugs, Sandler, ed., University Park Press, Baltimore, (1980)). Additional proteases such as plasmin, C-1 esterase, C-3 convertase, urokinase, plasminogen activator, acrosin, and kallikreins play key roles in normal biological functions of mammals. In many instances, it is beneficial to disrupt the function of one or more proteolytic enzymes in the course of therapeutically treating a mammal.
Serine proteases include such enzymes as elastase (human leukocyte), cathepsin G, plasmin, C-1 esterase, C-3 convertase, urokinase, plasminogen activator, acrosin, chymotrypsin, trypsin, thrombin, factor Xa and kallikreins.
Human leukocyte elastase is released by polymorphonuclear leukocytes at sites of inflammation and thus is a contributing cause for a number of disease states. Cathepsin G is another human neutrophil serine protease. Compounds with the ability to inhibit the activity of these enzymes are expected to have an anti-inflammatory effect useful in the treatment of gout, rheumatoid arthritis and other inflammatory diseases, and in the treatment of emphysema. Chymotrypsin and trypsin are digestive enzymes. Inhibitors of these enzymes are useful in treating pancreatitis. Inhibitors of urokinase and plasminogen activator are useful in treating excessive cell growth disease states, such as benign prostatic hypertrophy, prostatic carcinoma and psoriasis.
The serine protease thrombin occupies a central role in hemostasis and thrombosis, and as a multifactorial protein, induces a number of effects on platelets, endothelial cells, smooth muscle cells, leukocytes, the heart, and neurons. Activation of the coagulation cascade through either the intrinsic pathway (contact activation) or the extrinsic pathway (activation by exposure of plasma to a non-endothelial surface, damage to vessel walls or tissue factor release) leads to a series of biochemical events that converge on thrombin. Thrombin cleaves fibrinogen ultimately leading to a hemostatic plug (clot formation), potently activates platelets through a unique proteolytic cleavage of the cell surface thrombin receptor (Coughlin, Seminars in Hematology 31(4):270-277 (1994)), and autoamplifies its own production through a feedback mechanism. Thus, inhibitors of thrombin function have therapeutic potential in a host of cardiovascular and non-cardiovascular diseases.
Factor Xa is another serine protease in the coagulation pathway. Factor Xa associates with factor Va and calcium on a phospholipid membrane thereby forming a prothrombinase complex. This prothrombinase complex then converts prothrombin to thrombin (Claeson, Blood Coagulation and Fibrinolysis 5:411-436 (1994); Harker, Blood Coagulation and Fibrinolysis 5 (Suppl 1):S47-S58 (1994)). Inhibitors of factor Xa are thought to offer an advantage over agents that directly inhibit thrombin since direct thrombin inhibitors still permit significant new thrombin generation (Lefkovits and Topol, Circulation 90(3):1522-1536 (1994); Harker, Blood Coagulation and Fibrinolysis 5 (Suppl 1):S47-S58 (1994)).
In vivo diagnostic imaging methods for intravascular thrombi have been previously reported. These imaging methods use compounds that are detectably labeled with radioactive or paramagnetic atoms. For example, platelets labeled with the gamma emitter, In-111, can be employed as an imaging agent for detecting thrombi (Thakur, M. L. et al., Thromb Res. 9:345 (1976); Powers et al., Neurology 32:938 (1982)). The thrombolytic enzyme streptokinase labeled with Tc-99m has been proposed as an imaging agent (Wong, U.S. Pat. No. 4,418,052(1983)). The fibrin-binding domains of Staphylococcus aureus derived protein A labeled with the gamma emitters, I-125 and I-131, have been proposed as imaging agents (Pang, U.S. Pat. No. 5,011,686 (1991)). Monoclonal antibodies having specificity for fibrin (in contrast to fibrinogen) and labeled with Tc-99m have been proposed as imaging agents (Berger et al., U.S. Pat. No. 5,024,829 (1991); Dean et al., U.S. Pat. No. 4,980,148 (1990)). The use of the paramagnetic contrasting agent, gadolinium diethylenetriaminepentaacetic acid in magnetic resonance imaging of patients treated by thrombolysis for acute myocardial infarction has been reported (De Roos, A. et al., Int. J. Card. Imaging 7:133 (1991)). Radiolabeled and paramagnetically labeled alpha-ketoamide derivatives have also been proposed as thrombus imaging agents (Abelman et al., U.S. Pat. No. 5,656,600).
A need continues to exist for non-peptidic compounds that are potent and selective protease inhibitors, and which possess greater bioavailability and fewer side-effects than currently available protease inhibitors. Accordingly, new classes of potent protease inhibitors, characterized by potent inhibitory capacity and low mammalian toxicity, are potentially valuable therapeutic agents for a variety of conditions, including treatment of a number of mammalian proteolytic disease states.
The present invention is directed to novel aminoguanidine (hydrazinoamidine) and alkoxyguanidine (alkoxyaminoamidine) pyrazinones having Formula I (below). Also provided are processes for preparing compounds of Formula I. The novel compounds of the present invention are potent inhibitors of proteases, especially trypsin-like serine proteases, such as chymotrypsin, trypsin, thrombin, plasmin and factor Xa. Certain of the compounds exhibit antithrombotic activity via direct, selective inhibition of thrombin, or are intermediates useful for forming compounds having antithrombotic activity. Also provided are methods of inhibiting or treating aberrant proteolysis in a mammal and methods of treating thrombosis, ischemia, stroke, restenosis or inflammation in a mammal by administering an effective amount of a compound of Formula I.
The invention includes a composition for inhibiting loss of blood platelets, inhibiting formation of blood platelet aggregates, inhibiting formation of fibrin, inhibiting thrombus formation, and inhibiting embolus formation in a mammal, comprising a compound of the invention in a pharmaceutically acceptable carrier. These compositions may optionally include anticoagulants, antiplatelet agents, and thrombolytic agents. The compositions can be added to blood, blood products, or mammalian organs in order to effect the desired inhibitions.
Also provided are methods of inhibiting or treating aberrant proteolysis in a mammal, and methods for treating myocardial infarction; unstable angina; stroke; restenosis; deep vein thrombosis; disseminated intravascular coagulation caused by trauma, sepsis or tumor metastasis; hemodialysis; cardiopulmonary bypass surgery; adult respiratory distress syndrome; endotoxic shock; rheumatoid arthritis; ulcerative colitis; induration; metastasis; hypercoagulability during chemotherapy; Alzheimer""s disease; Down""s syndrome; fibrin formation in the eye; and wound healing. Other uses of compounds of the invention are as anticoagulants either embedded in or physically linked to materials used in the manufacture of devices used in blood collection, blood circulation, and blood storage, such as catheters, blood dialysis machines, blood collection syringes and tubes, blood lines and stents.
The invention also includes a method for reducing the thrombogenicity of a surface in a mammal by attaching to the surface, either covalently or noncovalently, a compound of the invention.
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, such as 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.
Compounds of the present invention include compounds of Formula I:
or a solvate, hydrate or pharmaceutically acceptable salt thereof; wherein:
W is hydrogen, R1, R1OCO, R1CO, R1(CH2)sNHCO, or (R1)2CH(CH2)sNHCO,
wherein s is 0-4;
R1 is
R2,
R2(CH2)tC(R12)2, where t is 0-3, and each R12 can be the same or different,
(R2)(OR12)CH(CH2)p, where p is 1-4,
(R2)2(OR12)C(CH2)p, where p is 1-4,
R2C(R12)2(CH2)t, wherein t is 0-3, and each R12 can be the same or different, wherein (R12)2 can also form a ring with C represented by C3-7 cycloalkyl,
R2CF2C(R12)2(CH2)q, wherein q is 0-2, and each R12 can be the same or different, wherein (R12)2 can also form a ring with C represented by C3-7 cycloalkyl,
R2CH2C(R12)2(CH2)q, wherein q is 0-2, and each R12 can be the same or different, wherein (R12)2 can also form a ring with C represented by C3-7 cycloalkyl,
(R2)2CH(CH2)r, where r is 0-4 and each R2 can be the same or different, and wherein (R2)2 can also form a ring with CH represented by C3-7 cycloalkyl, C7-12 bicylic alkyl, C10-16 tricylic alkyl, or a 5- to 7-membered mono- or bicyclic heterocyclic ring which can be saturated or unsaturated, and which contains from one to three heteroatoms selected from the group consisting of N, O and S,
R2O(CH2)p, wherein p is 2-4,
(R2)2CF(CH2)r, wherein r is 0-4 and each R12 can be the same different, wherein (R12)2 can also form a ring with C represented by C3-7 cycloalkyl, C7-12 bicyclic alkyl, C10-16 tricyclic alkyl, or a 5- to 7-membered mono- or bicyclic heterocyclic ring which can be saturated or unsaturated, and which contains from one to three heteroatoms selected from the group consisting of N, O and S,
xe2x80x83where s is 0 or 1, or
R2CF2C(R12)2;
R2 is
phenyl, naphthyl, or biphenyl, each of which is unsubstituted or substituted with one or more of C1-4 alkyl, C1-4 alkoxy, halogen, hydroxy, CF3, OCF3, COOH, CONH2, or SO2NH2,
a 5- to 7-membered mono- or a 9- to 10-membered bicyclic heterocyclic ring or non-heterocyclic ring which can be saturated or unsaturated, wherein the heterocyclic ring contains from one to four heteroatoms selected from the group consisting of N, O and S, and wherein the heterocyclic or non-heterocyclic ring is unsubstituted or substituted with halogen or hydroxy,
C1-7 alkyl, unsubstituted or substituted with one or more of hydroxy, COOH, amino, aryl, C3-7 cycloalkyl, CF3, N(CH3)2, xe2x80x94C1-3alkylaryl, heteroaryl, or heterocycloalkyl,
CF3,
C3-7 cycloalkyl, unsubstituted or substituted with aryl,
C7-12 bicyclic alkyl, or
C10-16 tricyclic alkyl;
R3 is
hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, optionally substituted aryl, optionally substituted aralkyl, optionally substituted heteroaryl, trifluoromethyl, halogen, hydroxyalkyl, cyano, nitro, carboxamido, xe2x80x94CO2Rx, xe2x80x94CH2ORx or xe2x80x94ORx,
where Rx, in each instance, is independently one of hydrogen, alkyl or cycloalkyl wherein said alkyl or cycloalkyl groups may optionally have one or more unsaturations;
R4 is
hydrogen or halogen;
R12 is
hydrogen,
phenyl, naphthyl, or biphenyl, each of which is unsubstituted or substituted with one or more of C1-4 alkyl, C1-4 alkoxy, halogen, hydroxy, CF3, OCF3, COOH, or CONH2,
a 5- to 7-membered mono- or a 9- to 10-membered bicyclic heterocyclic ring which can be saturated or unsaturated, and which contains from one to four heteroatoms selected from the group consisting of N, O and S,
C1-4 alkyl, unsubstituted or substituted with one or more of hydroxy, COOH, amino, aryl, heteroaryl, or heterocycloalkyl,
CF3,
C3-7 cycloalkyl,
C7-12 bicyclic alkyl, or
C10-16 tricyclic alkyl;
R5 is hydrogen, C1-4alkyl, or C2-4 alkenyl;
R6 is hydrogen, alkyl, alkenyl, aralkyl, aryl, hydroxyalkyl, aminoalkyl, monoalkylamino(C2-10)alkyl, dialkylamino(C2-10)alkyl or carboxyalkyl;
R7, R8, R9 and R10 are independently hydrogen, alkyl, aralkyl, aryl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl or carboxyalkyl;
