Macrocycles as factor XIa inhibitors

The present invention provides compounds of Formula (Ia): or a stereoisomer, a tautomer, or a pharmaceutically acceptable salt thereof, wherein all the variables are as defined herein. These compounds are selective factor XIa inhibitors or dual inhibitors of FXIa and plasma kallikrein. This invention also relates to pharmaceutical compositions comprising these compounds and methods of treating thromboembolic and/or inflammatory disorders using the same.

FIELD OF THE INVENTION

The present invention relates generally to novel macrocyclic compounds, and their analogues thereof, which are inhibitors of factor XIa and/or plasma kallikrein, compositions containing them, and methods of using them, for example, for the treatment or prophylaxis of thromboembolic disorders.

BACKGROUND OF THE INVENTION

Thromboembolic diseases remain the leading cause of death in developed countries despite the availability of anticoagulants such as warfarin (COUMADIN®), heparin, low molecular weight heparins (LMWH), and synthetic pentasaccharides and antiplatelet agents such as aspirin and clopidogrel (PLAVIX®). The oral anticoagulant warfarin, inhibits the post-translational maturation of coagulation factors VII, IX, X and prothrombin, and has proven effective in both venous and arterial thrombosis. However, its usage is limited due to its narrow therapeutic index, slow onset of therapeutic effect, numerous dietary and drug interactions, and a need for monitoring and dose adjustment. Thus discovering and developing safe and efficacious oral anticoagulants for the prevention and treatment of a wide range of thromboembolic disorders has become increasingly important.

One approach is to inhibit thrombin generation by targeting the inhibition of coagulation factor XIa (FXIa). Factor XIa is a plasma serine protease involved in the regulation of blood coagulation, which is initiated in vivo by the binding of tissue factor (TF) to factor VII (FVII) to generate factor VIIa (FVIIa). The resulting TF:FVIIa complex activates factor IX (FIX) and factor X (FX) that leads to the production of factor Xa (FXa). The generated FXa catalyzes the transformation of prothrombin into small amounts of thrombin before this pathway is shut down by tissue factor pathway inhibitor (TFPI). The process of coagulation is then further propagated via the feedback activation of Factors V, VIII and XI by catalytic amounts of thrombin. (Gailani, D. et al.,Arterioscler. Thromb. Vasc. Biol.,27:2507-2513 (2007).) The resulting burst of thrombin converts fibrinogen to fibrin that polymerizes to form the structural framework of a blood clot, and activates platelets, which are a key cellular component of coagulation (Hoffman, M.,Blood Reviews,17:S1-S5 (2003)). Therefore, factor XIa plays a key role in propagating this amplification loop and is thus an attractive target for anti-thrombotic therapy.

SUMMARY OF THE INVENTION

The present invention provides novel macrocyclic compounds, their analogues, including stereoisomers, tautomers, pharmaceutically acceptable salts, or solvates thereof, which are useful as selective inhibitors of serine protease enzymes, especially factor XIa and/or plasma kallikrein.

The present invention also provides processes and intermediates for making the compounds of the present invention.

The present invention also provides pharmaceutical compositions comprising a pharmaceutically acceptable carrier and at least one of the compounds of the present invention or stereoisomers, tautomers, pharmaceutically acceptable salts, or solvates thereof.

The compounds of the invention may be used in the treatment and/or prophylaxis of thromboembolic disorders.

The compounds of the present invention may be used in therapy.

The compounds of the present invention may be used for the manufacture of a medicament for the treatment and/or prophylaxis of a thromboembolic disorder.

The compounds of the invention can be used alone, in combination with other compounds of the present invention, or in combination with one or more, preferably one to two, other agent(s).

These and other features of the invention will be set forth in expanded form as the disclosure continues.

DETAILED DESCRIPTION OF THE INVENTION

I. Compounds of the Invention

In a first aspect, the present invention provides compounds of Formula (I):

ring A is selected from aryl and a 5- to 6-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NH, N(C1-4alkyl), S(O)p, and O, wherein said aryl and heterocycle are optionally substituted with one or more R1as valence allows;

ring B is a 5- to 6-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NH, S(O)p, and O, wherein said heterocycle are optionally substituted with one or more R10as valence allows;

ring C is a 4- to 5-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NR9, S(O)p, and O, wherein said heterocycle are optionally substituted with one or more R2as valence allows;

X1is selected from C1-4alkylene and C2-4alkenylene; optionally one or more of the carbon atoms of said alkylene and alkenylene may be replaced by O, S(O)p, NH, and N(C1-4alkyl);

R3is selected from H and C1-4alkyl;

alternatively, R2and R3, together with the atoms to which they are directly or indirectly attached, form a ring wherein said ring is optionally substituted with ═O;

R5is selected from H and C1-4alkyl;

R9is selected from H and C1-4alkyl;

p is, independently at each occurrence, selected from 0, 1, and 2;

provided the following compounds are excluded

In a second aspect, the present invention provides compounds of Formula (II):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, within the scope of the first aspect, wherein:

ring A is selected from aryl and a 6-membered heterocycle comprising: carbon atoms and 1-3 heteroatoms selected from N, NH, and N(C1-4alkyl);

ring B is selected from imidazole, pyridine, pyridone, and pyridazine;

W and Q are each independently selected from N, NR9, CR2, and CHR2; and

In a third aspect, the present invention provides compounds of Formula (II), or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, within the scope of the second aspect, wherein:

ring A is selected from phenyl and piperidine;

is independently selected from

In a fourth aspect, the present invention provides compounds of Formula (III):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, within the scope of the third aspect, wherein:

is independently selected from

W and Q are each independently selected from N and CR2;

R1aand R1bare each independently selected from H and halogen;

R2is independently at each occurrence, selected from H and C1-4alkyl optionally substituted with OH;

R4is selected from H and C1-4alkyl;

R5is selected from H and C1-4alkyl;

R10is selected from H, halogen and CN.

In a fifth aspect, the present invention provides compounds of Formula (IV):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, within the scope of the fourth aspect, wherein:

W and Q are each independently selected from N and CH;

R10is selected from H and CN.

In a sixth aspect, the present invention provides compounds of Formula (V):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, within the scope of the fifth aspect, wherein:

R1ais selected from H and F;

In a seventh aspect, the present invention provides compounds of Formula (VI):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, within the scope of the fourth aspect, wherein;

is independently selected from

W is selected from N and CH;

Q is selected from N and CH;

R1aand R1bare each independently selected from F and Cl;

R4is selected from H, methyl, and ethyl; and

ring A is selected from aryl and a 5- to 6-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NH, N(C1-4alkyl), S(O)p, and O, wherein said aryl and heterocycle are optionally substituted with R1;

ring B is a 5- to 6-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NH, S(O)p, and O, wherein said heterocycle is optionally substituted with R10;

ring C is a 4- to 6-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NR9, S(O)p, and O, wherein said heterocycle is optionally substituted with R2;

X is selected from C4-8alkylene and C4-8alkenylene, wherein said alkylene and alkenylene are substituted with R4and R5; alternatively one or more of the carbon atoms of said alkylene and alkenylene may be replaced by O, C═O, S(O)p, NH, and N(C1-4alkyl);

Y is selected from —CR6R7—NH— and —NH—CR6R7—;

R3is selected from H and C1-4alkyl;

alternatively, R2and R3, together with the atoms to which they are directly or indirectly attached, form a ring;

R4and R5are independently selected from H, halogen, C1-6alkyl, OH, NH2, —CH2NH2, C1-4haloalkyl, —OCH2F, —OCHF2, —OCF3, NH(C1-4alkyl), N(C1-4alkyl)2, C1-4alkoxy, —CH2OH, and —CH2O(C1-4alkyl); when R4and R5are not attached to the same carbon atom, they may be taken together with the carbon atoms to which they are attached to form a carbocycle;

R9is selected from H and C1-4alkyl;

p is, independently at each occurrence, selected from 0, 1, and 2;

provided the following compounds are excluded

In another aspect, the present invention provides compounds of Formula (Ia), or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein:

ring C is a 6-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, and NR9, wherein said heterocycle is optionally substituted with R2and wherein all the variables have the meanings as defined in Formula (Ia).

In another aspect, the present invention provides compounds of Formula (Ia), or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein:

R5is selected from H, F, and C1-4alkyl; when R4and R5are not attached to the same carbon atom, they may be taken together with the carbon atoms to which they are attached to form a carbocycle;

n is, independently at each occurrence, selected from 0, 1, 2, and 3, and

wherein all the variables have the meanings as defined in Formula (Ia).

ring A is selected from aryl and a 5- to 6-membered heterocycle comprising: carbon atoms and 1-3 heteroatoms selected from N, NH, and N(C1-4alkyl);

ring B is selected from imidazole, pyridine, pyridone, pyrimidine, and pyridazine;

X1ais selected from C2-4alkylene and C2-4alkenylene wherein said C2-4alkylene and C2-4alkenylene are optionally substituted with R4and R5; alternatively, one or more of the carbon atoms of said alkylene may be replaced by O and C═O;

——— is an optional bond;

R4and R5are independently selected from H, halogen, C1-6alkyl, OH, and NH2; when R4and R5are not attached to the same carbon atom, they may be taken together with the carbon atoms to which they are attached to form a carbocycle;

R9is selected from H and C1-4alkyl;

n is, independently at each occurrence, selected from 0, 1, 2, and 3.

In another aspect, the present invention provides compounds of Formula (IIa), or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein:

ring A is selected from phenyl, piperidine, and pyridine;

is selected from

In another aspect, the present invention provides compounds of Formula (IIIa):

is independently selected from

X1ais selected from —CR4R5—CR4R5—, —CR4R5—CR4R5—CR4R5—, and —CR4═CR5CR4R5—, wherein one or more —CR4R5— may be replaced by O or C═O;

R9is selected from H and C1-4alkyl;

n is, independently at each occurrence, selected from 0, 1, 2, and 3.

In another aspect, the present invention provides compounds of Formula (IVa):

is independently selected from

W and Q are each independently selected from N and CR2;

R5is H and F;

n is, independently at each occurrence, selected from 0, 1, 2, and 3.

is independently selected from

Q is selected from N and CR2;

R1ais selected from H and F;

R5is H and F;

n is, independently at each occurrence, selected from 0, 1, 2, and 3.

In another aspect, the present invention provides compounds of Formula (Vab):

R8is selected from C(O)OH and a 4-10-membered heterocycle optionally substituted with R13.

is independently selected from

R1ais selected from H and F;

R5is H and F;

n is, independently at each occurrence, selected from 0, 1, 2, and 3.

is independently selected from

W is selected from N and CR2;

Q is selected from N and CR2;

R1ais selected from H and F;

R5is selected from H and F;

n is, independently at each occurrence, selected from 0, 1, 2, and 3.

In another aspect, the present invention provides compounds of Formula (VIIa) or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, a solvate thereof, wherein:

is independently selected from

W is selected from N and CR2;

Q is selected from N and CR2;

R1ais selected from H and F;

R5is selected from H and F;

ring B is a 5- to 6-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NH, S(O)p, and O, wherein said heterocycle is optionally substituted with R10;

ring C is a 4- to 6-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NR9, S(O)p, and O, wherein said heterocycle is optionally substituted with R2;

X is selected from C4-8alkylene and C4-8alkenylene, wherein said alkylene and alkenylene are substituted with R4and R5; alternatively one or more of the carbon atoms of said alkylene and alkenylene may be replaced by O, C═O, S(O)p, NH, and N(C1-4alkyl);

Y is selected from —CR6R7—NH— and —NH—CR6R7—;

R1bis selected from H and Cl;

R3is selected from H and C1-4alkyl;

R4and R5are independently selected from H, halogen, C1-6alkyl, OH, NH2, —CH2NH2, C1-4haloalkyl, —OCH2F, —OCHF2, —OCF3, NH(C1-4alkyl), N(C1-4alkyl)2, C1-4alkoxy, —CH2OH, and —CH2O(C1-4alkyl); when R4and R5are not attached to the same carbon atom, they may be taken together with the carbon atoms to which they are attached to form a carbocycle;

R9is selected from H and C1-4alkyl;

p is, independently at each occurrence, selected from 0, 1, and 2.

In another aspect, the present invention provides compounds of Formula (IIa) or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein

ring A is

X1ais selected from —CR4R5—CR4R5—, —CR4R5—CR4R5—CR4R5—, and —CR4═CR5CR4R5—, wherein one or more —CR4R5— may be replaced by O or C═O. All other variables have the meanings as defined in Formula (IIa).

In another aspect, the present invention provides compounds of Formula (IXa):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein the variables have the meanings as defined in Formula (IIa).

In one embodiment, the present invention provides compounds of Formula (I) or (II), or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein:

ring A is selected from piperidine and phenyl optionally substituted with R1;

In another embodiment, ring A is selected from

wherein R1is, independently at each occurrence, selected from H, halogen, and C1-6alkyl.

In another embodiment, ring A is

and is selected from

In another embodiment, ring A is

and is selected from

In another embodiment, ring A is

In yet another embodiment, the present invention provides compounds of Formula (I), (II), or (III), or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein ring B is selected from imidazole, oxadiazole, pyridine, pyridinone, pyridazine, pyridazinone, and phenyl.

In another embodiment,

is selected from

In another embodiment,

is selected from

In another embodiment,

is selected from

In still another embodiment,

is selected from

In another embodiment,

In another embodiment,

In another embodiment,

In another embodiment,

In another embodiment, ring C is a 4-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NR9, S(O)p, and O.

In another embodiment, ring C is

wherein the nitrogen in the azetidine ring is attached to ring A.

In another embodiment, ring C is a 5-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NR9, S(O)p, and O.

In another embodiment, ring C is

wherein W and Q are each independently selected from C, N, O, and S, whereby carbon is tetravalent, nitrogen is trivalent, and sulfur and oxygen are divalent; andis a single or double bond.

In another embodiment, ring C is

wherein W and Q are each independently selected from N, NR9, CR2, and CHR2;

In another embodiment, ring C is a 5-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NR9, S(O)p, and O.

In another embodiment, ring C is

wherein U, V, W, and Q are each independently selected from the group consisting of C, N, O, and S, whereby carbon is tetravalent, nitrogen is trivalent, and sulfur and oxygen are divalent; andis a single or double bond.

In another wherein U, V, W, and Q are each independently selected from N, NR9, S, O, C, CR2, and CHR2.

In another embodiment, ring C is

In another embodiment, ring C is

In another embodiment, ring C is

In another embodiment, ring C is

In another embodiment, ring C is

In another embodiment, ring C is

In another embodiment, ring C is

In another embodiment, ring C is

wherein W and Q are each independently selected from N, NR9, CR2, and CHR2;

In another embodiment, ring C is

wherein Q is selected from N and CR2;

In another embodiment, ring C is

In another embodiment, ring C is

W is selected from N and CR2.

In another embodiment, ring C is

In another embodiment, ring C is

In another embodiment, ring C is

In another embodiment, ring C is 6-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NR9, S(O)p, and O.

In another embodiment, ring C is

In one embodiment, X1ais selected from C2-3alkylene and C2-4alkenylene; wherein said alkylene and alkenylene are optionally substituted with F, OH and C1-4alkyl; alternatively, one or two of the carbon atoms of said alkylene and alkenylene may be replaced by O, NH, and N(C1-4alkyl).

In another embodiment, X1ais selected from —CH2CH2— and —CH═CHCH2—.

In one embodiment, X1is selected from C1-3alkylene and C2-4alkenylene; wherein said alkylene and alkenylene are optionally substituted with OH and C1-4alkyl; alternatively, one or two of the carbon atoms of said alkylene and alkenylene may be replaced by O, S(O)p, NH, N(C1-4alkyl), CONH, or CON(C1-4alkyl).

In another embodiment, X1is selected from —CH2—, —CH═CH—, and —C(Me)═CH—.

In another embodiment, X1is selected from —CH2— and —CH═CH—.

In another embodiment, R1is, independently at each occurrence, selected from H and halogen.

In another embodiment, ring A is

wherein R1aand R1bare each independently selected from H and halogen.

In another embodiment, R1ais selected from H, F and Cl.

In another embodiment, R1ais selected from H and F.

In another embodiment, R1ais F and R1bis Cl.

In another embodiment, R3is selected from H, and C1-4alkyl.

In another embodiment, R4is selected from H, C1-4alkyl, and hydroxyl.

In another embodiment, R4is selected from H and C1-4alkyl.

In another embodiment, R4is selected from H and methyl, ethyl, isopropyl, and C3-6cycloalkyl.

In another embodiment, R5is selected from H and C1-4alkyl.

In another embodiment, R5is selected from H and methyl.

Rbis selected from H and C1-6alkyl;

Rcis selected from H and C1-6alkyl;

alternatively, Rband Rcare taken together with the nitrogen atom to which they are attached to form a 4- to 7-membered heterocycle optionally substituted with OH, OMe, and halogen.

In another embodiment, R7is selected from H, C1-4alkyl, and CF3.

In another embodiment, R6and R7are taken together to be ═O.

In another embodiment, R12is selected from methyl, —(CH2)n—OC1-4alkyl, —(CH2)n—OH, —(CH2)n—C3-6cycloalkyl, and —(CH2)n-5-membered heterocycle optionally substituted with R13and selected from

In another embodiment, the present invention provides compounds of Formula (I), or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein:

ring A is a 6-membered aryl or piperidine, said ring moieties are optionally substituted with R1;

ring B is selected from imidazole, oxadiazole, pyridine, pyridinone, pyridazine, pyridazinone, pyrimidine, and phenyl, said ring moieties are optionally substituted with R10; and

ring C is selected from imidazole, pyrazole, pyrrole, and triazole, said ring moieties are optionally substituted with R2.

In one embodiment, the present invention provides compounds of Formula (VII):

ring B is a 5- to 6-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NH, S(O)p, and O, wherein said heterocycle are optionally substituted with one or more R19as valence allows;

ring C is a 4- to 5-membered heterocycle comprising: carbon atoms and 1-4 heteroatoms selected from N, NR9, S(O)p, and O, wherein said heterocycle are optionally substituted with one or more R2as valence allows;

X1is selected from C1-4alkylene, and C2-4alkenylene wherein said alkylene and alkenylene are optionally substituted with OH and C1-4alkyl; alternatively one or more of the carbon atoms of said alkylene and alkenylene may be replaced by O, S(O)p, NH, and N(C1-4alkyl);

R1aand R1bare each independently selected from H and halogen;

R3is selected from H and C1-4alkyl;

alternatively, R2and R3, together with the atoms to which they are directly or indirectly attached, form a ring wherein said ring is optionally substituted with ═O;

R5is selected from H and C1-4alkyl;

R9is selected from H and C1-4alkyl;

R12is selected from H and C1-6alkyl;

R13is selected from H and C1-6alkyl;

alternatively, R12and R13are taken together with the nitrogen atom to which they are attached to form a 4- to 7-membered heterocycle, optionally substituted with OH, OMe, and halogen; and

p is, independently at each occurrence, selected from 0, 1, and 2.

In another embodiment, the present invention provides compounds of Formula (VII), or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein:

is selected from

ring C is

R2is selected from H and NH2; and

R4is selected from H, methyl, and ethyl; and

In another embodiment, the present invention provides compounds of Formula (VIII):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein the variables have the meanings as defined in Formula (VII).

In another embodiment, the present invention provides compounds of Formula (IX):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein the variables have the meanings as defined in Formula (VII).

In another embodiment, the present invention provides compounds of Formula (IXa):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein the variables have the meanings as defined in Formula (IVa).

In another embodiment, the present invention provides compounds of Formula (X):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein the variables have the meanings as defined in Formula (VII).

In another embodiment, the present invention provides compounds of Formula (XI):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein the variables have the meanings as defined in Formula (VII).

In another embodiment, the present invention provides compounds of Formula (XIa):

or stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, or prodrugs thereof, wherein the variables have the meanings as defined in Formula (IVa).

In another aspect, the present invention provides a compound selected from any subset list of compounds exemplified in the present application.

In another embodiment, the compounds of the present invention have Factor XIa Ki values ≦10 μM.

In another embodiment, the compounds of the present invention have Factor XIa Ki values ≦1 μM.

In another embodiment, the compounds of the present invention have Factor XIa Ki values ≦0.5 μM.

In another embodiment, the compounds of the present invention have Factor XIa Ki values ≦0.1 μM.

II. Other Embodiments of the Invention

In another embodiment, the present invention provides a composition comprising at least one of the compounds of the present invention or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a solvate thereof.

In another embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and at least one of the compounds of the present invention or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a solvate, thereof.

In another embodiment, the present invention provides a pharmaceutical composition, comprising: a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one of the compounds of the present invention or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a solvate thereof.

In another embodiment, the present invention provides a process for making a compound of the present invention.

In another embodiment, the present invention provides an intermediate for making a compound of the present invention.

In another embodiment, the present invention provides a pharmaceutical composition further comprising additional therapeutic agent(s). In a preferred embodiment, the present invention provides pharmaceutical composition, wherein the additional therapeutic agent(s) are an anti-platelet agent or a combination thereof. Preferably, the anti-platelet agent(s) are clopidogrel and/or aspirin, or a combination thereof.

In another embodiment, the present invention provides a method for the treatment and/or prophylaxis of a thromboembolic disorder comprising administering to a patient in need of such treatment and/or prophylaxis a therapeutically effective amount of at least one of the compounds of the present invention or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a solvate thereof.

In another embodiment, the present invention provides a compound of the present invention or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a solvate thereof, for use in therapy.

In another embodiment, the present invention provides a compound of the present invention or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a solvate thereof, for use in therapy for the treatment and/or prophylaxis of a thromboembolic disorder.

In another embodiment, the present invention also provides the use of a compound of the present invention or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a solvate thereof, for the manufacture of a medicament for the treatment and/or prophylaxis of a thromboembolic disorder.

In another embodiment, the present invention provides a method for treatment and/or prophylaxis of a thromboembolic disorder, comprising: administering to a patient in need thereof a therapeutically effective amount of a first and second therapeutic agent, wherein the first therapeutic agent is a compound of the present invention or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a solvate thereof, and the second therapeutic agent is at least one agent selected from a second factor Xa inhibitor, an anti-coagulant agent, an anti-platelet agent, a thrombin inhibiting agent, a thrombolytic agent, and a fibrinolytic agent. Preferably, the second therapeutic agent is at least one agent selected from warfarin, unfractionated heparin, low molecular weight heparin, synthetic Pentasaccharide, hirudin, argatroban, aspirin, ibuprofen, naproxen, sulindac, indomethacin, mefenamate, droxicam, diclofenac, sulfinpyrazone, piroxicam, ticlopidine, clopidogrel, tirofiban, eptifibatide, abciximab, melagatran, desulfatohirudin, tissue plasminogen activator, modified tissue plasminogen activator, anistreplase, urokinase, and streptokinase. Preferably, the second therapeutic agent is at least one anti-platelet agent. Preferably, the anti-platelet agent(s) are clopidogrel and/or aspirin, or a combination thereof.

The thromboembolic disorder includes arterial cardiovascular thromboembolic disorders, venous cardiovascular thromboembolic disorders, arterial cerebrovascular thromboembolic disorders, and venous cerebrovascular thromboembolic disorders. Examples of the thromboembolic disorder include, but are not limited to, unstable angina, an acute coronary syndrome, atrial fibrillation, first myocardial infarction, recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, or procedures in which blood is exposed to an artificial surface that promotes thrombosis.

In another embodiment, the present invention provides a method for the treatment and/or prophylaxis of an inflammatory disorder comprising: administering to a patient in need of such treatment and/or prophylaxis a therapeutically effective amount of at least one of the compounds of the present invention or a stereoisomer, a tautomer, a pharmaceutically acceptable salt, or a solvate thereof. Examples of the inflammatory disorder include, but are not limited to, sepsis, acute respiratory distress syndrome, and systemic inflammatory response syndrome.

In another embodiment, the present invention provides a combined preparation of a compound of the present invention and additional therapeutic agent(s) for simultaneous, separate or sequential use in therapy.

In another embodiment, the present invention provides a combined preparation of a compound of the present invention and additional therapeutic agent(s) for simultaneous, separate or sequential use in treatment and/or prophylaxis of a thromboembolic disorder.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. This invention encompasses all combinations of preferred aspects of the invention noted herein. It is understood that any and all embodiments of the present invention may be taken in conjunction with any other embodiment or embodiments to describe additional embodiments. It is also to be understood that each individual element of the embodiments is its own independent embodiment. Furthermore, any element of an embodiment is meant to be combined with any and all other elements from any embodiment to describe an additional embodiment.

