Aminopyridinyl-, aminoguanidinyl- and alkoxyguanidinyl- substituted phenyl acetamides as protease inhibitors

ES 2 269 474 T3

DESCRIPTION

Phenylarethamides substituted with aminopyridinyl, aminoguanidinyl and alkoxyguanidinyl used as protease inhibitors.

Background of the invention

Field of the invention

The present invention relates to novel compounds that function as proteolytic enzyme inhibitors and specifically to a new class of thrombin inhibitors.

Related art

Proteases are enzymes that cleave proteins at specific, single 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 for a variety of biological activities, such as digestion, the formation and dissolution of blood clots, reproduction, and the immune reaction to foreign cells and organisms. Aberrant proteolysis is associated with numerous disease states in man or other mammals. Human neutrophil proteases, elastase and cathepsin G, have been implicated in 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 mammalian biological functions. In many cases, it is beneficial to disrupt the function of one or more proteolytic enzymes in the course of therapeutic treatment of a mammal.

Serine proteases include enzymes such as elastase (human leukocytes), cathepsin G, plasmin, esterase C-1, convertase C-3, urokinase, plasminogen activator, acrosin, chymotrypsin, trypsin, thrombin, factor Xa, and kallikreins. Human leukocyte elastase is released by polymorphonuclear leukocytes at sites of inflammation and is therefore a contributing cause of various 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 a useful anti-inflammatory effect 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. Urokinase inhibitors and plasminogen activator are useful in treating disease states with excessive cell growth, such as benign prostatic hypertrophy, prostatic carcinoma, and psoriasis.

Serine protease thrombin plays a central role in homeostasis and thrombosis, and as a multifactorial protein, it induces various effects on platelets, endothelial cells, smooth muscle cells, leukocytes, the heart, and neurons. Activation of the coagulation cascade through the intrinsic pathway (contact activation) or the extrinsic pathway (activation by exposure of plasma to a non-endothelial surface, damage to vessel walls, or release of tissue factor) leads to a series of biochemical events that converge on thrombin. Thrombin clears fibrinogen ultimately leading to a hemostatic plug (clot formation), potently activating platelets through a unique proteolytic cleavage of the cell surface thrombin receptor (Coughlin, Seminars in Hematology 31 (4): 270- 277 (1994)), and self-amplifies 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)). Factor Xa inhibitors are thought to offer an advantage over agents that directly inhibit thrombin since direct thrombin inhibitors still allow significant generation of new thrombin (Lefkovits and Topol, Circulation 90 (3): 15221536 (1994); Harker, Blood Coagulation and Fibrinolysis 5 (Suppl 1): S-47-S58 (1994)).

In vivo diagnostic imaging methods for intravascular thrombi have been previously reported. Compounds that are detectably labeled with radioactive or paramagnetic atoms are used in these imaging methods. For example, platelets labeled with the gamma emitter, In-111, can be used as an imaging agent for the detection of thrombi (Thakur, ML et al., Throm Res. 9: 345 (1976); Powers et al., Neurology 32: 938 (1982)). The Tc-99m-labeled streptokinase thrombolytic enzyme has been proposed as an imaging agent (Wong, US Patent No. 4,418,052 (1983)). The fibrin-binding domains of Staphylococcus aureus-derived protein A labeled with gamma emitters, I-125 and I-131, have been proposed as imaging agents (Pang, US Patent No. 5,011. 686 (1991)). Monoclonal antibodies that have specificity for fibrin (in contrast to fibrinogen) and labeled with Tc-99 have been proposed as imaging agents (Berger et al., US Patent No. 5,024,829 (1991)) ; Dean et al., US Patent No.

ES 2 269 474 T3

4,980,148 (1990)). The use of the paramagnetic contrast agent, gadolinium-diethylenetriaminepentaacetic acid has been reported in magnetic resonance imaging of patients treated by thrombolysis for acute myocardial infarction (De Roos, A. et al., J. Card. Imaging . 7: 133 (1991)). Radiolabeled and paramagnetically labeled alfacetoamide derivatives have also been proposed as thrombus imaging agents (Abelman et al., US Patent No. 5,656,600).

There remains a need for non-peptide compounds that are potent and selective protease inhibitors, and that possess higher bioavailability and fewer side effects than currently available protease inhibitors. Accordingly, new classes of potent protease inhibitors, characterized by strong inhibitory capacity and little toxicity to mammals, are potentially valuable therapeutic agents for a variety of conditions, including the treatment of various proteolytic disease states in mammals.

Compendium of the invention

The present invention is directed to aminopyridinyl, aminoguanidinyl, and alkoxyguanidinyl substituted phenyl-aretamides having Formula I (below). Processes for preparing the compounds of Formula I are also provided. The novel compounds of the present invention are potent inhibitors of proteases, especially of trypsin-like serine proteases, such as chymotrypsin, trypsin, thrombin, plasmin and factor. For. Some of the compounds show antithrombotic activity through selective, direct inhibition of thrombin, or are useful intermediates for forming compounds that have antithrombotic activity. Also provided is the use of a compound of Formula I in the manufacture of a medicament for inhibiting or treating aberrant proteolysis in a mammal, and for treating thrombosis, ischemia, stroke, restenosis, or inflammation in a mammal.

The invention includes a composition for inhibiting platelet loss, inhibiting platelet aggregate formation, inhibiting fibrin formation, inhibiting thrombus formation, and inhibiting emboli formation in a mammal, comprising a compound of the invention in a pharmaceutically acceptable carrier. These compositions can optionally include anticoagulants, antiplatelet agents, and thrombolytic agents. The compositions can be added to the blood, blood products, or mammalian organs in order to effect the desired inhibitions.

Also provided is the use of a compound of the invention to inhibit or treat aberrant proteolysis in a mammal, and to treat myocardial infarction; unstable angina; stroke; restenosis; deep vein thrombosis; disseminated intravascular coagulation caused by trauma, sepsis or tumor metastasis: hemodialysis; cardiopulmonary bypass surgery; respiratory fatigue syndrome in adults; endotoxic shock; rheumatoid arthritis; ulcerative colitis; induration; metastasis; hypercoagulability during chemotherapy, Alzheimer's disease; Dawn syndrome; the formation of fibrin in the eye; and wound healing. Other uses of the compounds of the invention are as anticoagulants embedded in, or physically anchored to, materials used in the manufacture of devices used in blood collection, blood circulation, and blood storage, such as catheters, blood pressure machines. blood dialysis, blood collection tubes and syringes, blood lines and stents.

The invention also relates to reducing the thrombogenicity of a surface in a mammal by anchoring to the surface, covalently or non-covalently, a compound of the invention.

In another aspect, the present invention includes compositions that are useful for in vivo imaging of thrombi in a mammal, comprising a compound of the present invention that is detectable outside of 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 that are useful for in vivo imaging of thrombi in a mammal, comprising a pharmaceutically acceptable carrier and a diagnostic effective amount of a compound or composition of the present invention.

In another aspect, the present invention relates to in vivo imaging of thrombi in a mammal.

WO 96 40100 is a published PCT application, and describes arylsulfonylaminobenzene derivatives and the use thereof as factor Xa inhibitors.

WO 99 51571 is a published PCT application, and describes benzamide and sulfonamide substituted aminoguanidines and alkoxyguanidines and the use thereof as protease inhibitors.

WO 98 23565 is a published PCT application, and describes aminoguanidines and alkoxyguanidines and the use thereof as protease inhibitors.

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Detailed description of the preferred embodiments

The compounds of the present invention include the compounds of Formula I:

<img file="ES2269474T3_D0001.tif" />

or a solvate, hydrate or pharmaceutically acceptable salt thereof; where:

W is hydrogen, R¹, R¹OC (O), R¹C (O), R^!(CH2) SNHC (O), R^!S (O) 2, or (R¹ )₂CH (CH₂)_SCNH (O), where s is 0-4; R¹ it 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, where t is 0-3, and each R¹² can be the same or different, where (R¹²) 2 can also form a ring with C represented by cycloalkyl C₃-C₉,

R²CF2C (R¹²) 2 (CH₂)_what, where q is 0-2, and each R¹² can be the same or different, where (R¹²)₂ can also form a ring with C represented by cycloalkyl C₃-C₉,

R²CH2C (R¹²) 2 (CH₂)_what, where q is 0-2, and each R¹² can be the same or different, where (R¹²)₂ can also form a ring with C represented by cycloalkyl C₃-C₉, (R² ) 2CH (CH2) r, where r is 0-4 and each R² can be the same or different, and where (R²) 2 can also form a ring with CH represented by cycloalkyl C₃-C₉, C bicyclic alkyl₇-C₁₂, C tricyclic alkyl₁₀-C₁₆, or a 5- to 7-membered monocyclic or bicyclic heterocyclic ring that can be saturated or unsaturated, and that contains one to three heteroatoms selected from the groups consisting of N, O and S, R²O (CH₂)_p, where p is 2-4, (R₂ )₂CF (CH₂)_r, where r is 0-4 and each R² can be the same or different, (R²)₂ can also form a ring with C represented by cycloalkyl C₃-C₉, C bicyclic alkyl₇-C₁₂, C tricyclic alkyl₁₀-C₁₆, or a 5- to 7-membered monocyclic or bicyclic heterocyclic ring that can be saturated or unsaturated, and that contains one to three heteroatoms selected from the groups consisting of N, O and S,

<img file="ES2269474T3_D0002.tif" />

where s is 0 or 1, or R²CF2C (R¹²)2;

ES 2 269 474 T3

R² is phenyl, naphthyl, or biphenyl, each of which is unsubstituted or substituted with one or more C alkyl₁-C₄, C1-C4 alkoxy, halogen, hydroxy, CF₃, OCF₃, COOH, CONH₂, bear₂NH₂, a 5- to 7-membered monocyclic or 9 to 10-membered bicyclic heterocyclic ring or a non-heterocyclic ring that can be saturated or unsaturated, where the heterocyclic ring contains one to four heteroatoms selected from the group consisting of N, O and S, and where the heterocyclic or non-heterocyclic ring is unsubstituted or substituted with halogen or hydroxy, C alkyl₁-C₁₂, unsubstituted or substituted with one or more of hydroxyl, COOH, amino, aryl optionally substituted with C alkyl₁-C₃, cycloalkyl C₃-C₉, CF₃, N (CH₃)₂, heteroaryl, or heterocycloalkyl,

CF3, C cycloalkyl₃-C₉, unsubstituted or substituted with aryl, C bicyclic alkyl₇-C₁₂, or C tricyclic alkyl₁₀_C₁₆;

Y is -NH- or -O-;

R³, R⁴, R⁵ and R⁶ are independently hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heteroaryl, haloalkyl, hydroxy, alkoxy, aryloxy, heteroaryloxy, halogen, haloalkoxy, hydroxyalkyl, cyano, nitro, -CO2R^x, -CH2R^x or -OR^x, where R^x, in each case, is independently one of hydrogen, C alkyl₁-C₁₂ or C cycloalkyl₃C₉ where said C alkyl groups₁-C₁₂ or C cycloalkyl₃-C₉ they can optionally have one or more unsaturations;

R¹¹ is hydrogen, alkyl, or alkenyl;

_R¹²_{it is} hydrogen or halogen, phenyl, naphthyl, or biphenyl, each of which is unsubstituted or substituted with one or more C alkyl₁-C₄, C alkoxy₁-C₄, halogen, hydroxy, CF₃, OCF₃, COOH, or CONH₂, a 5- to 7-membered monocyclic or 9 to 10-membered bicyclic heterocyclic ring that can be saturated or unsaturated, and contains one to four heteroatoms selected from the group consisting of N, O and S, C alkyl₁-C₁₂, unsubstituted or substituted with one or more of hydroxyl, COOH, amino, aryl C₆C₁₄, heteroaryl, or heterocycloalkyl,

CF3, C cycloalkyl₃-C₉, C7-C12 bicyclic alkyl, or C tricyclic alkyl_10-C₁₆;

B is selected from the group consisting of:

<img file="ES2269474T3_D0003.tif" />

R⁷, R⁸, R⁹, and R¹⁰ they are independently hydrogen, alkyl, aralkyl, aryl, hydroxyalkyl, aminoalkyl, monoaminoalkyl, dialkylaminoalkyl, or carboxyalkyl;

ES 2 269 474 T3 or R⁷ and R⁸ are taken together to form - (CH₂)_or-, where u is from 2 to 7, while R⁹ and R¹⁰ are defined as before;

or R⁹ and R¹⁰ are taken together to form - (CH₂)_v-, where v is from 2 to 7, while R⁷ and R⁸ are defined as before;

or R⁷ and R⁹ are taken together to form - (CH₂)_Y-, where y is 0 (a bond) or 1 to 7, while R⁸ and R¹⁰are defined as before;

X is -O-, -NR¹⁸-, or -CH = N- (where N is attached to NR¹³) where R¹⁸ is hydrogen, alkyl, cycloalkyl, or aryl, wherein said alkyl, cycloalkyl, or aryl are optionally substituted with amino, monoalkylamino, dialkylamino, alkoxy, hydroxy, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, aryl, heteroaryl, acylamino, cyano, or trifluoromethyl;

R^to, R^b and R^c are independently hydrogen, alkyl, hydroxy, alkoxy, aryloxy, aralkoxy, alkoxycarbonyloxy, cyano, or -CO2R^w, where R^w is C1-C alkyl₁₂, cycloalkyl C₃-C₉, aryl C₆-C₁₄, ar (C₆-C₁₄) C alkyl₁-C₁₂,

<img file="ES2269474T3_D0004.tif" />

where R^and and R^F are independently hydrogen, C1-C6 alkyl, C2-C6 alkenyl, or C6-C14 aryl, R⁸ is hydrogen, C1-C alkyl₆, alkenyl C₂-C₆, or C aryl₆-C₁₄, R^h is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, or C6-C14 aryl, and R¹ is ar (C6-C₁₄) C alkyl₁-C₁₂ or C alkyl₁-C₁₂, n is zero to 8; ym is from zero to 6;

R¹³ is hydrogen, alkyl, alkenyl, aralkyl, aryl, hydroxyalkyl, aminoalkyl, monoalkylaminoalkyl, dialkylaminoalkyl, carboxyalkyl, alkoxy C₁-C₂₀, aril (C₆-C₁₄) oxy, or alkoxy (C₁-C₂₀) carbonyl;

R¹⁴ and R¹⁵ they are independently hydrogen, alkyl, cycloalkyl, halogen or alkoxy; Y

R¹⁶ and R¹⁷ are independently hydrogen, alkyl, hydroxy, alkoxy, aryloxy, alkoxycarbonyl, cyano or CO2R^j, where R^j is C1-C alkyl₁₂, cycloalkyl C₃-C₉, aryl C₆-C₁₄, ar (C₆-C₁₄) C alkyl₁-C₁₂, halo (C₁-C₁₂) alkyl or

<img file="ES2269474T3_D0005.tif" />

where R^and, R^F and R^g are independently hydrogen or C alkyl₁-C₁₂, and where, unless otherwise defined, and when used by itself or as part of another group:

the term "alkyl" refers to both straight and branched chain radicals of up to 12 carbon atoms;

the term "alkenyl" refers to a straight or branched chain radical of 2-20 carbon atoms;

the term "alkynyl" refers to a straight or branched chain radical of 2-20 carbon atoms, where there is at least one triple bond between two of the carbon atoms in the chain;

ES 2 269 474 T3 the term "alkoxy" refers to a straight or branched chain radical of 1-20 carbon atoms, attached to a carbon atom;

the term "aryl" refers to monocyclic or bicyclic aromatic groups containing 6-14 carbon atoms in the ring portion;

the term "heteroaryl" refers to groups having 5 to 14 ring atoms; 6, 10, or 14 π electrons shared in a cyclic arrangement; and containing carbon atoms and 1,2 or 3 oxygen, nitrogen or sulfur heteroatoms;

the term "aralkyl" refers to a Ci-C alkyl group₁₂ having an aryl substituent;

the term "cycloalkyl" refers to cycloalkyl groups containing 3 to 9 carbon atoms;

the term "monoalkylamine" refers to an amino group that is substituted with an alkyl group having 1 to 12 carbon atoms;

the term "dialkylamine" refers to an amino group that is substituted with two alkyl groups, each having 1 to 12 carbon atoms.

Compounds within the scope of the present invention include those for which:

R³, R⁴, R⁵ and R⁶ are independently hydrogen, C alkyl_i-C_i2, cycloalkyl C₃-C₉, halogen, alkenyl C₂-C₂₀, C alkynyl₂-C₂₀, aryl C₆-C_i4, ar (C₆-C_i4) C alkyl_i-C_i2, heteroaryl, haloalkyl C_i-C_i2, C alkoxy_iC_i2, aril (C₆-C_i4) oxy, heteroaryloxy, halo (C_i-C₂₀) C alkoxy or hydroxyalkyl_i-C_i2;

R¹¹ is hydrogen, C alkyl_i-C_i2 or alkenyl C₂-C₂₀;

R⁷, R⁸, R⁹ and Rⁱ⁰ are independently hydrogen, C alkyl_i-C_i2, ar (C₆-C_i4) C alkyl_i-C_i2, aryl C₆C_i4, hydroxyalkyl C_i-C_i2; aminoalkyl C_i-C_i2; monoalkyl (C_i-C_i2) aminoalkyl C_i-C_i2, dialkyl (C_i-C_i2) aminoalkyl C_i-C_i2, or carboxyalkyl C_i-C_i2;

Rⁱ⁸ is C alkyl_i-C_i2, cycloalkyl C₃-C₉ or C aryl₆-C_i4, each of which is optionally substituted with amino, monoalkyl (C_i-C_i2) amino, dialkyl (C_i-C_i2) amino, C alkoxy_i-C₂₀, hydroxy, carboxy, alkoxy (C_iC₂₀) carbonyl, aryl (C₆-C_i4) oxycarbonyl, ar (C₆-C_i4) alkoxy (C_i-C₂₀) carbonyl, aryl C₆-C_i4, heteroaryl C₅C_i0, acylamino, cyano, or trifluoromethyl;

R^to, R^b and R^c are independently C alkyl_i-C_i2, C alkoxy_i-C₂₀, aril (C₆-C_i4) oxy, ar (C₆-C_i4) C alkoxy_i-C₂₀, or alkoxy (C_i-C₂₀) carbonyloxy;

Rⁱ³ is C alkyl_i-C_i2, C alkoxy_i-C₂₀, aril (C₆-C_i4) oxy or alkoxy (C_i-C₂₀) carbonyl;

Rⁱ⁴ and Rⁱ⁵ are independently C alkyl_i-C_i2, cycloalkyl C₃-C₉ or C alkoxy_i-C₂₀;

Rⁱ⁶ and Rⁱ⁷ are independently C alkyl_i-C_i2, C alkoxy_i-C₂₀, aril (C₆-C_i4) oxy or alkoxy (C_i-C₂₀) carbonyl.

Preferred compounds of Formula I above are those for which Y is -NH- or -SO2NH-.

A subgenus of preferred compounds of Formula I above are those for which B is

<img file="ES2269474T3_D0006.tif" />

where R⁷-Rⁱ⁰, Rⁱ³ and R^to-R^c are defined as before.

ES 2 269 474 T3

Another subgenus of preferred compounds of Formula I above are those for which B is

<img file="ES2269474T3_D0007.tif" />

where R⁹, R¹⁰ and R¹⁴-R¹⁷ are defined as before.

Preferred compounds of Formula I above are those for which WesR¹, where R¹ is R² and R² is either phenyl, naphthyl or C alkyl₁-C₇ substituted with aryl.

Preferred compounds of Formula I above are those for which R¹ is R²CF2C (R¹²) 2 (CH2) q.

Preferred compounds of Formula I above are those for which R⁶ is C alkyl₁-C₆ or halogen. The most preferred compounds within the third preferred subgenus are those for which R⁶ is methyl or chlorine, including compounds for which R⁶ is chlorine at the same time as R³ it is fluorine.

Preferred compounds of Formula I above are those for which R¹¹ it is hydrogen.

The preferred values ​​of R^to, R^b and R^c in Formula I are independently hydrogen, hydroxy, C1-C6 alkyl, C1-C6 alkoxy, cyano or -CO2R^w, where R^w, in each case, is preferably one of C1-C alkyl₄, cycloalkyl C₄-C₇ or benzyloxycarbonyl. Between the appropriate values ​​of R^to, R^b and R^c include hydrogen, methyl, ethyl, propyl, n-butyl, hydroxy, methoxy, ethoxy, cyano, -CO2CH3, -CO2CH2CH3, and -CO2CH2CH2CH3. In the most preferred embodiments, each of R^to, R^b and R^c it is hydrogen.

Also preferred in R^to, R^b and R^c is the -CO group₂R^w, where R^w is one of

<img file="ES2269474T3_D0008.tif" />

where IR'-R¹ are defined as before. When R^to, R^b and R^c are -CO₂R^w, where R^w is one of these three radicals, the resulting compounds are prodrugs that possess desirable formulation and bioavailability characteristics. A preferred value for each of R^and, R^F and R^h is hydrogen, R^g is methyl, and among the preferred values ​​for R¹ benzyl and t-butyl are included.

Preferred compounds are those of Formula I, where R⁷, R⁸, R⁹ and R¹⁰ they are independently one of hydrogen, C1-C6 alkyl, C1-C6 aralkyl, C6-C10 aryl, C2-C10 hydroxyalkyl or C2-C7 carboxyalkyl. Between the 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. Additional preferred compounds are those in which R⁷ and R⁸ or R⁹ and R¹⁰ are taken together to form (-CH2)_Y- where y is 2.

Preferred Compounds When X is NR¹⁸ are those in which R¹⁸ is hydrogen or C alkyl₁-C₆, optionally substituted with 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.

Between the appropriate values ​​of R¹⁸ include hydrogen, methyl, ethyl, propyl, n-butyl, phenethyl, 2-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl, carboxymethyl, and carboxyethyl.

The most preferred compounds are those in which X is oxygen.

R⁶ may represent hydrogen, C1-C3 alkyl, halogen, or C1-C2 alkoxy, R⁶ is preferably C1-C alkyl₃eg methyl, or halogen, eg chlorine, bromine or fluorine.

ES 2 269 474 T3

R³, R⁴, R⁵ and R⁶ may independently represent hydrogen, hydroxy, C alkyl₁-C₃, halogen or C alkoxy_iC₂. Preferably R³ it is fluorine and hydroxy.

Preferred values ​​of n in Formula I include zero to 6, more preferably zero to 4, and most preferably zero, 1 or 2. Preferred values ​​of m include zero to 4, more preferably zero, 1, 2, or 3.