or R7 and R8 are taken together to form xe2x80x94(CH2)uxe2x80x94, where u is 2 to 7, preferably 2 to 5, while R9 and R10 are defined as above;
or R9 and R10 are taken together to form xe2x80x94(CH2)vxe2x80x94, where v is 2 to 7, preferably 2 to 5, while R7 and R8 are defined as above;
or R7 and R9 are taken together to form xe2x80x94(CH2)yxe2x80x94, where y is 0 (a bond) or 1 to 7, preferably 0-4, while R8 and R10 are defined as above;
X is oxygen, NR11, or CH=N (where N is bonded to NR6)
where R11 is hydrogen, alkyl, cycloalkyl or aryl, wherein said alkyl, cycloalkyl or aryl can be optionally substituted with amino, monoalkylamino, dialkylamino, alkoxy, hydroxy, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, aryl, heteroaryl, acylamino, cyano or trifluoromethyl;
Ra, Rb and Rc are independently hydrogen, alkyl, hydroxy, alkoxy, aryloxy, aralkoxy, alkoxycarbonyloxy, cyano or xe2x80x94CO2Rw, where
Rw is alkyl, cycloalkyl, phenyl, benzyl,
where Rd and Re are independently hydrogen, C1-6 alkyl, C2-6 alkenyl or phenyl, Rf is hydrogen, C1-6 alkyl, C2-6 alkenyl or phenyl, Rg is hydrogen, C1-6 alkyl, C2-6 alkenyl or phenyl, and Rh is aralkyl or C1-6 alkyl;
n is from zero to 8; and
m is from zero to 6.
In one class of compounds and pharmaceutically acceptable salts thereof, R3 is hydrogen, C1-4 alkyl, C3-7 cycloalkyl, or CF3; preferably C1-4 alkyl, and m and n are each 0 to 4.
In a subclass of this class of compounds and pharmaceutically acceptable salts thereof, R4 is hydrogen or halogen.
In a group of this subclass of compounds and pharmaceutically acceptable salts thereof, W is H or R1.
In a subgroup of this group of compounds and pharmaceutically acceptable salts thereof, R1 is
R2,
R2(CH2)tC(R12)2, where t is 0-3, and each R12 can be the same or different,
R2C(R12)2(CH2)t, wherein t is 0-3, and each R12 can be the same or different, wherein (R12)2 can also form a ring with C represented by C3-7 cycloalkyl,
R2CH2C(R12)2(CH2)q, wherein q is 0-2, and each R12 can be the same or different, wherein (R12)2 can also form a ring with C represented by C3-7cycloalkyl,
(R2)2CH(CH2)r, where r is 0-4 and each R2 can be the same or different, and wherein (R2)2 can also form a ring with CH represented by C3-7 cycloalkyl, C7-12 bicylic alkyl, C10-16 tricylic alkyl, or a 5- to 7-membered mono- or bicylic heterocyclic ring which can be saturated or unsaturated, and which contains from one to three hetero atoms selected from the group consisting of N, O and S,
R2CF2C(R12)2(CH2)q, wherein q is 0-2, and each R12 can be the same or different, wherein (R12)2 can also form a ring with C represented by C3-7cycloalkyl, or
R2O(CH2)p, wherein p is 2-4;
R2 is
phenyl or naphthyl, each of which is unsubstituted or substituted with one or more of C1-4 alkyl, C1-4 alkoxy, halogen, hydroxy, CF3, OCF3, or SO2NH2,
a 5- to 7-membered mono- or a 9- to 10-membered bicyclic heterocyclic ring or non-heterocyclic ring which can be saturated or unsaturated, wherein the heterocyclic ring contains from one to four heteroatoms selected from the group consisting of N, O and S, and wherein the heterocyclic or non-heterocyclic ring is unsubstituted or substituted with halogen or hydroxy,
C1-7 alkyl, unsubstituted or substituted with one or more of hydroxy, COOH, C3-7 cycloalkyl, CF3, N(CH3)2, xe2x80x94C1-3alkylaryl, heteroaryl, or heterocycloalkyl,
CF3, or
C3-7 cycloalkyl, unsubstituted or substituted with aryl; and
R12 is
hydrogen, or
C1-4 alkyl, unsubstituted or substituted with one or more of hydroxy, COOH, amino, aryl, heteroaryl, or heterocycloalkyl.
In a family of this subgroup of compounds and pharmaceutically acceptable salts thereof,
R3 is H, CH3, or CH2CH3;
R4 is H or chloro; and
W is PhCH2CH2, (CH3)3Cxe2x80x94, HOOCCH2, CF3CH2, (CH3)2N(CH2)2, PhCH2O(CH2)2, PhCH(CH3), PhCH2CH(COOH), CH3(CH2)5, PhCH2, H, CH3(CH2)4, CH3CH2CH(CH3)CH2, (Ph)2CHCH2, PhCH2CH(CH3), PhCH(CH3)CH2, (CH3)2CH, PhCH(OH)CH2, PhC(CH3)2CH2, (Ph)2CHCH2, or
A preferred R5 group is hydrogen.
Preferred compounds when X is NR11 are those wherein R11 is hydrogen or C1-6 alkyl, optionally substituted by one, two or three, preferably one, of amino, monoalkylamino, dialkylamino, alkoxy, hydroxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carboalkoxy, phenyl, cyano, trifluoromethyl, acetylamino, pyridyl, thiophenyl, furyl, pyrrolyl or imidazolyl.
Suitable values of R11 include hydrogen, methyl, ethyl, propyl, n-butyl, benzyl, phenethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, carboxymethyl and carboxyethyl.
Most preferred compounds are those where X is oxygen.
Preferred compounds are those of Formula I, where R6 is hydrogen or C1-6 alkyl.
Preferred compounds are those of Formula I, where R7, R8, R9 and R10 are independently one of hydrogen, C1-6 alkyl, C6-10 ar(C1-6)alkyl, C6-10 aryl, C2-10 hydroxyalkyl or C2-7 carboxyalkyl. Useful values of R7, R8, R9 and R10 include hydrogen, methyl, ethyl, propyl, n-butyl, benzyl, phenylethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, 2-carboxymethyl, 3-carboxyethyl and 4-carboxypropyl.
Also preferred are compounds where either R7 and R8, or R9 and R10 combine as an alkylene linker, xe2x80x94(CH2)uxe2x80x94 and xe2x80x94(CH2)vxe2x80x94 respectively, where u and v are each 2-5, most preferably 2 or 3.
Preferred values of Ra, Rb and Rc in Formula I are hydrogen, hydroxy, C1-6 alkyl, C1-6 alkoxy, cyano or xe2x80x94CO2Rw, where Rw, in each instance, is preferably one of C1-4alkyl, C4-7cycloalkyl or benzyloxycarbonyl. Suitable values of Ra, Rb and Rc include hydrogen, methyl, ethyl, propyl, n-butyl, hydroxy, methoxy, ethoxy, cyano, xe2x80x94CO2CH3, xe2x80x94CO2CH2CH3 and xe2x80x94CO2CH2CH2CH3. In the most preferred embodiments, Ra, Rb and Rc are each hydrogen.
Also preferred at Ra, Rb and Rc is the group xe2x80x94CO2Rw, where Rw is one of
where Rd-Rh are defined as above. When Ra, Rb and Rc are xe2x80x94CO2Rw, where Rw is one of these moieties, the resulting compounds are prodrugs that possess desirable formulation and bioavailability characteristics. A preferred value for each of Rd, Re and Rg is hydrogen, Rw is methyl, and preferred values for Rh include benzyl and tert-butyl.
Preferred values of n in Formula I include from zero to 6, more preferably from zero to 4, and most preferably zero, 1 or 2.
Preferred values of m are from zero to 4, most preferably zero, 1 or 2.
Especially preferred compounds are represented by Formulae II, III and IV:
or a solvate, hydrate or pharmaceutically acceptable salt thereof; wherein
W is as defined, and has the preferred values, as for Formula I, above;
X is xe2x80x94Oxe2x80x94, xe2x80x94NHxe2x80x94 or xe2x80x94CHxe2x95x90Nxe2x80x94 (an amidinohydrazone group, where the NH is attached to the NH group of the parent formula). X is preferably attached to the 3- or 4- position of the ring, most preferably the 4- position.
R3 is hydrogen, C1-3 alkyl, halogen or C1-2 alkoxy;
R4 is hydrogen or halogen;
n is 0, 1, 2 or 3, preferably 0, 1 or 2;
m is 0, 1, 2 or 3, preferably 0 or 1;
nxe2x80x2 is 0, 1, 2 or 3, preferably 0, 1 or 2; and
mxe2x80x2 is 0, 1, 2 or 3, preferably 0 or 1.
Specific compounds within the scope of the invention include the following:
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(phenethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2,2-diphenylethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonymethyl-6-methyl-3-(4-methylphenethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(4-methoxyphenethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(1-phenylcyclobutyl)methylamino-pyrazinone,
1-{N-[2-(amidinoaaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-[2-(1-naphthalene)ethyl]amino-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-phenyl-1-butylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[3,4-methylenedioxyphenyl]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[2-pyridyl]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[2-methylphenyl]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[3-methylphenyl]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[2-trifluoromethylphenyl]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[3-trifluoromethylphenyl]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[4-trifluoromethylphenyl]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[3,5-dimethylphenyl]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-indanylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[3,4-difluorophenyl]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[5-indanyl]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-fluorophenethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(3,4-dimethoxyphenethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(4-fluorophenethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(4-ethylphenethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-phenylpropylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(3,4-dimethylphenethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-naphthaleneethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2,2-diphenylpropylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-(3-indolyl)-ethylamino-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[1-(4-methylnaphthalene)]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[2,4-difluorophenyl]ethylamino)-pyrazinone.
1-{N-[2-(amidino-Nxe2x80x2-methylaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-[4-methylphenyl]ethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-5-chloro-6-methyl-3-(phenethylamino)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-5-chloro-6-methyl-3-(2,2-diphenylethylamino)-pyrazinone,
1-{N-[2-(amidinohydrazino)ethyl]amino}carbonylmethyl-6-methyl-3-(phenethyl)-pyrazinone,
1-{N-[2-(amidinohydrazino)ethyl]amino}carbonylmethyl-6-methyl-3-(1-methyl-2-phenylethyl)-pyrazinone,
1-{N-[2-(amidinohydrazino)ethyl]amino}carbonylmethyl-6-ethyl-3-(phenethyl)-pyrazinone,
1-{N-[2-(amidinohydrazino)ethyl]amino}carbonylmethyl-6-methyl-3-(4-methylphenylethyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(1-methyl-2-(4-methylamino-3-pyridyl)ethyl-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(1-methyl-2-(3-pyridyl)ethyl-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-cyclopropyl-2-(3,4-dimethoxyphenyl)ethyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-cyclobutylethyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-cyclobutyl-2,2-difluoroethyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-(3-fluorophenyl)ethyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2,2-difluoro-2-phenylethyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-phenylcyclopropyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-(4-chlorophenyl)-2-cyclopropylethyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-cyclopropyl-2-(3-pyridyl)ethyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-benzylcylopropyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-cyclopropyl-2-(3-fluorophenyl)ethyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2-cyclopropyl-2-(3,4-difluorophenyl)ethyl)-pyrazinone,
1-{N-[2-(amidinoaminooxy)ethyl]amino}carbonylmethyl-6-methyl-3-(2,2-difluoro-2-phenylethylamino)-pyrazinone,
as well as pharmaceutically acceptable salts thereof, for example the hydrochloride, acetate and trifluoroacetate salts thereof.