Throughout the specification and the appended claims, a given chemical formula or name shall encompass all stereo and optical isomers and racemates thereof where such isomers exist. Unless otherwise indicated, all chiral (enantiomeric and diastereomeric) and racemic forms are within the scope of the invention. Many geometric isomers of C═C double bonds, C═N double bonds, ring systems, and the like can also be present in the compounds, and all such stable isomers are contemplated in the present invention. Cis- and trans- (or E- and Z-) geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. The present compounds can be isolated in optically active or racemic forms. Optically active forms may be prepared by resolution of racemic forms or by synthesis from optically active starting materials. All processes used to prepare compounds of the present invention and intermediates made therein are considered to be part of the present invention. When enantiomeric or diastereomeric products are prepared, they may be separated by conventional methods, for example, by chromatography or fractional crystallization. Depending on the process conditions the end products of the present invention are obtained either in free (neutral) or salt form. Both the free form and the salts of these end products are within the scope of the invention. If so desired, one form of a compound may be converted into another form. A free base or acid may be converted into a salt; a salt may be converted into the free compound or another salt; a mixture of isomeric compounds of the present invention may be separated into the individual isomers. Compounds of the present invention, free form and salts thereof, may exist in multiple tautomeric forms, in which hydrogen atoms are transposed to other parts of the molecules and the chemical bonds between the atoms of the molecules are consequently rearranged. It should be understood that all tautomeric forms, insofar as they may exist, are included within the invention.

The term “stereoisomer” refers to isomers of identical constitution that differ in the arrangement of their atoms in space. Enantiomers and diastereomers are examples of stereoisomers. The term “enantiomer” refers to one of a pair of molecular species that are mirror images of each other and are not superimposable. The term “diastereomer” refers to stereoisomers that are not mirror images. The term “racemate” or “racemic mixture” refers to a composition composed of equimolar quantities of two enantiomeric species, wherein the composition is devoid of optical activity.

The symbols “R” and “S” represent the configuration of substituents around a chiral carbon atom(s). The isomeric descriptors “R” and “S” are used as described herein for indicating atom configuration(s) relative to a core molecule and are intended to be used as defined in the literature (IUPAC Recommendations 1996, Pure and Applied Chemistry,68:2193-2222 (1996)).

The term “chiral” refers to the structural characteristic of a molecule that makes it impossible to superimpose it on its mirror image. The term “homochiral” refers to a state of enantiomeric purity. The term “optical activity” refers to the degree to which a homochiral molecule or nonracemic mixture of chiral molecules rotates a plane of polarized light.

As used herein, the term “alkyl” or “alkylene” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms. For example, “C1to C10alkyl” or “C1-10alkyl” (or alkylene), is intended to include C1, C2, C3, C4, C5, C6, C7, C8, C9, and C10alkyl groups. Additionally, for example, “C1to C6alkyl” or “C1-C6alkyl” denotes alkyl having 1 to 6 carbon atoms.

Alkyl group can be unsubstituted or substituted with at least one hydrogen being replaced by another chemical group. Example alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), and pentyl (e.g., n-pentyl, isopentyl, neopentyl). When “C0alkyl” or “C0alkylene” is used, it is intended to denote a direct bond.

“Alkenyl” or “alkenylene” is intended to include hydrocarbon chains of either straight or branched configuration having the specified number of carbon atoms and one or more, preferably one to two, carbon-carbon double bonds that may occur in any stable point along the chain. For example, “C2to C6alkenyl” or “C2-6alkenyl” (or alkenylene), is intended to include C2, C3, C4, C5, and C6alkenyl groups. Examples of alkenyl include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3, pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 2-methyl-2-propenyl, and 4-methyl-3-pentenyl.

“Alkynyl” or “alkynylene” is intended to include hydrocarbon chains of either straight or branched configuration having one or more, preferably one to three, carbon-carbon triple bonds that may occur in any stable point along the chain. For example, “C2to C6alkynyl” or “C2-6alkynyl” (or alkynylene), is intended to include C2, C3, C4, C5, and C6alkynyl groups; such as ethynyl, propynyl, butynyl, pentynyl, and hexynyl.

The term “alkoxy” or “alkyloxy” refers to an —O-alkyl group. “C1to C6alkoxy” or “C1-6alkoxy” (or alkyloxy), is intended to include C1, C2, C3, C4, C5, and C6alkoxy groups. Example alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), and t-butoxy. Similarly, “alkylthio” or “thioalkoxy” represents an alkyl group as defined above with the indicated number of carbon atoms attached through a sulphur bridge; for example methyl-S— and ethyl-S—.

“Halo” or “halogen” includes fluoro (F), chloro (Cl), bromo (Br), and iodo (I). “Haloalkyl” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, substituted with 1 or more halogens. Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, pentafluoroethyl, pentachloroethyl, 2,2,2-trifluoroethyl, heptafluoropropyl, and heptachloropropyl. Examples of haloalkyl also include “fluoroalkyl” that is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms, substituted with 1 or more fluorine atoms.

“Haloalkoxy” or “haloalkyloxy” represents a haloalkyl group as defined above with the indicated number of carbon atoms attached through an oxygen bridge. For example, “C1to C6haloalkoxy” or “C1-6haloalkoxy”, is intended to include C1, C2, C3, C4, C5, and C6haloalkoxy groups. Examples of haloalkoxy include, but are not limited to, trifluoromethoxy, 2,2,2-trifluoroethoxy, and pentafluorothoxy. Similarly, “haloalkylthio” or “thiohaloalkoxy” represents a haloalkyl group as defined above with the indicated number of carbon atoms attached through a sulphur bridge; for example trifluoromethyl-S—, and pentafluoroethyl-S—.

The term “carboxy” refers to the group —C(═O)OH.

The term “alkoxycarbonyl” refers to the group —C(═O)ORwwhere Rwis an alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.

The term “cycloalkyl” refers to cyclized alkyl groups, including mono-, bi- or poly-cyclic ring systems. “C3to C7cycloalkyl” or “C3-7cycloalkyl” is intended to include C3, C4, C5, C6, and C7cycloalkyl groups. Example cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and norbornyl. Branched cycloalkyl groups such as 1-methylcyclopropyl and 2-methylcyclopropyl are included in the definition of “cycloalkyl”.

As used herein, “carbocycle” or “carbocyclic residue” is intended to mean any stable 3-, 4-, 5-, 6-, 7-, or 8-membered monocyclic or bicyclic or 7-, 8-, 9-, 10-, 11-, 12-, or 13-membered bicyclic or tricyclic ring, any of which may be saturated, partially unsaturated, unsaturated or aromatic. Examples of such carbocycles include, but are not limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl, adamantyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, [3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane (decalin), [2.2.2]bicyclooctane, fluorenyl, phenyl, naphthyl, indanyl, adamantyl, anthracenyl, and tetrahydronaphthyl (tetralin). As shown above, bridged rings are also included in the definition of carbocycle (e.g., [2.2.2]bicyclooctane). Preferred carbocycles, unless otherwise specified, are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, phenyl, and indanyl. When the term “carbocycle” is used, it is intended to include “aryl”. A bridged ring occurs when one or more carbon atoms link two non-adjacent carbon atoms. Preferred bridges are one or two carbon atoms. It is noted that a bridge always converts a monocyclic ring into a tricyclic ring. When a ring is bridged, the substituents recited for the ring may also be present on the bridge.

As used herein, the term “bicyclic carbocycle” or “bicyclic carbocyclic group” is intended to mean a stable 9- or 10-membered carbocyclic ring system that contains two fused rings and consists of carbon atoms. Of the two fused rings, one ring is a benzo ring fused to a second ring; and the second ring is a 5- or 6-membered carbon ring which is saturated, partially unsaturated, or unsaturated. The bicyclic carbocyclic group may be attached to its pendant group at any carbon atom which results in a stable structure. The bicyclic carbocyclic group described herein may be substituted on any carbon if the resulting compound is stable. Examples of a bicyclic carbocyclic group are, but not limited to, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, and indanyl.

The term “arylalkyloxy” refers to an arylalkyl bonded through an oxygen linkage (—O-arylalkyl).

The term “benzyl”, as used herein, refers to a methyl group on which one of the hydrogen atoms is replaced by a phenyl group, wherein said phenyl group may optionally be substituted with 1 to 5 groups, preferably 1 to 3 groups, OH, OCH3, Cl, F, Br, I, CN, NO2, NH2, N(CH3)H, N(CH3)2, CF3, OCF3, C(═O)CH3, SCH3, S(═O)CH3, S(═O)2CH3, CH3, CH2CH3, CO2H, and CO2CH3.

As used herein, the term “heterocycle” or “heterocyclic group” is intended to mean a stable 3-, 4-, 5-, 6-, or 7-membered monocyclic or bicyclic or 7-, 8-, 9-, 10-, 11-, 12-, 13-, or 14-membered polycyclic heterocyclic ring that is saturated, partially unsaturated, or fully unsaturated, and that contains carbon atoms and 1, 2, 3 or 4 heteroatoms independently selected from the group consisting of N, O and S; and including any polycyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O)p, wherein p is 0, 1 or 2). The nitrogen atom may be substituted or unsubstituted (i.e., N or NR wherein R is H or another substituent, if defined). The heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure. The heterocyclic rings described herein may be substituted on carbon or on a nitrogen atom if the resulting compound is stable. A nitrogen in the heterocycle may optionally be quaternized. It is preferred that when the total number of S and O atoms in the heterocycle exceeds 1, then these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in the heterocycle is not more than 1. When the term “heterocycle” is used, it is intended to include heteroaryl.

As used herein, the term “bicyclic heterocycle” or “bicyclic heterocyclic group” is intended to mean a stable 9- or 10-membered heterocyclic ring system which contains two fused rings and consists of carbon atoms and 1, 2, 3, or 4 heteroatoms independently selected from the group consisting of N, O and S. Of the two fused rings, one ring is a 5- or 6-membered monocyclic aromatic ring comprising a 5-membered heteroaryl ring, a 6-membered heteroaryl ring or a benzo ring, each fused to a second ring. The second ring is a 5- or 6-membered monocyclic ring which is saturated, partially unsaturated, or unsaturated, and comprises a 5-membered heterocycle, a 6-membered heterocycle or a carbocycle (provided the first ring is not benzo when the second ring is a carbocycle).

The bicyclic heterocyclic group may be attached to its pendant group at any heteroatom or carbon atom which results in a stable structure. The bicyclic heterocyclic group described herein may be substituted on carbon or on a nitrogen atom if the resulting compound is stable. It is preferred that when the total number of S and O atoms in the heterocycle exceeds 1, then these heteroatoms are not adjacent to one another. It is preferred that the total number of S and O atoms in the heterocycle is not more than 1.

As used herein, the term “aromatic heterocyclic group” or “heteroaryl” is intended to mean stable monocyclic and polycyclic aromatic hydrocarbons that include at least one heteroatom ring member such as sulfur, oxygen, or nitrogen. Heteroaryl groups include, without limitation, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl, quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrroyl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4-thiadiazolyl, isothiazolyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, benzodioxolanyl, and benzodioxane. Heteroaryl groups are substituted or unsubstituted. The nitrogen atom is substituted or unsubstituted (i.e., N or NR wherein R is H or another substituent, if defined). The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., N→O and S(O)p, wherein p is 0, 1 or 2).

Bridged rings are also included in the definition of heterocycle. A bridged ring occurs when one or more atoms (i.e., C, O, N, or S) link two non-adjacent carbon or nitrogen atoms. Examples of bridged rings include, but are not limited to, one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms, and a carbon-nitrogen group. It is noted that a bridge always converts a monocyclic ring into a tricyclic ring. When a ring is bridged, the substituents recited for the ring may also be present on the bridge.

The term “counterion” is used to represent a negatively charged species such as chloride, bromide, hydroxide, acetate, and sulfate.

When a dotted ring is used within a ring structure, this indicates that the ring structure may be saturated, partially saturated or unsaturated.

As referred to herein, the term “substituted” means that at least one hydrogen atom is replaced with a non-hydrogen group, provided that normal valencies are maintained and that the substitution results in a stable compound. When a substituent is keto (i.e., ═O), then 2 hydrogens on the atom are replaced. Keto substituents are not present on aromatic moieties. When a ring system (e.g., carbocyclic or heterocyclic) is said to be substituted with a carbonyl group or a double bond, it is intended that the carbonyl group or double bond be part (i.e., within) of the ring. Ring double bonds, as used herein, are double bonds that are formed between two adjacent ring atoms (e.g., C═C, C═N, or N═N).

In cases wherein there are nitrogen atoms (e.g., amines) on compounds of the present invention, these may be converted to N-oxides by treatment with an oxidizing agent (e.g., mCPBA and/or hydrogen peroxides) to afford other compounds of this invention. Thus, shown and claimed nitrogen atoms are considered to cover both the shown nitrogen and its N-oxide (N→O) derivative.

When any variable occurs more than one time in any constituent or formula for a compound, its definition at each occurrence is independent of its definition at every other occurrence. Thus, for example, if a group is shown to be substituted with 0-3 R groups, then said group may optionally be substituted with up to three R groups, and at each occurrence R is selected independently from the definition of R. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, and/or other problem or complication, commensurate with a reasonable benefit/risk ratio.

In addition, compounds of Formula (I) may have prodrug forms. Any compound that will be converted in vivo to provide the bioactive agent (i.e., a compound of Formula (I)) is a prodrug within the scope and spirit of the invention. Various forms of prodrugs are well known in the art. For examples of such prodrug derivatives, see:

Compounds containing a carboxy group can form physiologically hydrolyzable esters that serve as prodrugs by being hydrolyzed in the body to yield formula I compounds per se. Such prodrugs are preferably administered orally since hydrolysis in many instances occurs principally under the influence of the digestive enzymes. Parenteral administration may be used where the ester per se is active, or in those instances where hydrolysis occurs in the blood. Examples of physiologically hydrolyzable esters of compounds of formula I include C1-6alkyl, C1-6alkylbenzyl, 4-methoxybenzyl, indanyl, phthalyl, methoxymethyl, C1-6alkanoyloxy-C1-6alkyl (e.g., acetoxymethyl, pivaloyloxymethyl or propionyloxymethyl), C1-6alkoxycarbonyloxy-C1-6alkyl (e.g., methoxycarbonyl-oxymethyl or ethoxycarbonyloxymethyl, glycyloxymethyl, phenylglycyloxymethyl, (5-methyl-2-oxo-1,3-dioxolen-4-yl)-methyl), and other well known physiologically hydrolyzable esters used, for example, in the penicillin and cephalosporin arts. Such esters may be prepared by conventional techniques known in the art.

Preparation of prodrugs is well known in the art and described in, for example, King, F. D., ed.,Medicinal Chemistry: Principles and Practice, The Royal Society of Chemistry, Cambridge, UK (1994); Testa, B. et al.,Hydrolysis in Drug and Prodrug Metabolism. Chemistry, Biochemistry and Enzymology, VCHA and Wiley-VCH, Zurich, Switzerland (2003); Wermuth, C. G., ed.,The Practice of Medicinal Chemistry, Academic Press, San Diego, Calif. (1999).

The present invention is intended to include all isotopes of atoms occurring in the present compounds. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include deuterium and tritium. Isotopes of carbon include13C and14C. Isotopically-labeled compounds of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed. Such compounds have a variety of potential uses, e.g., as standards and reagents in determining the ability of a potential pharmaceutical compound to bind to target proteins or receptors, or for imaging compounds of this invention bound to biological receptors in vivo or in vitro.

“Stable compound” and “stable structure” are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. It is preferred that compounds of the present invention do not contain a N-halo, S(O)2H, or S(O)H group.

The term “solvate” means a physical association of a compound of this invention with one or more solvent molecules, whether organic or inorganic. This physical association includes hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. The solvent molecules in the solvate may be present in a regular arrangement and/or a non-ordered arrangement. The solvate may comprise either a stoichiometric or nonstoichiometric amount of the solvent molecules. “Solvate” encompasses both solution-phase and isolable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods of solvation are generally known in the art.

Abbreviations as used herein, are defined as follows: “1×” for once, “2×” for twice, “3×” for thrice, “° C.” for degrees Celsius, “eq” for equivalent or equivalents, “g” for gram or grams, “mg” for milligram or milligrams, “L” for liter or liters, “mL” for milliliter or milliliters, “μL” for microliter or microliters, “N” for normal, “M” for molar, “mmol” for millimole or millimoles, “min” for minute or minutes, “h” for hour or hours, “rt” for room temperature, “RT” for retention time, “atm” for atmosphere, “psi” for pounds per square inch, “conc.” for concentrate, “sat” or “saturated” for saturated, “MW” for molecular weight, “mp” for melting point, “ee” for enantiomeric excess, “MS” or “Mass Spec” for mass spectrometry, “ESI” for electrospray ionization mass spectroscopy, “HR” for high resolution, “HRMS” for high resolution mass spectrometry, “LCMS” for liquid chromatography mass spectrometry, “HPLC” for high pressure liquid chromatography, “RP HPLC” for reverse phase HPLC, “TLC” or “tic” for thin layer chromatography, “NMR” for nuclear magnetic resonance spectroscopy, “nOe” for nuclear Overhauser effect spectroscopy, “H” for proton, “δ” for delta, “s” for singlet, “d” for doublet, “t” for triplet, “q” for quartet, “m” for multiplet, “br” for broad, “Hz” for hertz, and “α”, “β”, “R”, “S”, “E”, and “Z” are stereochemical designations familiar to one skilled in the art.

Me Methyl

The compounds of the present invention can be prepared in a number of ways known to one skilled in the art of organic synthesis.

While blood coagulation is essential to the regulation of an organism's hemostasis, it is also involved in many pathological conditions. In thrombosis, a blood clot, or thrombus, may form and obstruct circulation locally, causing ischemia and organ damage. Alternatively, in a process known as embolism, the clot may dislodge and subsequently become trapped in a distal vessel, where it again causes ischemia and organ damage. Diseases arising from pathological thrombus formation are collectively referred to as thromboembolic disorders and include acute coronary syndrome, unstable angina, myocardial infarction, thrombosis in the cavity of the heart, ischemic stroke, deep vein thrombosis, peripheral occlusive arterial disease, transient ischemic attack, and pulmonary embolism. In addition, thrombosis occurs on artificial surfaces in contact with blood, including catheters, stents, artificial heart valves, and hemodialysis membranes.

Some conditions contribute to the risk of developing thrombosis. For example, alterations of the vessel wall, changes in the flow of blood, and alterations in the composition of the vascular compartment. These risk factors are collectively known as Virchow's triad. (Colman, R. W. et al., eds.,Hemostasis and Thrombosis, Basic Principles and Clinical Practice,5th Edition, p. 853, Lippincott Williams & Wilkins (2006))

Antithrombotic agents are frequently given to patients at risk of developing thromboembolic disease because of the presence of one or more predisposing risk factors from Virchow's triad to prevent formation of an occlusive thrombus (primary prevention). For example, in an orthopedic surgery setting (e.g., hip and knee replacement), an antithrombotic agent is frequently administered prior to a surgical procedure. The antithrombotic agent counterbalances the prothrombotic stimulus exerted by vascular flow alterations (stasis), potential surgical vessel wall injury, as well as changes in the composition of the blood due to the acute phase response related to surgery. Another example of the use of an antithrombotic agent for primary prevention is dosing with aspirin, a platelet activation inhibitor, in patients at risk for developing thrombotic cardiovascular disease. Well recognized risk factors in this setting include age, male gender, hypertension, diabetes mellitus, lipid alterations, and obesity.

Antithrombotic agents are also indicated for secondary prevention, following an initial thrombotic episode. For example, patients with mutations in factor V (also known as factor V Leiden) and additional risk factors (e.g., pregnancy), are dosed with anticoagulants to prevent the reoccurrence of venous thrombosis. Another example entails secondary prevention of cardiovascular events in patients with a history of acute myocardial infarction or acute coronary syndrome. In a clinical setting, a combination of aspirin and clopidogrel (or other thienopyridines) may be used to prevent a second thrombotic event.

Antithrombotic agents are also given to treat the disease state (i.e., by arresting its development) after it has already started. For example, patients presenting with deep vein thrombosis are treated with anticoagulants (i.e., heparin, warfarin, or LMWH) to prevent further growth of the venous occlusion. Over time, these agents also cause a regression of the disease state because the balance between prothrombotic factors and anticoagulant/profibrinolytic pathways is changed in favor of the latter. Examples on the arterial vascular bed include the treatment of patients with acute myocardial infarction or acute coronary syndrome with aspirin and clopidogrel to prevent further growth of vascular occlusions and eventually leading to a regression of thrombotic occlusions.

Thus, antithrombotic agents are used widely for primary and secondary prevention (i.e., prophylaxis or risk reduction) of thromboembolic disorders, as well as treatment of an already existing thrombotic process. Drugs that inhibit blood coagulation, or anticoagulants, are “pivotal agents for prevention and treatment of thromboembolic disorders” (Hirsh, J. et al.,Blood,105:453-463 (2005)).

An alternative way of initiation of coagulation is operative when blood is exposed to artificial surfaces (e.g., during hemodialysis, “on-pump” cardiovascular surgery, vessel grafts, bacterial sepsis), on cell surfaces, cellular receptors, cell debris, DNA, RNA, and extracellular matrices. This process is also termed contact activation. Surface absorption of factor XII leads to a conformational change in the factor XII molecule, thereby facilitating activation to proteolytic active factor XII molecules (factor XIIa and factor XIIf). Factor XIIa (or XIIf) has a number of target proteins, including plasma prekallikrein and factor XI. Active plasma kallikrein further activates factor XII, leading to an amplification of contact activation. Alternatively, the serine protease prolylcarboxylpeptidase can activate plasma kallikrein complexed with high molecular weight kininogen in a multiprotein complex formed on the surface of cells and matrices (Shariat-Madar et al.,Blood,108:192-199 (2006)). Contact activation is a surface mediated process responsible in part for the regulation of thrombosis and inflammation, and is mediated, at least in part, by fibrinolytic-, complement-, kininogen/kinin-, and other humoral and cellular pathways (for review, Coleman, R., “Contact Activation Pathway”,Hemostasis and Thrombosis, pp. 103-122, Lippincott Williams & Wilkins (2001); Schmaier, A.H., “Contact Activation”,Thrombosis and Hemorrhage, pp. 105-128 (1998)). The biological relevance of the contact activation system for thromboembolic diseases is supported by the phenotype of factor XII deficient mice. More specifically, factor XII deficient mice were protected from thrombotic vascular occlusion in several thrombosis models as well as stroke models and the phenotype of the XII deficient mice was identical to XI deficient mice (Renne et al.,J. Exp. Med.,202:271-281 (2005); Kleinschmitz et al.,J. Exp. Med.,203:513-518 (2006)). The fact that factor XI is down-stream from factor XIIa, combined with the identical phenotype of the XII and XI deficient mice suggest that the contact activation system could play a major role in factor XI activation in vivo.

Factor XI is a zymogen of a trypsin-like serine protease and is present in plasma at a relatively low concentration. Proteolytic activation at an internal R369-I370 bond yields a heavy chain (369 amino acids) and a light chain (238 amino acids). The latter contains a typical trypsin-like catalytic triad (H413, D464, and S557). Activation of factor XI by thrombin is believed to occur on negatively charged surfaces, most likely on the surface of activated platelets. Platelets contain high affinity (0.8 nM) specific sites (130-500/platelet) for activated factor XI. After activation, factor XIa remains surface bound and recognizes factor IX as its normal macromolecular substrate. (Galiani, D.,Trends Cardiovasc. Med.,10:198-204 (2000))

In addition to the feedback activation mechanisms described above, thrombin activates thrombin activated fibrinolysis inhibitor (TAFI), a plasma carboxypeptidase that cleaves C-terminal lysine and arginine residues on fibrin, reducing the ability of fibrin to enhance tissue-type plasminogen activator (tPA) dependent plasminogen activation. In the presence of antibodies to FXIa, clot lysis can occur more rapidly independent of plasma TAFI concentration. (Bouma, B. N. et al.,Thromb. Res.,101:329-354 (2001).) Thus, inhibitors of factor XIa are expected to be anticoagulant and profibrinolytic.