It should be understood that the present invention is considered to include stereoisomers as well as optical isomers, eg mixtures of enantiomers as well as individual enantiomers and diastereomers, which appear as a consequence of structural asymmetry in selected compounds of the present series. . The compounds of the present invention may also have polymorphous crystalline forms, all polymorphous crystalline forms being included in the present invention.

The compounds of Formula I can also be solvated, especially hydrated. Hydration can occur during the manufacture of the compounds or compositions comprising the compounds, or hydration can 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 term "prodrug" denotes a derivative of a known direct-acting drug, the derivative of which has enhanced release characteristics and therapeutic value compared to the drug, and is transformed into the active drug by a chemical or enzymatic process. Useful prodrugs are those in which R^to'R^b, R^c and / or R^dare -CO₂R^w, where R^w has been defined before. See, US Patent No. 5,466,811 and Saulnier et al., Bioorg. Med. Chem. Lett. 4: 1985-1990 (1994).

When any variable exists more than once in any constituent in Formula I, its definition at each occurrence is independent of its definition at each other occurrence. Also, combinations of substituents and / or variables are permissible only if such combinations produce stable compounds.

In another aspect, the present invention includes compositions that are useful for in vivo imaging of thrombi in a mammal, comprising a compound of the present invention capable of being detected outside the body. Compositions comprising a compound of the present invention and a detectable label, such as a radioactive or paramagnetic atom, are preferred.

In another aspect, the present invention provides diagnostic compositions that are used for in vivo imaging of thrombi in a mammal, comprising a pharmaceutically acceptable carrier and a diagnostic effective amount of a compound or composition of the present invention.

In another aspect, the present invention relates to in vivo imaging of thrombi in a mammal.

According to a preferred aspect, useful compounds are those in which the substituent Rⁱ it is substituted with a detectable label, such as a radioactive iodine atom, such as I-125, I-131, or I-123. In this regard, R¹ it is preferably phenyl, having a para I-123, para I-125 or para I-131 substitution, or benzyl having a meta 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 a substituent R¹, either directly or through a divalent connecting group A ”. Alternatively, the group -A "-L replaces the group W in Formula I. By suitable ligand is meant an organic radical that is capable of chelating a radioactive or paramagnetic metal ion.

In these compounds, the divalent linking group A "includes groups that are capable of covalently bonding with a free amino group and chelating media. For example, A "can be -C (= S) -, -C (= O) -, -C (= NH) - (CH₂)₆C - (= NH) -, -C (= O) - (CH2) 6-C- (O) -,

<img file="ES2269474T3_D0009.tif" />

and the like.

ES 2 269 474 T3

Likewise, in the compounds represented by Formula I, the chelating ligand, L, includes groups capable of covalently binding or non-covalently binding to a radioactive or paramagnetic atom. Chelating media include those that are commonly used to complex with radioactive or paramagnetic atoms. These include chelating media containing 3 to 12, preferably 3 to 8, methylene phosphonic acid groups, methylenecarbohydroxamic acid groups, carboxyethylidene groups, or especially carboxymethylene groups, which are attached to a nitrogen atom. If only one or two of the acidic groups are attached to the nitrogen atom, the nitrogen atom is attached to another nitrogen atom having such groups by an optionally substituted ethylene group or by up to four ethylenic units separated by a nitrogen atom or oxygen or sulfur. Diethylenetriamine N, N, N ', N ", N" -pentaacetic acid (DTPA) is preferred as the medium for complexing. DTPA is well known in the art as a chelating medium for the radioactive atoms of indium-111 (In-111), technetium-99m (Tc-99m), and the paramagnetic gadolinium (Gd) atom. Khaw, et al., Science 209: 295 (1980); Paik CH et al., US Patent No. 4,652,440 (1987); Gries, H. et al., US Patent No. 4,957,939 (1990). A preferred chelating ligand, L, is 1- (p-aminobenzyl) diethylenetriaminepentaacetic acid. Also included as chelating means are compounds having sulfhydryl or amine radicals, the total of which in any combination is at least four. These sulfhydryl or amine radicals are separated from each other by at least two atoms which can be carbon, nitrogen, oxygen, or sulfur. Especially preferred for chelating medium, L, is metallothionein which is well known in the art as a chelating agent for Tc-99m.

The term "alkyl" as used 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. Preferably, alkyl has 1 to 6 carbon atoms.

The term "alkenyl" as used herein means a straight or branched chain radical of 2-20 carbon atoms, if 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 2 to 4 carbon atoms in length.

The term "alkenyl" is used here to mean a straight or branched chain radical of 2-20 carbon atoms, if the chain length is limited to this, where 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 2 to 4 carbon atoms in length.

In all cases where there is an alkenyl or alkynyl radical as a substituent group, the unsaturated bond, that is, the vinylene or acetylene bond, is preferably not directly attached to the nitrogen, oxygen or sulfur radical.

The term "alkoxy" is used herein to mean a straight or branched chain radical of 1-20 carbon atoms, if the chain length is limited thereto, attached to an oxygen atom, including, but not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, and the like. Preferably the alkoxylated chain is 1 to 10 carbon atoms in length, preferably 1 to 8 carbon atoms in length.

The term "aryl" as used herein by itself or as part of another group refers to bicyclic aromatic groups containing 6 to 14 carbon atoms in the ring portion, such as phenyl, naphthyl, or tetrahydronaphthyl.

The term "heteroaryl" as used herein refers to groups having 5 to 14 ring atoms; 6, 10, or 14 π electrons shared in a cyclic arrangement; 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, thiantrenyl, furyl, pyranyl, isobenzofuranyl, benzoxazolyl, chromenyl, xanthenyl, phenoxythinyl, 2H-pyrrolyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl groups pyridazinyl, indolizinyl, isoindolyl, 3Hindolyl, indolyl, indazolyl, purinyl, 4H-quinolizinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinazolinyl, cinolinyl, pteridinyl, 4H-carbazolyl, phenanthrylinyl, 3-carbazolinyl, 3-carbazolinyl, acridinyl, perimidinyl, phenanthroline, phenazinyl, isothiazolyl, phenothiazinyl, isoxazolyl, furazanil, and phenoxazinyl).

The term "aralkyl" or "arylalkyl" as used herein by itself or as part of another group, refers to C-alkyl groups.₁-C₁₂ preferably Ci-C₆, as discussed above that have an aryl substituent, such as benzyl, phenylethyl, or 2-naphthylmethyl.

The term "cycloalkyl" as used 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 "C alkyl₇-C₁₂ "bicyclic" 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.

ES 2 269 474 T3

The term "C alkyl₁₀-C₁₆ tricyclic ”is intended to include tricyclo [5,2,1,0²'⁶] decyl, adamantyl, and the like.

The term "halogen" or "halo" as used herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine with chlorine and fluorine being more preferred.

The term "monoalkylamine" as used herein by itself or as part of another group refers to an amino group that is substituted with an alkyl group having 1 to 12, preferably 1 to 6, carbon atoms.

The term "dialkylamine" as used herein by itself or as part of another group refers to an amino group that is substituted with two alkyl groups, each having 1 to 12, preferably 1 to 6, carbon atoms.

The term "hydroxyalkyl" as used herein refers to any of the above alkyl groups substituted with one or more hydroxyl radicals.

The term "carboxyalkyl" as used herein refers to any of the above alkyl groups substituted with one or more carboxylic acid radicals.

The term "heterocycle" or "heterocyclic ring", as used herein except where indicated, represents a stable 5- to 7-membered bicyclic heterocyclic ring system, either ring of which may be saturated or unsaturated, and consisting of carbon atoms. and one to three heteroatoms selected from the group consisting of N, O and S, and where the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally 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 an oxygen or sulfur, one to three nitrogen atoms, or an oxygen or sulfur combined with one or two nitrogen atoms. The heterocyclic ring can be attached to any heteroatom or carbon atom that causes 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, imidazolidinyl, pyrazolidinyl, imidazolidinyl, pyrazolidinyl, , 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, thiamorpholinylsulfoxide, thiamorpholinylsulfone, and oxadiazolyl. Morpholino is the same as morpholinyl.

The term "heteroatom" is used herein to mean an oxygen atom ("O"), a sulfur atom ("S"), or a nitrogen atom ("N"). It should be noted that when the heteroatom is nitrogen, it can form a radical NR^toR^b, where R^to and R^b are, independently of the other, hydrogen or C-alkyl₁ to C₈, or together with the nitrogen atom to which they are attached, they form a saturated or unsaturated 5, 6, or 7 membered ring.

Schemes 1-8 outline a synthetic route for the compounds of Formula 1.

(Scheme goes to next page)

ES 2 269 474 T3

Scheme 1

<img file="ES2269474T3_D0010.tif" />

In Scheme 1, an acetic acid side chain is introduced into a benzene ring by reacting a fluorinated nitrobenzene 1, such as 1,2,3, -trifluoro-4-nitrobenzene, with the metal salt of a substituted malonate ester or unsubstituted, such as diethyl malonate, in a suitable solvent such as tetrahydrofuran (THF), followed by acid hydrolysis and subsequent decarboxylation on heating, to produce compound 2 (Yokomoto, MW, et al., European Published Patent Application No. 0,470,578 A1 (1991)). The carboxyl group of 2 is converted to a hydroxyl group under typical reducing conditions, for example with borane (BH3) -TBF complex and sodium borohydride (NaBH₄), in a suitable solvent such as THF, to give alcohol 3 (Yokomoto, MW, et al., ditto). The introduction of a suitable functionality in para relative to the nitro group in the ring is achieved by nucleophilic substitution of the fluoride in compound 3 with a suitable nucleophile, such as t-butylamine, in suitable solvents such as dimethylsulfoxide (DMSO) and toluene. at reflux to provide compound 4 (Yokomoto, MW, et al., ditto). The nitrogen protecting group, such as t-butyl, in compound 4 is removed under standard conditions, for example with concentrated hydrochloric acid (HCl) under reflux, to give compound 5 (Yokomoto, MW, et al., Ditto) . The hydroxyl group of compound 5 is masked with a suitable protecting group, such as acetyl, under standard conditions well known in the art (Greene, TW, and Wuts, PGM, Protecting Groups in Organic Synthesis, 2^to ed., John Wiley and Sons, Inc., New York (1991)), for example with acetyl chloride in dichloromethane (DCM) in the presence of a base such as triethylamine or diisopropylethylamine (DIEA), to give compound 6. The Coupling an activated carbonyl compound ACOCI with compound 6 in a suitable solvent, such as DCM, produces compound 7.

ES 2 269 474 T3

Scheme 2

<img file="ES2269474T3_D0011.tif" />

In Scheme 2, the reaction of an arylnitro compound 7 under typical conditions such as catalytic hydrogenation with hydrogen in the presence of palladium on activated carbon in ethanol or methanol produces the arylamine 8. The acetyl protecting group of compound 8 is removed (with the in order to increase the solubility of the compound before manipulation of the amino group) by hydrolysis under alkaline conditions, for example with an aqueous solution of potassium carbonate (K₂CO₃) in methanol, to release the hydroxyl protecting group, yielding compound 9. The R⁶ The desired compound is introduced into the central scaffold of compound 9 by a Sandmeyer-type reaction ((a) Gunstone, FD, et al., Org. Syn. Collect Vol. 1, Wiley, New York, NY (1941), p. 170 ; (b) Yokomoto, MW, et al., European Published Patent Application No. 0,470,578 A1 (1991)) with suitable reagents, such as sodium nitrite (NaNO₂) and HCl followed by copper chloride (CuCl) (I), or by substitutive deamination (Doyle, MP, et al. J. Org. Chem. 42: 2426 (1977)) with the appropriate reagents, such as nitrile of t -butyl (t-BuONO) and copper chloride (CuCl₂) (II) to give compound 10. The amino group of arylamine 9 can be converted to a methyl group under carbon-carbon coupling conditions in the presence of a palladium catalyst through an arenediazonium salt intermediate (Kikukawa, K., et al., J. Org. Chem. 48: 1333 (1983)). Compound 10 in turn is reduced with a suitable reducing agent, such as BH₃, to generate the WY fragment of compound 11 where Y is -NH-. The oxidation of 11 with an oxidizing agent, such as sulfur trioxide-pyridine complex (SO₃-pyridine) in DCM, yields aldehyde 12. Construction of the center and left fragment is finally achieved by further oxidation of aldehyde 12 to carboxylic acid 13 under suitable oxidation conditions, for example with sodium chlorite (NaClO2) in the presence of sodium dihydrogen phosphate (NaH2PO4) and DMSO (Dalcanale, E., et al., J. Org. Chem. 51: 567 (1986)).

ES 2 269 474 T3

Scheme 3

<img file="ES2269474T3_D0012.tif" />

In Scheme 3, acid 13 is coupled to a suitable amine 14, such as protected O-guanidylamine (Tianbao Lu, et al., WO 99/26926 (1999), or aminopyridinylamine (Sanderson, PE, et al., WO 97/01338 (1997)) in the presence of a typical peptide coupling agent, such as Castro's reagent (BOP), and a base, such as DIEA, in a suitable solvent, such as N, N-dimethylformamide (DMF), to produce amide 15. Optionally, protecting groups, such as t- (butoxy) carbonyl (Boc), can be removed under typical deprotection conditions, for example with a solution of trifluoroacetic acid (TFA) in DCM when B is O-guanidine, or a solution of HCl in 1,4-dioxane when B is aminopyridine, to generate O-guanidine, or aminopyridine, respectively.

Scheme 4

<img file="ES2269474T3_D0013.tif" />

ES 2 269 474 T3

In Scheme 4, the phenylacetic acid derivative 16 is nitrated at the meta position of the benzene ring using standard conditions, for example with 96% nitric acid in concentrated sulfuric acid (Sindelar et al., Coll. Czechoslov. Chem. Commun 42: 2231 (1977)), to give nitro compound 17. The carboxylic acid group of compound 17 is then protected using standard conditions well known in the art (Greene, TW, and Wuts, PGM, Protective Groups in Organic Synthesis, 2^to ed., John Wiley and Sons, Inc., New York (1991)), such as conversion to the ester by reaction with oxalyl chloride followed by alcohol POH, to provide ester 18 (where P is an acid protecting group typical carboxylic). The reduction of the nitro group is completed using a suitable reagent, for example, tin (II) chloride, in an appropriate solvent, such as ethanol, and the resulting amine 19 is reacted with an acylating agent (W = RⁱC (O)) or a sulfonylating agent (W = RⁱSW)₂), such as benzylsulfonyl chloride, and of a suitable base, such as N-methylmorpholine, in a solvent, such as DCM, to provide the N-substituted aminophenylacetate (Y = -NH-). The carboxylic acid group is deprotected using standard conditions well known in the art (Greene, TW and Wuts, PGM, Protective Groups in Organic Synthesis, 2^to ed., John Wiley and Sons, Inc., New York (1991)), for example by hydrolysis with aqueous hydroxide, to give acid 13 (Y = -NH-). This is then coupled to amine 14 and deprotected, as in Scheme 3, to produce phenylacetamide 15 (Y = NH).

Scheme 5

<img file="ES2269474T3_D0014.tif" />

In Scheme 5, nitrophenylacetic acid 17 is coupled to an amino alcohol 21, such as ethanolamine, using a standard peptide coupling procedure, such as in Scheme 3, to give alcohol 22. The alcohol is converted to the protected alkoxyamine by coupling to N-hydroxyphthalimide using reacti15

ES 2 269 474 T3 standardized (Mitsunobu, O., Synthesis 1: 1 (1981)), such as triphenylphosphine and diethyl azodicarboxylate, in a suitable solvent, such as THF, to provide compound 23, which is then converted to the aniline 24 under typical reducing conditions, for example by hydrogenation over palladium (0) on carbon, in a suitable solvent, such as ethanol. The amine is then acylated or sulfonylated as in Scheme 4 to give intermediate 25, and the deprotected alkoxyamine using standard conditions well known in the art (Greene, TW and Wuts, PGM, Protective Groups in Organic Synthesis, 2^to ed., John Wiley and Sons, Inc., New York (1991)), for example with aqueous methylamine in ethanol / THF. Guanidinylation of the resulting alkoxyamine 26 is completed with a standard guanidinylation reagent, such as N, N'-bis (t-butoxycarbonyl) -S-methylthiourea (Bergeron, RJ and McManis, JSJ Org. Chem. 52: 1700 (1987 )) or NR^to-N'-R^b, R^c-1H-pyrazole-1-carboxamide (Bernatowicz, MS et al. Tetrahedron Lett. 34: 3389 (1983)), and guanidine is optionally deprotected as in Scheme 3, to provide the end goal 27.

Scheme 6

<img file="ES2269474T3_D0015.tif" />

In Scheme 6, the ketone, the aldehyde (R¹¹ = H), or carboxylic acid (R¹¹ = OH) starting material 28 is reduced with a suitable reagent, such as borane-THF, to give alcohol 29, which is then converted to a better removable group by reaction with a sulfonyl chloride such as methanesulfonyl chloride, in a suitable solvent, such as DCM, to produce compound 30. The sulfonate is displaced by cyanide under standard conditions, for example with potassium cyanide in refluxing acetonitrile to give nitrile 31, which is then hydrolyzed with a typical reagent, such as aqueous hydroxide. Coupling of the resulting acid 17 with amine 14 is completed as in Scheme 3 to give intermediate 32, and the nitro group is reduced as in Scheme 4 or 5 to provide aniline 33. This is acylated or sulfonylated as in Scheme 4 and guanidine is optionally deprotected as in Scheme 3 to give the end goal 15 (Y = NH).

ES 2 269 474 T3

Scheme 7

<img file="ES2269474T3_D0016.tif" />

In Scheme 7, nitrophenol 34 is alkylated with an allyl halide 35 and a suitable base, such as cesium carbonate in a polar aprotic solvent, such as DMF, giving intermediate 36, which is then incorporated into compound 37 through Claisen's transposition heating up. The phenol is protected using typical reagents, such as benzyl bromide and cesium carbonate, in a solvent, such as DMF, to give 38 (where P is a typical hydroxyl protecting group) and the nitro group is reduced as in the Scheme 4 or 5 to produce the aniline 39. Aniline 39 is converted to intermediate 40 as in Scheme 4 and the alkane is oxidatively cleaved using standard conditions, for example with sodium periodate and osmium tetroxide in dioxane / water followed by Jones's reagent, to provide acid 41. This is then coupled to amine 14, the guanidine is optionally deprotected as in Scheme 3, and the phenol group is optionally deprotected using standard conditions, for example by hydrogenation on palladium (0) on carbon, in a suitable solvent, such as ethanol, to produce the target compound 42.

ES 2 269 474 T3

Scheme 8

<img file="ES2269474T3_D0017.tif" />

In Scheme 8, monoprotected catechol 43 is sulfonylated with a W-Cl reagent, such as methatoluenesulfonyl chloride, in a solvent, such as DCM, in the presence of a base, such as triethylamine, to give compound 44. The protecting group is removed using standard conditions, for example with boron tribromide in DCM, and the resulting phenol 45 is alkylated with allyl halide 35 to give 46, rearranged to phenol 47, and protected to provide intermediate 48 as in Scheme 7. The alkene is oxidatively cleaved using standard conditions, for example with sodium periodate and ruthenium (III) chloride in acetonitrile / water (Ashby, EC and Goel, AB, J. Org. Chem. 46: 3936 (1981)) followed by Jones's reagent, yielding acid 49, which is then coupled to amine 14 and optionally deprotected as in Schemes 3 and 7 to provide target compound 50. Among 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 of quaternary ammonium salts which are formed, eg from inorganic or organic acids or bases. Examples of such acid addition salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, citrate, camphorate, camphorsulfonate, cyclopentane-propionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, 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, trifluoroacetate, and undecanoate. Bases 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, etc., including salts with a guanidinyl radical. Likewise, the alkaline nitrogen-containing salts can be quaternized with agents such as lower alkyl halides, such as the chlorides, bromides and iodides of methyl, ethyl, propyl, and butyl dialkyl sulfates such as dimethyl, diethyl, dibutyl sulfates, and diamyl; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; halides of

ES 2 269 474 T3 aralkyl such as benzyl and phenethyl bromides and others. Preferred acids to form acid addition salts include HCl, acetic acid, and trifluoroacetic acid.

The compounds of the present invention represent a novel class of potent inhibitors of metalloproteases and of thiol, acid and serine proteases. Examples of serine proteases inhibited by compounds within the scope of the invention include neutrophil leukocyte elastase, a proteolytic enzyme involved in the pathogenesis of ephysema; 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 clotting 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 used to inhibit trypsin-like proteases.

For their end use application, the potency and other biochemical parameters of the enzyme inhibitory characteristics of the compounds of the present invention are readily ascertained by standard biochemical techniques known to those skilled in the art. For example, one end-use application of compounds that inhibit chymotrypsin and trypsin is in the treatment of pancreatitis. Actual dosage ranges for your end-use application will, of course, depend on the nature and severity of the disease state of the patient or animal to be treated, as determined by the diagnosing physician. A useful dosage range is expected to be 0.01 to 10 mg per kg per day for effective therapeutic effect.

Compounds of the present invention that are distinguished by their ability to inhibit thrombin can be used for various therapeutic purposes. As thrombin inhibitors, the compounds of the present invention inhibit thrombin production. Accordingly, these compounds are useful for the treatment or prophylaxis of conditions characterized by abnormal venous or arterial thrombosis involving thrombin production or action. These conditions include, but are not limited to, deep vein thrombosis; disseminated intravascular coagulopathy that occurs during septic shock, viral infections, and cancer; myocardial infarction; stroke; coronary artery bypass; the formation of fibrin in the eye; hip replacement; and thrombus formation resulting from thrombolytic therapy or percutaneous transluminal coronary angioplasty (PCTA). Other uses include the use of such thrombin inhibitors as anticoagulants embedded in, or physically attached to, materials used in the manufacture of devices used in blood collection, blood circulation, and blood storage, such as catheters. , blood dialysis machines, syringes and tubes for blood collection and blood lines. The compounds of the present invention can also be used as anticoagulants in extracorporeal blood circuits.