It is also to be understood that the present invention is considered to include stereoisomers as well as optical isomers, e.g. mixtures of enantiomers as well as individual enantiomers and diastereomers, which arise as a consequence of structural asymmetry in selected compounds of the present series. The compounds of the present invention may also have polymorphic crystalline forms, with all polymorphic crystalline forms being included in the present invention.
The compounds of Formula I may also be solvated, especially hydrated. Hydration may occur during manufacturing of the compounds or compositions comprising the compounds, or the hydration may occur over time due to the hygroscopic nature of the compounds.
Certain compounds within the scope of Formula I are derivatives referred to as prodrugs. The expression xe2x80x9cprodrugxe2x80x9d denotes a derivative of a known direct acting drug, which derivative has enhanced delivery characteristics and therapeutic value as compared to the drug, and is transformed into the active drug by an enzymatic or chemical process. Useful prodrugs are those where Ra, Rb and/or Rc are xe2x80x94CO2Rw, where Rw is defined above. See, U.S. Pat. No. 5,466,811 and Saulnier et al., Bioorg. Med. Chem. Lett. 4:1985-1990 (1994).
When any variable occurs more than one time in any constituent or in Formula I, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.
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, such as a radioactive or paramagnetic atom.
In another aspect, the present invention provides diagnostic compositions which are used 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.
According to a preferred aspect, useful compounds are those wherein the R1 substituent is substituted with a detectable label, such as a radioactive iodine atom, such as I-125, I-131 or I-123. In this aspect, R1 is preferably phenyl, having a para I-123, para I-125 or para I-131 substitution, or benzyl, having a metal I-123, meta I-125 or meta I-131 substitution.
The detectable label can also be a radioactive or paramagnetic chelate in which a suitable ligand (L) is attached to an R1 substituent, either directly or via a divalent linking group Axe2x80x3. Alternatively, the group xe2x80x94Axe2x80x3xe2x80x94L substitutes for the groups W in Formula I. By suitable ligand is meant an organic moiety that is capable of chelating a radioactive or paramagnetic metal ion.
In these compounds, the divalent linking group Axe2x80x3 includes groups that are capable of covalently bonding with a free amino group and the chelating means. For example, Axe2x80x3 may be xe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90NH)xe2x80x94(CH2)6xe2x80x94C(xe2x95x90NH)xe2x80x94, xe2x80x94C(xe2x95x90O)xe2x80x94(CH2)6xe2x80x94C(xe2x95x90O)xe2x80x94,
and the like.
Also, in the compounds represented by Formula I, the chelating ligand, L, includes groups capable of covalently bonding to or noncovalently binding to either a radioactive or paramagnetic atom. The chelating means including 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 groups or by up to four separated ethylene units separated by a nitrogen or oxygen or sulfur atom. Preferred as a completing means is diethylenetrimine-N,N,Nxe2x80x2,Nxe2x80x3,Nxe2x80x3-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. No. 4,652,440 (1987); Gries, H. et al., U.S. Pat. No. 4,957,939 (1990). An preferred chelating ligand, L, is 1-(p-aminobenzyl)-diethylenetriaminepentaacetic acid. Also included as chelating means are compounds which contain sulfhdryl 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 term xe2x80x9calkylxe2x80x9d as employed herein by itself or as part of another group refers to both straight and branched chain radicals of up to 12 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl.
The term xe2x80x9calkenylxe2x80x9d is used herein to mean a straight or branched chain radical of 2-20 carbon atoms, unless the chain length is limited thereto, including, but not limited to, ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Preferably, the alkenyl chain is 2 to 10 carbon atoms in length, more preferably, 2 to 8 carbon atoms in length most preferably from 2 to 4 carbon atoms in length.
The term xe2x80x9calkynylxe2x80x9d is used herein to mean a straight or branched chain radical of 2-20 carbon atoms, unless the chain length is limited thereto, wherein there is at least one triple bond between two of the carbon atoms in the chain, including, but not limited to, acetylene, 1-propylene, 2-propylene, and the like. Preferably, the alkynyl chain is 2 to 10 carbon atoms in length, more preferably, 2 to 8 carbon atoms in length, most preferably from 2 to 4 carbon atoms in length.
In all instances herein where there is an alkenyl or alkynyl moiety as a substituent group, the unsaturated linkage, i.e., the vinylene or acetylene linkage is preferably not directly attached to a nitrogen, oxygen or sulfur moiety.
The term xe2x80x9calkoxyxe2x80x9d is used herein to mean a straight or branched chain radical of 1 to 20 carbon atoms, unless the chain length is limited thereto, bonded to an oxygen atom, including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the like. Preferably the alkoxy chain is 1 to 10 carbon atoms in length, more preferably 1 to 8 carbon atoms in length.
The term xe2x80x9carylxe2x80x9d as employed herein by itself or as part of another group refers to monocyclic or bicyclic aromatic groups containing from 6 to 12 carbons in the ring portion, preferably 6-10 carbons in the ring portion, such as phenyl, naphthyl or tetrahydronaphthyl.
The term xe2x80x9cheteroarylxe2x80x9d as employed herein refers to groups having 5 to 14 ring atoms; 6, 10 or 14xcfx80 electrons shared in a cyclic array; and containing carbon atoms and 1, 2 or 3 oxygen, nitrogen or sulfur heteroatoms (where examples of heteroaryl groups are: thienyl, benzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl, phenoxathiinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimindinyl, pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinazolinyl, cinnolinyl, pteridinyl, 4xcex1H-carbazolyl, carbazolyl, xcex2-carbolinyl, phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanyl and phenoxazinyl groups).
The term xe2x80x9caralkylxe2x80x9d or xe2x80x9carylalkylxe2x80x9d as employed herein by itself or as part of another group refers to C1-6alkyl groups as discussed above having an aryl substituent, such as benzyl, phenylethyl or 2-naphthylmethyl.
The term xe2x80x9ccycloalkylxe2x80x9d as employed herein by itself or as part of another group refers to cycloalkyl groups containing 3 to 9 carbon atoms, preferably 3 to 7 carbon atoms. Typical examples are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and cyclononyl.
The term xe2x80x9cC7-12 bicyclic alkylxe2x80x9d is intended to include bicyclo[2.2.1]heptyl (norbornyl), bicyclo[2.2.2]octyl, 1,1,3-trimethylbicyclo[2.2.1]heptyl(bornyl), and the like.
The term xe2x80x9cC10-16 tricyclic alkylxe2x80x9d is intended to include tricyclo[5, 2, 1, 02,6]decyl, adamantyl, and the like.
The term xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d as employed herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine with chlorine being preferred.
The term xe2x80x9cmonoalkylaminexe2x80x9d as employed herein by itself or as part of another group refers to an amino group which is substituted with one alkyl group having from 1 to 6 carbon atoms.
The term xe2x80x9cdialkylaminexe2x80x9d as employed herein by itself or as part of another group refers to an amino group which is substituted with two alkyl groups, each having from 1 to 6 carbon atoms.
The term xe2x80x9chydroxyalkylxe2x80x9d as employed herein refers to any of the above alkyl groups substituted by one or more hydroxyl moieties.
The term xe2x80x9ccarboxyalkylxe2x80x9d as employed herein refers to any of the above alkyl groups substituted by one or more carboxylic acid moieties.
The term xe2x80x9cheterocyclexe2x80x9d or xe2x80x9cheterocyclic ringxe2x80x9d, as used herein except where noted, represents a stable 5- to 7-membered mono- or bicyclic or stable 7- to 10-membered bicyclic heterocyclic ring system any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. Especially useful are rings containing one oxygen or sulfur, one to three nitrogen atoms, or one oxygen or sulfur combined with one or two nitrogen atoms. The hetero cyclic ring may be attached at any hetero atom or carbon atom which results in the creation of a stable structure. Examples of such heterocyclic groups include piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, pyrrolyl, 4-piperidonyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, thiadiazoyl, benzopyranyl, benzothiazolyl, benzoxazolyl, furyl, tetrahydrofuryl, tetrahydropyranyl, thienyl, benzothienyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and oxadiazolyl. Morpholino is the same as morpholinyl.
The term xe2x80x9cheteroatomxe2x80x9d is used herein to mean an oxygen atom (xe2x80x9cOxe2x80x9d), a sulfur atom (xe2x80x9cSxe2x80x9d) or a nitrogen atom (xe2x80x9cNxe2x80x9d). It will be recognized that when the heteroatom is nitrogen, it may form an NRaRb moiety, wherein Ra and Rb are, independently from one another, hydrogen or C1 to C8 alkyl, or together with the nitrogen to which they are bound, form a saturated or unsaturated 5-, 6-, or 7-membered ring.
Schemes 1 and 2 outline the synthetic steps to produce compounds of Formula I.
In Schemes 1 and 2, W, R3, R7, R8, R9, R10, Ra, Rb, Rc, n and m are as defined above; R4=Cl or Br; P is an amino protecting group, such as benzyloxycarbonyl (Cbz), and Pxe2x80x2 is an ester protecting group, such as benzyl.
In Scheme 1, protected aminoalcohol 1 is coupled to N-hydroxyphthalimide 2 using a Mitsunobu coupling procedure (Mitsunobu, O. Synthesis 1 (1981)) to provide compound 3. Preferred coupling conditions include using a trialkylphosphine or triarylphosphine, such as triphenylphosphine, in a suitable solvent, such as tetrahydrofuran or methylene chloride, and a dialkyl azodicarboxylate, such as diethyl azodicarboxylate. Unveiling of the phthalimide protecting group is accomplished using standard conditions well known in the art (Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd edition, John Wiley and Sons, Inc. New York (1991)), such as methylamine or hydrazine, in an appropriate solvent, such as ethanol or isopropanol. Guanidinylation of the resulting alkoxyamine to 4 using substituted guanidinylating reagents such as N,Nxe2x80x2-bis(tert-butoxycarbonyl)-S-methylisothiourea (Bergeron, R. J. and McManis, J. S., J Org. Chem. 52:1700 (1987)) or N-Ra, N-Rb, N-Rc-1H-pyrazole-1-carboxamidine (Bernatowicz, M. S. et al., Tetrahedron Letter 34:3389 (1993)). Deprotection of the amino group to give intermediate 5 is accomplished using standard procedure well known in the art (Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd edition, John Wiley and Sons, Inc. New York (1991)), such as palladium on carbon, in a suitable solvent, such as methanol or ethanol. In some cases, it is advantageous to add an acid, such as hydrochloric acid.