Further evidence for the anti-thromboembolic effects of targeting factor XI is derived from mice deficient in factor XI. It has been demonstrated that complete fXI deficiency protected mice from ferric chloride (FeCl3)-induced carotid artery thrombosis (Rosen et al.,Thromb. Haemost.,87:774-777 (2002); Wang et al.,J. Thromb. Haemost.,3:695-702 (2005)). Also, factor XI deficiency rescues the perinatal lethal phenotype of complete protein C deficiency (Chan et al.,Amer. J. Pathology,158:469-479 (2001)). Furthermore, baboon cross-reactive, function blocking antibodies to human factor XI protect against baboon arterial—venous shunt thrombosis (Gruber et al., Blood, 102:953-955 (2003)). Evidence for an antithrombotic effect of small molecule inhibitors of factor XIa is also disclosed in published U.S. Patent Application No. 2004/0180855 A1. Taken together, these studies suggest that targeting factor XI will reduce the propensity for thrombotic and thromboembolic diseases.

Genetic evidence indicates that factor XI is not required for normal homeostasis, implying a superior safety profile of the factor XI mechanism compared to competing antithrombotic mechanisms. In contrast to hemophilia A (factor VIII deficiency) or hemophilia B (factor IX deficiency), mutations of the factor XI gene causing factor XI deficiency (hemophilia C) result in only a mild to moderate bleeding diathesis characterized primarily by postoperative or posttraumatic, but rarely spontaneous hemorrhage. Postoperative bleeding occurs mostly in tissue with high concentrations of endogenous fibrinolytic activity (e.g., oral cavity, and urogenital system). The majority of the cases are fortuitously identified by preoperative prolongation of aPTT (intrinsic system) without any prior bleeding history.

The increased safety of inhibition of XIa as an anticoagulation therapy is further supported by the fact that Factor XI knock-out mice, which have no detectable factor XI protein, undergo normal development, and have a normal life span. No evidence for spontaneous bleeding has been noted. The aPTT (intrinsic system) is prolonged in a gene dose-dependent fashion. Interestingly, even after severe stimulation of the coagulation system (tail transection), the bleeding time is not significantly prolonged compared to wild-type and heterozygous litter mates. (Gailani, D.,Frontiers in Bioscience,6:201-207 (2001); Gailani, D. et al.,Blood Coagulation and Fibrinolysis,8:134-144 (1997).) Taken together, these observations suggest that high levels of inhibition of factor XIa should be well tolerated. This is in contrast to gene targeting experiments with other coagulation factors, excluding factor XII.

In vivo activation of factor XI can be determined by complex formation with either C1 inhibitor or alpha 1 antitrypsin. In a study of 50 patients with acute myocardial infarction (AMI), approximately 25% of the patients had values above the upper normal range of the complex ELISA. This study can be viewed as evidence that at least in a subpopulation of patients with AMI, factor XI activation contributes to thrombin formation (Minnema, M. C. et al.,Arterioscler. Thromb. Vasc. Biol.,20:2489-2493 (2000)). A second study establishes a positive correlation between the extent of coronary arteriosclerosis and factor XIa in complex with alpha 1 antitrypsin (Murakami, T. et al.,Arterioscler. Thromb. Vasc. Biol.,15:1107-1113 (1995)). In another study, Factor XI levels above the 90th percentile in patients were associated with a 2.2-fold increased risk for venous thrombosis (Meijers, J. C. M. et al.,N. Engl. J. Med.,342:696-701 (2000)).

Plasma kallikrein is a zymogen of a trypsin-like serine protease and is present in plasma at 35 to 50 μg/mL. The gene structure is similar to that of factor XI. Overall, the amino acid sequence of plasma kallikrein has 58% homology to factor XI. Proteolytic activation by factor XIIa at an internal I 389-R390 bond yields a heavy chain (371 amino acids) and a light chain (248 amino acids). The active site of plasma kallikrein is contained in the light chain. The light chain of plasma kallikrein reacts with protease inhibitors, including alpha 2 macroglobulin and C1-inhibitor. Interestingly, heparin significantly accelerates the inhibition of plasma kallikrein by antithrombin III in the presence of high molecular weight kininogen (HMWK). In blood, the majority of plasma kallikrein circulates in complex with HMWK. Plasma kallikrein cleaves HMWK to liberate bradykinin. Bradykinin release results in increase of vascular permeability and vasodilation (for review, Coleman, R., “Contact Activation Pathway”,Hemostasis and Thrombosis, pp. 103-122, Lippincott Williams & Wilkins (2001); Schmaier A. H., “Contact Activation”,Thrombosis and Hemorrhage, pp. 105-128 (1998)).

Also, it is preferred to find new compounds with improved activity in in vitro clotting assays, compared with known serine protease inhibitors, such as the activated partial thromboplastin time (aPTT) or prothrombin time (PT) assay. (for a description of the aPTT and PT assays see, Goodnight, S. H. et al., “Screening Tests of Hemostasis”,Disorders of Thrombosis and Hemostasis: A Clinical Guide,2nd Edition, pp. 41-51, McGraw-Hill, New York (2001)).

It is also desirable and preferable to find compounds with advantageous and improved characteristics compared with known serine protease inhibitors, in one or more of the following categories that are given as examples, and are not intended to be limiting: (a) pharmacokinetic properties, including oral bioavailability, half life, and clearance; (b) pharmaceutical properties; (c) dosage requirements; (d) factors that decrease blood concentration peak-to-trough characteristics; (e) factors that increase the concentration of active drug at the enzyme; (f) factors that decrease the liability for clinical drug-drug interactions; (g) factors that decrease the potential for adverse side-effects, including selectivity versus other biological targets; and (h) factors that improve manufacturing costs or feasibility.

Pre-clinical studies demonstrated significant antithrombotic effects of small molecule factor XIa inhibitors in rabbit and rat model of arterial thrombosis, at doses that preserved hemostasis. (Wong P. C. et al.,American Heart Association Scientific Sessions, Abstract No. 6118, Nov. 12-15, 2006; Schumacher, W. et al.,Journal of Thrombosis and Haemostasis,3(Suppl. 1):P1228 (2005); Schumacher, W. A. et al.,European Journal of Pharmacology, pp. 167-174 (2007)). Furthermore, it was observed that in vitro prolongation of the aPTT by specific XIa inhibitors is a good predictor of efficacy in our thrombosis models. Thus, the in vitro aPTT test can be used as a surrogate for efficacy in vivo.

As used herein, the term “patient” encompasses all mammalian species.

As used herein, “treating” or “treatment” cover the treatment of a disease-state in a mammal, particularly in a human, and include: (a) inhibiting the disease-state, i.e., arresting it development; and/or (b) relieving the disease-state, i.e., causing regression of the disease state.

As used herein, “prophylaxis” or “prevention” cover the preventive treatment of a subclinical disease-state in a mammal, particularly in a human, aimed at reducing the probability of the occurrence of a clinical disease-state. Patients are selected for preventative therapy based on factors that are known to increase risk of suffering a clinical disease state compared to the general population. “Prophylaxis” therapies can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment in a subject that has not yet presented with a clinical disease state, whereas secondary prevention is defined as preventing a second occurrence of the same or similar clinical disease state.

As used herein, “risk reduction” covers therapies that lower the incidence of development of a clinical disease state. As such, primary and secondary prevention therapies are examples of risk reduction.

“Therapeutically effective amount” is intended to include an amount of a compound of the present invention that is effective when administered alone or in combination to inhibit factor XIa and/or plasma kallikrein and/or to prevent or treat the disorders listed herein. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the preventive or therapeutic effect, whether administered in combination, serially, or simultaneously.

The term “thrombosis”, as used herein, refers to formation or presence of a thrombus (pl. thrombi); clotting within a blood vessel that may cause ischemia or infarction of tissues supplied by the vessel. The term “embolism”, as used herein, refers to sudden blocking of an artery by a clot or foreign material that has been brought to its site of lodgment by the blood current. The term “thromboembolism”, as used herein, refers to obstruction of a blood vessel with thrombotic material carried by the blood stream from the site of origin to plug another vessel. The term “thromboembolic disorders” entails both “thrombotic” and “embolic” disorders (defined above).

The term “thromboembolic disorders” as used herein includes arterial cardiovascular thromboembolic disorders, venous cardiovascular or cerebrovascular thromboembolic disorders, and thromboembolic disorders in the chambers of the heart or in the peripheral circulation. The term “thromboembolic disorders” as used herein also includes specific disorders selected from, but not limited to, unstable angina or other acute coronary syndromes, atrial fibrillation, first or recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, or procedures in which blood is exposed to an artificial surface that promotes thrombosis. The medical implants or devices include, but are not limited to: prosthetic valves, artificial valves, indwelling catheters, stents, blood oxygenators, shunts, vascular access ports, ventricular assist devices and artificial hearts or heart chambers, and vessel grafts. The procedures include, but are not limited to: cardiopulmonary bypass, percutaneous coronary intervention, and hemodialysis. In another embodiment, the term “thromboembolic disorders” includes acute coronary syndrome, stroke, deep vein thrombosis, and pulmonary embolism.

In another embodiment, the present invention provides a method for the treatment of a thromboembolic disorder, wherein the thromboembolic disorder is selected from unstable angina, an acute coronary syndrome, atrial fibrillation, myocardial infarction, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, or procedures in which blood is exposed to an artificial surface that promotes thrombosis. In another embodiment, the present invention provides a method for the treatment of a thromboembolic disorder, wherein the thromboembolic disorder is selected from acute coronary syndrome, stroke, venous thrombosis, atrial fibrillation, and thrombosis resulting from medical implants and devices.

In another embodiment, the present invention provides a method for the primary prophylaxis of a thromboembolic disorder, wherein the thromboembolic disorder is selected from unstable angina, an acute coronary syndrome, atrial fibrillation, myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, or procedures in which blood is exposed to an artificial surface that promotes thrombosis. In another embodiment, the present invention provides a method for the primary prophylaxis of a thromboembolic disorder, wherein the thromboembolic disorder is selected from acute coronary syndrome, stroke, venous thrombosis, and thrombosis resulting from medical implants and devices.

In another embodiment, the present invention provides a method for the secondary prophylaxis of a thromboembolic disorder, wherein the thromboembolic disorder is selected from unstable angina, an acute coronary syndrome, atrial fibrillation, recurrent myocardial infarction, transient ischemic attack, stroke, atherosclerosis, peripheral occlusive arterial disease, venous thrombosis, deep vein thrombosis, thrombophlebitis, arterial embolism, coronary arterial thrombosis, cerebral arterial thrombosis, cerebral embolism, kidney embolism, pulmonary embolism, and thrombosis resulting from medical implants, devices, or procedures in which blood is exposed to an artificial surface that promotes thrombosis. In another embodiment, the present invention provides a method for the secondary prophylaxis of a thromboembolic disorder, wherein the thromboembolic disorder is selected from acute coronary syndrome, stroke, atrial fibrillation and venous thrombosis.

The term “stroke”, as used herein, refers to embolic stroke or atherothrombotic stroke arising from occlusive thrombosis in the carotid communis, carotid interna, or intracerebral arteries.

It is noted that thrombosis includes vessel occlusion (e.g., after a bypass) and reocclusion (e.g., during or after percutaneous transluminal coronary angioplasty). The thromboembolic disorders may result from conditions including but not limited to atherosclerosis, surgery or surgical complications, prolonged immobilization, arterial fibrillation, congenital thrombophilia, cancer, diabetes, effects of medications or hormones, and complications of pregnancy.

Thromboembolic disorders are frequently associated with patients with atherosclerosis. Risk factors for atherosclerosis include but are not limited to male gender, age, hypertension, lipid disorders, and diabetes mellitus. Risk factors for atherosclerosis are at the same time risk factors for complications of atherosclerosis, i.e., thromboembolic disorders.

Similarly, arterial fibrillation is frequently associated with thromboembolic disorders. Risk factors for arterial fibrillation and subsequent thromboembolic disorders include cardiovascular disease, rheumatic heart disease, nonrheumatic mitral valve disease, hypertensive cardiovascular disease, chronic lung disease, and a variety of miscellaneous cardiac abnormalities as well as thyrotoxicosis.

Diabetes mellitus is frequently associated with atherosclerosis and thromboembolic disorders. Risk factors for the more common type 2 include but are not limited to are family history, obesity, physical inactivity, race/ethnicity, previously impaired fasting glucose or glucose tolerance test, history of gestational diabetes mellitus or delivery of a “big baby”, hypertension, low HDL cholesterol, and polycystic ovary syndrome.

Risk factors for congenital thrombophilia include gain of function mutations in coagulation factors or loss of function mutations in the anticoagulant- or fibrinolytic pathways.

Thrombosis has been associated with a variety of tumor types, e.g., pancreatic cancer, breast cancer, brain tumors, lung cancer, ovarian cancer, prostate cancer, gastrointestinal malignancies, and Hodgkins or non-Hodgkins lymphoma. Recent studies suggest that the frequency of cancer in patients with thrombosis reflects the frequency of a particular cancer type in the general population (Levitan, N. et al.,Medicine(Baltimore), 78(5):285-291 (1999); Levine M. et al.,N. Engl. J. Med.,334(11):677-681 (1996); Blom, J. W. et al.,JAMA,293(6):715-722 (2005)). Hence, the most common cancers associated with thrombosis in men are prostate, colorectal, brain, and lung cancer, and in women are breast, ovary, and lung cancer. The observed rate of venous thromboembolism (VTE) in cancer patients is significant. The varying rates of VTE between different tumor types are most likely related to the selection of the patient population. Cancer patients at risk for thrombosis may possess any or all of the following risk factors: (i) the stage of the cancer (i.e., presence of metastases), (ii) the presence of central vein catheters, (iii) surgery and anticancer therapies including chemotherapy, and (iv) hormones and antiangiogenic drugs. Thus, it is common clinical practice to dose patients having advanced tumors with heparin or low molecular heparin to prevent thromboembolic disorders. A number of low molecular heparin preparations have been approved by the FDA for these indications.

There are three main clinical situations when considering the prevention of VTE in a medical cancer patient: (i) the patient is bedridden for prolonged periods of time; (ii) the ambulatory patient is receiving chemotherapy or radiation; and (iii) the patient is with indwelling central vein catheters. Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are effective antithrombotic agents in cancer patients undergoing surgery. (Mismetti, P. et al.,British Journal of Surgery,88:913-930 (2001).)

A. In Vitro Assays

The effectiveness of compounds of the present invention as inhibitors of the coagulation Factors XIa, VIIa, IXa, Xa, XIIa, plasma kallikrein or thrombin, can be determined using a relevant purified serine protease, respectively, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at room temperature or at 37° C. Hydrolysis of the substrate resulted in the release of pNA (para nitroaniline), which was monitored spectrophotometrically by measuring the increase in absorbance at 405 nm, or the release of AMC (amino methylcoumarin), which was monitored spectrofluorometrically by measuring the increase in emission at 460 nm with excitation at 380 nm. A decrease in the rate of absorbance or fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.

Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.

Factor VIIa determinations were made in 0.005 M calcium chloride, 0.15 M sodium chloride, 0.05 M HEPES buffer containing 0.1% PEG 8000 at a pH of 7.5. Determinations were made using purified human Factor VIIa (Haematologic Technologies) or recombinant human Factor VIIa (Novo Nordisk) at a final assay concentration of 0.5-10 nM, recombinant soluble tissue factor at a concentration of 10-40 nM and the synthetic substrate H-D-11e-Pro-Arg-pNA (S-2288; CHROMOGENIX® or BMPM-2; AnaSpec) at a concentration of 0.001-0.0075 M.

Factor IXa determinations were made in 0.005 M calcium chloride, 0.1 M sodium chloride, 0.0000001 M Refludan (Berlex), 0.05 M TRIS base and 0.5% PEG 8000 at a pH of 7.4. Refludan was added to inhibit small amounts of thrombin in the commercial preparations of human Factor IXa. Determinations were made using purified human Factor IXa (Haematologic Technologies) at a final assay concentration of 20-100 nM and the synthetic substrate PCIXA2100-B (CenterChem) or Pefafluor IXa 3688 (H-D-Leu-Ph′Gly-Arg-AMC; CenterChem) at a concentration of 0.0004-0.0005 M.

Factor Xa determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human Factor Xa (Haematologic Technologies) at a final assay concentration of 150-1000 pM and the synthetic substrate S-2222 (Bz-11e-Glu (gamma-OMe, 50%)-Gly-Arg-pNA; CHROMOGENIX®) at a concentration of 0.0002-0.00035 M.

Factor XIIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000. Determinations were made using purified human Factor XIIa at a final concentration of 4 nM (American Diagnostica) and the synthetic substrate SPECTROZYME® #312 (H-D-CHT-Gly-L-Arg-pNA.2AcOH; American Diagnostica) at a concentration of 0.00015 M.

Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.

Thrombin determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human alpha thrombin (Haematologic Technologies or Enzyme Research Laboratories) at a final assay concentration of 200-250 pM and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.0004 M.

The Michaelis constant, Km, for substrate hydrolysis by each protease, was determined at 25° C. or 37° C. Values of Kiwere determined by allowing the protease to react with the substrate in the presence of the inhibitor. Reactions were allowed to go for periods of 20-180 minutes (depending on the protease) and the velocities (rate of absorbance or fluorescence change versus time) were measured. The following relationships were used to calculate Kivalues:
(vo−vs)/vs=I/(Ki(1+S/Km)) for a competitive inhibitor with one binding site; or
vs/vo=A+((B−A)/(1+((IC50/(I))n))); and
Ki=IC50/(1+S/Km) for a competitive inhibitor,
where:

vois the velocity of the control in the absence of inhibitor;

vsis the velocity in the presence of inhibitor;

I is the concentration of inhibitor;

A is the minimum activity remaining (usually locked at zero);

B is the maximum activity remaining (usually locked at 1.0);

n is the Hill coefficient, a measure of the number and cooperativity of potential inhibitor binding sites;

IC50is the concentration of inhibitor that produces 50% inhibition under the assay conditions;

Kiis the dissociation constant of the enzyme:inhibitor complex;

S is the concentration of substrate; and

Kmis the Michaelis constant for the substrate.

The selectivity of a compound may be evaluated by taking the ratio of the Kivalue for a given protease with the Kivalue for the protease of interest (i.e., selectivity for FXIa versus protease P=Kifor protease P/Kifor FXIa). Compounds with selectivity ratios >20 are considered selective. Compounds with selectivity ratios >100 are preferred, and compounds with selectivity ratios >500 are more preferred.

The effectiveness of compounds of the present invention as inhibitors of coagulation can be determined using a standard or modified clotting assay. An increase in the plasma clotting time in the presence of inhibitor is indicative of anticoagulation. Relative clotting time is the clotting time in the presence of an inhibitor divided by the clotting time in the absence of an inhibitor. The results of this assay may be expressed as IC1.5× or IC2×, the inhibitor concentration required to increase the clotting time by 50 or 100 percent, respectively. The IC1.5× or IC2× is found by linear interpolation from relative clotting time versus inhibitor concentration plots using inhibitor concentration that spans the IC1.5× or IC2×.

Clotting times are determined using citrated normal human plasma as well as plasma obtained from a number of laboratory animal species (e.g., rat, or rabbit). A compound is diluted into plasma beginning with a 10 mM DMSO stock solution. The final concentration of DMSO is less than 2%. Plasma clotting assays are performed in an automated coagulation analyzer (Sysmex, Dade-Behring, Illinois). Similarly, clotting times can be determined from laboratory animal species or humans dosed with compounds of the invention.

Activated Partial Thromboplastin Time (aPTT) is determined using ALEXIN® (Trinity Biotech, Ireland) or ACTIN® (Dade-Behring, Illinois) following the directions in the package insert. Plasma (0.05 mL) is warmed to 37° C. for 1 minute. ALEXIN® or ACTIN® (0.05 mL) is added to the plasma and incubated for an additional 2 to 5 minutes. Calcium chloride (25 mM, 0.05 mL) is added to the reaction to initiate coagulation. The clotting time is the time in seconds from the moment calcium chloride is added until a clot is detected.

Prothrombin Time (PT) is determined using thromboplastin (Thromboplastin C Plus or INNOVIN®, Dade-Behring, Illinois) following the directions in the package insert. Plasma (0.05 mL) is warmed to 37° C. for 1 minute. Thromboplastin (0.1 mL) is added to the plasma to initiate coagulation. The clotting time is the time in seconds from the moment thromboplastin is added until a clot is detected.

The exemplified Examples disclosed below were tested in the Factor XIa assay described above and found having Factor XIa inhibitory activity. A range of Factor XIa inhibitory activity (Ki values) of ≦10 μM (10000 nM) was observed. Table 1 below lists Factor XIa Ki values measured for the following examples.

The effectiveness of compounds of the present invention as antithrombotic agents can be determined using relevant in vivo thrombosis models, including In Vivo Electrically-induced Carotid Artery Thrombosis Models and In Vivo Rabbit Arterio-venous Shunt Thrombosis Models.

The rabbit ECAT model, described by Wong et al. (J. Pharmacol. Exp. Ther.,295:212-218 (2000)), can be used in this study. Male New Zealand White rabbits are anesthetized with ketamine (50 mg/kg+50 mg/kg/h IM) and xylazine (10 mg/kg+10 mg/kg/h IM). These anesthetics are supplemented as needed. An electromagnetic flow probe is placed on a segment of an isolated carotid artery to monitor blood flow. Test agents or vehicle will be given (i.v., i.p., s.c., or orally) prior to or after the initiation of thrombosis. Drug treatment prior to initiation of thrombosis is used to model the ability of test agents to prevent and reduce the risk of thrombus formation, whereas dosing after initiation is used to model the ability to treat existing thrombotic disease. Thrombus formation is induced by electrical stimulation of the carotid artery for 3 min at 4 mA using an external stainless-steel bipolar electrode. Carotid blood flow is measured continuously over a 90-min period to monitor thrombus-induced occlusion. Total carotid blood flow over 90 min is calculated by the trapezoidal rule. Average carotid flow over 90 min is then determined by converting total carotid blood flow over 90 min to percent of total control carotid blood flow, which would result if control blood flow had been maintained continuously for 90 min. The ED50(dose that increased average carotid blood flow over 90 min to 50% of the control) of compounds are estimated by a nonlinear least square regression program using the Hill sigmoid Emaxequation (DeltaGraph; SPSS Inc., Chicago, Ill.).

b. In Vivo Rabbit Arterio-Venous (AV) Shunt Thrombosis Model

The rabbit AV shunt model, described by Wong et al. (Wong, P. C. et al.,J. Pharmacol. Exp. Ther.292:351-357 (2000)), can be used in this study. Male New Zealand White rabbits are anesthetized with ketamine (50 mg/kg+50 mg/kg/h IM) and xylazine (10 mg/kg+10 mg/kg/h IM). These anesthetics are supplemented as needed. The femoral artery, jugular vein and femoral vein are isolated and catheterized. A saline-filled AV shunt device is connected between the femoral arterial and the femoral venous cannulae. The AV shunt device consists of an outer piece of tygon tubing (length=8 cm; internal diameter=7.9 mm) and an inner piece of tubing (length=2.5 cm; internal diameter=4.8 mm). The AV shunt also contains an 8-cm-long 2-0 silk thread (Ethicon, Somerville, N.J.). Blood flows from the femoral artery via the AV-shunt into the femoral vein. The exposure of flowing blood to a silk thread induces the formation of a significant thrombus. Forty minutes later, the shunt is disconnected and the silk thread covered with thrombus is weighed. Test agents or vehicle will be given (i.v., i.p., s.c., or orally) prior to the opening of the AV shunt. The percentage inhibition of thrombus formation is determined for each treatment group. The ID50values (dose that produces 50% inhibition of thrombus formation) are estimated by a nonlinear least square regression program using the Hill sigmoid Emaxequation (DeltaGraph; SPSS Inc., Chicago, Ill.).

The anti-inflammatory effect of these compounds can be demonstrated in an Evans Blue dye extravasation assay using C1-esterase inhibitor deficient mice. In this model, mice are dosed with a compound of the present invention, Evans Blue dye is injected via the tail vein, and extravasation of the blue dye is determined by spectrophotometric means from tissue extracts.