Stents have been shown to reduce restenosis, but they are thrombogenic. One strategy to reduce the thrombogenicity of stents is to coat, embed, adsorb, or covalently anchor a thrombin inhibiting agent to the surface of the stent. The compounds of the present invention can be used for this purpose. The compounds of the invention can be anchored to, or embedded in soluble and / or biodegradable polymers and thereafter coated onto the stent materials. Such polymers may include polyvinylpyrrolidone, polyhydroxy-polymethacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethylene oxide-polylysine substituted with palmitoyl moieties, polylactic acid, polyglycolic acid, copolymers of polylactic acid and polyglycolic acid, kaproyethylene-kaproyethyl acid , polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels. See European Application No. 761,251, European Application No. 604,022, Canadian Patent No. 2,164,684 and PCT Published Application Nos. WO 96/11668, WO 96/32143 and WO 96/38136.

By virtue of the effects of thrombin on a host of cell lines, such as smooth muscle cells, endothelial cells, and neutrophils, the compounds of the present invention find additional use in the treatment or prophylaxis of fatigue syndrome. respiratory in adults; inflammatory responses; wound healing; reperfusion injury; atherosclerosis; and restenosis following injury such as balloon angioplasty, atherectomy, and arterial stenting.

The compounds of the present invention may be useful for treating neoplasia and metastasis as well as neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.

When used as thrombin inhibitors, the compounds of the present invention can be administered in an effective amount within the dosage range of 0.1 to 500 mg / kg, preferably between 0.1 and 10 mg / kg of body weight, in a regimen of daily unit doses or divided in 2-4.

When used as thrombin inhibitors, the compounds of the present invention can be used in combination with thrombolytic agents such as tissue plasminogen activator, streptokinase, and urokinase. Additionally, the compounds of the present invention can be used in combination with other antithrombotic or anticoagulant drugs such as, but not limited to, fibrinogen antagonists and thromboxane receptor antagonists.

Thrombin inhibitors can also be coupled to soluble polymers as targetable drug carriers. Such polymers may include polyvinylpyrrolidone, pyran copolymer, polyhydroxypolymethacrylamide-phenol, polyhydroxyethyl-aspartamide-phenol, or polyethylene oxide-polylysine substituted with pal19 moieties.

ES 2 269 474 T3 mitoyl. Additionally, thrombin inhibitors can be coupled to a class of biodegradable polymers useful to achieve controlled drug release, eg, polylactic acid, polyglycolic acid, polylactic and polyglycolic acid copolymers, polypsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates, and cross-linked or amphipathic block copolymers of hydrogels.

Human leukocyte elastase is released by polymorphonuclear leukocytes at sites of inflammation and thus is a contributing cause of various disease states. The 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 leukocyte elastase inhibitory properties of the 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 are readily determined by standard biochemical mechanisms that are well known in the art.

The cathepsin G inhibitory properties of the compounds within the scope of the present invention are determined by the following method. A partially purified cathepsin G preparation is obtained by the method 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). The leukocytes are lysed and the granules are isolated. The leukocyte granules are extracted with 0.20 M sodium acetate, pH 4.0, and the extracts are dialyzed against a 0.05 M Tris buffer, pH 8.0 containing 0.05 M NaCl at 4 ° C. A protein fraction precipitates during dialysis and is isolated by centrifugation. This fraction contains most of the chymotrypsin-like activity of the leukocyte granules. Specific substrates are prepared for each enzyme, this is N-Suc-Ala-Ala-Pro-Val-p-nitroanilide. The latter is not hydrolyzed by leukocyte elastase. The 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 N-Suc-Ala-Ala-Pro-Val- 0.0020 M p-nitroanilide as substrate. The hydrolysis of the p-nitroanilide substrate is verified at 405 nm and at 25 ° C.

The useful dosage range for the application of the compounds of the present invention as inhibitors of neutrophil elastase and as inhibitors of Cathepsin G depends on the nature and severity of the disease state, as determined by the diagnosing physician, being A range of 0.01 to 10 mg / kg of body weight per day is useful for the aforementioned disease states.

Compounds of the present invention that inhibit urokinase or plasminogen activator are potentially useful in the treatment of disease states with excessive cell growth. As such compounds of the present invention they may also be useful in the treatment of benign prostatic hypertrophy and prostatic carcinoma, the treatment of psoriasis, and as an abortifacient. For its end use application, the potency and other biochemical parameters of the enzyme that inhibits the characteristics of the compounds of the present invention are readily determined by standard biochemical mechanisms well known in the art. Actual dosage ranges for this application will depend on the nature and severity of the disease state of the patient or animal to be treated as determined by the diagnosing physician. A general dosage range is expected to be 0.01 to 10 mg per kg per day for effective therapeutic effect.

Additional uses for the compounds of the present invention include testing commercial reactive enzymes for concentration at the active site. For example, chymotrypsin is supplied as a standard reagent for use in the clinical quantification of chymotrypsin activity in pancreatic juices and feces. Such tests are diagnostic for gastrointestinal and pancreatic disorders. Pancreatic elastase is also supplied commercially as a reagent for the quantification of α-1 antitrypsin in plasma. The concentration of α-1 antitrypsin in plasma increases in the course of various inflammatory diseases, and α-1 antitrypsin deficiencies are associated with an increased incidence of pulmonary diseases. The compounds of the present invention can be used to increase the accuracy and reproducibility of these analyzes by titration standardization of the commercial elastase supplied as a reagent. See, US Patent No. 4,499,082.

Protease activity in certain protein extracts during the purification of particular proteins is a recurring problem that can complicate and compromise the results of protein isolation procedures. Certain proteases present in such extracts can be inhibited during the purification steps by the compounds of the present invention, which are tightly bound to different 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. Chief 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 achieves the intended purpose. For example, administration can be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, or ocular routes. Alternatively, or concurrently, administration may be by the oral route. The dose administered will depend on the age, health, and weight of the recipient, the kind of concurrent treatment, if any, the frequency of treatment, and the nature of the effect desired.

ES 2 269 474 T3

In addition to the pharmacologically active compounds, the new pharmaceutical preparations can contain suitable pharmaceutically acceptable carriers comprising excipients and adjuvants that facilitate the transformation 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 known per se, 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 transforming the mixture into granules, after the addition of suitable adjuvants, if desired or necessary, to obtain tablets or dragee nuclei.

For compositions of the present invention suitable for administration to a human, the term "excipient" is intended to include, but is not limited to, those excipients described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, 2^to Ed. (1994). 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, corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, disintegrating agents can be added, such as the aforementioned starches and also carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate. The adjuvants 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 which, if desired, are resistant to gastric juices. For this purpose, concentrated saccharide solutions can be used, which may optionally contain acacia, talc, polyvinylpyrrolidone, polyethylene glycol, and / or titanium dioxide, varnish solutions and suitable organic solvents or mixtures thereof. In order to produce coatings resistant to gastric juices, solutions of suitable cellulose preparations are used, such as acetylcellulose phthalate or hydroxypropylmethylcellulose phthalate. Dyes or pigments can be added to the coatings of tablets or lozenges, for example, for identification or in order to characterize combinations of active compound doses.

Other pharmaceutical preparations that can be used orally include hard capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The hard capsules can contain the active compounds in the form of granules that can 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 can be added.

Formulations suitable for parenteral administration include aqueous solutions of the active compounds in a water soluble form, eg, water soluble salts, alkaline solutions, and inclusion complexes of cyclodextrins. Especially preferred alkali salts are the ammonium salts prepared, for example, with Tris, choline hydroxide, Bis-Tris propane, N-methylglucamine, or arginine. One or more modified or unmodified cyclodextrins may be employed to stabilize and increase the water solubility of the compounds of the present invention. Useful cyclodextrins for this purpose are described in US Patent Nos. 4,727,064, 4,764,604, and 5,024,998.

In addition, suspensions of the active compounds may be administered, as appropriate, oily injectable suspensions. Lipophilic solvents or vehicles include fatty oils, eg, sesame oil, or synthetic fatty acid esters, eg, ethyl oleate or triglycerides or polyethylene glycol-400 (the compounds are soluble in PEG-400). Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol and / or dextran. Optionally, the suspension can also contain stabilizers.

Compounds of Formula I can be labeled with radioactive iodine using an exchange reaction. The exchange of hot iodine for cold iodine is well known in the art. Alternatively, it can be prepared from the corresponding bromine compound via a tributylstannyl intermediate. See, for example, US Patent No. 5,122,361.

Also included in the present invention are compositions that are useful for in vivo imaging of thrombi in a mammal, where the compositions consist of a compound of Formula I complexed with a radioactive atom.

For compounds of Formula I, suitable radioactive atoms include, Co-57, Cu-67, Ga-67, Ga-68, Ru-97, Tc-99m, In-111, In-113, Hg-197 , Au-198, and Pb-203. Some radioactive atoms have superior properties for use in radiochemical imaging techniques. Specifically, technetium-99m (Tc-99m) is an ideal radioactive atom for imaging due to its nuclear properties. Rhenium-186 and 188 also produce gamma emission which allows them to form images. Preferred compositions contain the radioactive atom, Tc-199m.

ES 2 269 474 T3

The compounds of Formula I by any of the many mechanisms 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 depicted compound of the present invention. by Formula I. For example, the imaging compounds of this invention are made by reacting technetium-99m (in the oxidized state) with the compounds of the present invention having a chelating medium in the presence of a reducing agent to form a stable complex between the technetium-99m in a reduced state (valence state IV or V).

One embodiment of the composition of the present invention is prepared by labeling a compound of Formula I having a DTPA chelating medium with technetium-99m. This can be accomplished by combining a predetermined amount (eg, 5 pg to 0.5 mg) of a compound of the present invention with an aqueous solution containing citrate buffer and a stannous reducing agent, then adding freshly eluted sodium pertechnetate containing a predetermined level of radioactivity (for example 15 mCi). After allowing the mixture to incubation at room temperature, the reaction mixture is loaded into a shielded syringe through a sterile filter (0.2-0.22 microns), then dispensed into 0.9 saline. % for your 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 medium with technetium-99m. This can be achieved by combining aqueous sodium pertechnetate-99m with aqueous stannous glucoheptonate to form a soluble complex of technetium-99m (in the reduced state) with two glucoheptonate molecules, then combining this solution with a compound of Formula I that has a metallothionein attached. . After incubation of the mixture for a period of time and under conditions that allow an exchange of the technetium-99m of the glucoheptonate complex for the metallothionein of the compound of Formula I, the technetium-labeled composition of the present invention is formed.

Reducing agents for use in the method are physiologically acceptable to reduce technetium-99m from its oxidized state to valence state IV or V or to reduce rhenium from its oxidized state. Reducing agents that can be used are stannous chloride, stannous fluoride, stannous glucoheptonate, stannous tartrate, and sodium dithionine. Preferred agents are stannous reducing agents, especially stannous chloride or stannous glucoheptonate. The amount of reducing agent is the amount necessary to reduce technetium-99m to provide binding to the chelating medium of a compound of Formula I in this reduced radioisotope state. For example, stannous chloride (SnCl₂) is the reducing agent and can be used in the range of 1-1,000 µg / ml.

Citric acid complexes with technetium-99m rapidly form a stable technetium-99m complex. Upon contact with a compound of Formula I, a substantially quantitative transfer of technetium-99m from its complex with citrate to the chelating medium of the compound of Formula is achieved rapidly and under mild conditions.

I. The amount of citric acid (in the form of sodium citrate) can range from 0.5 mg / ml to the amount that is most soluble in the medium. The preferred amounts of citric acid range from 15 to 30 µg / ml.

The amount of the compound of Formula I having a chelating medium can range from 0.001 to 3 mg / ml, preferably from 0.017 to 0.15 mg / ml. Finally, technetium-99m in the form of pertechnetate can be used in amounts of preferably 1-50 mCi. The amount of mCi per mg of compound of the present invention is preferably 30-150.

The reaction between the compound of Formula I and the metal ion / transfer ligand complex is preferably carried out in an aqueous solution at a pH at which the compound of Formula I is stable. By "stable", it is meant that the compound remains soluble and retains its α-thrombin inhibitory activity. Typically the pH for the reaction will be between 5 and 9, with the preferred pH being above 6-8. The technetium-99m-citrate complex and a compound of Formula I are incubated, preferably at 20 to 60 ° C, most preferably 20 to 37 ° C, for a sufficient amount of time to allow transfer of the metal ion from the citrate complex to the chelating medium 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.

Also included in the present invention are compositions of the compounds of the present invention that are useful for in vivo imaging of thrombi in a mammal, consisting of a compound represented by Formula I complexed with a paramagnetic atom.

Preferred paramagnetic atoms are divalent or trivalent ions of elements with an atomic number of 21 to 29, 42, 22 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). Due to their very high magnetic moments, gadolinium (III), terbium (III), disoprosium (III), holmium (III), and erbium (III) are preferred. Gadolinium (III) is especially preferred for the paramagnetic atom.

ES 2 269 474 T3

The compositions of the present invention can be prepared by combining a compound of Formula I with a paramagnetic atom. For example, the metal oxide or a metal salt (eg, nitrate, chloride, or sulfate) of a suitable paramagnetic atom is dissolved or suspended in a medium consisting 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 can be moderately heated until the reaction is complete. The insoluble compositions formed can be isolated by filtration, while the soluble compositions can be isolated by evaporation of the solvent. If the acid groups of the chelating media are still present in the composition of the present invention, inorganic or organic bases, and even amino acids, can be added to convert the acid complex into a neutral complex to facilitate the isolation or purification of the composition. homogeneous. Organic bases or alkali amino acids can be used as neutralizing agents, as well as inorganic bases such as sodium, potassium or lithium hydroxide carbonates or bicarbonates.

Also included in the present invention are diagnostic compositions that are useful for in vivo imaging of thrombi in a mammal, comprising a pharmaceutically acceptable carrier and a diagnostic effective amount of compositions derived from the compounds of Formula I.

The "diagnostic effective amount" of the composition required as a dose will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration. These factors and their relationship to determining this dose are well known to those skilled in the arts of medical diagnosis. Also, the effective amount for diagnosis and the method of administration can be adjusted to achieve maximum efficacy but will depend on factors such as weight, diet, concurrent medication, and other factors that will be recognized by those skilled in the medical arts. In either case, the imaging dose should be sufficient to detect the presence of imaging agent at the site of a thrombus in question. Typically, radiological imaging will require that the dose provided by the pharmaceutical composition of the present invention be 5 to 20 // Ci, preferably about 10 // Ci. Magnetic resonance imaging will require the delivered dose to be 0.001 to 5 mmol / kg, preferably 0.005 to 0.5 mmol / kg of a compound of formula I complexed with a paramagnetic atom. In any event, it is known in the art that the actual dose will depend on the location of the thrombus.

"Pharmaceutically acceptable carriers" for use in vivo are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985). The pharmaceutical compositions of the present invention can 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 dissolution 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, injectable pharmaceutical compositions may contain minor amounts of non-toxic adjuvants, such as wetting agents, pH buffering agents, and the like. If desired, absorption enhancing preparations (eg liposomes) can be used.

The present invention also encompasses diagnostic compositions for storage or administration. These could additionally contain preservatives, stabilizers and colorants. For example, sodium benzoate, sorbic acid, and p-hydroxybenzoic acid esters can be added as preservatives. Ditto at 1449. In addition, antioxidants and suspending agents can be used.

The in vivo imaging methods of the present invention also offer several advantages over prior imaging techniques for the detection or verification of the presence, size, regression, or enlargement of a thrombus. In particular, the present invention provides compounds, compositions that bind closely to thrombin associated with a thrombus and thereby reduce the "background" due to circulating radioactivity or paramagnetism emanating from the unbound imaging agent. Furthermore, in vivo imaging by intracoronary injection of the diagnostic compounds, compositions or compositions of the present invention is expected to be almost instantaneous since these imaging agents could saturate thrombin bound thrombin immediately.

Accordingly, the present invention also relates to in vivo imaging of a thrombus in a mammal. A diagnostic acceptable amount of a compound, composition, or diagnostic composition of the present invention can be administered to a mammal in order to detect a thrombus in a blood vessel.

The term "in vivo imaging" as used herein refers to the detection of a thrombus in a mammal, as well as the verification of the size, location and number of thrombi in a mammal, as well as the dissolution or thrombus growth.

In employing the compounds, compositions, or in vivo diagnostic compositions by this method, "administration" is performed parenterally, in a generalized or locally targeted manner. Administration gene23

ES 2 269 474 T3 is performed by injecting the compounds, compositions, or diagnostic compositions of the present invention into a convenient and accessible vein or artery. This includes but is not limited to administration into the antecubital vein. Local targeted administration is accomplished by injecting the compounds, compositions, or diagnostic compositions of the present invention proximal in flow into a vein or artery that is presumed to contain thrombi distal to the injection site. This includes but is not limited to administration into the coronary artery vasculature to image coronary thrombi, into the carotid artery to image thrombi in the cerebral vasculature, or into the pedal vein to image venous thrombosis. deep leg.

Also, the manner of delivering a composition of the present invention to the site of a thrombus is considered within the scope of the term "administration". For example, the mammal can be injected with a compound represented by Formula I having an anchored chelating medium, ultimately followed by the radioactive atom, thereby forming in vivo at the thrombus site the composition comprising the compound of the formula forming complex with a radioactive atom. Alternatively, a composition comprising the compound of the formula complexed with a radioactive atom can be injected into the mammal.

The "diagnostic effective amount" of the diagnostic compounds, compositions or compositions used in the methods of the present invention will depend, as previously mentioned, 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 in determining this dose are well known to those skilled in the diagnostic arts. In either case, the in vivo imaging dose should be sufficient to detect the presence of the imaging agent at the thrombus site in question. Typically, radiological imaging will require that the doses provided by the diagnostic composition of the present invention be 5 to 20 pCi, preferably about 10 pCi. Magnetic resonance imaging will require the doses provided by the diagnostic composition to be 0.001 to 5 mmol / kg, preferably 0.005 to 0.5 mmol / kg of a compound of Formula I complexed with a paramagnetic atom. In any event, it is known in the art that the actual dose will depend on the location of the thrombus.

Detection of a thrombus by imaging is possible by the presence of radioactive or paramagnetic atoms located in such a thrombus.

The radioactive atoms associated with the diagnostic compositions and compositions of the present invention are 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 (where the high-energy gamma ray is absorbed, displacing an electron that emits a proton upon its return to the orbital state), photoelectric detectors arranged in a camera spatial detection (to detect the position of the emitted photons), and circuits to analyze the protons detected in the camera and produce an image.

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 localized paramagnetic atoms in a thrombus and an image of the patient is read as the nuclei return to equilibrium alignments.

The following examples are illustrative, but not limiting, of the method and compositions of the present invention.

Examples

Example 1

N- [2- (amidinoammooxy) ethyl] -2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl) acetamide trifluoroacetate salt

<img file="ES2269474T3_D0018.tif" />

1. Ethyl 2,2-Difluoro-2-phenylacetate (Middleton, W., et al., J. Org. Chem. 42: 2883 (1980))

PhCF2CÜ2Et

ES 2 269 474 T3

A mixture of ethyl benzoylformiate (12.5 g, 70.0 mmol) and (diethylamino) sulfur trifluoride (DAST, 18.5 mL, 140 mmol) was stirred for 48 hours at room temperature, and then poured over ice. The oil formed was taken up in dichloromethane (DCM), washed with H2O, dried over Na2SO4, concentrated, and filtered through a short column of silica gel eluting with 50% DCM / hexane. The filtrate was concentrated to give the title compound (12.3 g, 88% yield) as a brown liquid, 1H NMR (400 MHz, CDCl3) δ 7.65-7.63 (m, 2H), 7.52-7.43 (m, 3H), 4.30 (q, J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H).

2. 2,2-Difluoro-2-phenylacetic acid

FCF2CO2H

A suspension of ethyl 2,2-difluoro-2-phenylacetate (6.0 g, 30 mmol), prepared as in the previous step, in 1N NaOH (36 mL, 36 mmol) was stirred at room temperature. After 36 hours, the reaction became almost homogeneous. The mixture was acidified with 1N HCl (36 mL), and extracted with DCM twice. The extracts were combined, washed with H₂O, they dried over Na₂SW₄, and concentrated to give the title compound (3.85 g, 81% yield) as a pale yellow solid, which was used without further purification.

3. 2,2-Difluoro-2-phenylacetyl chloride

FCF2COCl

In a flask filled with 2,2-difluoro-2-phenylacetic acid (0.8 g, 5.06 mmol), prepared as in the previous step, under argon in an ice bath, oxalyl chloride (5 mL) was added , and the reaction mixture was stirred for 15 min. A solution of dimethylformamide (DMF) (37 mg, 0.506 mmol) in DCM (0.5 mL) was added. After 2 hours, the water bath was removed, and the mixture was stirred for 1 hour. The solvents were evaporated, DCM was added, and then evaporated in vacuo to give the title compound (0.88 g, 98% yield), which was used immediately without further purification.

Four. 2- (2,3-Difluoro-6-nitrophenyl) acetic acid (Yokomoto, M, W., et al. 1991, EP 0 470 578 A1).

<img file="ES2269474T3_D0019.tif" />

To a suspension of NaH (11.3 g, 60% oil dispersion, 282 mmol) in tetrahydrofuran (THF) (35 mL) in an ice bath was added a solution of diethyl malonate (45.2 g, 42 , 9 mL, 282 mmol) in THF (70 mL) over a period of one hour so that the reaction temperature was kept below 20 ° C. Some white solid precipitated during the addition. A solution of 1,2,3-trifluoro-4-nitrobenzene (25.0 g, 141 mmol) in THF (35 mL) was added to the above reaction mixture over a period of 1 hour so that the reaction temperature was kept below 10 ° C. The ice bath was removed and the mixture was stirred at room temperature for 2 hours. Acetic acid (18 mL) was added to the reaction solution, and THF was evaporated under reduced pressure. Chloroform (200 mL), H₂O (250 mL), and concentrated HCl (18 mL). The organic layer was separated, concentrated, mixed with 4N HCl (45 mL) and acetic acid (35 mL), and refluxed for 14 hours. The reaction mixture was allowed to cool to room temperature. The solid precipitated after cooling was filtered off, washed with diisopropyl ether, and dissolved in MeOH (70 mL). After treatment with activated charcoal, the solvent was evaporated, and the crystalline residue was washed with isopropyl ether, and filtered off to give the title compound (17.6 g, 58% yield) as a solid of White color. 1H NMR (400 MHz, CDCl3) δ 8.05-8.01 (m, 1H), 7.47 (dd, J = 17.4, 8.9 Hz, 1H), 4.10 (s, 2H) .