In Scheme 2, an ester protected glycine, such as Pxe2x80x2=benzyl, or ethyl, is condensed with an aldehyde, such as acetaldehyde, and a cyanide, such as cyanotrimethylsilane (TMSCN), in a suitable solvent, such as methylene chloride to afford the aminonitrile 6. The aminonitrile is reacted with oxalyl chloride or oxalyl bromide in an appropriate solvent, such as 1,2-dichlorobenzene to give the pyrazinone 7. The 3-chloro or 3-bromo of pyrazinone 7 is then displaced by an appropriate amine, such as phenethylamine, 2,2-diphenylethylamine or 4-methoxyphenethylamine, in an appropriate solvent, such as ethyl acetate, to give compound 8. The ester 8 is converted to the acid 9 by standard procedures well known in the art (Greene, T. W., Wuts, P. G. M., Protective Groups in Organic Synthesis, 2nd edition, John Wiley and Sons, Inc. New York (1991)), such as hydrolysis using base, such as LiOH or NaOH, in a suitable solvent, such as tetrahydrofuran/methanol/water. The remaining chlorine or bromine is then optionally removed by hydrogenolysis using a catalyst, such as palladium on carbon or Raney nickel, in an appropriate solvent, such as water to effort 9. Alternatively, in the case of Pxe2x80x2=benzyl, deprotection of the benzyl ester and removal of the remaining chlorine or bromine can be achieved simultaneously using palladium on carbon in a suitable solvent, such as tetrahydrofuran/ethanol to give 9. The acid is then coupled to amine 5 using typical anino acid coupling procedures, such as BOP or PyBOP, in an appropriate solvent, such as N,N-dimethylformamide, and base, such as di-isopropylethylamine (DIEA), followed by optional removing of Ra, Rb, Rc, in the case of Ra and Rb=tert-butyloxycarbonyl (Boc) and Rc=hydrogen. The Boc groups are removed by treatment with an acid, such as trifluoroacetic acid or hydrochloric acid, in an appropriate solvent, such as methylene chloride or dioxane to provide target compound 10.
Compounds wherein X is xe2x80x94CHxe2x95x90NR11xe2x80x94 or NR11 can be formed using the steps exhibited in Scheme 3.
Acid 9 (as synthesized in Scheme 2) is coupled with an aminoalcohol 11 using standard amino acid coupling procedures, such as BOP or PyBOP in a solvent such as N,N-dimethylformamide, and base, such as DIEA, to form compound 12. The corresponding aldehyde 13 is synthesized using routine procedures for the oxidation of alcohols (see for example Carey, F. A., and Sundberg, R. J., Advanced Organic Chemistry, Part B.: Reactions and Synthesis, 3rd edition, Plenum Press, New York (1990)) such as the Swern oxidation (Mancuso, A. J. et al., Journal of Organic Chemistry 3329 (1976)) pyridinium chlorochromate (Corey, E. J. and Suggs, J. W. Tetrahedron Letters 2647 (1975)) pyridinium dichromate (Corey, E. J. and Schmidt, G. Tetrahedron Letters 399 (1979)), or sulfur trioxide pyridine complex/dimethylsulfoxide (Tetrahedron Letters 28:1603 (1987)).
The aldehyde 13 is then converted to amidinohydrazone 14 using standard conditions, for example, treatment with an aminoguanidine or a substituted aminoguanidine, such as aminoguanidine or 2-hydrazinoimidazoline, optionally in the presence of an acid such as nitric acid, hydrogen chloride, or hydrogen bromide, in an appropriate solvent, for example, ethanol or methanol, which, in addition, may contain other solvents such as dichloromethane or tetrahydrofuran.
Conversion of the amidinohydrazone 14 to the aminoguanidine 15 is accomplished under reducing conditions well known in the art, for example, lithium borohydride in appropriate solvent such as tetrahydrofuran or methanol at various temperatures up to reflux. As an alternative method, catalytic hydrogenation with palladium on carbon catalyst can be employed.
Ra, Rb, and Rc in compounds 14 and 15 can optionally be removed by using conditions well known in the art. In the case of Ra and Rb=tert-butyloxycarbonyl (Boc) and Rc=hydrogen, the Boc groups are removed by treatment with an acid, such as trifluoro acetic acid or hydrochloric acid, in an appropriate solvent, such as methylene chloride or dioxane to provide compounds 16 and 17 respectively.
The pharmaceutically-acceptable salts of the compounds of Formula I (in the form of water- or oil-soluble or dispersible products) include the conventional non-toxic salts or the quaternary ammonium salts which are formed, e.g., from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, sulfate, tartrate, thiocyanate, tosylate, and undecanoate. Base salts include ammonium salts, alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups may be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl; and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides and others. Preferred acids for forming acid addition salts include HCl and acetic acid.
The compounds of the present invention represent a novel class of potent inhibitors of metallo, acid, thiol and serine proteases. Examples of the serine proteases inhibited by compounds within the scope of the invention include leukocyte neutrophil elastase, a proteolytic enzyme implicated in the pathogenesis of emphysema; chymotrypsin and trypsin, digestive enzymes; pancreatic elastase, and cathepsin G, a chymotrypsin-like protease also associated with leukocytes; thrombin and factor Xa, proteolytic enzymes in the blood coagulation pathway. Inhibition of thermolysin, a metalloprotease, and pepsin, an acid protease, are also contemplated uses of compounds of the present invention. The compounds of the present invention are preferably employed to ininbit trypsin-like proteases.
For their end-use application, the potency and other biochemical parameters of the enzyme-inhibiting characteristics of the compounds of the present invention is readily ascertained by standard biochemical techniques well known in the art. For example, an end use application of the compounds that inhibit chymotrypsin and trypsin is in the treatment of pancreatitis. Actual dose ranges for their specific end-use application will, of course, depend upon the nature and severity of the disease state of the patient or animal to be treated, as determined by the attending diagnostician. It is expected that a useful dose range will be about 0.01 to 10 mg per kg per day for an effective therapeutic effect.
Compounds of the present invention that are distinguished by their ability to inhibit thrombin may be employed for a number of therapeutic purposes. As thrombin inhibitors, compounds of the present invention inhibit thrombin production. Therefore, these compounds are useful for the treatment or prophylaxis of states characterized by abnormal venous or arterial thrombosis involving either thrombin production or action. These states include, but are not limited to, deep vein thrombosis; disseminated intravascular coagulopathy which occurs during septic shock, viral infections and cancer; myocardial infarction; stroke; coronary artery bypass; fibrin formation in the eye; hip replacement; and thrombus formation resulting from either thrombolytic therapy or percutaneous transluminal coronary angioplasty (PCTA). Other uses include the use of said thrombin inhibitors as anticoagulants either embedded in or physically linked to materials used in the manufacture of devices used in blood collection, blood circulation, and blood storage, such as catheters, blood dialysis machines, blood collection syringes and tubes, and blood lines. The compounds of the present invention may also be used as an anticoagulant in extracorporeal blood circuits.
Metal stents have been shown to reduce restenosis, but are thrombogenic. A strategy for reducing the thrombogenicity of stents is to coat, embed, adsord or covalently attach a thrombin-inhibiting agent to the stent surface. The compounds of the present invention can be employed for this purpose. Compounds of the invention can be attached to, or embedded within soluble and/or biodegradeable polymers as and thereafter coated onto stent materials. Such polymers can include polyvinylpyrrolidone, polyhydroxy-propylmethacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogels. See European Application 761 251, European Application 604,022, Canadian Patent 2,164,684 and PCT Published Applications WO 96/11668, WO 96/32143 and WO 96/38136.
By virtue of the effects of thrombin on a host of cell types, such as smooth muscle cells, endothelial cells and neutrophils, the compounds of the present invention find additional use in the treatment or prophylaxis of adult respiratory distress syndrome; inflammatory responses; wound healing; reperfusion damage; atherosclerosis; and restenosis following an injury such as balloon angioplasty, atherectomy, and arterial stent placement.
The compounds of the present invention may be useful in treating neoplasia and metastasis as well as neurodegenerative diseases, such as Alzheimer""s disease and Parkinson""s disease.
When employed as thrombin inhibitors, the compounds of the present invention may be administered in an effective amount within the dosage range of about 0.1 to about 500 mg/kg, preferably between 0.1 to 10 mg/kg body weight, on a regimen in single or 2-4 divided daily doses.
When employed as inhibitors of thrombin, the compounds of the present invention may be used in combination with thrombolytic agents such as tissue plasminogen activator, streptokinase, and urokinase. Additionally, the compounds of the present invention may be used in combination with other antithrombotic or anticoagulant drugs such as, but not limited to, fibrinogen antagonists and thromboxane receptor antagonists.
The thrombin inhibitors may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxy-propylmethacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the thrombin inhibitors may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross linked or amphipathic block copolymers of hydrogens.
Human leucocyte elastase is released by polymorphonuclear leukocytes at sites of inflammation and thus is a contributing cause for a number of disease states. Compounds of the present invention are expected to have an anti-inflammatory effect useful in the treatment of gout, rheumatoid arthritis and other inflammatory diseases, and in the treatment of emphysema. The leucocyte elastase inhibitory properties of compounds of the present invention are determined by the method described below. Cathepsin G has also been implicated in the disease states of arthritis, gout and emphysema, and in addition, glomerulonephritis and lung infestations caused by infections in the lung. In their end-use application the enzyme inhibitory properties of the compounds of Formula I is readily ascertained by standard biochemical techniques that are well-known in the art.
The Cathepsin G inhibitory properties of compounds within the scope of the present invention are determined by the following method. A preparation of partially purified human Cathepsin G is obtained by the procedure of Baugh et al., Biochemistry 15: 836 (1979). Leukocyte granules are a major source for the preparation of leukocyte elastase and cathepsin G (chymotrypsin-like activity). Leukocytes are lysed and granules are isolated. The leukocyte granules are extracted with 0.20 M sodium acetate, pH 4.0, and extracts are dialyzed against 0.05 M Tris buffer, pH 8.0 containing 0.05 M NaCl overnight at 4xc2x0 C. A protein fraction precipitates during dialysis and is isolated by centrifugation. This fraction contains most of the chymotrypsin-like activity of leukocyte granules. Specific substrates are prepared for each enzyme, namely Nxe2x80x94Sucxe2x80x94Alaxe2x80x94Alaxe2x80x94Proxe2x80x94Val-p-nitroanilide and Sucxe2x80x94Alaxe2x80x94Alaxe2x80x94Proxe2x80x94Phe-p-nitroanilide. The latter is not hydrolyzed by leukocyte elastase. Enzyme preparations are assayed in 2.00 mL of 0.10 M Hepes buffer, pH 7.5, containing 0.50 M NaCl, 10% dimethylsulfoxide and 0.0020 M Sucxe2x80x94Alaxe2x80x94Alaxe2x80x94Proxe2x80x94Phe-p-nitroanilide as a substrate. Hydrolysis of the p-nitroanilide substrate is monitored at 405 nm and at 25xc2x0 C.