The ability of the compounds of the current invention to reduce or prevent the systemic inflammatory response syndrome, for example, as observed during on-pump cardiovascular procedures, can be tested in in vitro perfusion systems, or by on-pump surgical procedures in larger mammals, including dogs and baboons. Read-outs to assess the benefit of the compounds of the present invention include for example reduced platelet loss, reduced platelet/white blood cell complexes, reduced neutrophil elastase levels in plasma, reduced activation of complement factors, and reduced activation and/or consumption of contact activation proteins (plasma kallikrein, factor XII, factor XI, high molecular weight kininogen, C1-esterase inhibitors).

The compounds of the present invention may also be useful as inhibitors of additional serine proteases, notably human thrombin, human plasma kallikrein and human plasmin. Because of their inhibitory action, these compounds are indicated for use in the prevention or treatment of physiological reactions, including blood coagulation, fibrinolysis, blood pressure regulation and inflammation, and wound healing catalyzed by the aforesaid class of enzymes. Specifically, the compounds have utility as drugs for the treatment of diseases arising from elevated thrombin activity of the aforementioned serine proteases, such as myocardial infarction, and as reagents used as anticoagulants in the processing of blood to plasma for diagnostic and other commercial purposes.

V. Pharmaceutical Compositions, Formulations and Combinations

The compounds of this invention can be administered in such oral dosage forms as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. They may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts. They can be administered alone, but generally will be administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

The term “pharmaceutical composition” means a composition comprising a compound of the invention in combination with at least one additional pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” refers to media generally accepted in the art for the delivery of biologically active agents to animals, in particular, mammals, including, i.e., adjuvant, excipient or vehicle, such as diluents, preserving agents, fillers, flow regulating agents, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms. Pharmaceutically acceptable carriers are formulated according to a number of factors well within the purview of those of ordinary skill in the art. These include, without limitation: the type and nature of the active agent being formulated; the subject to which the agent-containing composition is to be administered; the intended route of administration of the composition; and the therapeutic indication being targeted. Pharmaceutically acceptable carriers include both aqueous and non-aqueous liquid media, as well as a variety of solid and semi-solid dosage forms. Such carriers can include a number of different ingredients and additives in addition to the active agent, such additional ingredients being included in the formulation for a variety of reasons, e.g., stabilization of the active agent, binders, etc., well known to those of ordinary skill in the art. Descriptions of suitable pharmaceutically acceptable carriers, and factors involved in their selection, are found in a variety of readily available sources such as, for example,Remington's Pharmaceutical Sciences,18th Edition (1990).

By way of general guidance, the daily oral dosage of each active ingredient, when used for the indicated effects, will range between about 0.001 to about 1000 mg/kg of body weight, preferably between about 0.01 to about 100 mg/kg of body weight per day, and most preferably between about 0.1 to about 20 mg/kg/day. Intravenously, the most preferred doses will range from about 0.001 to about 10 mg/kg/minute during a constant rate infusion. Compounds of this invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three, or four times daily.

Compounds of this invention can also be administered by parenteral administration (e.g., intra-venous, intra-arterial, intramuscularly, or subcutaneously. When administered intra-venous or intra-arterial, the dose can be given continuously or intermittent. Furthermore, formulation can be developed for intramuscularly and subcutaneous delivery that ensure a gradual release of the active pharmaceutical ingredient.

Compounds of this invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using transdermal skin patches. When administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.

The compounds are typically administered in admixture with suitable pharmaceutical diluents, excipients, or carriers (collectively referred to herein as pharmaceutical carriers) suitably selected with respect to the intended form of administration, e.g., oral tablets, capsules, elixirs, and syrups, and consistent with conventional pharmaceutical practices.

Dosage forms (pharmaceutical compositions) suitable for administration may contain from about 1 milligram to about 1000 milligrams of active ingredient per dosage unit. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.1-95% by weight based on the total weight of the composition.

Suitable pharmaceutical carriers are described inRemington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

Where the compounds of this invention are combined with other anticoagulant agents, for example, a daily dosage may be about 0.1 to about 100 milligrams of the compound of the present invention and about 0.1 to about 100 milligrams per kilogram of patient body weight. For a tablet dosage form, the compounds of this invention generally may be present in an amount of about 5 to about 100 milligrams per dosage unit, and the second anti-coagulant in an amount of about 1 to about 50 milligrams per dosage unit.

Where the compounds of the present invention are administered in combination with an anti-platelet agent, by way of general guidance, typically a daily dosage may be about 0.01 to about 25 milligrams of the compound of the present invention and about 50 to about 150 milligrams of the anti-platelet agent, preferably about 0.1 to about 1 milligrams of the compound of the present invention and about 1 to about 3 milligrams of anti-platelet agents, per kilogram of patient body weight.

Where the compounds of the present invention are administered in combination with thrombolytic agent, typically a daily dosage may be about 0.1 to about 1 milligrams of the compound of the present invention, per kilogram of patient body weight and, in the case of the thrombolytic agents, the usual dosage of the thrombolyic agent when administered alone may be reduced by about 50-80% when administered with a compound of the present invention.

In another embodiment, the present invention provides a pharmaceutical composition further comprising additional therapeutic agent(s) selected from an antiarrhythmic agent, an anti-hypertensive agent, an anti-coagulant agent, an anti-platelet agent, a thrombin inhibiting agent, a thrombolytic agent, a fibrinolytic agent, a calcium channel blocker, a potassium channel blocker, a cholesterol/lipid lowering agent, or a combination thereof.

In another embodiment, the present invention provides pharmaceutical composition, wherein the additional therapeutic agent(s) are an anti-platelet agent or a combination thereof.

In another embodiment, the present invention provides a pharmaceutical composition, wherein the additional therapeutic agent is the anti-platelet agent clopidogrel.

The compounds of the present invention can be administered alone or in combination with one or more additional therapeutic agents. By “administered in combination” or “combination therapy” it is meant that the compound of the present invention and one or more additional therapeutic agents are administered concurrently to the mammal being treated. When administered in combination, each component may be administered at the same time or sequentially in any order at different points in time. Thus, each component may be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

Compounds that can be administered in combination with the compounds of the present invention include, but are not limited to, anticoagulants, anti-thrombin agents, anti-platelet agents, fibrinolytics, hypolipidemic agents, antihypertensive agents, and anti-ischemic agents.

Other anticoagulant agents (or coagulation inhibitory agents) that may be used in combination with the compounds of this invention include warfarin, heparin (either unfractionated heparin or any commercially available low molecular weight heparin, for example LOVENOX®), synthetic Pentasaccharide, direct acting thrombin inhibitors including hirudin and argatroban, as well as other factor VIIa inhibitors, factor IXa inhibitors, factor Xa inhibitors (e.g., ARIXTRA®, apixaban, rivaroxaban, LY-517717, DU-176b, DX-9065a, and those disclosed in WO 98/57951, WO 03/026652, WO 01/047919, and WO 00/076970), factor XIa inhibitors, and inhibitors of activated TAFI and PAI-1 known in the art.

Other examples of suitable anti-platelet agents for use in combination with the compounds of the present invention, with or without aspirin, are ADP (adenosine diphosphate) receptor antagonists, preferably antagonists of the purinergic receptors P2Y1and P2Y12, with P2Y12being even more preferred. Preferred P2Y12receptor antagonists include clopidogrel, ticlopidine, prasugrel, ticagrelor, and cangrelor, and pharmaceutically acceptable salts or prodrugs thereof. Ticlopidine and clopidogrel are also preferred compounds since they are known to be more gentle than aspirin on the gastro-intestinal tract in use. Clopidogrel is an even more preferred agent.

A preferred example is a triple combination of a compound of the present invention, aspirin, and another anti-platelet agent. Preferably, the anti-platelet agent is clopidogrel or prasugrel, more preferably clopidogrel.

The term thrombin inhibitors (or anti-thrombin agents), as used herein, denotes inhibitors of the serine protease thrombin. By inhibiting thrombin, various thrombin-mediated processes, such as thrombin-mediated platelet activation (that is, for example, the aggregation of platelets, and/or the secretion of platelet granule contents including serotonin) and/or fibrin formation are disrupted. A number of thrombin inhibitors are known to one of skill in the art and these inhibitors are contemplated to be used in combination with the present compounds. Such inhibitors include, but are not limited to, boroarginine derivatives, boropeptides, heparins, hirudin, argatroban, dabigatran, AZD-0837, and those disclosed in WO 98/37075 and WO 02/044145, and pharmaceutically acceptable salts and prodrugs thereof. Boroarginine derivatives and boropeptides include N-acetyl and peptide derivatives of boronic acid, such as C-terminal a-aminoboronic acid derivatives of lysine, ornithine, arginine, homoarginine and corresponding isothiouronium analogs thereof. The term hirudin, as used herein, includes suitable derivatives or analogs of hirudin, referred to herein as hirulogs, such as disulfatohirudin.

The term thrombolytic (or fibrinolytic) agents (or thrombolytics or fibrinolytics), as used herein, denotes agents that lyse blood clots (thrombi). Such agents include tissue plasminogen activator (TPA, natural or recombinant) and modified forms thereof, anistreplase, urokinase, streptokinase, tenecteplase (TNK), lanoteplase (nPA), factor VIIa inhibitors, thrombin inhibitors, inhibitors of factors IXa, Xa, and XIa, PAI-I inhibitors (i.e., inactivators of tissue plasminogen activator inhibitors), inhibitors of activated TAFI, alpha-2-antiplasmin inhibitors, and anisoylated plasminogen streptokinase activator complex, including pharmaceutically acceptable salts or prodrugs thereof. The term anistreplase, as used herein, refers to anisoylated plasminogen streptokinase activator complex, as described, for example, in European Patent Application No. 028,489, the disclosure of which is hereby incorporated herein by reference herein. The term urokinase, as used herein, is intended to denote both dual and single chain urokinase, the latter also being referred to herein as prourokinase.

The compounds of the present invention are also useful as standard or reference compounds, for example as a quality standard or control, in tests or assays involving the inhibition of thrombin, Factor VIIa, IXa, Xa, XIa, and/or plasma kallikrein. Such compounds may be provided in a commercial kit, for example, for use in pharmaceutical research involving thrombin, Factor VIIa, IXa, Xa, XIa, and/or plasma kallikrein. XIa. For example, a compound of the present invention could be used as a reference in an assay to compare its known activity to a compound with an unknown activity. This would ensure the experimentor that the assay was being performed properly and provide a basis for comparison, especially if the test compound was a derivative of the reference compound. When developing new assays or protocols, compounds according to the present invention could be used to test their effectiveness.

The compounds of the present invention may also be used in diagnostic assays involving thrombin, Factor VIIa, IXa, Xa, XIa, and/or plasma kallikrein. For example, the presence of thrombin, Factor VIIa, IXa, Xa XIa, and/or plasma kallikrein in an unknown sample could be determined by addition of the relevant chromogenic substrate, for example S2366 for Factor XIa, to a series of solutions containing test sample and optionally one of the compounds of the present invention. If production of pNA is observed in the solutions containing test sample, but not in the presence of a compound of the present invention, then one would conclude Factor XIa was present.

Extremely potent and selective compounds of the present invention, those having Kivalues less than or equal to 0.001 μM against the target protease and greater than or equal to 0.1 μM against the other proteases, may also be used in diagnostic assays involving the quantitation of thrombin, Factor VIIa, IXa, Xa, XIa, and/or plasma kallikrein in serum samples. For example, the amount of Factor XIa in serum samples could be determined by careful titration of protease activity in the presence of the relevant chromogenic substrate, S2366, with a potent and selective Factor XIa inhibitor of the present invention.

The present invention also encompasses an article of manufacture. As used herein, article of manufacture is intended to include, but not be limited to, kits and packages. The article of manufacture of the present invention, comprises: (a) a first container; (b) a pharmaceutical composition located within the first container, wherein the composition, comprises: a first therapeutic agent, comprising: a compound of the present invention or a pharmaceutically acceptable salt form thereof; and, (c) a package insert stating that the pharmaceutical composition can be used for the treatment of a thromboembolic and/or inflammatory disorder (as defined previously). In another embodiment, the package insert states that the pharmaceutical composition can be used in combination (as defined previously) with a second therapeutic agent to treat a thromboembolic and/or inflammatory disorder. The article of manufacture can further comprise: (d) a second container, wherein components (a) and (b) are located within the second container and component (c) is located within or outside of the second container. Located within the first and second containers means that the respective container holds the item within its boundaries.

The first container is a receptacle used to hold a pharmaceutical composition. This container can be for manufacturing, storing, shipping, and/or individual/bulk selling. First container is intended to cover a bottle, jar, vial, flask, syringe, tube (e.g., for a cream preparation), or any other container used to manufacture, hold, store, or distribute a pharmaceutical product.

The second container is one used to hold the first container and, optionally, the package insert. Examples of the second container include, but are not limited to, boxes (e.g., cardboard or plastic), crates, cartons, bags (e.g., paper or plastic bags), pouches, and sacks. The package insert can be physically attached to the outside of the first container via tape, glue, staple, or another method of attachment, or it can rest inside the second container without any physical means of attachment to the first container. Alternatively, the package insert is located on the outside of the second container. When located on the outside of the second container, it is preferable that the package insert is physically attached via tape, glue, staple, or another method of attachment. Alternatively, it can be adjacent to or touching the outside of the second container without being physically attached.

The package insert is a label, tag, marker, etc. that recites information relating to the pharmaceutical composition located within the first container. The information recited will usually be determined by the regulatory agency governing the area in which the article of manufacture is to be sold (e.g., the United States Food and Drug Administration). Preferably, the package insert specifically recites the indications for which the pharmaceutical composition has been approved. The package insert may be made of any material on which a person can read information contained therein or thereon. Preferably, the package insert is a printable material (e.g., paper, plastic, cardboard, foil, adhesive-backed paper or plastic, etc.) on which the desired information has been formed (e.g., printed or applied).

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments that are given for illustration of the invention and are not intended to be limiting thereof. The following Examples have been prepared, isolated and characterized using the methods disclosed herein.

VI. General Synthesis Including Schemes

The compounds of the present invention may be synthesized by many methods available to those skilled in the art of organic chemistry (Maffrand, J. P. et al.,Heterocycles,16(1):35-37 (1981)). General synthetic schemes for preparing compounds of the present invention are described below. These schemes are illustrative and are not meant to limit the possible techniques one skilled in the art may use to prepare the compounds disclosed herein. Different methods to prepare the compounds of the present invention will be evident to those skilled in the art. Additionally, the various steps in the synthesis may be performed in an alternate sequence in order to give the desired compound or compounds.

Examples of compounds of the present invention prepared by methods described in the general schemes are given in the intermediates and examples section set out hereinafter. Example compounds are typically prepared as racemic mixtures. Preparation of homochiral examples may be carried out by techniques known to one skilled in the art. For example, homochiral compounds may be prepared by separation of racemic products by chiral phase preparative HPLC. Alternatively, the example compounds may be prepared by methods known to give enantiomerically enriched products. These include, but are not limited to, the incorporation of chiral auxiliary functionalities into racemic intermediates which serve to control the diastereoselectivity of transformations, providing enantio-enriched products upon cleavage of the chiral auxiliary.

The compounds of the present invention can be prepared in a number of ways known to one skilled in the art of organic synthesis. The compounds of the present invention can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic organic chemistry, or by variations thereon as appreciated by those skilled in the art. Preferred methods include, but are not limited to, those described below. The reactions are performed in a solvent or solvent mixture appropriate to the reagents and materials employed and suitable for the transformations being effected. It will be understood by those skilled in the art of organic synthesis that the functionality present on the molecule should be consistent with the transformations proposed. This will sometimes require a judgment to modify the order of the synthetic steps or to select one particular process scheme over another in order to obtain a desired compound of the invention.

It will also be recognized that another major consideration in the planning of any synthetic route in this field is the judicious choice of the protecting group used for protection of the reactive functional groups present in the compounds described in this invention. An authoritative account describing the many alternatives to the trained practitioner is Greene et al. (Protective Groups in Organic Synthesis,4th Edition, Wiley-Interscience (2006)).

Certain 2-bromoacetophenone analogs (1b) that are not commercially available but used in the current invention may be synthesized from commercially available starting materials as described in Scheme 1. Acetophenone derivatives 1a can be treated with a brominating reagent such as bromine in a solvent such as CHCl3to give 1b. Alternatively, acetophenone derivatives la can be treated with either copper (II) bromide in a solvent such as EtOAc at elevated temperature or phenyltrimethylammonium tribromide in a solvent such as THF at low temperature to provide 1b. Benzoic acid derivatives 1c can be treated sequentially with oxalyl chloride in a suitable solvent, such as DCM, containing a few drops of DMF, and then treated with trimethylsilyldiazomethane in a suitable solvent or solvent combination, such as ACN and hexane. The intermediate diazoketone is isolated and treated with aqueous hydrobromic acid and DCM to provide 1b. Alternatively the benzoic acid derivatives 1c can be converted to the acetophenone derivatives 1a in three steps as described in Scheme 1. Alternatively, Stille coupling between a suitably substituted aryl halide or triflate and tributyl-(1-ethoxyvinyl) stannane with a palladium catalyst, such as bis-(triphenylphosphine)palladium dichloride, in a suitable solvent, such as toluene, at elevated temperature yields the enol ether 1e, which can then be converted to 1b with N-bromosuccinimide.

Triazole acids of this invention such as 2c, 2d, 2e, 2f can be easily prepared from readily accessible anilines in a three step process outlined in Scheme 2. Formation of the arylazide (2b) intermediate via diazotization and displacement with sodium azide followed by condensation with appropriate acetylenic compounds and removal of the protecting groups known to those in the art should afford intermediates such as 2c. Condensation of the arylazides with either malonates or ketoesters followed by hydrolysis should afford intermediates of this invention such as 2d, 2e and 2f. In cases wherein the anilines are not available, the corresponding arylcarboxylic acids can be used which are then converted to the anilines via the Curtius rearrangement. Alternatively haloaryl intermediates can be lithiated via BuLi and reacted with CO2to afford the corresponding carboxylic acids which can then be converted to the anilines as outlined below.

Substituted hydrazine (3a) of this invention can be obtained from commercial sources or can be made from the corresponding anilines via diazotization followed by reduction with tin chloride. These can be reacted either directly or after isolation with an appropriate malononitrile to afford aminopyrazole such as compound 3b. Treatment of 3b with isoamylnitrite in THF under elevated temperatures should provide the requisite pyrazole intermediate which is hydrolyzed to afford pyrazole acid intermediates of this invention such as 3c. Furthermore amino pyrazole intermediates of this invention can be obtained by hydrolysis of the ester will give 3d. Diazotization of the amino moiety as in 3b in fluoroboric acid followed by heating at high temperature should then provide fluoropyrazole intermediates such as 3e. Appropriately substituted hydrazines can be condensed with (E)-ethyl 2-((dimethylamino)methylene)-3-oxobutanoate to give, after hydrolysis, the methylpyrazole derivatives 3f.

Alternative approaches to pyrazoles can also be obtained via the Chan-Lam coupling as shown in Scheme 4. The requisite pyrazole (4b) and appropriately substituted boronic acids (4a) are commercially available. Alternatively these entities could be coupled via the Ullman coupling methodology with CuI, K2CO3in DMSO at 130° C. In these cases the boronic acid derivatives would be substituted with the arylbromides or iodides.

Imidazole acids of this invention such as 4af and 4ag can be prepared as outlined in Scheme 4a. Ullman coupling between an appropriately substituted imidazole 4aa and an appropriately substituted arylhalide 4ab can provide the imidazole derivatives 4ad and 4ae in one step. Hydrolysis of the ester will generate the imidazole acids 4af and 4ag. Alternatively, an appropriately substituted imidazole 4aa can be coupled to an appropriately substituted arylboronic acid 4ac using a modified procedure described by Sreedhar (Synthesis,5:795 (2008)). Alternative approaches to the imidazole derivatives 4ad and 4ae can be achieved using a modified procedure described by Gomez-Sanchez (J. Heterocyclic Chem.,24:1757 (1987)). Condensation of the ethyl nitroacetate, triethyl orthoformate, and an appropriately substituted aniline (4ah) can provide ethyl 3-arylamino-2-nitroacrylate 4ai. The ethyl 3-arylamino-2-nitrocrotonate derivatives 4aj can be prepared by reacting ethyl 3-ethoxy-2-nitrocrotonate with an appropriately substituted aniline 4ah. Reacting compounds 4ai and 4aj with triethyl orthoformate and platinum on carbon under a hydrogen atmosphere at elevated temperature can yield the imidazole derivatives 4ad and 4ae. Hydrolysis of the ester will generate the imidazole acids 4af and 4ag.

Bicyclic pyrazole intermediates of this invention can be constructed via the methodology outline in Scheme 5. Reaction of the hydrazine (3a) with an appropriate aldehyde should afford the hydrazone 5a which on chlorination with NCS followed by condensation with an appropriate malonate should lead to pyrazole such as 5b. Coupling of the requisite acid with macrocyclic amines of this invention should lead to carboxamide pyrazole 5c which can be converted to the compounds of this invention via methods outlined or known to those in the art.

Intermediates for preparation of compounds of this invention wherein ring B is an imidazole ring, can be prepared from an appropriately N-protected allylglycine (6a) according to the general method outlined in Scheme 6 (Contour-Galcera et al.,Bioorg. Med. Chem. Lett.,11(5):741-745 (2001)). Condensation of 6a with a suitably substituted bromoacetophenone (1b) in the presence of a suitable base such as potassium bicarbonate, K2CO3or Cs2CO3in a suitable solvent such as DMF provides a keto ester intermediate which can be cyclized to afford an imidazole (6c) by heating in the presence of excess ammonium acetate in a solvent such as toluene or xylene. This latter transformation can be conveniently carried out on small scale at 160° C. in a microwave reactor or on larger scale by refluxing the mixture while removing water via a Dean-Stark trap. The resulting imidazole intermediate (6c) is then protected by treatment with SEM-Cl in the presence of a base such as sodium hydride or dicyclohexylmethylamine in a solvent such as THF or DCM. The aryl bromide (6d) is then converted to the corresponding aniline (6e) by heating in a sealed vessel with excess ammonium hydroxide, in the presence of copper iodide, a base such as Cs2CO3and a catalytic amount of proline in DMSO as solvent. Acylation of 6e with the appropriate alkenoic acid and a coupling agent such as T3P or BOP reagent, or alternately, by treatment with an alkenoic acid chloride in the presence of a base such as TEA or DIEA provides diene 6f, which undergoes ring closing metathesis by heating in dilute solution in the presence of p-toluene sulfonic acid and Grubbs II catalyst in a suitable solvent such as DCM or DCE to provide the corresponding macrocycle (6g) (Tetrahedron Letters,44:1379 (2003)). Alternately, the RCM can be run in a microwave at elevated temperatures without pTsOH. Chlorination on the imidazole ring with NCS, or initial reduction of the double bond followed by chlorination, and deprotection provides intermediates 6h and 6i, respectively. Alternately, for compounds wherein R10═CN, catalytic hydrogenation of 6g followed by bromination with NBS at room temperature and subsequent palladium-catalyzed cyanation and deprotection provides intermediate 6j. Intermediates 6h-j can be converted to compounds of this invention following the steps described in Scheme 15.

Representative imidazole containing amide macrocycle intermediates useful for the synthesis of compounds of this invention are described in Scheme 7. The aniline 6e can be coupled with an appropriately substituted carboxylic acid 7a using propane phosphonic acid anhydride (T3P) to give the amide 7b (n=0) and 7c (n=1). Using a modified procedure described by Lovely (Tetrahedron Letters,44:1379 (2003)), 7b and 7c, following pretreatment with p-TsOH to form the imidazolinium ion, can be cyclized via ring-closing metathesis using a catalyst, such as Grubbs (II), in a suitable solvent, such as DCM, DCE, or toluene at elevated temperature, to give the imidazole-containing macrocycles 7d (n=0) and 7e (n=1). The alkene can then be reduced with hydrogen over either palladium on carbon or platinum oxide and subsequent deprotection with TFA in DCM provides amine 7f and 7g. Compounds of the formulae 7f and 7g can be converted to compounds in this invention according to Scheme 15.