5. 2- (2,3-Difluoro-6-nitrophenyl) ethanol (Yokomoto, M, W., et al., EP 0 470 578 A1 (1991))

<img file="ES2269474T3_D0020.tif" />

ES 2 269 474 T3

To a mixture of NaBH₄ (3.60 g, 95.4 mmol) in THF (12 mL) cooled below 10 ° C was added a solution of 2- (2,3-difluoro-6-nitrophenyl) acetic acid (10.9 g, 50.2 mmol), prepared as in the previous step, in THF (4 mL) over a period of 1 hour. To this mixture, a solution of boron trifluoride-diethyl etherate complex (16.5 mL, 131 mmol) in THF (24 mL) was added over a period of 1 hour, keeping the reaction temperature below 10 ° C. After the addition, the reaction was stirred on ice for 15 minutes, and then at room temperature for 20 minutes. To a mixture of DCM (180 mL) and H₂O (140 mL) NaHCO was added₃ (15 g, 179 mmol). The reaction mixture was slowly added to the NaHCO solution₃above and stirred overnight. The organic layer was separated, dried over Na₂SW₄, and concentrated to give the title compound (10.1 g, 99% yield) as a light brown oil. 1H NMR (400 MHz, CDCl3) δ 7.82 (dd, J = 9.1, 4.4 Hz, 1H), 7.27-7.18 (m, 1H), 3.95 (t, J = 6.3 Hz, 2H), 3.30-3.27 (m, 2H), 1.82 (s, 1H).

6. 2- {3 - [(t-Butyl) amino] -2-fluoro-6-nitrophenyl} ethanol (Yokomoto, M, W., et al. EP 0 470 578 A1 (1991))

<img file="ES2269474T3_D0021.tif" />

A mixture of 2- (2,3-difluoro-6-nitrophenyl) ethanol (6.00 g, 29.6 mmol), prepared as in the previous step, tbutylamine (18.6 mL, 1.77 mmol), DMSO (30 mL), and toluene (5 mL) was refluxed for 16 hours. After cooling to room temperature the brown solution was poured into H2O (300 mL), and the deposited yellow crystals were filtered and washed with H2O twice. The yellow solid was dissolved in CHCl3 (70 mL), dried over Na₂SW₄, concentrated, and crystallized from hexane to give the title compound (4.70 g, 62% yield) as a white solid. 1 H NMR (400 MHz, CDCl₃) δ 7.92 (dd, J = 9.3, 1.5 Hz, 1H), 6.79 (t, J = 8.7 Hz, 1H), 4.69 (s broad, 1H), 3, 96 (dd, J = 11.4, 5.9 Hz, 2H), 3.32 (dt, J = 6.5, 3.1 Hz, 2H), 1.75 (t, J =

5.3 Hz, 1H), 1.46 (s, 9H).

7. 2- (3-Amino-2-fluoro-6-nitrophenyl) ethanol (Yokomoto, M, W., et al. EP 0 470 578 A1 (1991))

<img file="ES2269474T3_D0022.tif" />

A solution of 2- {3 - [(t-butyl) amino] -2-fluoro-6-nitrophenyl} ethanol (3.9 g, 15 mmol), prepared as in the previous step, in concentrated HCl (40 mL) refluxed for 2 hours. After cooling to room temperature, the mixture was extracted with ethyl acetate (6 x 50 mL). The extracts were combined, washed with saturated NaHCO3 (2 times) and brine, dried over Na2SO4, and concentrated to give the crude product as a solid. The solid was triturated in hexane, filtered, and dried under high vacuum to yield the title compound (2.8 g, 93% yield) as a white solid. 1 H NMR (400 MHz, CD₃OD) δ 7.80 (dd, J = 9.1, 1.5 Hz, 1H), 6.70 (t, J = 9.0 Hz, 1H), 3.77 (t, J = 7.1 Hz, 2H), 3.26 (dt, J = 7.3, 2.8 Hz, 2H).

8. 2- (3-Amino-2-fluoro-6-nitrophenyl) ethyl acetate

<img file="ES2269474T3_D0023.tif" />

ES 2 269 474 T3

To a solution of DIEA (1.80 mL, 10.6 mmol) and 2- (3-amino-2-fluoro-6-nitrophenyl) ethanol (0.88 g, 4.40 mmol), prepared as in step above, in THF (10 mL) in an ice bath a solution of acetyl chloride (319 juL, 4.49 mmol) in THF (5 mL) was added. After stirring for 1.5 hours, the water bath was removed and the mixture was stirred at room temperature overnight. Additional acetyl chloride (63 µL, 0.88 mmol) was added, and the mixture was stirred for another 16 hours. Solvents were removed, and the mixture was partitioned between DCM and H₂O. The organic layer was separated, and the aqueous layer was re-extracted with DCM. The organic layers were combined, washed with H2O (twice), dried over Na2SO4, concentrated, and flash chromatographed on silica gel eluting with EtOAc / DCM (0, 1, 2, and 5%) to Give the title compound (0.73 g, 69% yield) as a white solid. RmN H1 (400 MHz, CDCl₃) δ 7.85 (dd, J = 9.0, 1.5 Hz, 1H), 6.68 (t, J = 8.9 Hz, 1H), 4.38-4.35 (m, 4H) , 3.38 (dt, J = 6.6, 2.8 Hz, 2H), 2.03 (s, 3H).

9. 2- (3- (2,2-Difluoro-2-phenylacetylamino) -2-fluoro-6-nitrophenyl] ethyl acetate

<img file="ES2269474T3_D0024.tif" />

To a solution of DIEA (1.49 mL, 8.55 mmol) and 2- (3-amino-2-fluoro-6-nitrophenyl) ethyl acetate (690 mg, 2.85 mmol), prepared as in step above, in dCm (6 mL) a solution of 2,2-difluoro-2-phenylacetyl chloride (0.99 g, 5.20 mmol), prepared according to the procedure of step 3 of Example 1, in DCM ( 3 mL). After stirring for 24 hours, the mixture was concentrated, and partitioned between DCM and H₂O. The organic layer was separated, and the aqueous layer was extracted with DCM. The organic layers were combined, washed with H2O and brine, dried over Na2SO4, concentrated, and flash chromatographed on silica gel eluting with EtOAc / DCM (0, 2.5, and 5%) to give the Title compound (1.04 g, 92% yield) as an orange oil. 1 H NMR (400 MHz, CDCl₃) δ 8.49-8.43 (m, 2H), 7.87 (d, J = 9.2 Hz, 1H), 6.68 (d, J = 7.1 Hz, 2H), 7.55 -7.49 (m, 3H), 4.35 (t, J = 6.4 Hz, 2H), 3.37 (dt, J = 6.3, 2.3 Hz, 2H), 2.01 (s, 3H).

10. 2- [2-amino-5- (2,2-difluoro-2-phenylacetylamino) -6-fluorophenyl] ethyl acetate

<img file="ES2269474T3_D0025.tif" />

A mixture of 2- [3- (2,2-difluoro-2-phenylacetylamino) -2-fluoro-6-nitrophenyl] ethyl acetate (0.84 g, 2.12 mmol), prepared as in the previous step, and palladium catalyst (226 mg, 10% on activated carbon, 0.212 mmol) in ethanol (17 mL) was hydrogenated in a hydrogen balloon for 3.5 hours. The mixture was filtered through Celite (diatomaceous earth) and washed with MeOH. The filtrate and washings were combined, concentrated, and flash chromatographed on silica gel eluting with EtOAc / DCM (5, 10, and 20%) to give the title compound (0.713 g, 92% yield). ) as a white solid. 1 H NMR (400 MHz, CDCl₃) δ 8.10 (s broad, 1H), 7.81 (t, J = 8.7 Hz, 1H), 7.68-7.66 (m, 2H), 7.52-7.44 (m , 3H), 6.45 (dd, J = 8.8, 1.2 Hz, 1H), 4.18 (t, J =

7.4Hz, 2H), 4.07 (bs, 2h), 2.90 (dt, J = 7.4.1.9Hz, 2H), 2.07 (s, 3H). Mass Spectrum (LCMS, ESI) calculated for C_i8H_i8F₃N₂OR₃ (M + h): 367.1. Found: 367.1.

ES 2 269 474 T3

eleven. N- (4-Amino-2-fluoro-3- (2-hydroxyethyl) feml] -2,2-difluoro-2-phenylacetamide

<img file="ES2269474T3_D0026.tif" />

To a solution of 2- [2-amino-5- (2,2-difluoro-2-phenylacetylamino) -6-fluorophenyl] ethyl acetate (0.67 g, 1.84 mmol), prepared as in the previous step , in MeOH (19 mL) a solution of K₂CO₃ (280 mg, 2.03 mmol) in H₂O (4.8 mL). The mixture was stirred for 45 minutes, then neutralized with 1N HCl. The MeOH was evaporated, and the mixture was extracted with EtOAc twice. The extracts were combined, washed with H2O, dried over Na₂SW₄, and concentrated to give the title compound (0.55 g, 92% yield) as a pale yellow solid. 1H NMR (400 MHz, CD3OD) δ 7.70-7.68 (m, 2H), 7.54-7.48 (m, 3H), 7.01 (t, J = 8.5 Hz, 1H) , 6.53 (dd, J = 8.6, 1.3 Hz, 1H), 3.70 (t, J = 6.7 Hz, 2H), 2.80 (dt, J = 6.7, 2 , 0 Hz, 2H). Mass Spectrum (LCMS, ESI) calculated for C₁₆H₁₆F₃N₂OR₂ (M + H): 325.1. Found: 325.3.

12. N- [4-Chloro-2-fluoro-3- (2-hydroxyethyl) phenyl] -2,2-difluoro-2-phenylacetamide (Yokomoto, M, W., et al. 1991, EP 0 470 578 A1)

<img file="ES2269474T3_D0027.tif" />

A suspension of N- [4-amino-2-fluoro-3- (2-hydroxyethyl) phenyl] -2,2-difluoro-2-phenylacetamide (1.63 g, 5.00 mmol), prepared according to the procedure of the previous step, in 6N HCl (9 mL) was cooled in an ice bath, and then a solution of NaNO2 (434 mg, 6.30 mmol) in H2O (2.4 mL) was added over 5 minute period. After 30 minutes, acetic acid (2.9 mL) and concentrated HCl (2.9 mL) were added and the reaction mixture was stirred for 1 hour. To this mixture was added a solution of CuCl (848 mg, 8.55 mmol) in concentrated HCl (5 mL) over a period of 20 minutes. After stirring in an ice bath for 3 hours, the reaction mixture was extracted with EtOAc (200 mL x 3). The extracts were combined, washed with H2O (2 times) and brine, dried over Na₂SW₄, concentrated, and flash chromatographed on silica gel eluting with EtOAc / DCM (0, 2.5, and 5%) to provide the title compound (0.845 g, 48% yield) as an oil. orange. 1 H NMR (400 MHz, CDCl₃) δ 8.31 (s, 1H), 8.14 (t, J = 8.6 Hz, 1H), 7.68-7.66 (m, 2H), 7.54-7.46 (m, 3H), 7.19 (dd, J = 8.9, 1.7 Hz, 1H), 3.86 (dd, J = 12.6, 6.5 Hz, 2H), 3.09 (dt, J = 6.7, 2.3 Hz, 2H), 1.58 (t, J = 5.7 Hz, 1H). Mass Spectrum (LCMS, ESI) calculated for C₁₆Hi₄C1F₃do not₂ (M + H): 344.1. Found: 344.2.

13. 2- {3 - [(2,2-Difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} ethanol

<img file="ES2269474T3_D0028.tif" />

To a solution of N- [4-chloro-2-fluoro-3- (2-hydroxyethyl) phenyl] -2,2-difluoro-2-phenylacetamide (1.05 g, 3.06 mmol), prepared according to the procedure from the previous step, in THF (12 mL) at 0 ° C under argon, a solution of

ES 2 269 474 T3 borane-THF complex in THF (12.3 mL, 12.3 mmol, 1.0 M) over a period of 10 minutes, and the reaction mixture was continued to stir until consumed the ice bath. The reaction mixture was refluxed for 20 hours, and allowed to cool to room temperature. A solution of K₂CO₃ (1.7 g, 12 mmol) in H2O (12 mL), THF was removed in vacuo, and the mixture was extracted with DCM (3 times). The extracts were combined, washed with brine, dried over Na₂SW₄, concentrated, and flash chromatographed on silica gel eluting with EtOAC / DCM (0 and 2.5%) to give the title compound (815 mg, 81% yield) as a colorless oil. ¹H-NNR (400 MHz, CdC1₃) δ 7.52-7.43 (m, 5H), 6.97 (dd, J = 8.8,

1.7 Hz, 1H), 6.51 (t, J = 8.9 Hz, 1H), 4.17 (s broad, 1H), 3.83 (t, J = 6.9 Hz, 2H), 3.74 (dt, J = 13.4, 6.6 Hz, 2H), 3.04 (dt, J = 6.9, 2.4 Hz, 2H), 1.43 (s, 1H). Mass Spectrum (LCMS, ESI) calculated for C₁₆H₁₆ClF₃NO (M + H): 330.1. Found: 330.3.

14. 2- {3 - [(2,2-Difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} ethanol

<img file="ES2269474T3_D0029.tif" />

To a solution of DMSO (1.03 mL, 14.5 mmol), DIEA (1.99 mL, 11.4 mmol), and 2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} ethanol (1.45 g, 4.4 mmol), prepared according to the procedure of the previous step, in DCM (140 mL) in an ice bath, sulfur trioxide complex was added- pyridine (1.82 g, 11.4 mmol) and stirred at the same temperature for 3.5 hours. The mixture was diluted with DCM (300 mL), washed with 10% citric acid (3 times), H₂O, and brine, dried over Na₂SW₄, and concentrated to give the title compound (1.43 g, 99% yield) as an orange oil, which was used without further purification.

fifteen. 2- {3 - [(2,2-Difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} acetic acid (Dalcanale, E., et al. J. Org. Chem., 51: 567 ( 1986))

<img file="ES2269474T3_D0030.tif" />

A solution of sodium chlorite (692 mg, 6.11 mmol) in H₂O (6.1 mL) over a 30 minute period to a stirred mixture of 2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} ethanol (1.43 g, 4.37 mmol), prepared as in the previous step, in DMSO (4.5 mL) and NaH₂PO₄ (141 mg, 1.18 mmol) in H₂O (1.7 mL). After the addition, the mixture was stirred at room temperature overnight, acidified with 10M HCl to pH1, and extracted with DCM (3 times). The extracts were combined, washed with H2O and brine, dried over Na2SO4, and concentrated in vacuo. The resulting residue was flash chromatographed on silica gel eluting with MeOH / DCM (0, 2, and 4%) to give the title compound (0.77 g, 51% yield) as a colored solid. brown. 1 H NMR (400 MHz, CD3OD) δ 7.53-7.51 (m, 2H), 7.44-7.41 (m, 3H), 6.93 (dd, J = 8.9, 1.8 Hz, 1H), 6.62 (t, J = 9.1 Hz, 1H), 3.80 (t, J = 13.7 Hz, 2H), 3.74 (d, J = 2.2 Hz, 2H). Mass Spectrum (LCMS, ESI) calculated for C₁₆H₁₄ClF₃DO NOT₂ (m + H): 344.1. Found: 344.4.

ES 2 269 474 T3

16. 2-aza-3 - {(2- (2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} acetylamino) ethoxy] amino} -3 - [( t-butyl t-butoxy) carbonylamino] prop-2-enate

<img file="ES2269474T3_D0031.tif" />

To a solution of 2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} acetic acid (28 mg, 82 mmol), prepared as in the previous step, in DMF (0.3 mL) in an ice bath was added BOP (58 mg, 130pinols), HCl salt of [N, N'-di (t-butoxycarbonyl)] - 2-aminoethoxyguanidine (36 mg, 102 pmol) (Tianbao Lu, et al., WO 99/26926 (1999)), and a solution of DIEA (42 mg, 33 pmol) in DMF (0.1 mL). After the ice bath was consumed, the reaction mixture was continued to stir at room temperature overnight. The solvents were evaporated, and the resulting residue was partitioned between NaHCO₃ saturated and DCM. The aqueous phase was extracted with DCM, and the organic layers were combined, washed with KHSO₄ at 10%, H₂O, and brine, dried over Na₂SW₄, concentrated, and flash chromatographed on silica gel eluting with MeOH / DCM (1%) to give the title compound (44 mg, 83% yield) as a colorless oil. NMR1 H (400 MHz, CdC1₃) δ 7.54-7.50 (m, 2H), 7.45-7.42 (m, 3H), 6.93 (dd, J = 8.8, 1.7 Hz, 1H), 6, 63 (t, J = 9.0 Hz, 1H), 4.03 (t, J = 4.8 Hz, 2H), 3.81 (t, J =

13.7 Hz, 2H), 3.71 (d, J = 1.9 Hz, 2H), 3.47 (t, J = 5.1 Hz, 2H), 1.50 (s, 9H), 1 , 48 (s, 9H). Mass Spectrum (LCMS, ESI) calculated for C₂₉H₃₈ClF₃N₅OR₆ (M + H): 644.2. Found: 644.1.

17. N- [2- (amidinoaminooxy) ethyl] -2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} acetamide trifluoroacetate salt

<img file="ES2269474T3_D0032.tif" />

A solution of 2-aza-3 - {[2- (2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} acetylamino) ethoxy] amino} -3 t-Butyl - [(t-butoxy) carbonylamino] prop-2-enoate (44 mg, 68 pmol), prepared as in the previous step, in TFA / DCM (2 mL, 2/3) was stirred at room temperature environment for 4 hours. The solvents were evaporated, and the resulting residue was flash chromatographed on silica gel eluting with 0.05% TFA in MeOH / DCM (5 and 10%) to provide the title compound (37 mg, 98% yield). ) as a white solid. 1 H NMR (400 MHz, CD3OD) δ 7.67-7.52 (m, 2H), 7.47-7.42 (m, 3H), 6.96 (dd, J = 8.9, 1.7 Hz, 1H), 6.66 (t, J = 9.1 Hz, 1H), 3.93 (t, J = 5.4 Hz, 2H), 3.82 (t, J = 13.8 Hz, 2H), 3.71 (d, J = 2.0Hz, 2H), 3.50 (t, J = 5.4Hz, 2H). Mass Spectrum (LCMS, ESI) calculated for Ci₉H₂₂C1F₃N₅OR₂ (M + H): 444.1. Found: 444.2.

Example 2

N - [(6-amino-2-methyl (3-pyridyl)) methyl] -2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl hydrochloride salt } acetamide

<img file="ES2269474T3_D0033.tif" />

ES 2 269 474 T3

1. 2- {3 - [(2,2-Difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} -N - ({6 - [(t-butoxy) carbonylamino] -2-methyl (3- pyridyl)} methyl) acetamide

<img file="ES2269474T3_D0034.tif" />

To a solution of 2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} acetic acid (516 mg, 1.5 mmol), as prepared in step 15 from Example 1, in DMF (8.0 mL) was added N- [5- (aminomethyl) -6-methyl- (2-pyridyl)] (t-butoxy) carboxamide (498 mg, 2.1 mmol) (Sanderson , PE, et al., WO 97/01338 (1997)), BOP (1.06 g,

2.4 mmol), and DIEA (0.78 mL, 4.5 mmol). After stirring for 18 hours, additional amine (107 mg, 450 µmol) was added, and the mixture was stirred for 18 hours. The solvents were evaporated, and the reaction mixture was partitioned between DCM and saturated NaHCO3. The organic layer was separated, and the aqueous layer was extracted with DCM. Organic layers were combined, washed with KHSO₄ at 10% (2 times), H₂O, and brine, dried over Na2SO4, concentrated, and flash chromatographed on silica gel eluting with MeOH / DCM (0, 1, 5, and 2.5%) to give the title compound (770 mg , 91% yield) as a pale brown foam. 1 H NMR (400 MHz, CDCE) δ 7.66 (d, J = 8.4 Hz, 1H), 7.51-7.41 (m, 6H), 7.23 (s broad, 1H), 7, 01 (dd, J = 8.8, 1.5 Hz, 1H), 6.57 (t, J = 9.0 Hz, 1H), 5.64 (s broad, 1H), 4.37 (d , J = 5.6 Hz, 2H), 4.26-4.22 (m, 1H), 3.793.70 (m, 4H), 2.35 (s, 3H), 1.50 (s, 9H) . Mass Spectrum (lCmS, ESI) calculated for C₁₈H₃₁ClF₃N₄OR₃ (M + H): 563.2. Found: 562.9.

2. N - [(6-amino-2-methyl (3-pyridyl)) methyl] -2- {3 - [(2,2-difluorO-2-phenylethyl) amino] -6-chlorO-2-fluorophenyl hydrochloride salt } acetamide

<img file="ES2269474T3_D0035.tif" />

To a flask charged with 2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} -N - ({6 - [(t-butoxy) carbonylamino] -2 -methyl (3-pyridyl)} methyl) acetamide (770 mg, 1.37 mmol), prepared as in the previous step, a solution of HCl in 1,4-dioxane (5 mL, 20 mmol, 4, 0 M). After stirring at room temperature for

1.5 hours, some solid precipitated. A solution of MeOH (1 mL) in DCM (3 mL) was added to dissolve the solid, and the mixture was stirred for an additional 4 hours. Solvents were removed, and the resulting residue was washed with DCM (5 mL x 2), ether (8 mL x 2), and dried under high vacuum to give the title compound (620 mg, 91% yield) in form of a brown solid. 1 H NMR (400 MHz, CD₃OD) δ 7.81 (d, J = 9.1 Hz, 1H), 7.54-7.51 (m, 2H), 7.457.42 (m, 3H), 6.94 (dd, J = 8 , 9, 1.6 Hz, 1H), 6.80 (d, J = 9.1 Hz, 1H), 6.65 (t, J = 9.1 Hz, 1H), 4.25 (s, 2H ), 3.81 (t, J =

13.8Hz, 2H), 3.70 (d, J = 1.8Hz, 2H), 2.50 (s, 3H). Mass Spectrum (LCMS, ESI) calculated for C₂₃H₂₃ClF₃N₄O (M + H): 463.1. Found: 463.7.