Useful dose range for the application of compounds of the present invention as neutrophil elastase inhibitors and as Cathepsin G inhibitors depend upon the nature and severity of the disease state, as determined by the attending diagnostician, with a range of 0.01 to 10 mg/kg body weight, per day, being useful for the aforementioned disease states.
Compounds of the present invention that inhibit urokinase or plasminogen activator are potentially useful in treating excessive cell growth disease state. As such compounds of the present invention may also be useful in the treatment of benign prostatic hypertrophy and prostatic carcinoma, the treatment of psoriasis, and as abortifacients. For their end-use application, the potency and other biochemical parameters of the enzyme inhibiting characteristics of compounds of the present invention are readily ascertained by standard biochemical techniques well known in the art. Actual dose ranges for this application will depend upon the nature and severity of the disease state of the patient or animal to be treated as determined by the attending diagnostician. It is to be expected that a general dose range will be about 0.01 to 10 mg per kg per day for an effective therapeutic effect.
Additional uses for compounds of the present invention include analysis of commercial reagent enzymes for active site concentration. For example, chymotrypsin is supplied as a standard reagent for use in clinical quantitation of chymotrypsin activity in pancreatic juices and feces. Such assays are diagnostic for gastrointestinal and pancreatic disorders. Pancreatic elastase is also supplied commercially as a reagent for quantitation of xcex11-antitrypsin in plasma. Plasma xcex11-antitrypsin increases in concentration during the course of several inflammatory diseases, and xcex11-antitrypsin deficiencies are associated with increased incidence of lung disease. Compounds of the present invention can be used to enhance the accuracy and reproducibility of these assays by titrametric standardization of the commercial elastase supplied as a reagent. See, U.S. Pat. No. 4,499,082.
Protease activity in certain protein extracts during purification of particular proteins is a recurring problem which can complicate and compromise the results of protein isolation procedures. Certain proteases present in such extracts can be inhibited during purification steps by compounds of the present invention, which bind tightly to various proteolytic enzymes.
The pharmaceutical compositions of the invention can be administered to any animal that can experience the beneficial effects of the compounds of the invention. Foremost among such animals are humans, although the invention is not intended to be so limited.
The pharmaceutical compositions of the present invention can be administered by any means that achieve their intended purpose. For example, administration can be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, or ocular routes. Alternatively, or concurrently, administration can be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
In addition to the pharmacologically active compounds, the new pharmaceutical preparations can contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically.
The pharmaceutical preparations of the present invention are manufactured in a manner that is, itself, known, for example, by means of conventional mixing, granulating, dragee-making, dissolving, or lyophilizing processes. Thus, pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as saccharides, for example, lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example, tricalcium phosphate or calcium hydrogen phosphate, as well as binders, such as, starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxy-propylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone. If desired, disintegrating agents can be added, such as, the above-mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as, sodium alginate. Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as, magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragee cores are provided with suitable coatings that, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions can be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol, and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations, such as, acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate, are used. Dye stuffs or pigments can be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as, glycerol or sorbitol. The push-fit capsules can contain the active compounds in the form of granules that may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds are preferably dissolved or suspended in suitable liquids, such as, fatty oils or liquid paraffin. In addition, stabilizers may be added.
Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts, alkaline solutions and cyclodextrin inclusion complexes. Especially preferred alkaline salts are ammonium salts prepared, for example, with Tris, choline hydroxide, Bis-Tris propane, N-methylglucamine, or arginine. One or more modified or unmodified cyclodextrins can be employed to stabilize and increase the water solubility of compounds of the present invention. Useful cyclodextrins for this purpose are disclosed in U.S. Pat. Nos. 4,727,064, 4,764,604, and 5,024,998.
In addition, suspensions of the active compounds as appropriate oily injection suspensions can be administered. Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400 (the compounds are soluble in PEG-400). Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally, the suspension may also contain stabilizers.
Compounds of Formula I can be labeled with radioactive iodine by using an exchange reaction. Exchange of hot iodine for cold iodine is well known in the art. Alternatively, a radio iodine labeled compound can be prepared from the corresponding bromo compound via a tributylstannyl intermediate. See, U.S. Pat. No. 5,122,361, herein incorporated by reference.
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 complexed with a radioactive atom.
For the compounds of Formula I, 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 ke V, 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 I with radioisotopes which are suitable for detection externally. The gamma emitters, indium-111m 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 of Formula I can be labeled by any of the many techniques known in the art to provide a composition of the present invention. For example, these compounds can be labeled through a chelating agent such as diethylene-triaminepentaacetic acid (DTPA) or metallothionein, both of which can be covalently attached to the compound of Formula I.
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 I. 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 I having a DTPA chelating means with technetium-99m. This may be accomplished by combining a predetermined amount (as 5 xcexcg 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 I 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 I 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 I, the technetium-labeled composition of the present invention is formed.
The source of technetium-99m should preferably be water soluble. Preferred sources are alkali and alkaline earth metal pertechnetate (TcO4xe2x80x94). 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 I in this radioisotope""s reduced state. For example, stannous chloride (SnCl2) is the reducing agent and can be used in range from 1-1,000 xcexcg/mL. Especially preferred concentrations are about 30-500 xcexcg/mL.
Citric acid complexes with technetium-99m quickly to form a stable technetium-99m-citrate complex. Upon contact with a compound of Formula I substantially quantitative transfer of technetium-99m from its citrate complex to the chelating means of the compound of Formula I 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 xcexcg/ml.
The amount of compound of Formula I 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 I and the metal ion-transfer ligand complex is preferably carried out in a aqueous solution at a pH at which the compound of Formula I is stable. By xe2x80x9cstablexe2x80x9d, it is meant that the compound remains soluble and retains its inhibitory activity against xcex1-thrombin. Normally, the pH for the reaction will be from about 5 to 9, the preferred pH being above 6-8. The technetium-99m-citrate complex and a compound of Formula I are incubated, preferably at a temperature from about 20xc2x0 C. to about 60xc2x0 C., most preferably from about 20xc2x0 C. to about 37xc2x0 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 I. Generally, less than one hour is sufficient to complete the transfer reaction under these conditions.
Alternative compositions of the present invention include an In-111 labeled compound of the present invention.
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 I 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), dysoprosium(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 I 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 I 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 present invention also include 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.
The xe2x80x9cdiagnostically effective amountxe2x80x9d 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 medial 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 position of the present invention be about 5 to 20 xcexcCi, preferably about 10 xcexcCi. 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 I 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.
xe2x80x9cPharmaceutically acceptable carriersxe2x80x9d 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 suspensions 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 auxiliary 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.
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 xe2x80x9cbackgroundxe2x80x9d 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 xe2x80x9cin vivo imagingxe2x80x9d 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, xe2x80x9cadministeringxe2x80x9d is accomplished parenterally, in either a systemic or local targeted manner. Systemic administration is accomplished by injecting the compounds, compositions by diagnostic compositions of the present invention into a convenient and accessible vein or artery. This includes but is not limited to administration by the ankecubutal 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 xe2x80x9cadministeringxe2x80x9d. For example, a compound represented by Formula I 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 complexed to radioactive atom. Alternatively, a composition comprising the compound of formula complexed to radioactive atom may be injected into the mammal.
The xe2x80x9cdiagnostically effective amountxe2x80x9d 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 xcexcCi, preferably about 10 xcexcCi. 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 I 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 are 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.
The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered and obvious to those skilled in the art are within the spirit and scope of the invention.