Representative regioisomeric imidazole containing amide macrocycle intermediates useful for the synthesis of compounds of this invention are described in Scheme 7a. An appropriately N-protected allylglycine 6a can be converted to the bromoketone 7ab in two steps. Condensation of 7ab with formamidine at elevated temperature generates the imidazole 7ac. The imidazole 7ac can be protected with SEM-Cl and then deprotonation with nBuLi and subsequent quenching with NBS provides the bromo imidazole 7ae. Suzuki-Miyaura coupling between bromo imidazole Tae and an appropriately substituted aryl or heteroaryl boronic acid or ester 11e in the presence of a base such as K3PO4using a precatalyst such as Pd(dppf)Cl2.CH2Cl2complex provides, after separation of the enantiomers, aniline 7af. Aniline 7af can be converted to 7ag and 7ah according to Scheme 7. Compounds of the formulae 7ag and 7ah can be converted to compounds in this invention according to Scheme 15.

Alternatively, imidazole containing macrocycles of this invention can be derived from intermediate 8e according to Scheme 8. Ullmann type coupling reaction of compound 6d and allyl glycine, followed by methylation of the acid would provide the extended aniline analog 8b. Ring closing metathesis of the diene 8b using Grubb II catalyst would provide the macrocyclic olefin 8c. Then, the macrocyclic olefin 8c may be converted to the key intermediate 8e via hydrogenation and selective deprotection of the amine protecting group from compound 8d. The amine 8e may be converted to the corresponding cyclic carbamate or other analogs following the procedures described in Scheme 15. The other diastereomer at the methyl ester position can also be made in the same way as described above.

The cyano or chloro imidazole analog of intermediate 8e may be obtained by a slightly modified sequence of Scheme 9. The aniline nitrogen in compound 8b may be protected with a trifluoroacetyl group (TFA) in order to suppress bromination/chlorination on the phenyl group during conversion of compound 9b to 9c. Following the same sequence as outlined in Scheme 8, the resulting protected aniline 9a may be converted to macrocyclic compound 9b. Bromination or chlorination of 9b with NBS or NCS respectively provides intermediates 9c. For compounds wherein R10is CN, bromide 9c is converted to cyanoimidazole 9d by palladium-catalyzed cyanation as described in Scheme 6 above. Selective removal of the amine protecting group from compound 9d provides amine intermediates 9e. For example a Boc protecting group can be selectively removed either under mild acidic conditions or thermally by heating in trifluoroethanol in a microwave at 150° C. Intermediate 9e can be converted to the final compounds described in this invention according to Scheme 15.

Alternatively, imidazole compounds of this invention can be derived from trifluoromethyl substituted macrocycle intermediates, 10c which can be prepared from aniline 6e following the sequence described in Scheme 10. A condensation reaction of the aniline 6e with trifluoroacetaldehyde ethyl hemiacetal provides the aminal 10a. Treatment of 10a with allyl Grignard reagent, provides aniline 10b, which is then converted to the target compound 10c via the sequence described in Scheme 6.

Representative compounds of this invention where ring B is a six-membered heterocycle (example—pyridine) can be derived from intermediates 111, the synthesis of which is described in Scheme 11. Condensation of aldehyde 11a (X═N) prepared according to a modified procedure described by Negi (Synthesis,991 (1996)), with (S)-2-methylpropane-2-sulfinamide in the presence of anhydrous copper sulfate in a solvent such as DCM gives the sulfinimine 11b (Ellman, J.,J. Org. Chem.,64:1278 (1999)). Using a modified procedure described by Kuduk (Tetrahedron Letters,45:6641 (2004)), suitably substituted Grignard reagents, for example allylmagnesium bromide, can be added to sulfinimine 11b to give a sulfinamide 11e, as a mixture of diastereomers which can be separated at various stages of the sequence. The diastereoselectivity for the addition of allymagnesium bromide to sulfinimine 11b can be improved by employing indium(III) chloride according to a modified procedure of Xu (Xu, M-H,Organic Letters,10(6):1259 (2008)). Suzuki-Miyaura coupling between 4-chloropyridine 1c and an appropriately substituted aryl or heteroaryl boronic acid or ester 11e in the presence of a base such as potassium phosphate, in a solvent mixture, such as DMSO and H2O, or DMF, using a precatalyst such as Pd(dppf)Cl2.CH2Cl2complex provides 11g. Alternatively, the Suzuki-Miyaura coupling between boronic acid 11d and an appropriately substituted aryl or heteroaryl halide 11f can be used to prepared 11g. Protecting group interconversion can be accomplished in two steps to give 11h. Alternatively, the protecting group interconversion can take place initially on 11e followed by the Suzuki-Miyaura coupling. The aniline 11h can then be coupled with an appropriately substituted carboxylic acid 11i using T3P and a base, such as pyridine, to give the amide 11j. Using a modified procedure described by Lovely (Tetrahedron Letters, 44:1379 (2003)), 11j, following pretreatment with p-toluenesulfonic acid to form the pyridinium ion, can be cyclized via ring-closing metathesis using a catalyst, such as Grubbs (II), in a suitable solvent, such as DCM, DCE, or toluene at elevated temperature, to give the pyridine-containing macrocycle 11k. The alkene can be reduced with hydrogen over either palladium on carbon or platinum oxide, and subsequent deprotection with TFA in DCM or 4M HCl in dioxane provides amine 11l. Compounds of the formulae 11l can be converted to compounds in this invention according to Scheme 15.

Additional pyridine containing macrocycles useful for the synthesis of compounds of this invention can also be prepared according to Scheme 11. In cases where the pyridine core is a 4-pyridine (Z═N) rather than the 2-pyridine (X═N), conversion of 11h to 11j can be easily accomplished by using an acid chloride of 11i. Intermediates of formulae 11g where R8═NO2may be modified further to give intermediates where R8═NH CO2—C1-4alkyl either before coupling with acid 11i or after coupling with acid. Reduction of the nitro group to an amino group may be accomplished with a reducing agent (e.g., Zn—NH4Cl) in an inert solvent (e.g., MeOH) to give an intermediate of formula 11g where R8═NH2. These anilino derivatives may be coupled with chloroalkanoates of the formula ClCO2—C1-4alkyl in the presence of a base (e.g., DIEA) in an inert solvent (e.g., DCM) to give intermediates where R8═NH CO2—C1-4alkyl.

The amino ester analog 13e was obtained from key intermediate 12j following the sequence described in Scheme 13. Step-wise imine formation of 12j with ethyl 2,3-dioxopropanoate followed by addition of allyltributyltin under tin (IV) chloride conditions afforded RCM precursor 13a. Following the same sequence in Scheme 12, 13a can be converted into critical intermediate 13e over several steps. Other macrocyclic intermediates such as 13e wherein the ester is replaced with a variety of substituents can also be similarly constructed and following the sequence of reactions outlined above can be converted to compounds of this invention according to Scheme 15.

Methods for synthesis of a large variety of substituted pyridine compounds useful as starting materials for the preparation of compounds of the present invention are well known in the art and have been extensively reviewed. (For examples of methods useful for the preparation of pyridine starting materials see: Kroehnke, F.,Synthesis,1 (1976); Abramovitch, R. A., ed., “Pyridine and Its Derivatives”,The Chemistry of Heterocyclic Compounds,14(Suppl. 1-4), John Wiley & Sons, New York (1974); Boulton, A. J. et al., eds.,Comprehensive Heterocyclic Chemistry,2:165-524, Pergamon Press, New York (1984); McKillop, A., ed.,Comprehensive Heterocyclic Chemistry,5:1-300, Pergamon Press, New York (1996)).

In cases where suitably substituted boronic acids are not commercially available, a modification to this approach may be adopted wherein an aryl halide is subjected to a palladium mediated coupling with a diboron species such as bis(pinacolato)diboron or bis(neopentyl glycolato)diboron to provide the corresponding 4,4,5,5-tetramethyl-[1,3,2]dioxaborolane or the 5,5-dimethyl-[1,3,2]dioxaborolane intermediates using the method of Ishiyama, T. et al. (J. Org. Chem.,60(23):7508-7510 (1995)). Alternately, this same intermediate can be prepared by reaction of the intermediate halide with the corresponding dialkoxyhydroborane as described by Murata et al. (J. Org. Chem.,62(19):6458-6459 (1997)). The boron pinacolate intermediates can be used in place of boronic acids for coupling to the aryl/heteroaryl halides or triflates or the boron pinacolate intermediate can be converted to the boronic acids. Alternately, the corresponding boronic acids can be prepared by metal-halogen exchange of the aryl/heteroaryl halide, quenching with a trialkoxyborate reagent, and aqueous workup to provide the boronic acids (Miyaura, N. et al.,Chem. Rev.,95:2457 (1995)).

It is also realized that the scope of intermediate synthesis can be further extended outside the use of Suzuki-Miyaura coupling methodology since the precursor aryl halides or triflates described above are also precursors for Stille, Negishi, Hiyama, and Kumada-type cross coupling methodologies (Tsuji, J.,Transition Metal Reagents and Catalysts: Innovations in Organic Synthesis, John Wiley & Sons (2000); Tsuji, J.,Palladium Reagents and Catalysts: Innovations in Organic Synthesis, John Wiley & Sons (1996)).

Additional pyridazine and pyridazinone containing macrocycles can be prepared according to Scheme 14. Condensation of the potassium salt of 14a with a suitably substituted α-ketoester 14b, which is either commercially available or prepared using a modified procedure described by Domagala (Tetrahedron Lett.,21:4997-5000), in a solvent such as THF generates the α,β-unsaturated ketone derivative which can then be condensed with a suitably substituted hydrazine derivative to give pyridazinone 14c. The nitro group can then be reduced to the aniline 14f with zinc and NH4Cl in methanol. The pyridazinone 14c can be converted to chloro-pyridazine 14d by deprotection of the amine protecting group, followed by treatment with POCl3, then reprotection. The nitro group can be reduced to the aniline 14e with iron and AcOH. The anilines 14e and 14f can then be coupled with an appropriately substituted carboxylic acid 7a using T3P to give the amide 14g (R10═Cl) and 14h (R10═OH), respectively. 14g and 14h can then be cyclized via ring-closing metathesis using a catalyst, such as Grubbs (II), in a suitable solvent, such as DCM, DCE, or toluene at elevated temperature, to give the macrocycle 14i (R10═Cl) and 14j (R10═OH), respectively. The resulting alkenes can then be reduced with hydrogen over either palladium on carbon or platinum oxide to give 14k and 14l. 14k can be reduced with ammonium acetate and palladium on carbon to reduce the chlorine to give 14m. Subsequent deprotection of 14m and 14l provides amines 14n (R10═H) and 14o (R10═OH). Compounds of the formulae 14n and 14o can be converted to compounds in this invention according to Scheme 15.

Representative regioisomeric pyridazine containing amide macrocycle intermediates useful for the synthesis of compounds of this invention are described in Scheme 14a. Using a modification of the Minisci reaction described by Cowden (Org. Lett.,5:4497-4499 (2003)), an appropriately protected glycine 14aa and 3,6-dichloropyridazine can be coupled at elevated temperature in the presence of silver nitrate, ammonium persulfate, and an acid, such as ammonium formate, in a solvent, such as water or a water/dimethylformamide mixture, to give compounds of the formulae 14ab. Compound 14ab can be further functionalized by deprotonation with nBuLi and subsequent alkylation with an appropriately substituted alkyl halide, for instance allyl bromide, to give compound 14ac. Suzuki-Miyaura coupling between chloropyridazine 14ac and an appropriately substituted aryl or heteroaryl boronic acid or ester 11e in the presence of a base such as sodium carbonate using a precatalyst such as (Ph3P)4Pd provides, after separation of the enantiomers, aniline 14ad. Aniline 14ad can be converted to 14ae and 14af according to Scheme 7. Hydrogenolysis of the chloro under transfer hydrogenation conditions and Boc-deprotection will give compounds 14ag and 14ah. Compounds of the formulae 14ag and 14ah can be converted to compounds in this invention according to Scheme 15.

Representative compounds of this invention can then be made as shown in Scheme 15 using intermediates made in Schemes 2 to 13. The various substituted acids represented by formula 15b can be coupled with both 6- and 5-membered macrocycle amines represented by 15a using either coupling reagents or by converting them to acid chloride (like Vilsmeier reagent) and then treating the mixture with a base to afford the desired macrocycles of this invention.

Unless otherwise stated, analysis of final products was carried out by reverse phase analytical HPLC.

Intermediate 1. 1-(3-Chloro-2-fluorophenyl)-1H-1,2,3-triazole-4-carboxylic acid: 3-chloro-2-fluoro aniline was dissolved in TFA (4 mL) and H2O (2 mL) was added to the above solution. The mixture was then cooled to 0° C. and to this was added a predissolved aqueous solution (2 mL) of NaNO2dropwise to ensure the temperature did not rise above 5° C. The reaction mixture was stirred at this temperature for 0.5 h followed by the addition of solid NaN3portionwise. The reaction mixture was stirred cold and then allowed to warm up to rt overnight. The reaction mixture was quenched with H2O (100 mL) and extracted the azide with EtOAc (2×50 mL), dried and evaporated to a solid mass (1.1 g). The product obtained was dissolved in DMSO (5 mL) in a microwave flask and to this was added L-proline (0.02 g), Cu(OAc)2(0.1 g), K2CO3(1.5 g) and sodium ascorbate (0.1 g) and excess t-butyl propiolate (3 mL). The flask was sealed and heated at 75° C. overnight. Aliquot LCMS showed the reaction to be complete. The reaction mixture was quenched with H2O (100 mL) and extracted the organic layer with EtOAc (2×100 mL), washed with brine (50 mL) and dried (MgSO4). The crude product was then purified using silica gel chromatography. The desired ester was isolated and concentrated, evaporated to a brown solid (0.98 g). The ester (0.2 g) was dissolved in DCM (2 mL) and to this was added TFA (1 mL) and stirred at rt overnight. Aliquot LCMS showed the reaction to be complete. The reaction mixture was then quenched with H2O (50 mL) and the organic layer was extracted with EtOAc (2×100 mL), dried and evaporated to a brown solid mass. MS(ESI) m/z: 342.1 (M+H)+.1H NMR (500 MHz, CDCl3) δ 8.20 (s, 1H), 7.60-7.55 (dt, 1H), 7.42-7.37 (dt, 1H), 7.28-7.23 (dt, 1H) ppm.

Intermediate 3A. Ethyl-5-amino-1-(3-chlorophenyl)-1H-pyrazole-4-carboxylate: (Ref:J. Heterocyclic Chem.,267 (1987)) To a mixture of (3-chlorophenyl)hydrazine hydrochloride (2.328 g, 13 mmol), (E)-ethyl 2-cyano-3-ethoxyacrylate (2.199 g, 13.00 mmol) and K2CO3(1.797 g, 13.00 mmol) was added EtOH (20 mL). The suspension was then warmed to reflux and stirred at refluxing temperatures for overnight. After 20 h, the reaction mixture was poured into ice-H2O. The suspension was then filtered and the solid collected by filtration was dried in vacuo (50° C.) for overnight to yield a brown solid (2.93 g). MS(ESI) m/z: 266.1 (M+H)+.

Intermediate 3. 5-Amino-1-(3-chlorophenyl)-1H-pyrazole-4-carboxylic acid: (Reference:J. Heterocyclic Chem.,773 (2003)) A solution of Intermediate 3A (0.652 g, 2.454 mmol) and NaOH (0.613 g, 15.34 mmol) in EtOH (1.534 mL) and H2O (13.80 mL) was refluxed until the reaction mixture became homogeneous. After 24 h, the reaction mixture was cooled to rt and filtered. The filtrate was acidified with concentrated HCl to give a suspension which was subjected to filtration. The solid collected by filtration was washed with H2O and dried in vacuo (50° C.) for 4 h to give a yellow solid (0.51 g) as the desired product. MS(ESI) m/z: 238.1 (M+H)+.1H NMR (500 MHz, MeOD) δ 7.77 (s, 1H), 7.64-7.61 (m, 1H), 7.58-7.51 (m, 2H), 7.50-7.46 (m, 1H) ppm.

Intermediate 5A: tert-Butyl (3-chloro-2,6-difluorophenyl)carbamate: 3-chloro-2,6-difluorobenzoic acid (4.85 g, 25.2 mmol) was dissolved in THF (50 mL) and cooled to 0° C. To this solution was then added ethylchloroformate (3.01 g, 27.7 mmol) followed by TEA (3.86 mL, 27.7 mmol) and stirred at the same temperature for 1 h. To the slurry that developed was then added NaN3in H2O (5 mL) dropwise and stirred the reaction mixture at 0° C. for 1.25 h. Solids separated out from the reaction mixture and allowed the solids to decant followed by separation of the decantant. The residue was dissolved in H2O (50 mL) and extracted with DCM (2×). The above organic layer was then combined with the decantant, dried (MgSO4) and concentrated to yield a residue. The residue was re-dissolved in toluene (50 mL) and heated at 110° C. To the above solution was added t-BuOH (1.5 g) and refluxed for overnight. The reaction mixture was concentrated and purified by silica gel chromatography to yield the desired product (2.86 g, 43%). MS(ESI) m/z: 286.0 (M+Na)+.1H NMR (400 MHz, CDCl3) δ 7.28 (s, 1H), 7.03-6.72 (m, 1H), 6.10-5.83 (m, 1H), 1.57-1.37 (m, 9H) ppm.

Intermediate 5B. tert-Butyl 1-(3-chloro-2,6-difluorophenyl)-1H-1,2,3-triazole-4-carboxylate: To a solution of Intermediate 5A in DCM (5 mL) was added TFA (1 mL) and stirred at rt for 1 h. The reaction mixture was then concentrated to yield a brown oil which was redissolved in TFA (5 mL) and cooled to 0° C. To this cooled solution was then added NaNO2(0.209 g, 3.03 mmol) in H2O (1 mL) dropwise. The reaction mixture was allowed to stir at 0° C. for 0.5 h followed by the addition of NaN3(0.394 g, 6.07 mmol) in H2O (1 mL). The reaction mixture was continued to stir for 2 h at the same temperature and then quenched with H2O (100 mL) and extracted the aqueous layer with EtOAc (2×). The combined organic layers were dried and evaporated to a brown oil. The azide from the above reaction was then dissolved in DMSO (5 mL) and to this solution was added t-butylpropiolate (1.5 mL, 1.517 mmol), Cu(OAc)2(0.055 g, 0.303 mmol) and K2CO3(0.839 g, 6.07 mmol). The reaction was stirred at rt for overnight. The reaction mixture was then quenched with H2O and solid mass separated. The reaction mixture was extracted with EtOAc (2×). The organic layer was dried and evaporated to a dark brownish-black oil. The crude product was then purified using silica gel chromatography. The desired product was isolated as reddish oil (0.3 g, 62%). MS(ESI) m/z: 316.0 (M+H)+.

Intermediate 6A. Ethyl 1-(3-chlorophenyl)-1H-1,2,3-triazole-4-carboxylate: Sodium nitrite (1.947 g, 28.2 mmol) dissolved in H2O (5 mL) was added to a cold (<5° C.) TFA (20 mL) solution of 3-chloro aniline (3.6 g, 28.2 mmol). After 0.5 h, sodium azide (1.835 g, 28.2 mmol) dissolved in H2O (1 mL) was added dropwise to the above reaction mixture. The reaction mixture was then stirred cold for 2 h and then quenched with H2O (100 mL) and extracted the organics with EtOAc (2×100 mL). The organic layers were then dried over MgSO4and concentrated to a brown oil (3.5 g). Approximately 1 g of the azide from the above crude product was taken in a microwave flask. To this was added ethyl propiolate (1.5 mL), DMSO (4 mL), sodium carbonate (0.1 g) and L-proline (0.1 g) and the reaction mixture was heated at 75° C. overnight. The reaction was then quenched with H2O to precipitated out the solids. Filtered the solids and washed with excess H2O followed by drying under vacuum to afford 1.3 g of the desired triazole ester. MS(ESI) m/z: 252.1 (M+H)+.

Intermediate 8. 5-Amino-1-(3-chlorophenyl)-1H-1,2,3-triazole-4-carboxylic acid: The azide was made as previously described (Intermediate 6) starting with 3-chloroaniline. The azide was then treated with tert-butyl 2-cyanoacetate under refluxing conditions for overnight. After overnight stirring, the reaction mixture was quenched with H2O (100 mL) and extracted with EtOAc (2×100 mL). The organic layers were then dried (MgSO4) and evaporated to a brown solid. The brown solid was then re-dissolved in DCM (2 mL) and to this solution was added TFA (2 mL) and stirred at rt for overnight. The reaction mixture was then concentrated and quenched with H2O to precipitate out a brown solid. The solids were filtered, washed with excess MeOH and dried to afford brownish white solid. MS(ESI) m/z: 238.9 (M+H)+.1H NMR (400 MHz, MeOD) δ 7.75 (bs, 1H), 7.37-7.34 (dd, J=1.7 & 8.3 Hz, 1H), 7.25-7.21 (t, 1H), 6.91-6.89 (bd, 1H) ppm.

Intermediate 9A: 3-((tert-Butyldiphenylsilyl)oxy)propanal: To a solution of tert-butyldiphehenylsilylchloride (2.20 g, 8.0 mmol) in DCM/DMF (95:1) was added 1,3 propanediol (2.010 g, 26.4 mmol) followed by TEA (1.053 g, 10.43 mmol) and catalytic DMAP (0.049 g, 0.4 mmol) and the reaction mixture was stirred at rt overnight. The reaction mixture was then quenched with H2O (200 mL) and extracted the organics with EtOAc (3×). The crude product in a solution of DCM (5 mL) was added slowly to a cooled solution (−78° C.) of oxalyl chloride (5.75 mL, 11.59 mmol) in DCM (20 mL). The reaction mixture was continued to stir at −78° C. for 20 min and then treated with TEA (5.34 mL, 38.3 mmol) and then raised to rt slowly. The reaction mixture was then diluted with ether, washed with 10% aqueous citric acid followed by brine. The organic layers were then dried and concentrated to give the desired product.1H NMR (400 MHz, CDCl3) δ 7.90-7.56 (m, 4H), 7.49-7.28 (m, 6H), 3.96-3.61 (m, 2H), 2.27 (br. s., 1H), 1.88-1.72 (m, 2H), 1.16-0.96 (m, 9H) ppm.

Intermediate 9B. Ethyl 5-amino-3-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-1-(3-chlorophenyl)-1H-pyrazole-4-carboxylate: To a solution of Intermediate 9A (1.34 g, 3.1 mmol) in DMF (5 mL) was added NCS (0.455, 3.41 mmol) and stirred at rt. After 4 h, the reaction mixture was quenched with H2O (100 mL) and extracted with EtOAc (2×). The organic layers were dried (MgSO4) and evaporated to a reddish oil. Separately ethylcyanoacetate (0.351 g, 3.10 mmol) was dissolved in EtOH (5 mL) and to this solution was added NaOEt (21%) (1.16 mL, 3.10 mmol) and the reaction was stirred at rt for 0.5 h followed by the introduction of the iminochloride crude mixture. The above mixture was then stirred at rt. After 2 h, the reaction was quenched with H2O and extracted with EtOAc (2×). The organic layers were dried (MgSO4) and evaporated to yield orange-red oil. The crude product was then purified using silica gel chromatography. Two peaks were isolated—one is the desired product and the other is the chlorinated pyrazoline compound and the two products were taken to the next step as a crude mixture. MS(ESI) m/z: 548.1 (M+H)+.

Intermediate 9C. Ethyl 3-(2-((tert-butyldiphenylsilyl)oxy)ethyl)-1-(3-chlorophenyl)-1H-pyrazole-4-carboxylate: Isoamylnitride (1 mL) was added to a THF solution of Intermediate 9B (crude mixture) and stirred at 70° C. overnight. The reaction mixture was concentrated and taken to the next step as a crude mixture. MS(ESI) m/z: 555.3 (M+Na)+.

Intermediate 11A. Methyl 1-(3-chlorophenyl)-5-oxopyrrolidine-3-carboxylate (Ref:Tetrahedron,62:4011-4017 (2006)). To a cold solution of MeOH (8.35 mL) (0° C.) was added thionyl chloride (0.335 mL, 4.59 mmol) dropwise. After 30 min, Intermediate 10 (1 g, 4.17 mmol) was added, and the reaction mixture was warmed to rt. The reaction mixture was then concentrated and the residue dissolved in EtOAc, washed with saturated NaHCO3, H2O and brine. The organic layers were dried over Na2SO4, filtered and concentrated to yield the desired product (1.04 g, 98%) as yellow oil. MS(ESI) m/z: 254.0 (M+H)+.