ES 2 269 474 T3

Example 3

N - [(6-amino-2,4-dimethyl (3-pyridyl)) methyl] -2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2 hydrochloride salt -fluorophenyl} acetamide

<img file="ES2269474T3_D0036.tif" />

1. 2- {3 - [(2,2-Difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} -N - ({6 - [(t-butoxy) carbonylamino] -2,4-dimethyl ( 3-pyridyl)} methyl) acetamide

<img file="ES2269474T3_D0037.tif" />

To a solution of 2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} acetic acid (25 mg, 73 // mol), as prepared in step 15 of Example 1, in DMF (0.25 mL) was added BOP (52 mg, 116 // moles), a solution of DIEA (38 mg, 295 moles) in DMF (0.1 mL), and N- [5- (aminomethyl) -4,6-dimethyl (2-pyridyl)] (t-butoxy) -carboxamide (23 mg, 91 // moles) (Sanderson, PE, et al. WO 97/01338 (1997)) . After stirring at room temperature for 2 days, additional amine (7 mg, 28 // mole), BOP (16 mg, 36 // mole), and DIEA (9 mg, 70 // mole) were added, and the The mixture was stirred for another 16 hours. The solvents were evaporated, and the resulting residue was partitioned between saturated NaHCO3 and DCM. The organic layer was separated, and the aqueous layer was extracted with DCM. The organic layers were combined, washed with 10% citric acid, H₂O, and brine, dried over Na₂SW₄, concentrated, and flash chromatographed on silica gel eluting with MeOH / DCM (0.1.2%) to yield the title compound (18.5 mg, 44% yield) as a solid of Dun. 1H NMR (400 MHz, CDCl3) δ 7.58 (s, 1H), 7.50-7.41 (m, 5H), 7.24 (s, 1H), 6.98 (dd, J = 8, 8, 1.1 Hz, 1H), 6.55 (t, J = 9.0 Hz, 1H), 5.33 (s broad, 1H), 4.40 (d, J = 4.7 Hz, 2H ), 4.24-4.20 (m, 1H), 3.78-3.70 (m, 4H), 2.39 (s, 3H), 2.28 (s, 3H), 1.50 ( s, 9H). Mass Spectrum (LCMS, ESI) calculated for C₂₉H₃₃ClF₃N₄OR₃ (M + h): 577.0. Found: 577.1.

2. Hydrochloride salt of N - [(6-Amino-2,4-dimethyl (3-pyridyl)) methyl] -2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl } acetamide

<img file="ES2269474T3_D0038.tif" />

A solution of HCl in 1,4-dioxane (4.0 M, 0.5 mL, 2 mmol) was added to 2- {3 - [(2,2-difluoro-2-phenylethyl) amino] 6-chloro- 2-fluorophenyl} -N - ({6 - [(t-butoxy) carbonylamino] -2,4-dimethyl (3-pyridyl)} methyl) acetamide (18.5 mg, 32 // mole), prepared as in previous stage. After stirring at room temperature for 3 hours, a solid precipitated. A solution of MeOH (0.1 mL) in DCM (1 mL) was added to dissolve the solid. After stirring for another 2 hours the reaction was concentrated to give a brown solid, which was washed with ether and DCM and dried in vacuo to give the title compound (12.6 mg, 77% yield) as of a brown solid. 1 H NMR (400

ES 2 269 474 T3

MHz, CD3OD) δ 8.40 (s broad, 1H), 7.53-7.51 (m, 2H), 7.47-7.40 (m, 3H), 6.93 (dd, J = 8 , 8, 1.3 Hz, 1H), 6.68 (s, 1H), 6.64 (t, J = 9.1 Hz, 1H), 4.31 (d, J = 4.6 Hz, 2H ), 3.81 (t, J = 13.8 Hz, 2H), 3.67 (d, J = 1.2 Hz, 2H), 2.54 (s, 3H), 2.42 (s, 3H ). Mass Spectrum (LCMS, ESI) calculated for C24H25ClF3N4O (M + H): 477.2. Found: 477.5. Example 4

N- [2- (Amidinoaminooxy) ethyl] -2- (3 - {(2,2-difluoro-2- (4-fluoronaphthyl) ethyl] amino) -6-chloro-2-fluorophenyl) acetamide trifluoroacetate salt

<img file="ES2269474T3_D0039.tif" />

<img file="ES2269474T3_D0040.tif" />

A solution of n-butyl lithium (2.5 M in THF, 20 mL, 50 mmol) was added, cooled to -78 ° C, and a solution of 1-bromo-4-fluoronaphthalene (11.25 g , 50 mmol) in THF (40 mL) and the mixture was stirred for 1 hour. The reaction mixture was warmed to -20 ° C, then added to a solution of diethyl oxalate (29.2 g, 200 mmol) in THF (40 mL) at -78 ° C. After slowly warming to room temperature, EtOAc (100 mL), 10% HCl (50 mL) and water (50 mL) were added and the phases were separated. The aqueous layer was extracted with EtOAc (2 x 100 mL), and the organic layers were combined, washed with brine (50 mL), and dried over Na₂SW₄. After evaporating the solvent and excess diethyl oxalate under high vacuum, the residue was purified by flash column chromatography (DCM: hexane 1: 1) to give the title compound (9.4 g, 76% yield) in form of a white solid. 1H NMR (400 MHz, CDCL) δ (9.13 (d, J = 8.6 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 8.01 (dd, J = 8.2, 5.4 Hz, 1H), 7.76 (t, J = 7.2 Hz, 1H), 7.67 (t, J = 8.1 Hz, 1H), 7.21 (t, J = 8.5 Hz, 1H), 4.49 (q, J = 7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H).

2. Ethyl 2,2-Difluoro-2- (4-fluoronaphthyl) acetate

<img file="ES2269474T3_D0041.tif" />

To a solution of ethyl 2- (4-fluoronaphthyl) -2-oxoacetate (9.4 g, 38.2 mmol), prepared as in the previous step, in DCM (60 mL) was added DAST (16.1 g, 100 mmol). The mixture was stirred at room temperature overnight, slowly poured onto ice, and extracted with DCM (3 x 50 mL). The organic layers were combined, washed with brine, and dried over Na₂SW₄. After evaporating the solvent, the residue was purified by flash column chromatography (DCM: hexane 1: 1) to give the title compound (9.7 g, 95% yield) as a light brown oil. 1 H NMR (400 MHz, CDCl₃) δ 8.20 (m, 2H), 7.82 (dd, J = 8.2, 5.3 Hz, 1H), 7.63 (m, 2H), 7.20 (t, J = 8, 4Hz, 1H), 4.28 (c, J = 7.1Hz, 2H), 1.24 (t, J = 7.1Hz, 3H).

ES 2 269 474 T3

3. 2,2-Difluoro-2- (4-fluoronaphthyl) acetic acid

<img file="ES2269474T3_D0042.tif" />

To a solution of ethyl 2,2-difluoro-2- (4-fluoronaphthyl) acetate (9.6 g, 35.8 mmol), prepared as in the previous step, in methanol (20 mL) and THF (20 mL ) A solution of NaOH (2.0 g, 50 mmol) in water (40 mL) was added. The reaction mixture was stirred at room temperature for 2 hours. After evaporating the methanol and THF in vacuo, the aqueous phase was acidified to pH 2 using 10% HCl, and extracted with DCM (3 x 50 mL). The extracts were combined, washed with brine, dried over Na2SO4, and concentrated to give the title compound (8.1 g, 94% yield) as an off-white solid. 1 H NMR (400 MHz, CDCl₃) δ 9.68 (s broad, 1H), 8.18 (m, 2H), 7.83 (dd, J = 8.1, 5.3 Hz, 1H), 7.62 (m, 2H), 7.19 (t, J = 8.3 Hz, 1H).

Four. 2- {3- [2,2-Difluoro-2- (4-fluoronaphthyl) acetylamino] -2-fluoro-6-nitrophenyl} ethyl acetate

<img file="ES2269474T3_D0043.tif" />

To a solution of DIEA (7.8 mL) and ethyl 2- (3-amino-2-fluoro-6-nitrophenyl) acetate (4.6 g, 19 mmol), prepared as in step 8 of Example 1, 2,2-Difluoro-2- (4-fluoronaphthyl) acetyl chloride (prepared by refluxing 2,2-difluoro-2- (4-fluoronaphthyl) acetic acid) was added in DCM (60 mL), prepared as in previous step, with oxalyl chloride) (7.8 g, 30 mmol) in DCM (40 mL). The mixture was stirred at room temperature for 1 hour. Additional DCM (100 mL) was added, and the resulting mixture was washed with 10% citric acid (3 x 40 mL) and brine, and dried over Na₂ SW₄. After evaporating the solvent, the residue was purified by flash column chromatography eluting with DCM to give the title compound (5.3 g, 61%) as a yellow oil. 1 H NMR (400 MHz, CDCl₃) δ 8.45 (m, 2H), 8.21 (d, J = 7.0 Hz, 2H), 7.87 (s, 1H), 7.85 (t, J = 4.1 Hz, 1H ), 7.66 (m, 2H), 7.22 (t, J = 8.3 Hz, 1H), 4.34 (t, J = 6.4 Hz, 2H), 3.36 (t, J = 6.4 Hz, 2H), 2.00 (s, 3H).

5. 2- {2-Amino-5- [2,2-difluoro-2- (4-fluoronaphthyl) acetylamino] -6-fluorophenyl} ethyl acetate

<img file="ES2269474T3_D0044.tif" />

A mixture of 2- {3- [2,2-difluoro-2- (4-fluoronaphthyl) acetylamino] -2-fluoro-6-nitrophenyl} ethyl acetate (4.9 g, 10.5 mmol), prepared as in the previous step, and Pd / C (10%, 500 mg) in ethanol (50 mL) and THF (50 mL) was stirred in hydrogen for 5 hours. The reaction mixture was filtered through Celite, and washed with THF and MeOH. The filtrate and washings were combined, concentrated in vacuo, and flash chromatographed on silica gel eluting with EtOAc / DCM (0 to 2%) to yield the title compound (3.8 g, 83% ) as an off-white solid. H1 NMR (400 MHz, CdC1₃) δ 8.27 (d, J = 8.2 Hz, 1H), 8.19 (d, J = 8.6 Hz, 1H), 8.10 (s,

ES 2 269 474 T3

1H), 7.82 (m, 2H), 7.63 (m, 2H), 7.19 (t, J = 8.6 Hz, 1H), 6.46 (d, J = 8.7 Hz, 1H), 4.19 (t, J = 7.4 Hz, 2H), 4.08 (s, 2H), 2.91 (t, J = 7.3 Hz, 2H), 2.08 (s, 3H).

6. N- (4-AminO-2-fluoro-3- (2-hydroxyethyl) feml] -2,2-difluoro-2- (4-fluoronaphthyl) acetamide

<img file="ES2269474T3_D0045.tif" />

To a solution of 2- {2-amino-5- [2,2-difluoro-2- (4-fluoronaphthyl) acetylamino] -6-fluorophenyl} ethyl acetate (3.8 g,

8.8 mmol), prepared as in the previous step, in MeOH (40 mL) and THF (20 mL) a solution of K₂CO₃(1.68 g, 12 mmol) in water (30 mL). The mixture was stirred at room temperature for 3 hours. Additional water (50 mL) was added, and the resulting mixture was extracted with EtOAc (3 x 50 mL). The extracts were combined, washed with brine, dried over Na2SO4, and concentrated in vacuo to give the title compound (3.3 g, 96%) as an off-white solid. 1 H NMR (400 MHz, CDCl₃) δ 8.27 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 8.2 Hz, 1H), 8.13 (s, 1H), 7.81 (m, 2H ), 7.64 (m, 2H), 7.19 (t, J = 8.7Hz, 1H), 6.47 (d, J = 8.7Hz, 1H), 4.07 (s, 2H ), 3.89 (t, J = 5.6 Hz, 2H), 2.85 (t, J = 5.5 Hz, 2H).

7. N- [4-Chloro-2-fluoro-3- (2-hydroxyethyl) phenyl] -2,2-difluoro-2- (4-fluoronaphthyl) acetamide (Doile, MP, et al. J. Org. Chem., 42: 2426 (1977))

<img file="ES2269474T3_D0046.tif" />

A solution of t-butyl nitrite (1.46 g, 12.8 mmol, 90%, Aldrich) in acetonitrile was added to a flask filled with copper (II) chloride (1.84 g, 13.7 mmol). (35 mL) under argon atmosphere. The resulting green reaction mixture was cooled in an ice bath to 0 ° C, and a solution of N- [4-amino-2-fluoro-3- (2-hydroxyethyl) phenyl] -2.2 was added -difluoro-2- (4-fluoronaphthyl) acetamide (3.58 g, 9.13 mmol), prepared according to the procedure of the previous step, in acetonitrile (60 mL) over a period of 45 minutes. After stirring for an additional 6 hours at 0 ° C, the resulting brown mixture was allowed to warm to room temperature, then poured into 20% aqueous HCl (160 mL), and extracted with DCM (3 times ). The extracts were combined, washed with 20% HCl, H2O, and brine, dried over Na2SO4, concentrated, and flash chromatographed on silica gel eluting with EtOAc / DCM (0 and 2.5%) to Provide the title compound (2.1 g, 56% yield) as a white solid. 1 H NMR (400 MHz, CDCl₃) δ 8.31 (s broad, 1H), 8.24-8.12 (m, 3H), 7.84 (dd, J = 8.2, 5.3 Hz, 1H). 7.68-7.60 (m, 2H), 7.22-7.20 (m, 2H), 3.87 (dd, J = 12.6, 6.5 Hz, 2H), 3.09 ( dt, J = 6.7, 2.2 Hz, 2H), 1.47 (t, J = 5.6 Hz, 2H). Mass Spectrum (LCMS, ESI) calculated for C₂₀H₁₅C1F₄Do not₂ (M + H): 412.1. Found: 412.6.

ES 2 269 474 T3

8. 2- (3 - {[2,2-Difluoro-2- (4-fluoronaphthyl) ethyl] am, ino} -6-chloro-2-fluorofeml) ethanol

<img file="ES2269474T3_D0047.tif" />

To a solution of N- [4-chloro-2-fluoro-3- (2-hydroxyethyl) phenyl] -2,2-difluoro-2- (4-fluoronaphthyl) acetamide (1.9 g, 4.6 mmol) , prepared as in the previous step, in THF (19 mL) at 0 ° C a solution of BH complex was added dropwise₃-THF (19.4 mL, 19.4 mmol, 1.0 M in THF) over a 20 minute period, and the reaction mixture continued to stir until the ice bath was consumed. The mixture was then refluxed in a 75-80 ° C oil bath for 3 hours, and stirring was continued at room temperature overnight. NaHCO solution was added₃ (1.63 g, 19.4 mmol) in H₂O (20 mL), THF was evaporated, and the resulting mixture was extracted with DCM twice. The extracts were combined, washed with H2O and brine, dried over Na2SO4, concentrated, and flash chromatographed on silica gel eluting with EtOAc / DCM (0.1, 1.5%) to give the compound from titer (805 mg, 44% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.24-8.19 (m, 2H), 7.70-7.61 (m, 3H), 7.14 (t, J = 9.3 Hz, 1H) , 6.90 (dd, J = 8.7, 1.6 Hz, 1H), 6.42 (t, J = 8.9 Hz, 1H), 4.22-4.16 (m, 1H), 4.00 (dt, J = 13.4, 6.8 Hz, 2H), 3.82 (dd, J = 12.1, 6.4 Hz, 2H), 3.02 (dt, J = 6, 8, 2.3 Hz, 2H), 1.39 (s broad, 1H). Mass Spectrum (LCMS, ESI) calculated for C₂₀H₁₇ClF₄NO (m + H): 398.1. Found: 398.3.

9. 2- (3 - {[2,2-Difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6-chloro-2-fluorophenyl) ethanal

<img file="ES2269474T3_D0048.tif" />

To a solution of DMSO (470 mg, 6.0 mmol), DIEA (823 // L, 4.74 mmol) and 2- (3 - {[2,2-difluoro-2- (4-fluoronaphthyl) ethyl} amino} -6-chloro-2-fluorophenyl) ethanol (723 mg, 1.82 mmol), prepared as in the previous step, in DCM (55 mL) in an ice bath, sulfur trioxide-pyridine complex was added (754 mg, 4.74 mmol). After stirring for 3.5 hours the reaction mixture was diluted with DCM (110 mL). The organic layer was separated, and the aqueous layer was extracted with DCM (100 mL). The organic layers were combined, washed with 10% citric acid (3 times), H₂O, and brine, dried over Na₂SW₄, and concentrated to give the title compound (722 mg, quantitative yield) as an orange oil. 1 H NMR (400 MHz, CDCl₃) δ 9.69 (d, J = 1.1 Hz, 1H), 8.22-8.18 (m, 2H), 7.67-7.60 (m, 3H), 7.14 (t, J = 9.2 Hz, 1H), 6.94 (d, J = 8.8 Hz, 1H), 6.49 (t, J = 8.7 Hz, 1H), 4.20 (s broad, 1H ), 4.00 (dt, J = 13.3.6.7 Hz, 2H), 3.83 (s, 2H).

ES 2 269 474 T3

10. 2- (3 - {[2,2-Difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6-chloro-2-fluorophenyl) acetic acid

<img file="ES2269474T3_D0049.tif" />

A solution of sodium chlorite (309 mg, 2.73 mmol, 80%) in H₂O (3.0 mL) dropwise over a 30 minute period to a stirred mixture of 2- (3 - {[2,2-difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6 -chloro-2-fluorophenyl) ethanal (722 mg, 1.82 mmol), prepared as in the previous step, in DMSO (3.6 mL) and NaH₂PO₄ (74 mg, 0.55 mmol) in H2O (0.9 mL). After the addition, the mixture was stirred at room temperature for 48 hours, then acidified with 10M HCl to pH 1, and extracted with DCM (3 times). The extracts were combined, washed with H2O and brine, dried over Na₂SW₄, and concentrated in vacuo. The resulting residue was flash chromatographed on silica gel eluting with MeOH / DCM (0, 1, 1.5, and 2%) to give the starting aldehyde 2- (3 - {[2,2-difluoro-2- ( 4-fluoronaphthyl) ethyl] -amino} -6-chloro-2-fluorophenyl) ethanal (165 mg, 23% yield) and the title compound (570 mg, 76% yield) as a solid. 1 H NMR (400 MHz, CDCl₃) δ 8.32 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.71-7.61 (m, 3H), 7.18 (dd, J = 9.8, 8.5 Hz, 1H), 6.78 (dd, J = 8.8, 1.5 Hz, 1H), 6.44 (t, J =

9.1 Hz, 1H), 4.05 (t, J = 13.5 Hz, 2H), 3.69 (d, J = 2.1 Hz, 2H).

eleven. 2-Aza-3 - ({2- [2- (3 - {[2,2-difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6-chloro-2-fluorophenyl) acetylamino] ethoxy} amino) T-butyl -3 - [(t-butoxy) carbonylamino] prop-2-enoate

<img file="ES2269474T3_D0050.tif" />

To a solution of 2- (3 - {[2,2-difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6-chloro-2-fluorophenyl) acetic acid (570 mg, 1.39 mmol), prepared as in the previous step, in DMF (7.5 mL) in an ice bath was added BOP (981 mg, 2.22 mmol), HCl salt of [N, N'-di (t-butoxycarbonyl)] - 2 -aminoethoxyguanidine (689 mg, 1.94 mmol), and DIEA (0.96 mL, 5.55 mmol). After the ice bath was consumed, the mixture was continued to stir at room temperature overnight. BOP (123 mg, 0.28 mmol) and the HCl salt of [N, N'-di (t-butoxycarbonyl)] 2-aminoethoxyguanidine (98 mg, 0.28 mmol) were added, and the reaction mixture was stirred for 24 hours. The solvents were evaporated, and the resulting residue was partitioned between saturated NaHCO3 and DCM. The organic layer was separated, and the aqueous layer was extracted with DCM. Organic layers were combined, washed with KHSO₄ 10%, H2O, and brine, dried over Na2SO4, concentrated, and flash chromatographed on silica gel eluting with MeOH / DCM (0, 0.5, and 1%) to give the title compound ( 720 mg, 73% yield) as a white foam. 1 H NMR (400 MHz, CD₃Od) δ 9.12 (s, 1H), 8.24-8.19 (m, 2H), 7.78-7.81 (m, 1H), 7.70-7.60 (m, 4H) , 7.14 (dd, J = 9.6, 8.4 Hz, 1H), 6.91 (dd, J = 8.8, 1.6 Hz, 1H), 6.44 (t, J = 8 , 8 Hz, 1H), 4.20-4.15 (m, 1H), 4.13-4.10 (m, 2H), 3.98 (dt, J = 13.4, 6.7 Hz, 2H), 3.78 (d, J = 1.9 Hz, 2H), 3.60 (dd, J = 8.6, 4.9 Hz, 2H), 1.51 (s, 9H), 1 , 48 (s, 9H). Mass Spectrum (LCMS, ESI) calculated for C₃₃H₃₉ClF₄N₅OR₆ (M + H): 712.2. Found: 712.3.

ES 2 269 474 T3

12. N- [2- (amidinoaminooxy) ethyl] -2- (3 - {[2,2-difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6-chloro2-fluorophenyl) acetamide trifluoroacetate salt

<img file="ES2269474T3_D0051.tif" />

A solution of 2-aza-3 - ({2- [2- (3 - {[2,2-difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6-chloro-2-fluorophenyl) acetylamino] ethoxy } amino) -3 - [(t-butoxy) carbonylamino] prop-2-ethyl enoate (720 mg, 1.01 mmol), prepared as in the previous step, in TFA / DCM (2: 3.30 mL) stirred at room temperature for 4 hours. The solvents were evaporated, and the resulting residue was flash chromatographed on silica gel eluting with 0.05% TFA in MeOH / DCM (5 and 10%) to give the title compound (626 mg, 99% yield ) in the form of a pale brown foam. 1 H NMR (400 MHz, CD₃OD) δ 8.33 (d, J = 8.5 Hz, 1H), 8.17-8.15 (m, 1H), 7.73-7.63 (m, 3H), 7.20 (dd , J = 10.0, 8.3 Hz, 1H), 6.82 (dd, J = 8.8, 1.6 Hz, 1H), 6.92 (t, J = 9.1 Hz, 1H) , 4.07 (t, J = 13.7 Hz, 2H), 3.92 (t, J = 5.4 Hz, 2H), 3.67 (d, J = 1.8 Hz, 2H), 3 , 52-3.51 (m, 2H). Mass Spectrum (LCMS, ESI) calculated for C23H23CIF4N5O2 (M + H): 512.1. Found: 512.2.