Standard tricycles are generally known in the art. Some tricycles include features such as a footrest for a seated child to use while an adult pushes the tricycle. Other tricycles include a substantially rigid restraining hoop and tray that extends around the seated child. Other tricycles also include a canopy for protecting the seated child from the sun. Further other tricycles allow for folding of the tricycle for storage purposes. However, the accessories or integrated features available on these and other known tricycles do not safely and purposefully include a quick release mechanism for allowing the accessories/features to quickly transition from the use to the storage position. Additionally, the folding mechanism on these and other known tricycles can make storing and transporting the tricycle difficult because they tend to enlarge the overall volumetric envelope or footprint the tricycle occupies. The present disclosure seeks to overcome certain of these limitations and other drawbacks of the prior art, and to provide new features not heretofore available. A full discussion of the features and advantages of the present disclosure is deferred to the following detailed description, which proceeds with reference to the accompanying drawings.


Our man emerged in the 1880s bedeviling the French from Kabilya where the French had already had to suppress a rebellion. Collective punishment for that rising, onerous taxes, and the empire’s confiscations to benefit colonists all fired continuing resentment.

To French eyes, El Bachir was simply a bandit. But for periods of his nearly 15 years’ activity his word was next to law where the triclor could not reach. Kabilya’s colonial officials lived in fear of his revenge.