Intermediate 11B. Methyl 1-(3-chlorophenyl)pyrrolidine-3-carboxylate: To a solution of Intermediate 11A (0.27 g, 1.064 mmol) in THF (3 mL) was added BH3-THF complex (1.596 mL, 1.596 mmol) (1 M in THF). The reaction mixture was stirred at rt.

After 17 h, the reaction mixture was quenched by adding 1 mL MeOH, then H2O. The above mixture was then extracted with EtOAc and the organic layers were washed with H2O, brine, dried over Na2SO4, filtered and concentrated. The crude product was then purified using silica gel chromatography to afford a colorless oil as the desired product (0.175 g, 68.6%). MS(ESI) m/z: 240.1 (M+H)+.

Intermediate 12A. Ethyl 1-(3-chlorophenyl)-1H-imidazole-4-carboxylate, 1 TFA: A mixture of 1-chloro-3-iodobenzene (0.170 g, 0.714 mmol), ethyl 1H-imidazole-4-carboxylate (0.1 g, 0.714 mmol), copper(I) iodide (0.027 g, 0.143 mmol), and K2CO3(0.296 g, 2.141 mmol) in DMSO (1.427 mL) was vacuumed and back-filled with argon for three times, then capped and heated at 110° C. After 16 h, the reaction was cooled to rt and was then diluted with EtOAc, washed with H2O followed by brine. The organic layer was then dried over Na2SO4, filtered, and concentrated. The crude product was purified using silica gel chromatography to afford a white solid as the desired product (0.118 g, 66%). MS(ESI) m/z: 251.0 (M+H)+.

Intermediate 12. 1-(3-Chlorophenyl)-1H-imidazole-4-carboxylic acid: To a solution of Intermediate 12A (0.118 g, 0.471 mmol) in MeOH (4.71 mL) was added 1 N NaOH (0.941 mL, 0.941 mmol) and the reaction mixture was stirred at rt. After 2 h, the reaction mixture was concentrated and the residue was dissolved in MeOH/H2O. To the above solution was then added 1 N HCl (1.5 mL) to afford a white suspension which was filtered to isolate the solid. The solid was rinsed with H2O and then dried in a vacuum oven (50° C.) for 4 h to yield a white solid as Intermediate 12 (0.09 g, 74%). MS(ESI) m/z: 223.0 (M+H)+.1H NMR (400 MHz, MeOD) δ 8.44-8.23 (m, 2H), 7.77 (t, J=1.9 Hz, 1H), 7.63-7.45 (m, 3H) ppm.

Intermediate 13. 1-(3-Chloro-2-fluorophenyl)-1H-imidazole-4-carboxylic acid: Intermediate 13 was made in the same way as Intermediate 12 by replacing Intermediate 12A with Intermediate 13A. MS(ESI) m/z: 241.0 (M+H)+.

Intermediate 14. 1-(1-(tert-Butoxycarbonyl)piperidin-3-yl)-1H-pyrazole-4-carboxylic acid: To a clear, colorless solution of Intermediate 14A (0.0646 g, 0.200 mmol) in MeOH (0.666 ml) was added dropwise 1.0 M sodium hydroxide (0.599 ml, 0.599 mmol). The resulting slightly cloudy reaction mixture was stirred at rt. After 6 h, the reaction was cooled to 0° C. and neutralized with 1.0 N HCl. The mixture was concentrated to give a white solid. The solid was partitioned between EtOAc and 0.5 N HCl and the layers were separated. The aqueous layer was extracted with EtOAc (2×). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated to give Intermediate 14 (0.0616 g, 104%) as a white foam. MS(ESI) m/z: 240.1 (M-C4H8+H)+.

Intermediate 15 was prepared in the same way as Intermediate 14. MS(ESI) m/z: 210.1 (M+H)+.

Intermediate 16A. Methyl 4-iodo-3-nitrophenylcarbamate: To a cooled (0° C.), yellow suspension of 4-iodo-3-nitroaniline (8.46 g, 32.0 mmol) in DCM (320 mL) and pyridine (2.85 mL, 35.2 mmol) was added dropwise methyl chloroformate (2.61 mL, 33.6 mmol) and the reaction was stirred for 1.5 h. The reaction mixture was diluted with DCM and washed with saturated NaHCO3solution followed by brine. The organic layer was then dried over MgSO4, filtered and concentrated. The residue was dissolved in minimal DCM (˜100 mL), then hexane (600 mL) was added to give a yellow suspension. The above suspension was then filtered and the solid was rinsed with hexane and air-dried to yield the desired product as a yellow solid (10.3 g, 100%). MS(ESI) m/z: 321.3 (M−H).

Intermediate 16B. Methyl 4-acetyl-3-nitrophenylcarbamate: A solution of Intermediate 16A (1 g, 3.11 mmol), tributyl(1-ethoxyvinyl)stannane (2.098 mL, 6.21 mmol), and bis(triphenylphosphine) palladium(II)chloride (0.218 g, 0.311 mmol) in toluene (6.21 mL) was heated at 110° C. in a sealed tube. After 3 h, the reaction mixture was cooled to rt and concentrated to dryness. The residue was then dissolved in THF (5 mL), added 1 N HCl solution (15.53 mL, 15.53 mmol), and the reaction was stirred at rt for 1 h. The reaction mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered and concentrated. The crude product was then purified by silica gel chromatography to yield the desired product as a brown solid (0.544 g, 74%). MS(ESI) m/z: 239.3 (M+H)+.

Intermediate 16. Methyl 4-(2-bromoacetyl)-3-nitrophenylcarbamate: To a yellow solution of Intermediate 16B (0.544 g, 2.284 mmol) in EtOAc (18.27 mL) was added copper (II) bromide (1.020 g, 4.57 mmol). The flask was equipped with a reflux condenser and then the reaction was warmed to 70° C. After 3 h, the reaction was stopped and cooled to rt. The reaction mixture was then filtered through a sintered glass funnel eluting with EtOAc. The green filtrate was washed with H2O (3×), brine, dried over Na2SO4, filtered and concentrated to yield the desired product as a brown foam (0.724 g, 100%). MS(ESI) m/z: 317.4 (M+H)+, 319.4 (M+2+H)+. The crude product was carried forward without any further purification.

An alternative procedure for Intermediate 16 is highlighted here.

Alternative Intermediate 16B. Methyl 4-(1-ethoxyvinyl)-3-nitrophenylcarbamate: A solution of Intermediate 16A (1 g, 3.11 mmol), tributyl(1-ethoxyvinyl)stannane (1.574 mL, 4.66 mmol) and bis(triphenylphosphine)palladium(II) chloride (0.109 g, 0.155 mmol) in toluene (6.21 mL) in a round bottom flask equipped with a condenser was heated at 110° C. After 2 h, the reaction was cooled to rt, filtered through a 0.45μ GMF filter and rinsed with EtOAc. The filtrate concentrated to dryness and purified by silica gel chromatography to obtain the desired product as a brown solid (0.56 g, 68%). MS(ESI) m/z: 267.3 (M+H)+.

Alternative Intermediate 16. (Reference:J. Med. Chem.,45:2127-2130 (2002)) To a solution of alternative intermediate 16B (0.56 g, 2.103 mmol) in THF (3.12 mL) and H2O (1.091 mL) was added NBS (0.374 g, 2.103 mmol). After stirring at rt for 20 min, the reaction mixture was partitioned between EtOAc and brine. The organic layer was then dried over Na2SO4, filtered, and concentrated to yield the desired product as a yellow oil (0.667 g, 100%). MS(ESI) m/z: 317.2 (M+H)+, 319.2 (M+2+H)+.

Intermediate 17. Benzyl 2-methylbut-3-enoate: To a solution of 2-methylbut-3-enoic acid (9.5 g, 95 mmol) in DCM (80 mL) was added phenylmethanol (10.26 g, 95 mmol), N,N′-methanediylidenedicyclohexanamine (19.58 g, 95 mmol) and DMAP (1.159 g, 9.49 mmol) (exothermic reaction) and the reaction was stirred at rt over weekend. The reaction mixture was filtered through a pad of CELITE® to remove the solids and the filtrate was concentrated. The residue was purified by silica gel chromatography to yield the desired product as a colorless oil.

Intermediate 18A. 2-Bromo-4-nitro-benzoic acid: To a warm (80° C.) solution of pyridine (500 mL) and water (1 L) was added 4-nitro-2-bromo toluene (100 g, 0.46 mol). The resulting suspension was stirred until it became a clear solution. To the above reaction mixture was then added KMnO4(600 g, 3.8 mol) in portions over 1.5 h and stirring was continued overnight. The reaction mixture was then cooled to rt and then 10% aqueous NaOH (200 mL) was added. After 15 min, the reaction was filtered and the solid was rinsed with 10% aqueous NaOH (5×100 mL). The filtrate was extracted with MTBE (3×250 mL). The clear aqueous layer was cooled to 10° C. and then it was acidified with concentrated HCl. The aqueous layer was again extracted with MTBE (4×500 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to afford 72 g of Intermediate 18A.1H NMR (400 MHz, DMSO-d6) δ 7.96 (d, J=8 Hz, 1H), 8.28-8.48 (m, 1H), 8.49 (d, J=2.4 Hz, 1H), 14.1 (br. s, 1H) ppm.

Intermediate 18B. 2-(2-Bromo-4-nitro-benzoyl)-malonic acid diethyl ester: To a solution of Intermediate 18A (50 g, 0.2 mol) in toluene (500 mL) was added TEA (24.6 g, 0.24 mol). The reaction was cooled to 15° C. and ethyl chloroformate (24 g, 0.22 mol) was added. After 45 min, the mixed anhydride solution was cooled to 0° C. In a separate flask: To a suspension of Mg turnings (5.4 g) in dry ether (300 mL) was added EtOH (3.0 mL), CCl4(2.0 mL), and diethyl malonate (34 mL, 0.22 mol). The mixture was stirred at 40° C. for an hour to ensure that the magnesium dissolved completely. After the reaction became a clear solution, it was added to the cooled solution of the mixed anhydride. After 2 h, the reaction was quenched with 2 N sulfuric acid (200 mL) and then extracted with EtOAc (4×100 mL). The combined organic layers were dried over sodium sulfate, filtered, and concentrated to afford 80 g of Intermediate 18B. This was used in the next step without further purification.

Intermediate 18D. 1-(4-Amino-2-bromophenyl)ethanone: To a solution of Intermediate 18C (19 g, 0.077 mol) in EtOH (400 mL) was added in portions tin(II) chloride (74 g, 0.39 mol). Following the addition, the reaction was heated to refluxing temperature overnight. The reaction mixture was then concentrated and the residue was dissolved in 10% aqueous NaOH (200 mL). The aqueous solution was extracted with ethyl acetate (2×200 mL). The combined organic layers were washed with brine and concentrated to afford an oil. Petroleum ether (25 mL) was added to the oil to afford a suspension that was decanted and the solid was suspended in 20% ethyl acetate/petroleum ether. The organic layer was filtered and the solids were collected to afford 14 g of Intermediate 18D.

Intermediate 18. [3-Bromo-4-(2-bromo-acetyl)-phenyl]-carbamic acid methyl ester: To a cooled (10° C.) solution of Intermediate 18E (90 g, 0.33 mol) in dry dioxane (900 mL) was added a solution of bromine (52.9 g, 0.33 mol) in dioxane (430 mL) dropwise over 1 h. After 2 h, ice cold water (500 mL) was added and the reaction was extracted with ethyl acetate (2×500 mL). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, and concentrated to afford 110 g of crude product. A suspension of the crude product in EtOH (1 L) was warmed to 50° C. After a clear solution had formed, water (1.0 L) was added dropwise and the mixture was gradually cooled to 35° C. The precipitated solid was collected by filtration, washed with EtOH (200 mL), air-dried, and then dried at 50° C. under vacuum for 30 min to yield 70 g of Intermediate 18.

Intermediate 19A. Ethyl 5-amino-1-(2,3-dichlorophenyl)-1H-pyrazole-4-carboxylate: A mixture of (2,3-dichlorophenyl)hydrazine, HCl (1 g, 4.68 mmol), (E)-ethyl 2-cyano-3-ethoxyacrylate (0.792 g, 4.68 mmol), and K2CO3(0.647 g, 4.68 mmol) in EtOH (10 mL) was added to a microwave vial and was heated at 85° C. for 20 h. The reaction mixture was then cooled to rt and then poured into ice-water. The suspension formed was then filtered and the solid was rinsed with water and dried in a vacuum oven (50° C.) for 4 h to afford a brown solid. The crude product was then purified by silica gel chromatography to yield a brown solid as ethyl 5-amino-1-(2,3-dichlorophenyl)-1H-pyrazole-4-carboxylate (0.93 g, 66% yield). MS(ESI) m/z: 300.0 (M+H)+.

Intermediate 19. 5-Amino-1-(2,3-dichlorophenyl)-1H-pyrazole-4-carboxylic acid: A clear yellow solution of Intermediate 19A (0.026 g, 0.087 mmol) in MeOH (2 mL) and 1.0 N NaOH (0.260 mL, 0.260 mmol) was stirred at rt followed by heating to 70° C. for 24 h. To the mixture was added additional 1 N NaOH (0.260 ml, 0.260 mmol), and the reaction mixture was warmed to 90° C. for 7 h. The reaction mixture was cooled to rt, and 1 N HCl (0.75 mL) was added and the reaction mixture was concentrated to afford a yellow solid as 5-amino-1-(2,3-dichlorophenyl)-1H-pyrazole-4-carboxylic acid (0.065 g, 99%). MS(ESI) m/z: 271.9 (M+H)+.

Intermediate 20A. Ethyl 1-(2,3-dichlorophenyl)-1H-pyrazole-4-carboxylate: To a solution of Intermediate 19A (0.23 g, 0.766 mmol) in THF (8 mL) was added isoamyl nitrite (0.206 mL, 1.533 mmol) and the reaction was heated in a microwave vial at 80° C. After 16 h, the reaction mixture was cooled to rt and concentrated. The crude product was then purified by silica gel chromatography to afford a yellow gummy oil as ethyl 1-(2,3-dichlorophenyl)-1H-pyrazole-4-carboxylate (0.187 g, 86%). MS(ESI) m/z: 285.0 (M+H)+.

Intermediate 20. 1-(2,3-Dichlorophenyl)-1H-pyrazole-4-carboxylic acid: To a clear yellow solution of Intermediate 20A (0.187 g, 0.656 mmol) in MeOH (8 mL) was added 1.0 N NaOH (1.968 mL, 1.968 mmol) and the reaction mixture was stirred at rt. After 18 h, the reaction mixture was concentrated to remove MeOH. To the above crude product was then added water to afford a yellow solution. To this solution was then added 1 N HCl (2.5 mL) to afford a white suspension which was filtered and the solid was rinsed with water, and then dried in a vacuum oven (50° C.) for 4 h. A yellow solid was obtained as 1-(2,3-dichlorophenyl)-1H-pyrazole-4-carboxylic acid (0.16 g, 95%). MS(ESI) m/z: 257.0 (M+H)+.1H NMR (500 MHz, MeOD) δ 8.51 (s, 1H), 8.13 (s, 1H), 7.75 (dd, J=8.3, 1.7 Hz, 1H), 7.61-7.56 (m, 1H), 7.54-7.49 (m, 1H) ppm.

Intermediate 21. 1-(3-Chloro-2-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid: To a solution of 3-chloro-2-fluoroaniline (1.7 g, 11.68 mmol) in TFA (10 mL) was added water (2 mL) and the reaction mixture was cooled to 0° C. To the above solution was then added sodium nitrite (0.806 g, 11.68 mmol) over 0.5 h. To the above mixture was then added slowly a solution of sodium azide (1.928 g, 29.7 mmol) in water. The reaction mixture was then stirred at 0° C. for 10 min, and then allowed to warm to rt. After 2 h, the reaction mixture was quenched by addition of water (100 mL) and the insoluble solids from the reaction mixture were filtered and dried under suction in the presence of nitrogen. To the azide was then added methyl acetoacetate (1.492 g, 12.85 mmol) in MeOH (12 mL) and methanol, sodium derivative (2.78 g, 12.85 mmol) and the mixture was heated at 65° C. in a sealed tube overnight. The reaction mixture was cooled to rt and then to 0° C. followed by addition of THF (50 mL). To the above mixture was then added NaOH (58.4 mL, 58.4 mmol), and the reaction was warmed to 50° C. After 2 h, the organics were concentrated and the remaining aqueous layer was acidified with 1.0 M HCl solution. The resulting suspension was filtered and the solids were washed with water followed by a small amount of cold MeOH and dried in an oven overnight (50° C.) to give 1-(3-chloro-2-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid (1.86 g, 62%) as an off-white solid. MS(ESI) m/z: 256.0 (M+H)+.1H NMR (400 MHz, DMSO-d6) δ 7.99-7.89 (m, 1H), 7.80-7.71 (m, 1H), 7.53 (td, J=8.2, 1.3 Hz, 1H), 2.44 (s, 3H) ppm.

Intermediate 22A. 2-(3-Chloro-2,6-difluorophenyl)-4-(trifluoromethyl)-1H-imidazole: (Reference: WO 2008/050244) To a solution of potassium acetate (0.872 g, 8.88 mmol) in H2O (3 mL) was added 3,3-dibromo-1,1,1-trifluoropropan-2-one (1.098 g, 4.07 mmol). The above reaction mixture was then heated at 100° C. for 0.5 h. The reaction mixture was then cooled to rt and to the mixture was then added a solution of 3-chloro-2,6-difluorobenzaldehyde (0.653 g, 3.7 mmol) in MeOH (4 mL) and THF (4 mL), followed by concentrated NH4OH (8 mL). The mixture was stirred overnight at rt. The reaction mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4and concentrated in vacuo to yield 2-(3-chloro-2,6-difluorophenyl)-4-(trifluoromethyl)-1H-imidazole (0.95 g, 91%). MS(ESI) m/z: 283.0 (M+H)+.

Intermediate 23. 5-Amino-1-(5-chloro-2-fluorophenyl)-1H-pyrazole-4-carboxylic acid: A cloudy, yellow suspension of Intermediate 23A (0.125 g, 0.441 mmol) in MeOH (2.203 mL) and 1.0 N NaOH (1.763 mL, 1.763 mmol) was warmed to 50° C. After 8 h, the reaction mixture was cooled to rt and the clear, yellow orange solution was concentrated to give a yellow solid. The yellow solid was dissolved in water and 1.0 N HCl was added to give a white suspension (pH 3-4). The mixture was then extracted with EtOAc (2×). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to give Intermediate 23 (0.096 g, 85%) as an off-white solid. MS(ESI) m/z: 256.0 (M+H)+and 258.0 (M+2+H)+.

Intermediate 24A. Ethyl 1-(3-chloro-2-fluorophenyl)-5-methyl-1H-imidazole-4-carboxylate: Using a modified procedure of Sreedhar. (Reference: Sreedhar, B.,Synthesis,795 (2008)). To a suspension of ethyl 4-methyl-1H-imidazole-5-carboxylate (0.530 g, 3.44 mmol) and (3-chloro-2-fluorophenyl)boronic acid (0.500 g, 2.87 mmol) in MeOH (5.74 mL) was added cuprous oxide (0.041 g, 0.287 mmol). The resulting purple suspension was stirred vigorously under an atmosphere of air (drying tube used). After 20 h, the reaction mixture was filtered to remove the solids and the clear blue filtrate was concentrated to give a blue solid. The blue solid was suspended in DCM and filtered to remove the solids and the blue filtrate was concentrated to give a pale blue solid weighing 0.187 g. Purification by normal phase chromatography gave ethyl 1-(3-chloro-2-fluorophenyl)-4-methyl-1H-imidazole-5-carboxylate (0.0187 g, 2%) as a clear, colorless residue and ethyl 1-(3-chloro-2-fluorophenyl)-5-methyl-1H-imidazole-4-carboxylate (Intermediate 24A) (0.0079 g, 1%) as a clear, colorless residue. MS(ESI) m/z: 283.1 (M+H)+.

Intermediate 24A can also be synthesized in three steps according to the following sequence:

Intermediate 24A (Alternative). Ethyl 1-(3-chloro-2-fluorophenyl)-5-methyl-1H-imidazole-4-carboxylate: Using a modified procedure described by Gomez-Sanchez. (Reference: Gomez-Sanchez, A. et al.,J. Heterocyclic Chem.,24:1757 (1987).) A clear, yellow solution of Intermediate 24A1 (2.90 g, 9.58 mmol) in triethylorthoformate (96 mL) was degassed with argon for 20 min. Next, platinum on carbon (0.935 g, 0.479 mmol) was added. The flask was equipped with a reflux condensor and the reaction was purged with hydrogen (balloon) for several minutes. The reaction was stirred under a hydrogen atmosphere and the reaction was warmed to 75° C. After a total of 4 h, the reaction was cooled to rt. The reaction was placed under vacuum for several minutes and then backfilled with argon. The process was repeated a total of 5 times. Next, CELITE® was added and the reaction was filtered, washing with ethanol. The filtrate was concentrated to give a clear, yellow-brown oil weighing 3.17 g. Purification by normal phase chromatography provided Intermediate 24A (Alternative) (1.64 g, 61%) as a white solid. MS(ESI) m/z: 283.0 (M+H)+.1H NMR (500 MHz, methanol-d4) δ 7.82 (d, J=0.8 Hz, 1H), 7.73 (ddd, J=8.3, 6.7, 1.8 Hz, 1H), 7.48 (ddd, J=8.0, 6.5, 1.7 Hz, 1H), 7.43-7.38 (m, 1H), 4.36 (q, J=7.2 Hz, 2H), 2.39 (d, J=1.1 Hz, 3H), 1.39 (t, J=7.2 Hz, 3H).

Intermediate 25. 1-(3-Chloro-2-fluorophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid: To a solution of Intermediate 25A (50 mg, 0.177 mmol) in MeOH (0.884 mL) was added 1 N NaOH (aqueous) (1.061 mL, 1.061 mmol) and the reaction was stirred at 50° C. in a sealed vial for 3 h. The reaction mixture was then cooled to rt and concentrated. The residue was then partitioned between 1 N HCl (aqueous) and EtOAc. The layers were separated and the aqueous layer was extracted with EtOAc. The organic layers were combined, washed with brine, and concentrated to give Intermediate 25 as an off-white solid (48 mg, 107%). MS(ESI) m/z: 255.0 (M+H)+.

Intermediate 26A. 2-Azido-4-chloro-1,3-difluorobenzene: To a solution of 3-chloro-2,6-difluoroaniline (1.7 g, 10.39 mmol) in TFA (10 mL) and water (2 mL) at 0° C. was added sodium nitrite (0.717 g, 10.39 mmol) over a period of 0.5 h. After completion of addition, sodium azide (1.716 g, 26.4 mmol) in water (5 mL) was added dropwise. The reaction mixture was stirred at 0° C. for 10 min and then allowed to warm to rt. The reaction was diluted with water (75 mL) and extracted with EtOAc. The organic layer was dried and concentrated to give the desired product (1.16 g, 56%) as a brown solid.

Intermediate 26. 1-(3-Chloro-2,6-difluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid: To a solution of Intermediate 26B (46 mg, 0.160 mmol) was added LiOH (0.320 mL, 0.320 mmol). The reaction was stirred at rt overnight and acidified with 1 N HCl. The mixture was extracted with EtOAc. The organic layer was dried and concentrated to yield the desired product (40 mg, 82%). MS(ESI) m/z: 274.0 (M+H)+.

Intermediate 27A. Ethyl 5-amino-1-(3-chloro-2-fluorophenyl)-1H-1,2,3-triazole-4-carboxylate: (PCT International Application No. 2006/047516 (2006)) To a solution of NaOEt (4.99 g, 15.39 mmol) in EtOH (10 mL) at 0° C. was added ethyl 2-cyanoacetate (1.501 ml, 14.11 mmol). The reaction was stirred at 0° C. for 10 min and added 1-azido-3-chloro-2-fluorobenzene (2.2 g, 12.82 mmol). The reaction was allowed to slowly warm up to rt and stirred for 14 h. The mixture was treated with water (3 mL) and extracted with EtOAc (3×30 mL). The combined extracts were concentrated and purified by silica gel chromatography to yield the desired product (2.1 g, 58%). MS(ESI) m/z: 285.1 (M+H)+.