Example 5

Hydrochloride salt of N - [(6-Amino-2-methyl (3-pyridyl)) methyl] -2- (3 - {[2,2-difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6- chloro-2-fluorophenyl) acetamide

<img file="ES2269474T3_D0052.tif" />

1. 2- (3 - {[2,2-Difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6-chloro-2-fluorophenyl) -N - ({6 - [(t-butoxy) carbonylamino] -2 -methyl (3-pyridyl)} methyl) acetamide

<img file="ES2269474T3_D0053.tif" />

To a solution of 2- (3 - {[2,2-difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6-chloro-2-fluorophenyl) acetic acid (15 mg, 37 // mol), prepared according to the procedure of step 10 of Example 4, BOP (26 mg, 58 // moles), N- [5- (aminomethyl) -6-methyl (2-pyridyl) were added in DMF (0.3 mL) ] (t-butoxy) carboxamide (12 mg, 51 // moles), and a solution of DIEA (19 mg, 146 // moles) in DMF (0.1 mL) (Sanderson, PE, et al., WO 97 / 01338 (1997)). The mixture was stirred overnight, the solvents were evaporated, and the resulting mixture was partitioned between NaHCO₃ saturated and DCM. The organic layer was separated, and the aqueous layer was extracted with DCM. Organic layers were combined, washed with KHSO₄ at 10%, H₂O, and brine, dried over Na₂SW₄, concentrated, and flash chromatographed on silica gel eluting with MeOH / DCM (0.3, 0.6, and 1%) to give the title compound (11 mg, 49% yield) as a white solid. 1 H NMR (400 MHz, CD₃OD) δ 8.33 (d, J = 8.4 Hz, 1H), 8.17-8.14 (m, 1H), 7.72-7.61 (m, 4H), 7.55 (d , J = 8.5 Hz, 1H), 7.19 (dd, J = 10.0, 8.3 Hz, 1H), 6.81 (dd, J = 8.8, 1.7

ES 2 269 474 T3

Hz, 1H), 6.47 (t, J = 9.1 Hz, 1H), 4.32 (s, 2H), 4.12-4.03 (m, 2H), 3.67 (d, J = 2.0 Hz, 2H), 2.40 (s, 3H), 1.50 (s, 9H). Mass Spectrum (LCMS, ESI) calculated for C₃₂H₃₂ClF₄N₄OR₃ (M + H): 631.2. Found: 631.1.

2. Hydrochloride salt of N - [(6-amino-2-methyl (3-pyridyl)) methyl] -2- (3 - {[2,2-difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6chloro- 2-fluorophenyl) acetamide

<img file="ES2269474T3_D0054.tif" />

A solution of 2- (3 - {[2,2-difluoro-2- (4-fluoronaphthyl) ethyl] amino} -6-chloro-2-fluorophenyl) -N - ({6 - [(t-butoxy) carbonylamino ] -2-methyl (3-pyridyl)} methyl) acetamide (10 mg, 16 // mole), prepared as in the previous step, in HCl (0.5 mL, 4.0 M in 1,4-dioxane) stirred for 2 hours at room temperature. A MeOH / DCM solution (25%, 0.4 mL) was added, and the mixture was continued to stir overnight. The solvents were evaporated, and the resulting brown residue was flash chromatographed on silica gel eluting with MeOH / DCM (2.5, 5, and 10%) to give a solid product. This was treated with a solution of HCl (0.01 mL, 4.0 M in 1,4-dioxane, 40 µmol) in DCM (0.5 mL), stirred for 5 minutes, and the solvents were evaporated to give the title compound (6.2 mg, 69% yield) as a white solid. 1 H NMR (400 MHz, CD₃OD) δ 8.26 (d, J = 8.4 Hz, 1H), 8.178.14 (m, 1H), 7.80 (d, J = 9.1 Hz, 1H), 7.73-7, 64 (m, 4H), 7.20 (dd, J = 10.0, 8.3 Hz, 1H), 6.82-6.79 (m, 1H), 6.49 (t, J = 9, 1Hz, 1H), 4.24 (s, 2H), 4.10-4.02 (m, 2H), 3.66 (d, J = 2.3Hz, 2H), 2.49 (s, 3H). Mass Spectrum (LCMS, ESI) calculated for C₂₇H₂₄ClF₄N₄O (M + H): 531.1. Found: 531.6.

Example 6

<img file="ES2269474T3_D0055.tif" />

<img file="ES2269474T3_D0056.tif" />

A solution of 3-nitrophenylacetic acid (3.21 g, 17.7 mmol), ethanolamine (2.8 g, 46 mmol), and triethylamine (3.0 mL, 22 mmol) in anhydrous DMF (110 mL) was A solution of benzotriazol-1-yloxy-tris-pyrrolidine-phosphonium hexafluorophosphate (PyBOP, 9.37 g, 18.0 mmol) in anhydrous DMF (80 mL) was added. After stirring for 16 hours at room temperature (under nitrogen), the reaction mixture was concentrated in vacuo, dissolved in DCM, and filtered. The filtrate was washed with 10% aqueous citric acid, NaHCO₃ aqueous saturated, pH 7 buffer, and brine, dried over Na2SO4 and filtered. The evaporated filtrate was then purified by flash chromatography (10% methanol in DCM) to give the title compound (1.02 g, 26%) as a light yellow solid. 1H NMR (300 MHz, CDCl₃/CD₃OD) δ 8.18 (m, 1H), 8.11 (ddd, 1H, J = 8.1 Hz, 2.4 Hz, 1.1 Hz), 7.72 (m, 1H), 7.60 (t, 1H, J = 7.8 Hz), 3.62 (s, 2H), 3.44 (t, 2H, J = 5.9 Hz), 3.16 (t, 2H, J = 5, 9 Hz).

ES 2 269 474 T3

2. N- [2- (N'-Phthalimidyl) hydroxyethyl] -2- (3-nitrophenyl) acetamide

<img file="ES2269474T3_D0057.tif" />

To a solution of the product from the previous step (1.02 g, 4.55 mmol), N-hydroxyphthalimide (0.76 g, 4.64 mmol), and triphenylphosphine (1.22 g, 4.65 mmol) in Anhydrous THF (100 mL) was added diethylazodicarboxylate (0.75 mL, 4.77 mmol) via syringe. After stirring overnight at room temperature (under nitrogen), the reaction was concentrated in vacuo and purified by flash chromatography (40% ethyl acetate in DCM) to give an impure product that was dissolved in DCM, cooled, and leaked. The evaporated filtrate was then purified by flash chromatography (ethyl acetate in hexane 66% -100%) to give the title compound (0.86 g, 51%) as a white solid. 1H NMR (300 MHz, CDCl₃) δ 8.26 (t, 1H, J = 1.7 Hz), 8.15 (ddd, 1H, J = 8.3 Hz, 2.3 Hz, 1.0 Hz), 7.82 (m, 4H), 7.74 (m, 1H), 7.53 (t, 1H, J = 7.9 Hz), 7.03 (s broad, 1H), 4.26 (m, 2H), 3.76 (s, 2H), 3.57 (dd, 2H, J = 9.8 Hz, 5.7 Hz).

3. N- [2- (N'-Phthalimidyl) hydroxyethyl] -2- (3-aminophenyl) acetamide

<img file="ES2269474T3_D0058.tif" />

A solution of the product from the previous step (0.66 g, 1.80 mmol) and 10% palladium on carbon (15 mg) in ethanol: degassed 1: 1 THF (40 mL) was stirred under hydrogen at room temperature . After 6 hours the reaction was filtered over Celite and the filtrate was evaporated and purified by flash chromatography (5% methanol in DCM) to give the title compound (0.20 g, 33%) as a solid. White color. 1H NMR (300 MHz, CDCl₃) δ 7.81 (m, 4H), 7.14 (t, 1H, J = 8.0 Hz), 6.72 (m, 2H), 6.61 (ddd, 1H, J = 8.0 Hz , 2.2 Hz, 1.0 Hz), 4.23 (m, 2H), 3.55 (m, 4H). Mass spectrum (MALDI-TOF, α-cyano-4-hydroxycinnamic acid matrix) calculated for C18H17N3O4: 362.1 (M + Na), 340.1 (M + H). Found: 362.2, 340.3.

<img file="ES2269474T3_D0059.tif" />

To an ice-cold solution of the product from the previous step (0.20 g, 0.58 mmol) in anhydrous DCM (50 mL) was added a solution of α-toluenesulfonyl chloride (0.11 g, 0.58 mmol) in anhydrous DCM (20 mL) followed by N-methylmorpholine (0.10 mL, 0.91 mmol). After stirring for 16 hours at room temperature, more α-toluenesulfonyl chloride (0.07 g, 0.36 mmol) and N-methylmorpholine (0.10 mL, 0.91 mmol) were added and the reaction was stirred for an additional 4 hours and evaporated in vacuo. The residue was dissolved in DCM, washed with 10% aqueous citric acid, pH 7 buffer and brine, dried over Na₂SW₄, filtered, and the filtrate was evaporated to give the title compound (0.20 g, 69%) as a light yellow solid. 1H NMR (300 MHz, CDCl₃) δ 7.77 (m, 4H), 7.35 (m, 1H), 7.22 (s, 5H), 7.17 (m, 1H), 7.11 (m, 1H), 7.05 (s, 1H), 6.71 (bm, 1H), 4.37 (s, 2H), 4.25 (m, 2H), 3.64 (s, 2H), 3.64 (dd, 2H , J = 10 Hz, 5.5 Hz).

ES 2 269 474 T3

5. N- [2- (Aminooxy) ethyl ] -2- (3 - {[benzylsulfonyl] amino} phenyl) acetamide

<img file="ES2269474T3_D0060.tif" />

The product from the previous step (0.19 g, 0.39 mmol) was dissolved in ethanol: THF 1: 1 (20 mL) and reacted with 40% aqueous methylamine (10 mL) for 1 hour at room temperature. ambient. The reaction was evaporated in vacuo and purified on Waters Sep-Pak (5g silica, DCM: ethyl acetate 1: 1) yielding an impure yellow solid. This was then purified by preparative thin layer chromatography (10% methanol in DCM) to give the title compound (0.10 g, 72%) as a white solid. 1H NMR (300 MHz, CDCl₃/CD₃OD) δ 7.34 (m, 3H), 7.28 (m, 3H), 7.10 (m, 1H), 7.05 (m, 2H), 4.35 (s, 2H), 3, 69 (t, 2H, J = 5Hz), 3.51 (s, 2H), 3.43 (t, 2H, J = 5Hz). Mass spectrum (MALDI-TOF, a-cyano-4-hydroxycinnamic acid matrix) calculated for C₁₇H₂₁N₃OR₄S:

386.1 (M + Na). Found: 386.6.

6. N- [2 - ({N, N'-Di [t-butoxycarbonyl]} guanidinooxy) ethyl] -2- (3 - {[benzylsulfonyl] amino} phenyl) acetamide

<img file="ES2269474T3_D0061.tif" />

A solution of the product from the previous step (89 mg, 0.24 mmol) and [N, N'-di (t-butoxycarbonyl)] amidinopyrazole (86 mg, 0.28 mmol) in DMF (5 mL) was stirred for 4 days at room temperature. The reaction was evaporated in vacuo and the crude product was purified by flash chromatography (5% methanol in DCM) yielding an impure yellow oil. This was then purified by preparative thin layer chromatography (5% methanol in DCM) to give the title compound (72mg, 49%) as a colorless solid. 1H NMR (300 MHz, CDCl3) δ 9.19 (s, 1H), 8.22 (broad t, 1H, J = 5.0 Hz), 7.62 (s, 1H), 7.23 (m, 10H (Ar + NH)), 4.27 (s, 2H), 4.08 (m, 2H), 3.57 (m, 4H), 1.51 (s, 9H), 1.49 (s, 9H).

7. N- [2- (guanidinooxy) ethyl] -2- (3 - {[benzylsulfonyl] amino} phenyl) acetamide trifluoroacetate salt

<img file="ES2269474T3_D0062.tif" />

The product from the previous step (72 mg, 0.12 mmol) was dissolved in DCM (5 mL) and reacted with trifluoroacetic acid (2 mL) for 4 hours at room temperature. The reaction was concentrated in vacuo and the crude product was purified by preparative thin layer chromatography (20% methanol in DCM) to give the title compound (44mg, 71%) as a pale yellow wax. 1H NMR (300 MHz, CDCl₃/CD₃OD) δ 7.34 (m, 3H), 7.28 (m, 3H), 7.11 (m, 1H), 7.05 (m, 2H), 4.35 (s, 2H), 3, 90 (t, 2H, J = 4.9Hz), 3.52 (s, 2H), 3.47 (t, 2H, J = 4.8Hz). Mass spectrum (MALDI-TOF, a-cyano-4-hydroxycinnamic acid matrix) calculated for C₁₈H₂₃N₅OR₄S:

428.1 (M + Na), 406.2 (M + H). Found: 428.4, 406.4.

ES 2 269 474 T3

Example 7

N- [2- (guanidinooxy) ethyl] -2- (2-chloro-5 - {[benzylsulfonyl] amino} phenyl) acetamide trifluoroacetate salt

<img file="ES2269474T3_D0063.tif" />

1. 2-Chloro-5-nitrophenylacetic acid monohydrate

<img file="ES2269474T3_D0064.tif" />

A solution of 2-chlorophenylacetic acid (10.0 g, 58.6 mmol) in concentrated sulfuric acid (40 mL) was cooled to -10 ° C and slowly reacted with a steaming nitric acid solution (2.80 mL) , 66.7 mmol) in concentrated sulfuric acid (7.2 mL). After 2.5 hours the reaction was slowly poured into ice water (400 mL), filtered on a coarse filter frit, washed once with cold water, and dried on the frit overnight to give the compound from titer (13.5 g, 98%) as a white solid. Integration of the proton NMR spectrum showed that the product contained approximately 0.2 equivalents of 2-chlorophenylacetic acid, but thin layer chromatography showed that it was inseparable from the product. 1 H NMR (300 MHz, CDCl3 / CD3OD) δ 8.25 (d, 1H, J = 2.7 Hz), 8.13 (dd, 1H, J = 8.7 Hz, 2.7 Hz), 7, 61 (d, 1H, J = 8.8Hz), 3.89 (s, 2H).

2. Ethyl 2- (3-Amino-6-chlorophenyl) acetate

<img file="ES2269474T3_D0065.tif" />

The product from the previous step (4.14 g, 17.7 mmol) was suspended in DCM (70 mL) and reacted with oxalyl chloride (4.0 mL, 46 mmol) and a few drops of DMF. After stirring 1 hour at room temperature the reaction became homogeneous, reagent grade ethanol (30 mL) was added, the reaction was stirred another 30 minutes. The crude product was evaporated in vacuo and purified by flash chromatography (10-15% ethyl acetate in hexane) to give the title compound (4.6 g) as a pale yellow oil. Proton NMR showed that the product contained approximately 0.8 equivalents of diethyl oxalate which could not be located by thin layer chromatography. 1H NMR (300 MHz, CDCl₃) δ 8.21 (d, 1H, J = 2.7 Hz), 8.11 (dd, 1H, J = 8.8 Hz, 2.7 Hz), 7.57 (d, 1H, J = 8 , 8 Hz), 4.21 (q, 2H, J = 7.2 Hz), 3.87 (s, 2H), 1.28 (t, 3H, J = 7.2 Hz).

3. Ethyl 2- (3-Amino-6-chlorophenyl) acetate

<img file="ES2269474T3_D0066.tif" />

A solution of the product from the previous step (2.00 g, 8.21 mmol) in reagent grade ethanol (50 mL) was reacted with tin (II) chloride dihydrate (9.40 g, 41.7 mmol ) at room temperature. After 16 hours the reaction was concentrated in vacuo, dissolved in DCM, and filtered. The filtrate was washed with water and brine, dried over Na2SO4, and filtered. The evaporated filtrate was then purified by flash chromatography (40% ethyl acetate in hexane) to give the title compound (0.53 g, 30%) as a yellow oil.

ES 2 269 474 T3 pale. 1 H NMR (300 MHz, CDCI3) δ 7.12 (d, 1H, J = 8.5 Hz), 6.60 (d, 1H, J = 2.8 Hz), 6.53 (dd, 1H, 8 , 5 Hz, 2.9 Hz), 4.17 (c, 2H, J = 7.1 Hz), 3.65 (s, 2H), 1.26 (t, 3H, J = 7.1 Hz) .

Four. Ethyl 2- (2-Chloro-5 - {[benzylsulfonyl] amino} phenyl) acetate

<img file="ES2269474T3_D0067.tif" />

A solution of the product from the previous step (0.50 g, 2.32 mmol) and α-toluenesulfonyl chloride (0.74 g, 3.88 mmol) in DCM (40 mL) and N-methylmorpholine (0.80 mL, 7.3 mmol) was stirred at room temperature for 3 hours, washed with dilute aqueous HCl, water, and brine, dried over Na₂SW₄, and leaked. The evaporated filtrate was purified by flash chromatography (5% ethyl acetate in DCM) to give the title compound (0.693 g, 81%) as a pale yellow solid. 1H NMR (300 MHz, CDCl₃) δ 7.36 (m, 4H), 7.26 (m, 2H), 7.04 (d, 1H, J = 2.7 Hz), 6.99 (dd, 1H, J = 8.5 Hz , 2.8 Hz), 6.47 (s, 1H), 4.33 (s, 2H), 4.20 (q, 2H, J = 7.1 Hz), 3.73 (s, 2H) , 1.29 (t, 3H, J = 7.1 Hz). Mass spectrum (MALDI-TOF, α-cyano-4-hydroxycinnamic acid matrix) calculated for C₁₇H₁₈DO NOT₄SCl: 390.1 (M + Na). Found: 390.7.

5. 2- (2-Chloro-5 - {[benzylsulfonyl] amino} phenyl) acetic acid

<img file="ES2269474T3_D0068.tif" />

A solution of the product from the previous step (0.69 g, 1.87 mmol) in water / THF 1: 1 (20 mL) was reacted with potassium hydroxide (0.52 g, 9.32 mmol) at room temperature for 20 hours. After evaporating the THF in vacuo, the remaining aqueous layer was acidified to pH 3 with 1N HCl and extracted with DCM and ether. The combined organic layers were washed with brine, dried over Na₂SW₄, and leaked. The filtrate was then evaporated in vacuo to give the title compound (0.586 g, 92%) as a white solid. 1H NMR (300MHz, CDCl3 / CD3OD) δ 7.34 (m, 4H), 7.28 (m, 2H), 7.07 (m, 2H), 4.34 (s, 2H), 3.73 (s, 2H). Mass spectrum (MALDI-TOF, a-cyano-4-hydroxycinnamic acid matrix) calculated for Ci₅H₁₄Do not₄SC1: 378.0 (M + K), 362.0 (M + Na). Found: 378.8, 362.9.

6. N- [2 - ({N, N'-Di- (t-butoxycarbonyl]} guanidinooxy) ethyl] -2- (2-chloro-5 - {[benzylsulfonyl] amino} phenyl) acetamide

<img file="ES2269474T3_D0069.tif" />

A solution of the product from the previous step (0.21 g, 0.60 mmol) and [N, N'-di (t-butoxycarbonyl)] - 2-aminoethoxyguanidine (Tianbao Lu, et al., WO 99/26926 ( 1999)) (0.19 g, 0.60 mmol), in anhydrous THF (50 mL) it was reacted with BOP (0.33 g, 0.75 mmol) and triethylamine (0.25 mL, 1.8 mmol ) at room temperature for 16 hours. The reaction was evaporated in vacuo, dissolved in DCM, washed with pH 7 buffer and brine, dried over Na₂SW₄, and leaked. The evaporated filtrate was purified by flash chromatography (5% methanol in DCM) to give the title compound (0.380 g, 98%) as an orange solid. 1 H NMR (300 MHz, CDCI3) δ 9.14 (s, 1H), 8.22 (broad t, 1H, J = 5.0 Hz), 7.60 (s, 1H), 7.34 (m, 3H), 7.28 (m, 3H), 7.10 (d, 1H, J = 2.6 Hz), 7.03 (dd, 1H, J = 8.6 Hz, 2.7 Hz), 6 , 84 (s broad, 1H), 4.29 (s, 2H), 4.13 (m, 2H), 3.75 (s, 2H), 3.62 (dd, 2H, J = 8.8 Hz , 5.1 Hz), 1.51 (s, 9H), 1.46 (s, 9H). Mass spectrum (MALDI-TOF, gentisic acid matrix) calculated for C28H38N5O8SCI: 662.2 (M + Na), 440.1 (M-2 Boc + H). Found: 661.7, 439.9.

ES 2 269 474 T3

<img file="ES2269474T3_D0070.tif" />

A solution of the product from the previous step (0.375 g, 0.586 mmol) in DCM (10 mL) was reacted with trifluoroacetic acid (5 mL) at room temperature for 16 hours. The reaction was evaporated in vacuo, and the crude product was purified by flash chromatography (20% methanol in DCM) to give the title compound (0.326g, 100%) as a pale yellow solid. 1H NMR (300 MHz, CDCI3 / CD3OD) δ 7.34 (m, 3H), 7.29 (m, 3H), 7.06 (m, 2H), 4.35 (s, 2H), 3.94 (broad t, 2H, J = 5Hz), 3.65 (s, 2H), 3.49 (broad t, 2H, J = 5Hz). Mass spectrum (MALDI-TOF, a-cyano-4-hydroxycinnamic acid matrix) calculated for C₁₈H₂₂N₅OR₄SCl: 462.1 (M + Na),

440.1 (M + H). Found: 4.61.9.439.9.

Example 8

N- [2- (guanidinooxy) ethyl] -2- (2-methyl-5 - {[benzylsulfonyl] amino} phenyl) acetamide trifluoroacetate salt

<img file="ES2269474T3_D0071.tif" />

1. (2-Methyl-5-nitrophenyl) methanol

<img file="ES2269474T3_D0072.tif" />

The 2-methyl-5-nitrobenzoic acid (2.00 g, 11.0 mmol) was warmed under nitrogen, dissolved in anhydrous THF (25 mL), and treated with a 1 N solution of borane in THF (16, 5 mL). After stirring 18 hours at room temperature the reaction was quenched with a solution of potassium carbonate (1.8 g, 13 mmol) in water (50 mL), and the THF was removed in vacuo. The remaining aqueous solution was extracted with DCM, and the organic layer was washed with pH 7 buffer and brine, dried over sodium sulfate, and filtered. The evaporated filtrate yielded the title compound as a pale yellow solid (1.76 g, 95%). NMR 1 H (300 MHz, CdC1₃) δ 8.29 (d, 1H, J = 2.5 Hz), 8.04 (dd, 1H, J = 8.3 Hz, 2.5 Hz), 7.31 (d, 1H, J = 8 , 31 Hz), 4.77 (d, 2H, J = 5.5 Hz), 2.41 (s, 3H), 2.09 (t, 1H, J = 5.6 Hz).