1. Introduction {#sec1}
===============

Hydrogen sulfide (H~2~S) is a colorless, flammable gas with the characteristic odor of rotten eggs. It was widely considered to be just a toxic gas for nearly 300 years mostly due to its unpleasant smell. The breakthrough in the effort to link endogenous H~2~S levels and functional changes came when the possible role of H~2~S as an endogenous neuromodulator in the brain was reported \[[@B1]\]. The focus on enzymes generating H~2~S was another breakthrough in 2001 \[[@B2]\]. The initial work concluded that H~2~S was a physiological vasodilator and regulator of blood pressure, which stimulated research on H~2~S physiology \[[@B3]\]. In plants, H~2~S has been revealed as a crucial player in the regulation of normal plant physiological processes, including seed germination, root morphogenesis, photosynthesis, and flower senescence \[[@B4]--[@B8]\]. It was also shown to be an important messenger in plant defense signaling against various abiotic stresses at physiological concentrations \[[@B9]--[@B13]\]. In this review, we discuss recent progress that increases our understanding of H~2~S synthesis and signaling functions in plants.

2. H~**2**~S Synthesis {#sec2}
======================

In mammalian cells, H~2~S is physiologically generated by pyridoxal-5′-phosphate-dependent enzymes, including cystathionine beta-synthase, cystathionine gamma-lyase, and 3-mercaptopyruvate sulfurtransferase (3-MST), during cysteine (Cys) metabolism \[[@B3], [@B14]\]. H~2~S is generated in plants via both enzymatic and nonenzymatic pathways, although the latter only accounts for a small portion of H~2~S production. [Figure 1](#fig1){ref-type="fig"}, with the enzymes highlighted, demonstrates the production of H~2~S in*Arabidopsis thaliana*.

Several candidate Cys-degrading enzymes have been reported to exist in different plant species (shown in [Table 1](#tab1){ref-type="table"}). In the model plant*A. thaliana*, the enzymes that produce H~2~S can be roughly divided into two categories. One class of these enzymes is Cys desulfhydrases (CDes), which degrade Cys into H~2~S, ammonia, and pyruvate in a stoichiometric ratio of 1 : 1 : 1 and require pyridoxal 5′-phosphate as a cofactor \[[@B15]\]. L-Cys desulfhydrase is one of the enzymes that decompose L-Cys and was first discovered in the sulfur metabolism of tobacco cultured cells \[[@B16]\]. D-Cys desulfhydrase 1 specifically uses D-Cys as its substrate, and D-Cys desulfhydrase 2 degrades L/D-Cys simultaneously \[[@B17], [@B18]\]. The production of H~2~S by CDes has been confirmed in various areas of biology \[[@B9], [@B11], [@B14], [@B15], [@B19], [@B20]\]. CDes are Cys desulfhydrases with singular functions in desulfuration. Their mRNA levels were significantly higher in the stems and cauline leaves than in the roots, rosette leaves, and flowers of*A. thaliana* \[[@B9]\].

Another class of the enzymes is O-acetyl-L-serine (thiol) lyase (OAS-TL), which is responsible for the incorporation of inorganic S into Cys, and free H~2~S appears to be released only in a minor reaction \[[@B21]\]. During an incubation period, the enzyme formed about 25 times more Cys than H~2~S, in a molar ratio, per mg protein \[[@B22]\]. Nine OAS-TL genes have been identified in*A. thaliana*, which are located in the cytosol, mitochondria, or plastid \[[@B23]\]. Recently, DES1 was reported as a frequent novel L-Cys desulfhydrase, which, based on sequence feature alignments, belongs to the OAS-TL family \[[@B24]--[@B28]\]. The*Km* value for L-Cys in the DES1 reaction is 13-fold lower than that for OAS in the OAS-TL reaction, indicating a much higher affinity of DES1 for L-Cys as a substrate \[[@B2]\]. The biochemical characterization of the T-DNA insertion mutant*des1* reveals that the total intracellular Cys concentration increased by approximately 25% \[[@B28]\]. However, as a member of the OAS-TL family, its function in synthesizing H~2~S has not been clearly studied.*In vitro*, the reaction of OAS-TL is a net H~2~S-consuming reaction \[[@B22]\]. Thus, the statement that DES1 is the only enzyme involving in the degradation of Cys is open to question \[[@B24], [@B28], [@B29]\].

In addition, Nifs/NFS, with L-Cys desulfhydrase-like activity, is also potentially involved in H~2~S production \[[@B30], [@B31]\]. Two genes, At5g26600 and At1g01010, in*A. thaliana* have been identified that encode proteins with CDes structural features \[[@B15]\], and 3-MST is also related to H~2~S production in plants \[[@B32]\].

3. Physiological Functions of H~**2**~S in Plants {#sec3}
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H~2~S has been reported to play important roles in diverse physiological processes in plants. Research on the endogenous H~2~S of higher plants can be traced back to 1978, when H~2~S was observed to be released from leaves of cucumber, corn, and soybean \[[@B33]\]. Leaves of older plants contain higher H~2~S concentrations than younger plants \[[@B34]\]. A recent study showed that the mRNA levels of*CDes* were gradually elevated in a developmental stage-dependent manner \[[@B9]\]. The importance of H~2~S in the regulation of plant growth, development, and senescence has emerged.

The improvement in seed germination rates due to exogenous H~2~S treatments was confirmed. H~2~S or HS^−^, rather than other sulfur-containing components derived from the exogenous H~2~S donor, NaHS, contributed to the promotion of seed germination \[[@B4]\]. NaHS preferentially affects the activity of endosperm *β*-amylase and maintains lower levels of malondialdehyde and hydrogen peroxide (H~2~O~2~) in germinating seeds \[[@B7]\]. In addition, the application of NaHS to seedling cuttings of sweet potato promoted the number and length of adventitious roots \[[@B5]\]. At the same time, H~2~S modulates the expression of genes involved in photosynthesis and thiol redox modification to regulate its photosynthesis \[[@B35]\]. It is hypothesized that an increase in the stomatal density also contributes to this process \[[@B36]\]. The osmotic-induced decrease in the chlorophyll concentration could be alleviated by spraying the NaHS solution \[[@B6]\]. H~2~S was also found to delay flower opening and senescence in cut flowers and branches \[[@B8]\]. These effects occur in a dose-dependent manner. In the cytosol, H~2~S negatively regulates autophagy and modulates the transcriptional profile of*A. thaliana* using*des1* \[[@B37]\]. H~2~S strongly affects plant metabolism at most stages of life and causes statistically significant increases in biomass, including higher fruit yields \[[@B38]\].

H~2~S also plays pivotal roles in plant responses or adaptation under biotic and abiotic stress conditions. Early studies concerning H~2~S emissions in plants were associated with plant responses to pathogens as part of sulfur-induced resistance \[[@B39]\]. In 2008, H~2~S was found to be an important cellular signal for the first time, highlighting the protective effect of H~2~S against copper stress \[[@B4]\]. Thereafter, a stream of publications on various positive effects of H~2~S and H~2~S signaling in plants emerged. Soon, H~2~S was shown to alleviate the effects of aluminum, cadmium, chromium and boron toxicity, drought and osmotic stress, heat stress, hypoxia, and other stresses \[[@B9], [@B11]--[@B13], [@B20], [@B40]--[@B42]\]. Most of these reports discussed, as analogies with animal systems, how H~2~S signaling is important for plant protection against stress.

Stomatal movement is very important in plant responses to environmental stimuli, and a key target of H~2~S signaling in plants is the specialized guard cell. Recent studies have reported that H~2~S is responsible for drought stress relief by inducing stomatal closure in*A. thaliana* \[[@B9], [@B20]\]. These observations are consistent with a previous report in both*Vicia faba* and*Impatiens walleriana* \[[@B43]\]. Similarly, H~2~S was confirmed to be a novel downstream indicator of nitric oxide (NO) during ethylene-induced stomatal closure \[[@B44]\]. However, the effect of H~2~S on stomatal movement has been a controversial topic. Another research group reported that exogenous H~2~S induced stomatal opening by reducing the accumulation of NO in guard cells of*A. thaliana* and a crop plant,*Capsicum annuum* \[[@B45], [@B46]\]. The reasons for these different observations are not clear and require further study. The difference may simply be due to the different experimental materials and methods. The purpose of stomatal closure is to reduce the moisture loss under drought stress, and the induction of stomatal opening is to enhance photosynthesis and reduce the photorespiration.

4. Cross-talk of H~**2**~S with Other Signals {#sec4}
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Plants perceive and respond to H~2~S, but studies on the mechanisms of H~2~S functioning in plant responses to stress are very limited. An overview of our current understanding of plant H~2~S signaling is shown in [Figure 2](#fig2){ref-type="fig"}. H~2~S is particularly active and may interact with and modify numerous other signals. Thus, there may be multiple routes of H~2~S perception and signaling to be unraveled.