Intermediate 27. 5-Amino-1-(3-chloro-2-fluorophenyl)-1H-1,2,3-triazole-4-carboxylic acid: To a solution of Intermediate 27A (100 mg, 0.351 mmol) in THF (15 mL) and MeOH (15.0 mL) was added NaOH (70 mg, 1.756 mmol). The reaction was stirred at 50° C. for 2 h and then concentrated. The mixture was acidified to pH ˜5 with 1 N HCl. The resulting solid was filtered and dried to yield the desired product (69 mg, 77%). MS(ESI) m/z: 257.0 (M+H)+.

Intermediate 28A. Ethyl 5-chloro-1-(3-chloro-2-fluorophenyl)-1H-1,2,3-triazole-4-carboxylate: (Can. J. Chem.,37:118-119 (1959)). To a solution of Intermediate 27A (1.1 g, 3.86 mmol) in EtOH (30 mL) at 0° C. was passed HCl gas until all of the solid dissolved. To the solution was added isoamyl nitrite (0.520 mL, 3.86 mmol) in one portion and the resulting solution was kept at 0-5° C. for 48 h. The reaction mixture was diluted in EtOAc and washed with aq NaHCO3and brine. The organic layer was concentrated and purified by reverse phase HPLC to yield the desired product. MS(ESI) m/z: 304.0 (M+H)+.

Intermediate 28. 1-(3-Chloro-2,6-difluorophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid: To a solution of Intermediate 28A (50 mg, 0.164 mmol) in THF (6 mL) and MeOH (3.00 mL) was added LiOH (39.4 mg, 1.644 mmol). The reaction was stirred at rt for 1 h and concentrated. The residue was purified by reverse phase HPLC to yield Intermediate 28 (23 mg, 51%). MS(ESI) m/z: 276.0 (M+H)+.

Intermediate 29. 1-(3-Chloro-2-fluorophenyl)-5-methoxy-1H-1,2,3-triazole-4-carboxylic acid: To a solution of Intermediate 28A (50 mg, 0.164 mmol) in THF (6 mL) and MeOH (3.00 mL) was added LiOH (39.4 mg, 1.644 mmol). The reaction was stirred at rt for 1 h and concentrated. The residue was purified by reverse phase HPLC to yield Intermediate 29 (12 mg, 27%). MS(ESI) m/z: 272.0 (M+H)+.

Intermediate 30. 1-(3-Chloro-2-fluorophenyl)-5-methoxy-1H-1,2,3-triazole-4-carboxylic acid: To a solution of 2-fluoro-3-methoxyaniline (1 g, 7.09 mmol) in TFA (10 mL) and water (5 mL) at 0° C. was added an aq. solution of NaNO2(0.733 g, 10.63 mmol) dropwise. The resulting mixture was stirred at 0° C. for 0.5 h and NaN3(0.921 g, 14.17 mmol) was added portionwise. The reaction mixture was gradually warmed to rt and stirred for 4 h. The reaction was quenched with water (150 mL) and extracted with EtOAc (2×100 mL). The organic layer was washed with sodium phosphate solution (10%) and brine (50 mL), dried, and concentrated. The resulting brown oil was re-dissolved in DMSO (20 mL) and added t-butylpropiolate (1 mL) followed by K2CO3(1 g), Cu(OAc)2(0.2 g), and sodium ascorbate (100 mg). The resulting mixture was stirred at rt overnight. The reaction was quenched with water (200 mL) and extracted with EtOAc (2×100 mL). The organic layer was dried and concentrated. The residue was purified by silica gel chromatography to yield the desired product as brown oil.1H NMR (400 MHz, CDCl3) δ 13.19 (br. s., 1H), 7.36-7.20 (m, 2H), 7.17-6.95 (m, 1H), 6.76 (td, J=8.1, 1.3 Hz, 1H), 3.91 (s, 3H), 1.74-1.49 (m, 10H). MS(ESI) m/z: 238.0 (M+H)+.

Intermediate 32A. (E)-N′-(3-Chloro-2-fluorophenyl)-2-oxopropanehydrazonoyl chloride: To a solution of 3-chloro-2-fluoroaniline (1.511 mL, 13.74 mmol) in HCl (116 mL, 116 mmol) at 0° C. was added a solution of sodium nitrite (1.896 g, 27.5 mmol) in water (12 mL) dropwise while maintaining the temperature at 0° C. After completion of addition, the reaction was stirred at the same temperature for additional 30 mins. The pH of the reaction mixture was adjusted to 4.5 using solid sodium acetate. The resultant mixture was then treated dropwise with 3-chloropentane-2,4-dione (2.129 mL, 17.86 mmol) in methanol (12 mL). After completion of addition, the reaction mixture was allowed to warm to room temperature and stirred at room temperature overnight. The reaction mixture was diluted with water and then extracted with ether. The crude product was then purified by silica gel chromatography to isolate the desired product. MS(ESI) m/z: 249.0 (M+2H)+.

Intermediate 32B. Ethyl 3-acetyl-1-(3-chloro-2-fluorophenyl)-5-methyl-1H-pyrazole-4-carboxylate: To a solution (E)-ethyl 3-(pyrrolidin-1-yl)but-2-enoate (36.8 mg, 0.201 mmol) in DCM (2 mL) was added DIEA (0.168 mL, 1.204 mmol) followed by 32A (50 mg, 0.201 mmol) and the reaction was stirred at refluxing temperatures overnight. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was dried over MgSO4and concentrated to give the crude product. The crude product was then purified using an ISCO normal phase system. MS(ESI) m/z: 325.0 (M+H)+.

Intermediate 32. 3-Acetyl-1-(3-chloro-2-fluorophenyl)-5-methyl-1H-pyrazole-4-carboxylic acid: To a solution of 32B (67 mg, 0.206 mmol) in THF (2 mL) was added LiOH (0.227 mL, 0.227 mmol) and the reaction was stirred at room temperature overnight. The reaction mixture was acidified with 1 N HCl and extracted with EtOAc. The crude product was taken to the next step without further purification. MS(ESI) m/z: 297.0 (M+H)+.

Intermediate 33A. N′-(3-Chloro-2-fluorophenyl)acetohydrazide: To a solution of (3-chloro-2-fluorophenyl)hydrazine, HCl (450 mg, 2.284 mmol) in ether (10 mL) and THF (1 mL) at 0° C. was added sodium hydroxide (0.228 mL, 2.284 mmol) and stirred at room temperature for 1 h. The reaction mixture was concentrated, diluted with EtOAc and washed with brine. The crude product was then dried under vacuum and taken to the next step. To a solution of the above obtained oil in ether (10 mL) at 0° C. was added dropwise a solution of acetic anhydride (0.215 mL, 2.284 mmol) in ether (5 mL) and stirred at 0° C. for 30 min. The reaction mixture was concentrated, diluted with ethyl acetate and washed with brine. The organic layer was dried over MgSO4and concentrated to yield the crude product. The crude product was then taken to the next step without further purification. MS(ESI) m/z: 203.1 (M+H)+.

Intermediate 33B. Ethyl 1-(3-chloro-2-fluorophenyl)-3-hydroxy-1H-pyrazole-4-carboxylate: To Intermediate 33A (261 mg, 1.288 mmol) was added phosphoryl trichloride (973 μL, 10.43 mmol) followed by diethyl 2-(ethoxymethylene)malonate (351 μL, 1.739 mmol) and the resulting solution was heated at 70° C. for overnight. To the reaction mixture was added water slowly (Caution: lot of heat generated) and allowed to stir until the reaction mixture cooled back to room temperature. The crude product was then diluted with ethyl acetate and washed with brine. The organic layer was dried over MgSO4and concentrated to yield the crude product which was then purified using silica gel chromatography. MS(ESI) m/z: 285.0 (M+H)+.

The reaction mixture was acidified using 1 N HCl and then extracted with EtOAc. The organic layer was dried over MgSO4and concentrated to yield the crude product. The crude product was taken further without any further purification. MS(ESI) m/z: 257.0 (M+H)+.

Intermediate 34A. 2-Azido-4-chlorobenzonitrile: To a solution of 2-amino-4-chlorobenzonitrile (2.0 g, 13.11 mmol) in TFA (12 mL) was added water (2.4 mL). After cooling to 0° C., sodium nitrite (0.904 g, 13.11 mmol) was added over a period of 0.5 h. After this addition, sodium azide (2.164 g, 33.3 mmol) in water (5 mL) was gradually added dropwise. The reaction was stirred at 0° C. for 10 min, and then allowed to warm to room temperature. After 2 h, quenched the reaction with water (100 mL) and insoluble solid was filtered and dried under suction and nitrogen. Aliquot LCMS analysis indicated starting material disappeared and a new peak formed which was not ionizing.

Intermediate 34. 1-(5-Chloro-2-cyanophenyl)-5-methyl-1H-1,2,3-triazole-4-carboxylic acid: To a mixture of Intermediate 34A (400 mg, 2.035 mmol) and methyl acetoacetate (0.241 mL, 2.238 mmol) in MeOH (12 mL) was added NaOMe (121 mg, 2.238 mmol). The mixture was heated at 65° C. in a sealed tube overnight. The reaction mixture was quenched with brine and extracted to give the ester product. The aqueous layer was acidified and then extracted with ethyl acetate to yield the desired hydrolyzed product which was used in the next step without further purification. MS(ESI) m/z: 262.9 (M+H)+.

Intermediate 37B. Ethyl 1-(3-chloro-2-fluorophenyl)-2-methyl-1H-imidazole-4-carboxylate, TFA: Intermediate 37B was prepared according to the procedure described for Intermediate 24A (Alternative), by replacing Intermediate 24A1 with Intermediate 37A, by replacing triethylorthoformate with triethylorthoacetate, and by running the reaction for 45 min. Purification by reverse phase chromatography gave Intermediate 37B (0.027 g, 17%). MS(ESI) m/z: 283.1 (M+H)+.

Intermediate 39. 1-(5-Chloro-2-hydroxyphenyl)-5-methyl-1H-pyrazole-4-carboxylic acid: To a cooled (0° C.), clear yellow solution of Intermediate 38A (0.100 g, 0.339 mmol) in DCM (3.39 mL) was added dropwise boron tribromide (0.321 ml, 3.39 mmol). The resulting clear light green solution was stirred at 0° C. for 30 min and then the reaction was allowed to warm to rt. After 45 min, the reaction was added dropwise to a vigorously stirred mixture of cold EtOAc and NaHCO3. After the addition, the mixture was stirred vigorously for 10 min. Then, the layers were separated and the aqueous layer was extracted with EtOAc. The organic layers were combined, washed with brine, dried over sodium sulfate, filtered and concentrated to give the phenol (0.105 g) as an orange residue. MS(ESI) m/z: 281.0 (M+H)+and 283.0 (M+2H)+. To a clear, yellow orange solution of the phenol in methanol (2 mL) was added 1.0 M NaOH (2.036 mL, 2.036 mmol). The resulting clear, burgundy solution was stirred overnight at RT. The reaction was warmed to 50° C. for 2.5 h. The reaction was cooled to rt and concentrated. The residue was partitioned between water and EtOAc and the layers were separated. The aqueous layer was extracted with EtOAc. The aqueous layer was acidified with 1.0 M HCl and then this was extracted with EtOAc (2×). The organic layers, following acidification, were combined and washed with brine, dried over sodium sulfate, filtered and concentrated to give Intermediate 39 (0.0657 g, 77%) as an orange-brown solid. MS(ESI) m/z: 253.0 (M+H)+and 254.9 (M+2H)+.

Intermediate 40A. (E)-1-Benzylidene-2-(3-chloro-2-fluorophenyl)hydrazine: Using a modified procedure described by Deprez-Poulain. (Deprez-Poulain, R. et al.,European Journal of Medicinal Chemistry,46:3867 (2011).) To a clear, orange brown solution of (3-chloro-2-fluorophenyl)hydrazine, HCl (3 g, 15.23 mmol) in methanol (60.9 ml) was added benzaldehyde (1.543 mL, 15.23 mmol) followed by the slow addition of 1.0 M NaOH (15.23 mL, 15.23 mmol). The resulting dark brown solution was stirred at rt. Over time, a precipitate formed. After 2.5 h, the reaction was stopped and the solid was collected by filtration. The solid was washed with water, air-dried, and dried under vacuum overnight to give Intermediate 40A (1.01 g, 27%) as an off-white solid. MS(ESI) m/z: 249.0 (M+H)+.

Intermediate 40B. Ethyl 3-amino-1-(3-chloro-2-fluorophenyl)-1H-pyrazole-4-carboxylate: A dark brown mixture of Intermediate 40A (1.10 g, 4.42 mmol) and 2-cyano-3-ethoxy-2-propenoic acid ethyl ester (0.786 g, 4.64 mmol) in xylene (5.90 ml) was warmed to 160° C. After 72 h, the reaction was stopped and cooled to rt. The reaction was concentrated to give a brown residue. Next, 30 mL of a solution of 37% HCl/EtOH (1:2) was added to give a suspension. The suspension was warmed to 100° C. At elevated temperature a brown solution formed. After 20 min., the reaction was cooled to rt and the solvent was removed to give a brown residue. The residue was partitioned between sat. NaHCO3and EtOAc and the layers were separated. The aqueous layer was extracted with EtOAc (2×). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated to give a brown liquid weighing 1.3 g. Purification by normal phase chromatography gave an off-white solid weighing 0.269 g. Purification by reverse phase chromatography gave Intermediate 40B (0.080 g, 6%) as a fluffy, white solid. MS(ESI) m/z: 284.0 (M+H)+and 286.0 (M+2+H)+.

Intermediate 40. 3-Amino-1-(3-chloro-2-fluorophenyl)-1H-pyrazole-4-carboxylic acid: To a white suspension of Intermediate 40B (0.075 g, 0.264 mmol) in methanol (2.64 mL) was added 1.0 M NaOH (1.058 mL, 1.058 mmol). The suspension was warmed to 50° C. After 3 h, the resulting clear, colorless solution was cooled to rt. Then, the reaction was concentrated to give a white solid. The solid was dissolved in water and acidified to pH 3-4 with 1.0 N HCl to give a white suspension. The suspension was extracted with EtOAc (3×). The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated to give Intermediate 40 (0.0708 g, 105%) as a white solid. MS(ESI) m/z: 256.0 (M+H)+and 258.0 (M+2+H)+.

Intermediate 101A. 2-Bromo-4-chloro-3-fluoropyridine: To a solution of 2,2,6,6-tetramethylpiperidine (1.54 mL, 9.12 mmol) in THF (40 mL) was added 1.6 M n-BuLi in hexanes (5.23 mL, 8.36 mmol) dropwise at −78° C. The resulting solution was stirred for 0.5 h at 0° C. It was then cooled to −78° C. and 4-chloro-3-fluoropyridine (0.769 mL, 7.60 mmol) in 5 mL THF was added dropwise over 30 min. The resulting solution was stirred at −78° C. for 30 min. To the solution was added NBS (1.624 g, 9.12 mmol) in THF (25 mL) dropwise and the resulting solution was stirred for 1 h at −78° C., then at ambient temperature for 12 h. The reaction mixture was then diluted with EtOAc and water. The organic layer was washed with brine, concentrated and purified on silica gel chromatography to give the desired product (0.541 g, 34%) as orange oil (volatile).1H NMR (400 MHz, CDCl3) δ 8.13 (d, J=5.0 Hz, 1H), 7.35 (t, J=5.1 Hz, 1H).

Intermediate 41B. 4-Chloro-3-fluoro-2-hydrazinylpyridine, 2HCl: In microwave vial was added toluene (3 mL) and purged with N2for 5 min. tert-Butyl carbazate (128 mg, 0.950 mmol), Intermediate 101A (200 mg, 0.950 mmol), Cs2CO3(310 mg, 0.950 mmol), DPPF (20 mg, 0.036 mmol), and Pd2(dba)3(25 mg, 0.027 mmol) were added sequentially into the solution. The sealed tube was heated at 100° C. for 12 h. The reaction was diluted with brine, and extracted with EtOAc (2×). The combined organic layer was concentrated in vacuo, yielding oily residue, which was purified by silica gel chromatography to provide tert-butyl 2-(4-chloro-3-fluoropyridin-2-yl)hydrazinecarboxylate (41 mg, 16%) as orange solid. MS(ESI) m/z: 262.1 (M+H)+. To the solid was added EtOH (1 mL) and 4 N HCl in dioxane (4 mL) and the reaction mixture was stirred for 2 h at rt. The mixture was concentrated to dryness to the desired product. MS(ESI) m/z: 162.1 (M+H)+.

Intermediate 42A. 4-Chloro-2-hydrazinylpyridine, 2HCl: In microwave vial was added toluene (4 mL) and purged with N2for 5 min. tert-Butyl carbazate (66.6 mg, 0.494 mmol), 2-bromo-4-chloropyridine (95 mg, 0.494 mmol), Cs2CO3(161 mg, 0.494 mmol), and PdCl2(dppf)-CH2Cl2adduct (4.03 mg, 4.94 μmol) were added. The sealed tube was heated at 100° C. for 5 h. To the reaction mixture was added water, brine and the mixture was extracted with EtOAc (2×). The combined organic layer was concentrated and purified by silica gel chromatography to provide tert-butyl 2-(4-chloropyridin-2-yl)hydrazinecarboxylate (42 mg, 35%) as red oil. MS(ESI) m/z: 244.2 (M+H)+. To the above oil was added 4 N HCl in dioxane (2 mL) and the reaction was stirred at rt for 2 h. The mixture was concentrated to dryness to give the desired (36 mg, 34%) as a white solid. MS(ESI) m/z: 144.0 (M+H)+.

Intermediate 42. 1-(4-Chloropyridin-2-yl)-5-methyl-1H-pyrazole-4-carboxylic acid: To a solution of tert-butyl 2-((dimethylamino)methylene)-3-oxobutanoate (0.032 g, 0.148 mmol) in acetonitrile (2 mL) was added TEA (0.021 mL, 0.148 mmol) and Intermediate 42A (0.024 g, 0.133 mmol). The dark brown solution was heated to 85° C. for 1 h. The reaction mixture was concentrated, and water (1 mL) and CH2Cl2(1 mL) were added. The organic layer was concentrated and purified by silica gel chromatography to give tert-butyl 1-(4-chloropyridin-2-yl)-5-methyl-1H-pyrazole-4-carboxylate (12 mg, 27%) as yellow oil. MS(ESI) m/z: 294.2 (M+H)+. To the oil was added 4 N HCl in dioxane (2 mL). The reaction was stirred at rt for 4 h, and the clear orange solution was evaporated to dryness followed by coevaporation with toluene (2×) to give the desired product as sticky pink solid. MS(ESI) m/z: 238.2 (M+H)+.

Intermediate 43A. 3-Fluoro-2-hydrazinyl-4-methylpyridine: In a microwave vial, a solution of hydrazine monohydrate (0.051 mL, 1.053 mmol), 2-bromo-3-fluoro-4-methylpyridine (200 mg, 1.053 mmol), DIEA (0.551 mL, 3.16 mmol) in isopropanol (2 mL) was heated at 50° C. overnight. Additional hydrazine monohydrate (0.100 mL) was added and the reaction was heated at 100° C. for 30 min, then 120° C. for 30 min in a microwave reactor. Additional hydrazine monohydrate (0.100 mL) was added and the reaction was heated at 120° C. overnight. The volatile organics were removed in vacuo and the residue was washed with CH2Cl2. The resulting solid was filtered to provide the desired product. MS(ESI) m/z: 142.0 (M+H)+.

Intermediate 44A. Methyl 1-(3-fluoro-4-methylpyridin-2-yl)-1H-imidazole-4-carboxylate: A suspension of methyl 1H-imidazole-4-carboxylate (83 mg, 0.658 mmol), 2-bromo-3-fluoro-4-methylpyridine (200 mg, 1.053 mmol), copper (I) iodide (125 mg, 0.658 mmol) and potassium carbonate (546 mg, 3.95 mmol) in DMSO (2 mL) was heated at 120° for 90 min under microwave conditions. The reaction mixture was quenched with H2O, and the solid was suspended in EtOAc and MeOH. The combined organic layer was concentrated in vacuo, yielding oily residue, which was purified by reverse phase HPLC to give the desired product (5 mg, 3%). MS(ESI) m/z: 236.1 (M+H)+.

Intermediate 44. 1-(3-Fluoro-4-methylpyridin-2-yl)-1H-imidazole-4-carboxylic acid: To a solution of Intermediate 44A (5 mg, 0.021 mmol) in THF (0.6 mL) and H2O (0.3 mL) was added LiOH (5 mg). The reaction was stirred for 12 h at rt. The THF was removed in vacuo and 1 N HCl aq. solution was added until the solution became acidic. The mixture was extracted with EtOAc (2×). The combined organic layer was concentrated to dryness to give the desired product (4 mg, 3%) as a white solid. MS(ESI) m/z: 222.1 (M+H)+.

Intermediate 46. 3-(3-Chloro-2-fluorophenyl)-4-methylisoxazole-5-carboxylic acid: To the solution of Intermediate 46B (0.011 g, 0.039 mmol) in MeOH (1 mL) was added 1 N NaOH (0.078 mL, 0.078 mmol). After 18 h, the reaction was quenched with 1 N HCl (0.1 mL) and then it was concentrated to give the desired product (10 mg, 100% yield) as a white solid. MS(ESI) m/z: 255.9 (M+H)+. The material was carried onto the next step without further purification.

Intermediate 47. 5-(3-Chloro-2-fluorophenyl)-4-methylisoxazole-3-carboxylic acid: To the solution of Intermediate 47D (0.009 g, 0.032 mmol) in MeOH (1 mL) was added 1 N NaOH (0.063 mL, 0.063 mmol). After 3 h, the reaction was quenched with 1 N HCl (0.1 mL) and then it was concentrated to give the desired product (8.1 mg, 100% yield) as a white solid. MS(ESI) m/z: 255.9 (M+H)+. The material was carried onto the next step without further purification.

Intermediate 48. (R)-2-Methylbut-3-enoyl chloride: To a cooled (0° C.) solution of (R)-2-methylbut-3-enoic acid (0.450 g, 4.49 mmol) in DCM was added dropwise oxalyl chloride (0.393 mL, 4.49 mmol). The reaction mixture was stirred at 0° C. for 30 min and then it was allowed to stir at rt for 80 min. The resulting solution of (R)-2-methylbut-3-enoyl chloride was used directly.

To a flame-dried flask, equipped with a reflux condenser, containing 2-bromo-5-nitroaniline (10.0 g, 46.1 mmol), bis(neopentyl glycolato)diboron (13.01 g, 57.6 mmol), potassium acetate (13.57 g, 138 mmol), and PdCl2(dppf)-CH2Cl2adduct (0.941 g, 1.152 mmol) was added DMSO (132 mL). The resulting dark red-brown suspension was degassed with argon for 30 min and then the reaction was warmed to 80° C. After 4 h, the reaction was stopped and cooled to rt. The reaction was poured slowly into vigorously stirred ice-cold water (300 mL) to give a brown suspension. After stirring for 10 min, the suspension was filtered to collect the solid. The solid was rinsed with water (3×125 mL), air-dried, and then dried under a vacuum to give a brown solid. Purification by normal phase chromatography gave 4.36 g of Intermediate 49 as an orange solid. MS(ESI) m/z: 183.1 (M-C5H8+H)+.

Intermediate 50A. Methyl 4,5-dichloroisothiazole-3-carboxylate: To a solution of 4,5-dichloroisothiazole-3-carboxylic acid (211 mg, 1.07 mmol) in toluene (3 mL)) and MeOH (1 mL) was added trimethylsilyldiazomethane (2 M in hexane) (0.7 mL, 1.400 mmol) solution dropwise. The pale yellow solution was stirred at rt for 0.5 h. The solution was concentrated under vacuum to give yellow solid, which was subjected to the following reaction without further purification. MS(ESI) m/z: 212.1 (M+H)+.