2. (2-Methyl-5-nitrophenyl) methyl methylsulfonate

<img file="ES2269474T3_D0073.tif" />

A solution of the product from the previous step (1.74 g, 10.4 mmol) in DCM (50 mL) was cooled to 0 ° C and treated with methanesulfonyl chloride (0.90 mL, 11.6 mmol) and triethylamine (1.75 mL, 12.6 mmol). After stirring for 30 minutes the reaction was warmed to room temperature, stirred another 30 minutes, and poured into a buffer solution of pH 7. The phases were separated and the organic layer was washed with brine, dried over sodium sulfate. sodium, and filtered. The evaporated filtrate yielded the title compound as a pale yellow oil (2.53 g, 99%). 1H NMR (300 MHz, CDCl₃) δ 8.26 (d, 1H, J = 2.5 Hz), 8.16 (dd, 1H, J = 8.4 Hz, 2.4 Hz), 7.42 (d, 1H, J = 8 , 4Hz), 5.31 (s, 2H), 3.08 (s, 3H), 2.51 (s, 3H).

ES 2 269 474 T3

3. 2- (2-Methyl-5-nitrophenyl) ethanonitrile

<img file="ES2269474T3_D0074.tif" />

The product from the previous step (2.48 g, 10.1 mmol) and potassium cyanide (2.00 g, 30.7 mmol) were refluxed in acetonitrile (100 mL) for 8 hours, then cooled to room temperature and stirred overnight. The reaction was evaporated in vacuo, dissolved in DCM, and filtered. The filtrate was washed with pH 7 buffer and brine, evaporated, and purified by flash column chromatography (hexane: ethyl acetate 1: 1 as eluent) to give the title compound (1.27 g, 71%) in the form of a white solid. 1H NMR (300 MHz, CDCl₃) δ 8.26 (d, 1H, J = 2.4 Hz), 8.13 (dd, 1H, J = 8.3 Hz, 2.4 Hz), 7.42 (d, 1H, J = 8 , 3Hz), 3.78 (s, 2H), 2.48 (s, 3H).

Four. 2- (2-Methyl-5-nitrophenyl) acetic acid

<img file="ES2269474T3_D0075.tif" />

To a solution of the product from the previous step (1.27 g, 7.21 mmol) in methanol (30 mL) was added a solution of potassium hydroxide (4.06 g, 72.4 mmol) in water (30 mL). The reaction was refluxed overnight and the methanol was removed in vacuo. The remaining aqueous layer was acidified with 3N HCl and filtered, the solid was washed with diethyl ether, and the filtrate was separated. The aqueous layer was washed with DCM and diethyl ether, and the combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The evaporated filtrate yielded the title compound (0.84 g, 60%) as an orange solid. 1H NMR (300 MHz, acetone6) δ 8.17 (d, 1H, J = 2.3 Hz), 8.06 (dd, 1H, J = 8.4 Hz, 2.5 Hz), 7.29 ( d, 1H, J = 8.4Hz), 3.88 (s, 2H), 2.45 (s, 3H).

5. N- [2 - ({N, N'-Di- (t-butoxycarbonyl]} guanidinooxy) ethyl] -2- (2-methyl-5-nitrophenyl) acetamide

<img file="ES2269474T3_D0076.tif" />

A solution of the product from the previous step (0.27 g, 1.40 mmol), BOP (0.70 g, 1.58 mmol), triethylamine (0.50 mL, 3.60 mmol), and [N, N'-di (t-butoxycarbonyl)] - 2-aminoethoxyguanidine (Tianbao Lu, et al., WO 99/26926 (1999)) (0.44 g, 1.38 mmol), in anhydrous DMF were stirred at temperature atmosphere at night. The reaction was concentrated in vacuo and the crude product was purified by flash column chromatography (5% methanol in DCM as eluent) to give the title compound as an orange solid (0.63 g, 92%). 1H NMR (300 MHz, CDCl₃) δ 9.21 (s, 1H), 8.42 (m, 1H), 8.17 (d, 1H, J = 2.4 Hz), 7.99 (dd, 1H, J = 8.4 Hz , 2.5 Hz), 7.59 (s, 1H), 7.29 (d, 1H, J = 8.4 Hz), 4.12 (m, 2H), 3.74 (s, 2H), 3.63 (m, 2H), 2.48 (s, 3H), 1.52 (s, 9H), 1.47 (s, 9H).

6. N- [2 - ({N, N'-Di- [t-butoxycarbonyl]} guanidinooxy) ethyl] -2- (3-amino-6-methylphenyl) acetamide

<img file="ES2269474T3_D0077.tif" />

ES 2 269 474 T3

The product of the previous step (0.29 g, 0.58 mmol) and palladium (0) on carbon at 10% (0.06 g) were dissolved in reactive ethanol (50 mL), degassed with nitrogen and vacuum, and stirred in a hydrogen balloon at room temperature. After 4 hrs. The reaction was filtered over Celite, the frit was washed with methanol, and the filtrate was evaporated in vacuo to give the title compound (0.27 g, 100%). 1H NMR (300 MHz, CDCl₃) δ 9.10 (s, 1H), 7.61 (s, 1H), 7.30 (m, 1H), 6.94 (d, 1H, J = 8.0 Hz), 6.63 (d , 1H, J = 2.4Hz), 6.51 (dd, 1H, J = 8.0Hz, 2.5Hz), 4.09 (m, 2H), 3.58 (m, 2H), 3.53 (s, 2H), 2.21 (s, 3H), 1.52 (s, 9H), 1.47 (s, 9H).

7. N- [2 - ({N, N'-Di- (t-butoxycarbonyl]} guanidinooxy) ethyl] -2- (2-methyl-5 - {[benzylsulfonyl] amino} phenyl) acetamide

<img file="ES2269474T3_D0078.tif" />

The product from the previous step (0.27 g, 0.58 mmol), α-toluenesulfonyl chloride (0.18 g, 0.96 mmol), and N-methylmorpholine (0.20 mL, 1.82 mmol) were stirred at room temperature in DCM (20 mL). After 2 hours the reaction was diluted with additional DCM and washed with dilute aqueous HCl, saturated aqueous sodium bicarbonate, pH 7 buffer, and brine. The organic layer was dried over sodium sulfate, filtered, and the filtrate was concentrated in vacuo to give the title compound as a pale yellow solid (0.35 g, 97%). 1H NMR (300 MHz, CDCI3) δ 9.15 (s, 1H), 8.04 (broad t, 1H, J = 5 Hz), 7.60 (s, 1H), 7.34 (m, 5H) , 7.12 (d, 1H, J = 8.0Hz), 7.03 (dd, 1H, J = 11Hz, 2.3Hz), 6.26 (s, 1H), 4.31 (s , 2H), 4.12 (m, 2H), 3.63 (m, 4H), 2.33 (s, 3H), 1.51 (s, 9H), 1.45 (s, 9H).

8. N- [2- (guanidinooxy) ethyl] -2- (2-methyl-5 - {[benzylsulfonyl] -amino} phenyl) acetamide trifluoroacetate salt

<img file="ES2269474T3_D0079.tif" />

The product from the previous step (0.35 g, 0.56 mmol) was dissolved in DCM (10 mL) and treated with trifluoroacetic acid (3 mL) at room temperature. After 16 hours the reaction was concentrated in vacuo and the crude product was purified on 10 g of Waters Silica Sep-Pak® (gradient elution of methanol in DCM 5 to 20%) to give the title compound as a pale yellow solid (0.27 g, 89%). NMR 1 H (300 MHz, CdC1₃/CD₃OD) δ 7.34 (m, 3H), 7.29 (m, 2H), 7.14 (d, 1H, J = 9.0 Hz), 7.02 (dd, 1H, J = 6.5 Hz, 2.3 Hz), 7.00 (s, 1H), 4.33 (s, 2H), 3.92 (t broad, 2H, J = 5 Hz), 3.55 (s, 2H), 3.48 (broad t, 2H, J = 5Hz), 2.28 (s, 3H). Mass spectrum (MALDI-TOF, gentisic acid matrix) calculated for C_i9H₂₅N₅OR₄S: 442.2 (M + Na), 420.2 (M + H). Found: 442.5.420.6. Example 9

N- [2- (guanidinooxy) ethyl] -2- (2-hydroxy-6-methyl-3 - {[(3-methylphenyl) sulfonyl] amino} phenyl) acetamide hydrochloride salt

<img file="ES2269474T3_D0080.tif" />

ES 2 269 474 T3

1. 4-Methyl-1-nitro-2-prop-2-enyloxybenzene

<img file="ES2269474T3_D0081.tif" />

A solution of 5-methyl-2-nitrophenol (2.00 g, 13.1 mmol), allyl bromide (1.30 mL, 15.0 mmol), and cesium carbonate (5.5 g, 17 mmol) in DMF (100 mL) stirred at room temperature. After 20 hrs. The reaction was filtered, the frit was washed with methanol, and the filtrate was evaporated in vacuo at 50 ° C. The residue was purified by flash column chromatography (hexane: ethyl acetate 4: 1 then 2: 1 as eluent) to give the title compound as a yellow oil (2.39 g, 95%) which crystallized after stand after 3 days at room temperature. 1H NMR (300 MHz, CDCl₃) δ 7.79 (d, 1H, J = 8.2 Hz), 6.86 (s, 1h), 6.82 (m, 1H), 6.05 (ddt, 1H, J = 17.3 Hz , 10.6 Hz, 5.0 Hz), 5.50 (ddd, 1H, J = 17.3 Hz, 3.3 Hz, 1.7 Hz), 5.33 (ddd, 1H, J = 10, 6Hz, 2.9Hz, 1.5Hz), 4.67 (dt, 2H, J = 4.9Hz, 1.6Hz), 2.40 (s, 3H).

2. 3-Methyl-6-nitro-2-prop-2-enylphenol

<img file="ES2269474T3_D0082.tif" />

The 4-methyl-1-nitro-2-prop-2-enyloxybenzene (7.11 g, 36.8 mmol), prepared as in the previous step, was heated neat at 200 ° C under nitrogen for 3 hours, cooled at room temperature, and purified by flash column chromatography (hexane: DCM 1: 1 as eluent) to give the title compound as an orange oil (5.04 g, 71%). 1H NMR (300 MHz, CDCI3) δ 11.07 (s, 1H), 7.90 (d, 1H, J = 8.7 Hz), 6.80 (d, 1H, J = 8.7 Hz), 5.93 (m, 1H), 5.03 (m, 1H), 4.96 (m, 1H), 3.50 (dt, 2H, J = 5.9 Hz, 1.7 Hz), 2, 36 (s, 3R).

3.1-Methyl-4-nitro-3- (phenylmethoxy) -2-prop-2-enylbenzene

<img file="ES2269474T3_D0083.tif" />

A solution of the product from the previous step (5.02 g, 26.0 mmol), benzyl bromide (3.40 mL, 28.6 mmol), and cesium carbonate (17.2 g, 52.8 mmol) were added mmol) in DMF (100 mL) was stirred at room temperature for 20 hours. The solution was filtered, the filtrate was concentrated in vacuo, and the crude product was adsorbed on silica. This was poured onto a short plug of silica and eluted with DCM, and the eluate was evaporated to give the title compound (7.20 g, 98%) as a yellow oil. 1H NMR (300 MHz, CDCl₃) δ 7.72 (d, 1H, J = 8.4 Hz), 7.46 (m, 2H), 7.37 (m, 3H), 7.06 (d, 1H, J = 8.5 Hz ), 5.93 (m, 1H), 5.08 (m, 1H), 4.98 (s, 2H), 4.88 (m, 1H), 3.51 (dt, 2H, J = 5, 4Hz, 1.9Hz), 2.36 (s, 3H).

ES 2 269 474 T3

Four. 4-Methyl-2- (phenylmethoxy) -3-prop-2-enylphenylamine

<img file="ES2269474T3_D0084.tif" />

The product from the previous step (0.55 g, 1.94 mmol) and tin (II) chloride dihydrate (2.89 g, 12.8 mmol) were stirred in reagent grade ethanol (40 mL) at room temperature. After 20 hours the reaction was concentrated in vacuo and the residue was partitioned between saturated sodium bicarbonate and DCM. The resulting emulsion was filtered, the solids and the aqueous layer were washed with additional DCM, and the combined organic layers were washed with brine, dried over sodium sulfate, and filtered. The filtrate was then evaporated to give the title compound as an orange oil (0.51 g, 90%). 1H NMR (300 MHz, CDCl₃) δ 7.47 (m, 2H), 7.38 (m, 3H), 6.78 (d, 1H, J = 8.1 Hz), 6.60 (d, 1H, J = 8.0 Hz ), 5.97 (m, 1H), 5.02 (m, 1H), 4.95 (m, 1H), 4.85 (s, 2H), 3.64 (s broad, 2H), 3, 47 (dt, 2H, J = 5.7Hz, 1.8Hz), 2.20 (s, 3H).

5. [4-Methyl-2- (phenylmethoxy) -3-prop-2-enylphenyl] [(3-methylphenyl) sulfonyl] amine

<img file="ES2269474T3_D0085.tif" />

The product from the previous step (0.49 g, 1.92 mmol) was dissolved in DCM (10 mL) and treated with m-toluenesulfonyl chloride (0.37 g, 1.96 mmol) and N-methylmorpholine ( 0.25 mL, 2.27 mmol) at room temperature. After 18 hours the reaction was concentrated in vacuo and the residue was purified by flash column chromatography (DCM as eluent) to give the title compound as a pale yellow oil (0.72 g, 92%). 1H NMR (300 MHz, CDCL) δ 7.54 (m, 2H), 7.35 (m, 8H), 6.89 (d, 1H, J = 8.3 Hz), 6.80 (s, 1H ), 5.87 (m, 1H), 4.99 (m, 1H), 4.79 (m, 1H), 4.43 (s, 2H), 3.36 (dt, 2H, J = 5, 3Hz, 1.9Hz), 2.31 (s, 3H), 2.19 (s, 3H).

6. 2- (6-Methyl-3 - {[(3-methylphenyl) sulfonyl] amino} -2- (phenylmethoxy) phenyl) ethanol

<img file="ES2269474T3_D0086.tif" />

The product from the previous step (0.71 g, 1.74 mmol) was dissolved in 1,4-dioxane (25 mL) and treated with a sodium periodate solution (1.50 g, 7.01 mmol) in water (12 mL) and a 2.5% by weight solution of osmium tetroxide (0.25 mL, 0.02 mmol) in 2-methyl-2-propanol. After stirring 4 hours at room temperature the reaction was diluted with DCM, washed with 5% aqueous sodium bisulfite, water, and brine, dried over sodium sulfate, and filtered. The filtrate was concentrated in vacuo to produce a light yellow oil which was used without further purification.

ES 2 269 474 T3

7. 2- (6-methyl-3 - {[(3-methylphenyl) sulfonyl] amino} -2- (phenylmethoxy) phenyl) acetic acid

<img file="ES2269474T3_D0087.tif" />

A solution of sodium dichromate (0.79 g, 2.64 mmol) and concentrated sulfuric acid (1.5 mL, 28 mmol) in water (25 mL) was added to a solution of the product from the previous step in acetone (25 mL), and the reaction was stirred at room temperature for 3 days. After adding methanol (3 mL) and stirring for an additional 15 minutes, the organic solvents were removed in vacuo and the remaining aqueous layer was extracted with DCM. The DCM layer was washed with brine, dried over sodium sulfate, filtered, and the filtrate was concentrated and purified by flash column chromatography (10% methanol in DCM) to give the title compound as a pale yellow solid (0.51 g, 69% from Step 5). 1H NMR (300 MHz, CDCl₃) δ 7.56 (m, 2H), 7.39 (m, 4H), 7.31 (m, 4H), 6.93 (d, 1H, J = 8.4 Hz), 6.78 (s , 1H), 4.52 (s, 2H), 3.67 (s, 2H), 2.33 (s, 3H), 2.21 (s, 3H). Mass Spectrum (LCMS, ESI pos.) Calculated for C₂₃H₂₃DO NOT₅S: 448.1 (M + Na), 425.1 (M + H). Found: 448.1,425.9.

8. N- [2 - ({N, N'-Di- [t-butoxycarbonyl]} guanidinooxy) ethyl] -2- (6-methyl-3 - {[(3-methylphenyl) sulfonyl] amino} -2- (phenylmethoxy ) phenyl) acetamide

<img file="ES2269474T3_D0088.tif" />

A solution of the product from the previous step (0.32 g, 0.75 mmol), BOP (0.34 g, 0.77 mmol), triethylamine (0.25 mL, 1.80 mmol), and [N, N'-di (t-butoxycarbonyl)] - 2-aminoethoxyguanidine (Tianbao Lu, et al. WO 99/26926 (1999)) (0.27 g, 0.76 mmol), in DMF (15 mL) was stirred at room temperature overnight. The reaction was concentrated in vacuo, the residue was dissolved in DCM, washed with saturated sodium bicarbonate, water, and brine, dried over sodium sulfate, and filtered. The filtrate was concentrated in vacuo and the crude product was purified by flash column chromatography (7% methanol in DCM as eluent) to give the title compound as a pale yellow solid (0.41 g, 76% ). 1H NMR (300 MHz, CDCl₃) δ 9.11 (s, 1H), 7.93 (m, 111), 7.55 (m, 3H), 7.36 (m, 8H), 6.90 (d, 1H, J = 8, 3 Hz), 6.84 (s, 1H), 4.60 (s, 2H), 4.06 (m, 2H), 3.71 (s, 2H), 3.56 (dd, 2H, J = 8.8Hz, 5.2Hz), 2.34 (s, 3H), 2.23 (s, 3H), 1.51 (s, 9H), 1.43 (s, 9H).

9. N- [2 - ({N, N'-Di- (t-butoxycarbonyl]} guanidinooxy) ethyl] -2- (2-hydroxy-6-methyl-3 - {[(3-methylphenyl) sulfonyl] amino} phenyl ) acetamide

<img file="ES2269474T3_D0089.tif" />

The product from the previous step (0.41 g, 0.57 mmol) and palladium (0) on carbon at 10% (60 mg) were dissolved in reagent grade ethanol (20 mL), degassed with nitrogen and vacuum, and stirred in a hydrogen balloon at room temperature for 4 hours. The reaction was filtered over Celite, the frit was washed with methanol, the product

ES 2 269 474 T3 filtrate was evaporated, and the residue was purified by preparative thin layer chromatography (5% methanol in DCM as eluent) to give the title compound as a pale yellow solid (13.0 mg, 4%). 1H NMR (300 MHz, CDCl₃) δ 9.25 (s, 1H), 8.62 (m, 1H), 7.61 (m, 3H), 7.29 (m, 3H), 6.61 (d, 1H, J = 8, 5Hz), 4.06 (m, 2H), 3.64 (s, 2H), 3.54 (dd, 2H, J = 8.7Hz, 5.0Hz), 2.34 (s, 3H ), 2.30 (s, 3H), 1.51 (s, 9H), 1.49 (s, 9H).

10. N- [2- (guanidinooxy) ethyl] -2- (2-hydroxy-6-methyl-3 - {[(3-methylphenyl) sulfonyl] amino} phenyl) acetamide hydrochloride salt

<img file="ES2269474T3_D0090.tif" />

The product from the previous step (13.0 mg, 0.02 mmol) was dissolved in DCM (5 mL) and treated with trifluoroacetic acid (1 mL) at room temperature. After 16 hours the reaction was concentrated in vacuo and the crude product was purified by preparative thin layer chromatography (12% methanol in DCM as eluent, saturated with ammonia). The resulting product was treated with 4N HCl in ethanol, filtered, the filtrate was evaporated, and the solid was washed with diethyl ether and dried in vacuo to give the title compound (5.0 mg, 52%) as of a tan colored solid. 1 H NMR (300 MHz, CD₃OD) δ 7.51 (m, 2H), 7.38 (m, 2H), 6.56 (m, 2H), 3.91 (t, 2H, J = 5.3 Hz), 3.61 ( s, 2H), 3.47 (t, 2H, J = 5.3Hz), 2.36 (s, 3H), 2.27 (s, 3H). Mass Spectrum (LCMS, ESI pos.) Calculated for C19H25N5O5S: 436.1 (M + H). Found: 436.2.

Example 10

N - [(6-amino-2-methyl (3-pyridyl)) methyl] -2- (2-hydroxy-6-methyl-3 - {[(3-methylphenyl) sulfonyl] amino} phenyl) acetamide hydrochloride salt

<img file="ES2269474T3_D0091.tif" />

1. N - [(6-Amino-2-methyl (3-pyridyl)) methyl] -2- (6-methyl-3 - {[(3-methylphenyl) sulfonyl] amino} -2- (phenylmethoxy) phenyl) acetamide

<img file="ES2269474T3_D0092.tif" />

The product from Example 9, step 7 (0.18 g, 0.42 mmol), BOP (0.21 g, 0.47 mmol), triethylamine (0.25 mL, 1.80 mmol), and dihydrochloride of 2 -amino-5-aminomethyl-6-methylpyridine (Sanderson, PE, et al. WO 97/01338 (1997)) (0.10 g, 0.48 mmol), dissolved in DMF (10 mL) and stirred at room temperature for 2 hours. The reaction was concentrated in vacuo and the crude product was purified by flash column chromatography (gradient elution: 10% to 15% methanol in DCM) to give an impure product that was dissolved in DCM, washed with saturated sodium bicarbonate, water, and brine, dried over sodium sulfate, and filtered. The evaporated filtrate then produced the title compound as a pale yellow solid (0.23 g, 99%). 1H NMR (300 MHz, CDCl₃) δ 7.64 (m, 1H), 7.56 (m, 1H), 7.35 (m, 10H), 7.11 (d, 1H, J = 8.3 Hz), 6.94 (d , 1H, J = 8.5 Hz),

ES 2 269 474 T3

6.22 (d, 1H, J = 8.1 Hz), 5.60 (t broad, 1H, J = 5.3 Hz), 4.49 (s, 2H), 4.37 (s broad, 2H ), 4.19 (d, 2H, J = 5.5 Hz), 3.56 (s, 2H), 2.34 (s, 3H), 2.23 (s, 3H), 2.22 (s , 3H). Mass Spectrum (LCMS, ESI pos.) Calculated for C₃₀H₃₂N₄OR₄S: 545.2 (M + H). Found: 545.2.