Several lines of evidence point to an interrelationship between H~2~S and plant hormones in plant defenses. Abscisic acid (ABA) is produced in large amounts in plants under various abiotic stresses. Under drought stress, the expression of*CDes* was significantly upregulated, and the production rate of H~2~S from these plants also increased \[[@B9]\]. Subsequently, the relationship between H~2~S and ABA was reported based on a deficiency of H~2~S in the*lcd* mutant that had a weakened ABA induction of stomatal closure, which indicated that the induction of stomatal closure by ABA was partially dependent on H~2~S. As H~2~S was also involved in the expression regulation of ion-channel genes, H~2~S may be a critical component of ABA-induced stomatal closure via ion channels. At the same time, H~2~S influenced the expression of ABA receptors, and the influence of H~2~S may have begun upstream of the ABA signaling pathway. Therefore, the above results showed that H~2~S interacted with ABA in the stomatal regulation responsible for drought stress in*A. thaliana* \[[@B20]\]. Indole acetic acid (IAA) showed a rapid increase in different plants treated by exogenous H~2~S \[[@B5]\], and ethylene (Eth) could induce H~2~S generation \[[@B44]\]. In addition, gibberellic acid (GA) and jasmonic acid (JA) were also involved in the H~2~S signal transduction process. H~2~S can alleviate the GA-induced programmed cell death in wheat aleurone cells \[[@B47]\], and H~2~S may function downstream of H~2~O~2~ in JA-induced stomatal closure in*V. faba* \[[@B48]\].

H~2~O~2~ is another signaling molecule in plants, especially in guard cells. Abiotic stress induces synthesis of both H~2~S and H~2~O~2~; yet it is unclear how these two molecules work in concert in the physiological process. H~2~S may represent a novel downstream component of the H~2~O~2~ signaling cascade during JA-induced stomatal movement in*V. faba* \[[@B48]\]. Pretreatment of H~2~O~2~ could improve the germination percentage of*Jatropha curcas* seeds, and this improvement was mediated by H~2~S \[[@B49]\]. These results suggest that H~2~O~2~ is upstream of H~2~S. However, there is plenty of evidence to the contrary. H~2~S inhibited the cadmium influx through the plasma membrane calcium channels, which were activated by H~2~O~2~ \[[@B50]\]. H~2~S can participate in enhancing plant resistance to abiotic stress via the improvement of antioxidant systems, such as heavy metal stress, osmotic stress, heat stress, and hypoxia stress \[[@B4]--[@B7], [@B10], [@B41], [@B42], [@B49]\].

Recent evidence suggests that H~2~S also plays a role in the NO and carbon monoxide (CO) signaling pathway. In bermudagrass, sodium nitroprusside (SNP, a NO donor) and NaHS combined treatments showed that NO signaling could be blocked by H~2~S inhibitors and scavengers, indicating that NO-activated H~2~S was essential for the cadmium stress response \[[@B51]\]. Additional evidence showed that both NaHS and GYY4137 reduced the NO accumulation to a large extent in*A. thaliana* epidermal cells \[[@B45]\]. In sweet potato seedlings, a rapid increase in endogenous H~2~S and NO was sequentially observed in shoot tips treated with NaHS. A similar phenomenon in H~2~S donor-dependent root organogenesis was observed in both excised willow shoots and soybean seedlings. These results indicated that the process of H~2~S-induced adventitious root formation was likely mediated by IAA and NO and that H~2~S acts upstream in IAA and NO signaling transduction pathways \[[@B5]\]. Similarly, heme oxygenase 1 functions as a downstream component in H~2~S-induced adventitious root formation by the modulation of expression of related genes, which suggested that CO was involved in H2S-induced cucumber adventitious root formation \[[@B52]\].

Additionally, growing evidence suggests that H~2~S signaling interacts with calcium (Ca) signaling pathways. Ca^2+^ confers structure and rigidity to the cell wall and regulates plant processes through calmodulin. Li et al. (2013) showed that NaHS pretreatment could improve the entry of extracellular Ca^2+^ into tobacco suspension cultured cells mediated by intracellular calmodulin to increase the heat tolerance \[[@B40]\]. At the level of transcription, the expression of Ca^2+^ channel coding genes decreased, whereas Ca^2+^-ATPase and Ca^2+^-H^+^ cation antiporters were elevated in the*lcd* mutant. This was in accordance with stronger Ca^2+^ fluorescence in the wild type than in the*lcd* mutant \[[@B20]\]. These results suggest that Ca signaling plays an important role in the mechanism of H~2~S.

Numerous studies showed that, during the enhancement of plant resistance, many substances changed simultaneously. H~2~S plays an ameliorative role in protecting plants by increasing the proline content against aluminum toxicity and heat stress \[[@B10], [@B12], [@B40]\]. Aluminum-induced citrate secretion was also significantly enhanced by NaHS pretreatment \[[@B10]\]. During the NaHS preincubation period the grain *β*-amylase activity increased, improving seed germination \[[@B7]\].

5. Conclusions and Perspectives {#sec5}
===============================

The mechanisms by which H~2~S is generated still remain unresolved, and elucidating how it is made by different plant cells under different conditions is clearly a research priority. H~2~S is a key factor in the tolerance of cells to the oxidative stress induced by a range of abiotic conditions, including heavy metal toxicity, drought and osmotic stress, hot stress, hypoxia and other stresses. This probably involves the activation of antioxidant defenses, the induction of stomatal closure, and the enhanced expression of genes encoding resistance-associated enzymes. In these processes, plant hormones, H~2~O~2~, NO, CO, and Ca signaling participate in H~2~S signal transduction, resulting in a complex signaling network.

There are numerous unanswered questions and important areas for further research, concentrated in the following areas. (1) Owing to the promiscuous chemical properties of H~2~S, it is problematic to achieve adequate specificity and selectivity for its measurement. At present, the physiological H~2~S level was measured by various techniques such as the methylene blue method, monobromobimane, gas chromatography, ion selective electrodes, and fluorescent probes \[[@B53]\]. The diverse detection methods resulted in magnitude differences in measured biological sulfide levels, which will certainly attract increasing attention. (2) The mechanism of H~2~S functions performed at the protein level. Until now, a great number of studies focused on protein S-sulfhydration, which is impossible to determine directly by chemical analyses. But in mammals, there have been many results indicating that this process might occur by the transition of intermediate links, such as positional changes and interactions with associated proteins. Moreover, if H~2~S can thiolate proteins, it may have the same effect on DNA. (3) Even though H~2~S is a short-lived molecule, it is an extremely active one. The mechanisms by which either H~2~S or other molecules participating in H~2~S signaling function are also important. Thus, elucidation of the H~2~S complex signaling network is clearly a research priority.

This work was supported by the National Natural Science Foundation of China (31372085 to Yanxi Pei; 31400237 to Zhuping Jin; 31300236 to Zhiqiang Liu) and Shanxi Province Science Foundation for Youths (2014021026-2, to Zhuping Jin).

Conflict of Interests
=====================

The authors declare that there is no conflict of interests regarding the publication of this paper.

![An overview of H~2~S generation in higher plants (adapted from Papenbrock et al., 2007). APS: adenosine 5′-phosphosulfate; Fd~red~, Fd~ox~: reduced and oxidized ferredoxin; SIR: sulfite reductase.](OMCL2015-397502.001){#fig1}

![Generalized model of H~2~S signaling in response to abiotic stress in plants. Solid line arrows depict stimulatory effect; dashed cents arrows represent the putative interaction; gray bold rectangle indicates cell membrane. ABA: abscisic acid; CBF: C-repeat binding factor; CO: carbon monoxide; DREB: dehydration responsive element; Eth: ethylene; GA: gibberellic acid; cGMP: cyclic guanosine monophosphate; GSH: glutathione; H~2~O~2~: hydrogen peroxide; H~2~S: hydrogen sulfide; JA: jasmonic acid; NO: nitric oxide; PCD: programmed cell death; RD: responsive to desiccation.](OMCL2015-397502.002){#fig2}

######

Enzymes and coding genes related to H~2~S generation in *Arabidopsis*.

  Substrate      Enzyme                                  Cellular localization   Locus        Reference
  -------------- --------------------------------------- ----------------------- ------------ ------------
  L-Cys          AtLCD                                   Cytoplasm               At3g62130    \[[@B16]\]
  AtNFS1/Nifs1   Mitochondria                            At5g65720               \[[@B27]\]
  AtNFS2/Nifs2   Plastid                                 At1g08490               \[[@B28]\]
  DES1           Cytoplasm                               At5g28030               \[[@B24]\]
  OASTL-A1       Cytoplasm                               At4g14880               \[[@B25]\]
  OASTL-A2       Cytoplasm                               At3g22460               \[[@B43]\]
  OASTL-B        Plastid                                 At2g43750               \[[@B26]\]
  OASTL-C        Mitochondria                            At3g59760               \[[@B27]\]

  D-Cys          AtDCD1                                  Mitochondria            At1g48420    \[[@B17]\]

  L/D-Cys        AtDCD2                                  Mitochondria            At3g26115    \[[@B18]\]

  Unknown        PLP-dependent transferase superfamily   Chloroplast             At5g26600    \[[@B15]\]

  Unknown        NAC domain containing protein 1         Unknown                 At1g01010    \[[@B15]\]

At:*Arabidopsis thaliana*, Cys: cysteine, DCD: D-Cys desulfhydrase, DES: desulfhydrase, LCD: L-Cys desulfhydrase, NAC: N-acetyl-L-cysteine, NFS: nitrogenase Fe-S cluster, OASTL: O-acetyl-L-serine(thiol)lyase, and PLP: pyridoxal 5′-phosphate.

[^1]: Academic Editor: David Pattison