2. N - [(6-amino-2-methyl (3-pyridyl)) methyl] -2- (2-hydroxy-6-methyl-3 - {[(3-methylphenyl) sulfonyl] amino} phenyl) acetamide hydrochloride salt

<img file="ES2269474T3_D0093.tif" />

The product from the previous step (0.22 g, 0.41 mmol) and palladium (0) on carbon at 10% (0.03 g) were dissolved in ethanol: THF 2: 1 (30 mL), degassed with nitrogen and vacuum, and stirred in a hydrogen balloon at room temperature. After 7 hours the reaction was filtered over Celite, the frit was washed with methanol, and the filtrate was concentrated in vacuo. The residue was treated with 4N HCl in ethanol (approx. 3 mL), evaporated in high vacuum, dissolved in DCM, filtered, and the filtrate evaporated in high vacuum again to give the title compound (0.14 g, 71%) as a beige solid Sure. NMR1 H (300 MHz, CDC1₃/CD₃OD) δ 7.60 (m, 3H), 733 (m, 2H), 6.82 (d, 1H, J = 8.2 Hz), 6.67 (d, 1H, J = 9.0 Hz) , 6.58 (d, 1H, J = 8.3Hz), 4.18 (s, 2H), 3.58 (s, 2H), 2.45 (s, 3H), 2.37 (s, 3H), 2.25 (s, 3H). Mass Spectrum (LCMS, ESI pos.) Calculated for C₂₃H₂₆N₄OR₄S: 455.2 (M + H). Found: 455.2.

Example 11

<img file="ES2269474T3_D0094.tif" />

<img file="ES2269474T3_D0095.tif" />

A solution of m-toluenesulfonyl chloride (0.53 g, 2.78 mmol) and 2-methoxy-4-methylphenol (0.38 g, 2.75 mmol) in DCM (10 mL) was treated with triethylamine (0 , 5 mL, 3.6 mmol) and stirred at room temperature. After 18 hours the reaction was concentrated in vacuo, the residue was dissolved in 1: 1 hexane: DCM, filtered, and the filtrate was evaporated in high vacuum to give the title compound as a white solid ( 0.79 g, 99%). 1H NMR (300 MHz, CDCl₃) δ 7.72 (s broad, 1H), 7.66 (d broad, 1H, J = 7.2 Hz), 7.45 (d broad, 1H, J = 7.2 Hz), 7.38 ( t, 1H, J = 7.6Hz), 7.00 (d, 1H, J = 8.1Hz), 6.67 (m, 1H), 3.54 (s, 3H), 2.42 ( s, 3H), 2.31 (s, 3H).

ES 2 269 474 T3

2. 2-Hydroxy-4-methylphenyl 3-Methylbenzenesulfonate

<img file="ES2269474T3_D0096.tif" />

The product from the previous step (0.79 g, 2.72 mmol) was dissolved in DCM (10 mL), cooled to -78 ° C, and treated with 1N boron tribromide in DCM (3.0 mL ) in nitrogen. After 10 min the dry ice bath was removed, and the reaction was stirred for another hour while warming to room temperature. After slowly quenching with water, the reaction was diluted with additional DCM, washed with brine, dried over sodium sulfate, and filtered. The filtrate was concentrated in vacuo and the residue was purified by flash column chromatography (gradient elution: hexane in DCM 50% to 33% to 0%) to give the title compound as a white crystalline solid ( 0.51 g, 68%). 1H NMR (300 MHz, CDCl₃) δ 7.71 (broad s, 1H), 7.67 (broad d, 1H, J = 7.7 Hz), 7.51 (broad d, 1H, J = 8.1 Hz), 7.43 ( t, 1H, J = 7.7 Hz), 6.81 (d, 1H, J = 1.7 Hz), 6.63 (d, 1H, J = 8.3 Hz), 6.55 (m, 1H), 5.87 (s, 1H), 2.43 (s, 3H), 2.26 (s, 3H).

3. 4-Methyl-2-prop-2-enyloxyphenyl 3-Methylbenzenesulfonate

<img file="ES2269474T3_D0097.tif" />

A solution of the product from the previous step (0.51 g, 1.83 mmol), allyl bromide (0.20 mL, 2.30 mmol), and cesium carbonate (0.77 g, 2.40 mmol) in DMF (25 mL) stirred for 16 hours at room temperature. The reaction was concentrated in vacuo, the residue was dissolved in DCM, filtered, and the filtrate was washed with 1N aqueous KOH, water, and brine, dried over sodium sulfate, and filtered. The evaporated filtrate then produced the title compound as a pale yellow oil (0.54 g, 93%). 1 H NMR (400 MHz, CDCl₃) δ 7.72 (s, 1H), 7.64 (d, 1H, J = 7.7 Hz), 7.42 (d, 1H, J = 7.7 Hz), 7.35 (t, 1H , J = 7.7 Hz), 7.04 (d, 1H, J = 8.2 Hz), 6.68 (m, 2H), 5.80 (ddt, 1H, J = 17.3 Hz, 10 , 6 Hz, 5.1 Hz), 5.28 (ddd, 1H, J = 17.3 Hz, 3.1 Hz, 1.6 Hz), 5.20 (ddd, 1H, J =

10.6 Hz, 2.8 Hz, 1.3 Hz), 4.29 (dt, 2H, J = 5.1 Hz, 1.5 Hz), 2.40 (s, 3H), 2.30 ( s, 3H).

Four. 2-Hydroxy-4-methyl-3-prop-2-enylphenyl 3-Methylbenzenesulfonate

<img file="ES2269474T3_D0098.tif" />

The product from the previous step (0.54 g, 1.70 mmol) was heated neat at 200 ° C for 6 hrs., Cooled to room temperature, and purified twice by flash column chromatography (first with DCM : hexane 2: 1, then with hexane: ethyl acetate 4: 1 as eluent) to give the title compound as a colorless oil (84 mg, 16%). 1 H NMR (400 MHz, CDCl₃) δ 7.74 (s, 1H), 7.67 (d, 1H, J = 7.8 Hz), 7.44 (d, 1H, J = 7.6 Hz), 7.36 (t, 1H , J = 7.7 Hz), 6.86 (d, 1H, J = 8.3 Hz), 6.55 (d, 1H, J = 8.3 Hz), 4.74 (m, 1H), 3.15 (dd, 1H, J = 15.5Hz, 8.9Hz), 2.61 (dd, 1H, J = 15.5Hz, 7.6Hz), 2.41 (s, 3H) , 2.16 (s, 3H), 1.24 (d, 2H, J = 6.3 Hz).

ES 2 269 474 T3

5. 4-Methyl-2- (phenylmethoxy) -3-prop-2-enylphenyl 3-methylbenzenesulfonate

<img file="ES2269474T3_D0099.tif" />

The product from the previous step (68 mg, 0.21 mmol) and cesium carbonate (0.19 g, 0.58 mmol) were dissolved in DMF (5 mL) and treated with benzyl bromide (0.05 mL 0.42 mmol) at room temperature. After 3 days the reaction was concentrated in vacuo and the residue was purified by flash column chromatography (DCM as eluent) to give the title compound as a pale yellow oil (50mg, 58%). 1 H NMR (400 MHz, CDCl₃) δ 7.60 (s broad, 1H), 7.54 (d broad, 1H, J = 7.8 Hz), 7.34 (m, 4H), 7.27 (m, 3H), 7.10 (d, 1H, J = 8.4 Hz), 6.91 (d, 1H, J = 8.4 Hz), 5.74 (ddt, 1H, J = 17.1 Hz, 10.2 Hz, 5 , 7 Hz), 4.93 (dc, 1H, J = 10.2 Hz, 1.7 Hz), 4.81 (s, 2H), 4.72 (dc, 1H, J = 17.1 Hz, 1.8Hz), 3.30 (dt, 2H, J = 5.7Hz, 1.7Hz), 2.24 (s, 3H), 2.23 (s, 3H).

6. 4-Methyl-3- (2-oxoethyl) -2- (phenylmethoxy) phenyl 3-Methylbenzenesulfonate

<img file="ES2269474T3_D0100.tif" />

To a solution of the product from the previous step (50 mg, 0.12 mmol) and sodium periodate (0.12 g, 0.56 mmol) in 5: 1 acetonitrile: water (12 mL) was added chloride hydrate ruthenium (III) (8 mg, 0.04 mmol). The reaction was stirred for 6 hours at room temperature, diluted with DCM, and washed with 5% aqueous sodium bisulfite, water, and brine. The organic solution was dried over sodium sulfate, filtered, and the filtrate was evaporated to give the title compound as a crude oil which was used without further purification.

7. 2- {6-methyl-3 - [(3-methylphenyl) sulfonyloxy] -2- (phenylmethoxy) phenyl} acetic acid

<img file="ES2269474T3_D0101.tif" />

The product from the previous step was dissolved in acetone (5 mL) and treated with a solution of sodium dichromate (65 mg, 0.22 mmol) and concentrated sulfuric acid (1 mL) in water (4 mL) at room temperature. . After stirring for 3 days the acetone was removed in vacuo, and the remaining aqueous layer was extracted with DCM. The organic phase was then washed with brine, dried over sodium sulfate, filtered, and the evaporated filtrate was purified by flash column chromatography (8% methanol in DCM as eluent) to give the title compound (45 mg, 88% from step 5) as a white solid. 1 H NMR (400 MHz, CDCl₃) δ 7.61 (s broad, 1H), 7.53 (d

ES 2 269 474 T3 broad, 1H, J = 7.8 Hz), 7.33 (d broad, 1H, J = 7.6 Hz), 7.27 (m, 6H), 7.12 (d, 1H , J = 8.4 Hz), 6.92 (d, 1H, J = 8.4 Hz), 4.87 (s, 2H), 3.60 (s, 2H), 2.24 (s, 3H ), 2.23 (s, 3H).

8. 3 - ({N- [2 - ({N, N'-Di- [t-butoxycarbonyl]} guanidinooxy) ethyl] carbamoyl} methyl) -4-methyl-2- (phenylmethoxy) phenyl 3-Methylbenzenesulfonate

<img file="ES2269474T3_D0102.tif" />

To a solution of the product from the previous step (45 mg, 0.11 mmol), BOP (48 mg, 0.11 mmol), and [N, N'-di (t-butoxycarbonyl)] - 2-aminoethoxyguanidine (Tianbao Lu, et al., WO 99/26926 (1999)) (39 mg, 0.11 mmol), in DMF (5 mL) was added triethylamine (0.2 mL, 1.4 mmol). After stirring for 18 hours at room temperature, the reaction was concentrated in vacuo and the residue was purified by flash column chromatography (DCM: ethyl acetate 3: 1 as eluent) to give the title compound as a colorless oil. (61mg, 79%). 1H NMR (300 MHz, CDCl3) δ 9.11 (s, 1H), 7.65 (s broad, 1H), 7.58 (d broad, 1H, J = 8.5 Hz), 7.48 (m , 1H), 7.36 (broad d, 1H, J =

7.6 Hz), 7.29 (m, 7H), 7.03 (d, 1H, J = 8.4 Hz), 6.90 (d, 1H, J = 8.5 Hz), 4.92 (s, 2H), 4.02 (m, 2H), 3.66 (s, 2H), 3.50 (dd, 2H, J = 9.2 Hz, 5.2 Hz), 2.28 (s , 3H), 2.26 (s, 3H), 1.51 (s, 9H), 1.45 (s, 9H).

9. 3 - ({N- [2 - ({N, N'-Di- [t-butoxycarbonyl]} guanidinooxy) ethyl] carbamoyl} methyl) -2-hydroxy-4-methylphenyl 3-Methylbenzenesulfonate

<img file="ES2269474T3_D0103.tif" />

The product from the previous step (61 mg, 0.08 mmol) and 10% palladium (0) on carbon (20 mg) were dissolved in a 1: 1: 1 mixture of THF, methanol, and water (50 mL) , degassed with nitrogen and vacuum, and vigorously shaken in a hydrogen balloon at room temperature. After 18 hours the reaction was filtered over Celite, the frit was washed with methanol, and the evaporated filtrate was purified by flash column chromatography (10% ethyl acetate in dCm as eluent) to give the title compound as of a colorless solid (40 mg, 74%). 1 H NMR (400 MHz, CDCl₃) δ 9.25 (s, 1H), 8.50 (broad t, 1H, J = 4.9 Hz), 7.79 (s, 1H), 7.57 (d, 1H, J =

7.7 Hz), 7.59 (s broad, 1H), 7.43 (d, 1H, J = 7.7 Hz), 7.38 (t, 1H, J = 7.6 Hz), 6, 90 (d, 1H, J = 8.3 Hz), 6.62 (d, 1H, J = 8.4 Hz), 4.08 (m, 2H), 3.69 (s, 2H), 3, 56 (dd, 2H, J = 8.7Hz, 5.0Hz), 2.41 (s, 3H), 2.35 (s, 3H), 1.51 (s, 9H), 1.50 ( s, 9H).

10. 3-Methylbenzenesulfonate hydrochloride salt of 3 - ({N- [2- (guanidinooxy) ethyl] carbamoyl} methyl) -2-hydroxy-4-me-

<img file="ES2269474T3_D0104.tif" />

The product from the previous step (40 mg, 0.06 mmol) was dissolved in DCM (4 mL) and treated with pure trifluoroacetic acid (1.5 mL) at room temperature. After 3 hours the reaction was concentrated in vacuo and the residue was purified by preparative thin layer chromatography (20% methanol in DCM saturated with ammonia gas as eluent), treated with 4N HCl in ethanol, and filtered. The evaporated filtrate was washed with diethyl ether and dried

ES 2 269 474 T3 under high vacuum to give the title compound as a pale yellow solid (17 mg, 57%). 1H NMR (400 MHz, DMSO-d6) δ 10.87 (s, 1H), 9.76 (s, 1H), 7.67 (m, 5H), 7.60 (d, 1H, J = 7, 6 Hz), 7.51 (t, 1H, J = 7.7 Hz), 6.70 (d, 1H, J = 8.3 Hz), 6.60 (d, 1H, J = 8.5 Hz ), 3.79 (t, 2H, J = 5.5Hz), 3.45 (m, 4H), 2.39 (s, 3H), 2.17 (s, 3H). Mass spectrum (LCMS, ESI pos.) Calcd. for C₁₉H₂₄N₄OR₆S: 437.1 (M + H). Found: 437.3.

Example 12

Tablet Preparation

Tablets containing 25.0, 50.0, and 100.0 mg, respectively, of the following active compounds are prepared as illustrated below:

to. N- [2- (amidinoaminooxy) ethyl] -2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} acetamide trifluoroacetate salt; Y

b. N - [(6-amino-2-methyl (3-pyridyl)) methyl] -2- {3 - [(2,2-difluoro-2-phenylethyl) amino] -6-chloro-2-fluorophenyl} acetamide hydrochloride salt .

Tablets for dosages containing 25-100 mg of the active compound

Active compound

Microcrystalline cellulose

Modified Edible Corn Starch Magnesium Stearate

Amount-mg

<td> 25,0</td><td> 50,0</td><td> 100,00</td>

<td> 37,25</td><td> 100,0</td><td> 200,0</td>

<td> 37,25</td><td> 4,25</td><td> 8,5</td>

<td> 0,50</td><td> 0,75</td><td> 1,5</td>

All the active compounds, the cellulose, and a portion of the cornstarch are mixed and granulated to a 10% cornstarch paste. The resulting granulation is sieved, dried and combined with the rest of the cornstarch and magnesium stearate. The resulting granulation is then compressed into tablets containing 25.0, 50.0, and 100.0 mg, respectively, of active ingredient per tablet.

Example 13

Preparation of Intravenous Solution

An intravenous dosage is prepared as follows from the active compounds indicated above in Examples 1 and 2:

Active compound 0.5-10.0 mg

Sodium citrate 5-50 mg

Citric acid 1-15 mg

Sodium chloride 1-8 mg

Water for injections (USP) qs for 1 ml

Using the above amounts, the active compound is dissolved at room temperature in a previously prepared solution of sodium chloride, citric acid, and sodium citrate in Water for Injections (USP, see page 1636 of the United States Pharmacopoeia / National Formulary of 1995, published by the United States Pharmacopoeia Convention Inc., Rockville, Maryland (1994).

Example 14

In vitro Inhibition of Purified Enzymes

Reagents: All buffer salts were obtained from Sigma Chemical Company (St. Louis, MO), and were of the highest available purity. All enzyme substrates N-benzoyl-Phe-Val-Arg-p-nitroanilide (Sigma B7632), N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide hydrochloride (Sigma B2291), Np-Tosil-Gly- Pro-Lys-p-nitroanilide (Sigma T6140), N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Sigma S7388) and N-CBZ-Val-Gly-Arg-p-nitroanilide (Sigma C7271) were obtained from Sigma, N-succinyl-Ala-Ala-Pro-Arg-p-nitroanilide (BACHEM L-1720) and N-succinyl-AlaAla-Pro-Val-p-nitroanilide (BACHEM-1770) were obtained from BACHEM (Kong of Prussia, PA).

Human α-thrombin, human factor Xa, and human plasmin were obtained from Enzyme Research Laboratories (South Bend, Indiana). Bovine α-chymotrypsin (Sigma C4129), bovine trypsin (Sigma T8642) and

ES 2 269 474 T3 human kidney cell urokinase (Sigma U5004) were obtained from Sigma. Human leukocyte elastase was obtained from Elastin Products (Pacific, MO).

K, determinations: All assays are based on the ability of the test compound to inhibit enzyme-catalyzed hydrolysis of a p-nitroanilide peptide substrate. In a typical Ki determination, the substrate is prepared in DMSO, and diluted in an assay buffer consisting of 50 mM HEPES, 200 mM NaCl, pH 7.5. Final concentrations for each of the substrates are listed below. In general, substrate concentrations are less than the K value_m determined experimentally. Test compounds are prepared as a 1.0 g / ml solution in DMSO. Dilutions are prepared in DMSO yielding 8 final concentrations spanning a 200-fold concentration range. Enzyme solutions are prepared at the concentrations listed below in Assay Buffer.

In a typical Ki determination, 280 ml of substrate solution, 10 ml of test compound solution are pipetted into each well of a 96-well plate, and the plate is allowed to thermally equilibrate at 37 ° C in a cell reader. Molecular Devices plate for more than 15 minutes. Reactions were started by adding a 10 ml aliquot of enzyme and the increase in absorbance at 405 nm was recorded for 15 minutes. Data corresponding to less than 10% of total substrate hydrolysis were used in the calculations. The rate of rate (rate of change in absorbance as a function of time) for a sample containing no test compound is divided by the rate of a sample containing a test compound, and plotted as a function of the concentration of the test compound. The data is fit for a linear regression, and the value of the slope of the line is calculated. The inverse of the slope is the experimentally determined Ki value.

Thrombin: Thrombin activity was evaluated as the ability to hydrolyze the substrate N-succinyl-Ala-AlaPro-Arg-p-nitroanilide. Substrate solutions were prepared at a concentration of 32 mM (32 mM &lt;&lt; Km = 180 mM) in analysis buffer. The final DMSO concentration was 4.3%. Purified human α-thrombin was diluted in assay buffer to a concentration of 15 nM. Final concentrations were: [thrombin] = 0.5 nM, [N-succinyl-Ala-Ala-Pro-Arg-p-nitroanilide substrate] = 32 mM.

Factor X [FXa]: FXa activity was evaluated as the ability to hydrolyze the substrate Nbenzoyl-Ile-Glu-Gly-Arg-p-nitroanilide hydrochloride. Substrate solutions were prepared at a concentration of 51 mM (51 &lt;&lt; K_m = 1.3 mM) in assay buffer. The final DMSO concentration was 4.3%. The purified activated human Factor X was diluted in assay buffer to a concentration of 300 nM. The final reagent concentrations were: [Fxa] = 10 nM, [N-benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide hydrochloride] = 51 mM.

Plasmin: Plasmin activity was evaluated as the ability to hydrolyze Np-Tosil-Gly-Pro-Lys-pnitroanilide. Substrate solutions were prepared at a concentration of 37 mM (37 mM &lt;&lt; K_m = 243 mM) in analysis buffer. The final DMSO concentration was 4.3%. Purified human plasmin was diluted in assay buffer to a concentration of 240 nM. Final reagent concentrations were: [Plasmin] = 8 nM, [N-pTosyl-Gly-Pro-Lys-p-nitroanilide] = 37 mM.

Chymotrypsin: Chymotrypsin activity was evaluated as the ability to hydrolyze N-succinyl-Ala-AlaPro-Phe-p-nitroanilide. Substrate solutions were prepared at a concentration of 14 mM (14 mM &lt;&lt; K_m = 62 mM) in analysis buffer. The final DMSO concentration was 4.3%. The purified bovine chymotrypsin was diluted in assay buffer to a concentration of 81 nM. The final reagent concentrations were: [Chymotrypsin] =

2.7 nM, [N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide] = 14 mM.

Trypsin: Trypsin activity was evaluated as the ability to hydrolyze N-benzoyl-Phe-Val-Arg-p-nitroanilide. Substrate solutions were prepared at a concentration of 13 mM (13 mM &lt;&lt; K_m = 291 mM) in analysis buffer. The final DMSO concentration was 4.3%. The purified bovine trypsin was diluted in assay buffer to a concentration of 120 nM. Final reagent concentrations were: [Trypsin] = 4 nM, [N-benzoyl-Phe-ValArg-p-nitroanilide] = 13 mM.

Elastase: Elatase activity was evaluated as the ability to hydrolyze N-succinyl-Ala-Ala-Pro-Val-pnitroanilide. Substrate solutions were prepared at a concentration of 19 mM (19 mM &lt;&lt; K_m = 89 mM) in analysis buffer. The final DMSO concentration was 4.3%. Purified human lucocytic elastase was diluted in assay buffer to a concentration of 750 nM. The final reagent concentrations were: [Elastase] = 25 nM, [Nsuccinyl-Ala-Ala-Pro-Val-p-nitroanilide] = 19 mM.

Urokinase: Urokinase activity was evaluated as the ability to hydrolyze N-CBZ-Val-Gly-Arg-pnitroanilide. Substrate solutions were prepared at a concentration of 100 mM (100 mM &lt;K_m = 1.2 mM) in assay buffer. The final DMSO concentration was 4.3%. Purified human kidney urokinase was diluted in assay buffer to a concentration of 1.2 nM. The final reagent concentrations were: [Urokinase] = 40 nM, [N-CBZ-Val-Gly-Arg-p-nitroanilide] = 100 mM.

The results indicate that the compounds of Examples 1 to 11 have Ki values ​​for human thrombin between 0.0028 and 20 µM. The compound of Example 5 has a Ki of 0.0028 // M.