Intermediate 50B. Methyl 4-chloro-5-(3-chloro-2-fluorophenyl)isothiazole-3-carboxylate: To a solution of Intermediate 50A (0.100 g, 0.472 mmol) and Cs2CO3(0.461 g, 1.415 mmol) in DME (3.02 mL) and water (0.605 mL) was added methyl 4,5-dichloroisothiazole-3-carboxylate (0.100 g, 0.472 mmol). The solution was purged with Ar for 0.5 h. To the solution was added Pd(PPh3)4(0.054 g, 0.047 mmol). The reaction mixture was then sealed and heated in microwave for 0.5 h at 100° C. The reaction mixture was then diluted with EtOAc and aqueous layer was decanted. The organic layer was concentrated in vacuo, yielding an oily residue which was purified by silica gel column chromatography to provide the desired product (53 mg, 37%) as a white solid. MS(ESI) m/z: 360.0 (M+H)+.

Intermediate 50. 4-Chloro-5-(3-chloro-2-fluorophenyl)isothiazole-3-carboxylic acid: To a solution of Intermediate 50B (53 mg, 0.173 mmol) in THF (2 mL) and water (1 mL) was added lithium hydroxide monohydrate (0.014 mL, 0.519 mmol). The resulting solution was stirred for 2 h at rt. The reaction mixture was concentrated in vacuo. The aqueous solution was acidified with 1 N HCl (pH=2-3) and extracted with EtOAc (2×). The organic solution was dried over Na2SO4, filtered and concentrated in vacuo to provide the desired product as a white solid (47 mg, 93%). MS(ESI) m/z: 291.1 (M+H)+.

Intermediate 51A. Ethyl 5-(3-chloro-2-fluorophenyl)nicotinate: To a solution of (3-chloro-2-fluorophenyl)boronic acid (148 mg, 0.848 mmol), ethyl 5-bromonicotinate (150 mg, 0.652 mmol), tetrabutylammonium bromide (315 mg, 0.978 mmol) and Cs2CO3(637 mg, 1.956 mmol) in dimethoxyethane (9 mL) and water (1 mL) was added Pd(Ph3P)4(113 mg, 0.098 mmol) and the resulting heterogeneous solution was purged with N2. It was then sealed and heated at 120° C. for 0.5 h in a microwave reactor. The reaction mixture was diluted with DCM and washed with brine (2×). The organic solution was dried over Na2SO4, filtered and concentrated in vacuo, yielding oily residue which was purified by silica gel column chromatography to provide the desired product (100 mg, 55%). MS(ESI) m/z: 280.1 (M+H)+.

Intermediate 51. 5-(3-Chloro-2-fluorophenyl)nicotinic acid: To a solution of Intermediate 51 A (100 mg, 0.358 mmol) in THF (4 mL) and water (3 mL) was added lithium hydroxide monohydrate (0.030 mL, 1.073 mmol) and the resulting solution was stirred for 2 h at rt. The reaction mixture was concentrated in vacuo. The aqueous solution was acidified with 1 N HCl (pH=2-3). At this point, a white solid was precipitated from the solution. The solid was collected by filtration and dried under vacuum to provide Intermediate 51 (102 mg, 99%) as a white solid. MS(ESI) m/z: 252.1 (M+H)+.

1A. (R,E)-N-((5-Bromo-2-fluoropyridin-3-yl)methylene)-2-methylpropane-2-sulfinamide: To the solution of 5-bromo-2-fluoronicotinaldehyde (5 g, 24.51 mmol), titanium (IV) ethoxide (15.42 ml, 73.5 mmol) in DCM (49.0 ml) was added (R)-2-methylpropane-2-sulfinamide (3.12 g, 25.7 mmol) and the reaction mixture was stirred at rt. After 48 h, the reaction mixture was poured into brine while rapidly stirring to form a suspension. The resulting suspension was filtered through a plug of CELITE®, and the filter cake was washed several times with DCM. The filtrate phases were separated, and the organic phase was washed with brine and dried over MgSO4. The organic layers were then concentrated to give 7.6 g crude product which was further purified using silica gel chromatography to yield the desired product (6.97 g, 93%) as an off white solid. MS(ESI) m/z: 330.8 (M+Na)+.

1B. (R)—N—((S)-1-(5-Bromo-2-fluoropyridin-3-yl)but-3-en-1-yl)-2-methylpropane-2-sulfinamide: To a saturated aqueous solution of sodium bromide (420 g, 4084 mmol) (app. 420 g in 450 ml H2O) was added 1A (6.97 g, 22.69 mmol) and indium (10.42 g, 91 mmol). To this mixture was then added 3-bromoprop-1-ene (7.85 ml, 91 mmol) dropwise, and the resulting cloudy white suspension was allowed to stir at rt for 10 h. The reaction was then quenched with saturated aqueous NaHCO3solution followed by extraction with EtOAc. The organic layer was dried over anhydrous MgSO4and concentrated to yield the crude product. The crude product was then purified using silica gel chromatography to give the desired product (8.8 g, 98%) as an off white solid. MS(ESI) m/z: 350.8 (M+H)+.

1C. (S)-1-(5-Bromo-2-fluoropyridin-3-yl)but-3-en-1-amine, 2HCl: To a solution of 1B (8.8 g, 25.2 mmol) in MeOH (100 mL) was added HCl (31.5 mL, 126 mmol) (4 M in dioxane). The reaction mixture was stirred at rt for 1 h and then concentrated to near dryness. Et2O was added to give a yellow suspension which was then filtered and the filtered solid was further washed with Et2O. The filtrate was concentrated and re-filtered with Et2O. The collected solid was then dried on the vacuum pump to give 1C (6.45 g, 80%) as a white solid. MS(ESI) m/z: 246.9 (M+H)+.

1D. (S)-tert-Butyl (1-(5-bromo-2-fluoropyridin-3-yl)but-3-en-1-yl)carbamate: To a solution of 1C (6.55 g, 20.60 mmol) in DCM (68.7 ml) at 0° C. was added TEA (11.48 ml, 82 mmol) and Boc2O (4.50 g, 20.60 mmol). The reaction mixture was stirred at 0° C. for 2 h, and then allowed to warm to rt. After stirring for 2 h, the reaction mixture was diluted with DCM and washed with saturated NaHCO3solution. The aqueous layer was re-extracted with DCM (2×). The combined organic layers were then washed with brine, dried over MgSO4to yield the crude product. The crude product was then purified using silica gel chromatography to yield the desired product (6.64 g, 87%) as a white solid. MS(ESI) m/z: 368.9 (M+Na)+.

1E. (S)-tert-Butyl (1-(5-(2-amino-4-nitrophenyl)-2-fluoropyridin-3-yl)but-3-en-1-yl)carbamate: To a RBF was added 1D (4.5 g, 13.04 mmol), 2-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-5-nitroaniline (6.52 g, 26.1 mmol), PdCl2(dppf)-DCM adduct (1.065 g, 1.304 mmol), and potassium phosphate, tribasic (5.53 g, 26.1 mmol). The RBF was equipped with a reflux condenser and the apparatus was vacuumed and back-filled with argon. Degassed DMSO (65.2 mL) was added followed by degassed H2O (1.174 mL, 65.2 mmol). The dark red reaction mixture was warmed to 90° C. for 1 h, and then allowed to cool to rt. The reaction mixture was then partitioned between EtOAc and brine, and the layers were separated. The aqueous layer was re-extracted with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated to give the crude product as thick black oil which was subjected to silica gel chromatography to yield the desired product (5.90 g, 100%) as yellow foam. MS(ESI) m/z: 403.0 (M+H)+.

1F. Methyl (3-amino-4-(5-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-6-fluoropyridin-3-yl)phenyl)carbamate: To a clear, orange solution of 1E (4.4 g, 9.95 mmol) in MeOH (100 mL) was added sequentially zinc (6.51 g, 99 mmol) and ammonium chloride (5.32 g, 99 mmol). The resulting yellow-orange suspension turned clear after a few minutes and was stirred at rt. After 2 h, the reaction mixture was filtered off to remove solid and concentrated to give a residue. The residue was diluted with EtOAc and washed with saturated NaHCO3solution. The organic layer was then dried over MgSO4and purified by silica gel chromatography to yield the desired bis amine product as peach colored foam. To a −78° C. clear, orange solution of the above bis amine product (5.08 g, 13.64 mmol) and pyridine (1.103 ml, 13.64 mmol) in DCM (136 mL) was added dropwise methyl chlorocarbonate (0.949 ml, 12.28 mmol) and the reaction mixture was stirred at −78° C. for 1.5 h. The reaction was then quenched with saturated NH4Cl solution and allowed to slowly warm to rt. The reaction mixture was diluted with DCM and the aqueous layer was re-extracted with DCM. The combined organic layers were washed with saturated NaHCO3solution followed by brine. The organic layer was then dried over MgSO4, filtered and concentrated to give the crude product as peach-colored foam which was then purified using silica gel chromatography. COSY and NOE analysis confirmed the site of addition. The desired product (4.77 g, 81%) was isolated as beige foam. MS(ESI) m/z: 431.1 (M+H)+.

1G. Methyl (4-(5-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-6-fluoropyridin-3-yl)-3-((2-methylbut-3-enoyl)amino)phenyl)carbamate: To a solution of 2-methylbut-3-enoic acid (0.216 mL, 2.091 mmol) and 1F (0.900 g, 2.091 mmol) in EtOAc (59.7 mL) was added DIEA (1.095 mL, 6.27 mmol) and the reaction was allowed to cool to −10° C. under argon. To this mixture was then added T3P (2.464 mL, 4.18 mmol) and the reaction was allowed to stir for 5 min at the same temperature and then allowed to warm to 0° C. followed by to rt slowly while stirring under argon at rt. After overnight stirring, the reaction mixture was concentrated and purified by silica gel chromatography to give 1G (887 mg, 83%) as a white solid. MS(ESI) m/z: 513.1 (M+H)+.

1H. tert-Butyl N-[(11E)-16-fluoro-9-hydroxy-5-[(methoxycarbonyl)amino]-10-methyl-8,17-diazatricyclo[13.3.1.02,7]nonadec-11-en-14-yl]carbamate: A clear, colorless solution of 11G (887 mg, 1.730 mmol) in DCE (100 mL) was degassed with argon, then split into 5 microwave vials. To the above mixture was then added Grubbs II (588 mg, 0.692 mmol) (118 mg to each vial) and heated each vial in the microwave at 120° C. for 25 min. The reaction mixture was then combined and washed with saturated NaHCO3followed by brine. The organic layer was then dried over MgSO4, filtered and concentrated to give the crude product which was purified by silica gel chromatography. Desired fractions were collected and concentrated to give 1H (568 mg, 68%) as a brown solid. MS(ESI) m/z: 485.1 (M+H)+.

1I. tert-Butyl N-[(14S)-16-fluoro-5-[(methoxycarbonyl)amino]-10-methyl-9-oxo-8,17-diazatricyclo[13.3.1.02,7]nonadeca-1(19),2,4,6,15,17-hexaen-14-yl]carbamate: To a solution of 1H (0.568 g, 1.172 mmol) in MeOH (39.1 mL) was added platinum (IV) oxide (0.027 g, 0.117 mmol). The reaction mixture was then charged with H2gas using a H2balloon and vacuumed with H2several times. The reaction was then stirred at rt under H2for 40 h. After stirring for 40 h, the reaction mixture was filtered through a pad of CELITE® and the filtrate was concentrated to yield the crude product. The crude product was then purified by silica gel chromatography to yield the Diastereomer A (1Ia) (178 mg, 25%) and Diastereomer-B (95 mg, 14%, 1Ib) as white solids. Diastereomer A—MS(ESI) m/z: 487.1 (M+H)+.

2A. (S,E)-N-((4-Chloropyridin-2-yl)methylene)-2-methylpropane-2-sulfinamide: Liu, G. et al.,J. Org. Chem.,64:1278 (1999). To a solution of S-(−)-t-butyl-sulfinamide (0.856 g, 7.06 mmol) in dichloromethane (14.13 mL) was added sequentially copper(II) sulfate (2.481 g, 15.54 mmol) and 4-chloropicolinaldehyde [1.0 g, 7.06 mmol, prepared according to a modified described by Negi (Synthesis,991 (1996))]. The white suspension was stirred at rt. After 3 h, the brown suspension was filtered through CELITE®, eluting with DCM, to give a clear brown filtrate. Concentration gave a brown oil weighing 1.85 g. Purification by normal phase chromatography gave 1.31 g of 2A as a clear, yellow oil. MS(ESI) m/z: 245.0 (M+H)+.

2B. (S)—N—((S)-1-(4-Chloropyridin-2-yl)but-3-enyl)-2-methylpropane-2-sulfinamide: To a cooled solution (−78° C.) of 2A (10 g, 40.9 mmol) in THF (204 mL) was added dropwise allyl magnesium bromide (44.9 mL, 44.9 mmol, 1M in Et2O). The reaction mixture was stirred at −78° C. After 2 h, the reaction mixture was quenched with the addition of saturated NH4Cl (25 mL) and then the reaction mixture was allowed to warm to rt. The reaction mixture was then diluted with EtOAc and water and the layers were separated. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated. Purification by normal phase chromatography gave 2B (9.23 g, 79%) as a clear, orange oil.1H NMR indicated a 4.7:1 mixture of diastereomers whereby the major diastereomer corresponds to the title compound. MS(ESI) m/z: 287.1 (M+H)+.

2C. (S)—N—((S)-1-(4-(2-Amino-4-nitrophenyl)pyridin-2-yl)but-3-enyl)-2-methylpropane-2-sulfinamide, Diastereomer A and 2D. (S)—N—((R)-1-(4-(2-amino-4-nitrophenyl)pyridin-2-yl)but-3-enyl)-2-methylpropane-2-sulfinamide, Diastereomer B: To a RBF was added 2B (9.23 g, 32.2 mmol), 2-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-5-nitroaniline (16.09 g, 64.4 mmol), potassium phosphate, tribasic (13.66 g, 64.4 mmol), DMSO (161 mL), and water (2.90 mL, 161 mmol). The RBF was equipped with a reflux condenser and then the apparatus was purged with argon for 30 minutes. Next, Pd(dppf)Cl2—CH2Cl2adduct (2.63 g, 3.22 mmol) was added and the reaction mixture was warmed to 90° C. After 4 h, the reaction was cooled to rt and then it was poured into water (1000 mL) to give a suspension. The solid was collected by filtration and then it was dissolved in EtOAc. The filtrate was extracted with EtOAc (1×). The organic layers were combined and washed with brine, dried over sodium sulfate, filtered, and concentrated. Purification by normal phase chromatography gave 2C (3.9 g) as an orange foam. An additional 3.84 g of material was obtained as a mixture of diastereomers 2C and 2D. The diastereomers were separated by chiral SFC prep HPLC (CHIRALCEL® OD-H; 20% methanol/80% carbon dioxide) which gave 2C (2.0 g) as an orange foam and 2D (0.90 g) as an orange foam. The total amount of 2C isolated was (5.9 g, 47%) as an orange foam. MS(ESI) m/z: 389.2 (M+H)+.

2E. (S)—N—((S)-1-(4-(2,4-Diaminophenyl)pyridin-2-yl)but-3-enyl)-2-methylpropane-2-sulfinamide: To a clear, orange solution of 2C (2 g, 5.15 mmol) in methanol (51.5 mL) was added sequentially zinc (3.37 g, 51.5 mmol) and ammonium chloride (2.75 g, 51.5 mmol). The resulting suspension was stirred vigorously. After 3 h, the reaction was stopped and it was filtered through a 0.45 micron GMF eluting with methanol to give a yellow filtrate. The filtrate was concentrated, then the residue was partitioned between EtOAc and water, and the layers were separated. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered, and concentrated to give 2E (1.86 g, 101%) as a yellow foam. This material was used in the next step without further purification. MS(ESI) m/z: 359.1 (M+H)+.

2G. (S)-Methyl 3-amino-4-(2-(1-aminobut-3-enyl)pyridin-4-yl)phenylcarbamate, 3HCl: To a clear, yellow solution of 2F (2.3 g, 5.52 mmol) in MeOH (55.2 mL) was added 4 M HCl in dioxane (13.80 mL, 55.2 mmol). The reaction mixture was stirred at rt. After 2 h, the reaction was concentrated to give a yellow residue. The residue was suspended in DCM and then it was concentrated. This was repeated one more time to give 2G (2.329 g, 100%) as a yellow solid. This material was used in the next step without further purification. MS(ESI) m/z: 313.1 (M+H)+.

2J. ((E)-(10R,14S)-5-Methoxycarbonylamino-10-methyl-9-oxo-8,16-diaza-tricyclo[13.3.1.02,7]nonadeca-1(19),2(7),3,5,11,15,17-heptaen-14-yl)-carbamic acid tert-butyl ester, Diastereomer A and 2K. ((E)-(10S,14S)-5-Methoxycarbonylamino-10-methyl-9-oxo-8,16-diaza-tricyclo[13.3.1.02,7]nonadeca-1(19),2(7),3,5,11,15,17-heptaen-14-yl)-carbamic acid tert-butyl ester, Diastereomer B: To a RBF was added 21 (1.57 g, 3.17 mmol), pTsOH (0.664 g, 3.49 mmol), and DCM (794 mL). The flask was then equipped with a reflux condenser and the clear yellow solution was degassed with argon for 30 min. The reaction mixture was then warmed to 40° C. for 1 h. Then a solution of Grubbs II (0.269 g, 0.317 mmol) in DCM (2 mL) was added dropwise to the reaction mixture. The reaction mixture was then stirred at 40° C. After 6 h, the reaction was cooled to rt. The reaction was washed with saturated sodium carbonate, brine, dried over magnesium sulfate, filtered, and concentrated to give the crude product as a dark brown solid. Purification by normal phase chromatography gave 2J, Diastereomer A (0.374 g, 25%) as a brown solid and a mixture of 2J, Diastereomer A and 2K, Diastereomer B (0.44 g, 30%) as a brown solid. MS(ESI) m/z: 466.9 (M+H)+.

2M (Alternative, 2HCl): To a flask containing 2L (0.880 g, 1.878 mmol) was added 4.0 M HCl in dioxane (21.13 ml, 85 mmol). The resulting suspension was sonicated to give a clear, yellow solution. After 5 to 10 min, a precipitate formed. After 1 h, the reaction was stopped and the precipitate was collected by filtration. The solid was rinsed with dioxane and air-dried to give a hygroscopic, yellow solid. The solid was dissolved in methanol, concentrated, and lyophilized to give 2M (Alternative, 2HCl) (0.7171 g, 87%) as a yellow solid. MS(ESI) m/z: 369.3 (M+H)+.

The preferred sequence for the preparation of compound 2J is described below:

2B (Alternative). (S)—N—((S)-1-(4-Chloropyridin-2-yl)but-3-enyl)-2-methylpropane-2-sulfinamide: To a cooled (0-5° C.) mixture of indium(III) chloride (13.56 g, 61.3 mmol) in tetrahydrofuran (170 mL) was added dropwise over 30 min. Allylmagnesium bromide (1 M in diethylether) (62 mL, 61.3 mmol). The reaction was allowed to warm to rt. After 1 h at rt, a solution of 2A (10 g, 40.9 mmol) in ethanol (170 mL) was added. After 2-3 h, the reaction was concentrated under vacuum at 50-55° C. The crude material was partitioned between ethyl acetate (200 mL) and water (50 mL) and the layers were separated. The aqueous layer was extracted with ethyl acetate (2×50 mL). The organic layers were combined and washed with brine (100 mL), dried over sodium sulfate, filtered and concentrated to give 2B (Alternative) (13.5 g, 106%) as a yellow oil. MS(ESI) m/z: 287.2 (M+H)+. This material was used in the next step without further purification.

2N. (S)-tert-Butyl 1-(4-Chloropyridin-2-yl)but-3-enylcarbamate: Compound 2B (Alternative) was converted to 2N in two steps by removal of the chiral auxiliary according to the procedure in step 2G and Boc-protection according to the procedure in step 2H. MS(ESI) 227.3 (M-C4H8+H)+and 305.4 (M+Na)+.

2O. (S)-tert-Butyl 1-(4-(2-amino-4-nitrophenyl)pyridin-2-yl)but-3-enylcarbamate: Compound 2O was prepared by following the procedure described in step 2C, by replacing 2B with 2N. MS(ESI) 385.1 (M+H)+.

2P. (S)-tert-Butyl 1-(4-(2,4-diaminophenyl)pyridin-2-yl)but-3-enylcarbamate: To a clear, orange solution of 2O (2.9 g, 7.54 mmol) in methanol (75 mL) was added sequentially zinc dust (4.93 g, 75 mmol) and ammonium chloride (4.04 g, 75 mmol). The resulting suspension was stirred vigorously for 4 h. The reaction was stopped and filtered through a 0.45 micron GMF eluting with methanol to give a clear, yellow filtrate. Concentration of the filtrate gave a yellow-black residue. The residue was partitioned between EtOAc and 0.25 M HCl (50 mL) and the layers were separated. The organic layer was extracted with 0.25 M HCl (50 mL). The combined aqueous layers were basified with 1.5 M K2HPO4and then extracted with EtOAc (3×). The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated to give 2P (2.63 g, 98%) as a brown foam. MS(ESI) m/z: 355.2 (M+H)+.

2Q. {3-Amino-4-[2-((S)-1-tert-butoxycarbonylamino-but-3-enyl)-pyridin-4-yl]-phenyl}-carbamic acid methyl ester: To a cooled (−78° C.) clear, brown solution of 2P (2.63 g, 7.42 mmol) and pyridine (0.600 ml, 7.42 mmol) in dichloromethane (74.2 ml) was added dropwise over 30 min methyl chloroformate (0.516 ml, 6.68 mmol). The reaction was stirred at −78° C. After 1.5 h, the reaction was quenched with sat. NH4Cl and allowed to warm to rt. The reaction was diluted with DCM and water and the layers were separated. The aqueous layer was extracted with DCM. The combined organic layers were washed with saturated NaHCO3, brine, dried over Na2SO4, filtered and concentrated. The residue dissolved in DCM (˜10 mL) and then hexane (˜300 mL) was added to give a brown suspension with brown gummy sticky substance at the bottom. The mixture was sonicated to give a mostly clear solution with the brown substance at the bottom. The solution decanted and the bottom substance rinsed with hexane, dried to give 2Q (2.7 g, 88%) as a slightly brown foam. MS(ESI) m/z: 413.2 (M+H)+.

The following Examples in Table 2 were made by using the same procedure as shown in Example 2. The acids used in the final step are as indicated in the below table in the Intermediate section. Various coupling reagents could be used other than the one described in Example 2 such as BOP, PyBop, EDC/HOBt or HATU. If needed, the coupled products are subjected to TFA deprotection condition to remove the tert-Butyl protecting group.

21D. Methyl ((7S)-7-amino-2-oxo-2,3,4,5,6,7-hexahydro-1H-8,12-(metheno)-1,9-benzodiazacyclotetradecin-15-yl)carbamate: A mixture of 21C (20 mg, 0.044 mmol) and HCl (550 μL, 2.200 mmol) (4 N in dioxane) was stirred at rt. After stirring for 2 h, the reaction mixture was concentrated to yield a tan yellow powder as the desired product (18 mg, 99%). MS(ESI) m/z: 355.2 (M+H)+.

22A. (S)-tert-Butyl 1-(dimethoxyphosphoryl)-2-oxohex-5-en-3-ylcarbamate: To a solution of dimethyl methylphosphonate (15.85 ml, 148 mmol) in THF (99 mL) at −78° C. was added n-butyllithium (93 mL, 148 mmol) dropwise. After completion of addition, the reaction was stirred at the same temperature for 30 min and then a solution of (S)-methyl 2-(tert-butoxycarbonylamino)pent-4-enoate (6.8 g, 29.7 mmol) in THF (15 mL) was added dropwise. The reaction mixture was then stirred for another 40 min at −78° C. The reaction was then quenched by adding H2O and diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO3solution and brine. The organic phase was dried over MgSO4, filtered and concentrated to give clear oil. The crude product was then purified by silica gel chromatography to give the desired product (9.3 g, 98%) as colorless oil. MS(ESI) m/z: 599.0 (M+Na)+.

22B. Methyl 4-iodo-3-nitrophenylcarbamate: To a cooled (0° C.), yellow suspension of 4-iodo-3-nitroaniline (1.320 g, 5 mmol) in DCM (50.0 mL) and pyridine (0.445 mL, 5.50 mmol) was added dropwise methyl chloroformate (0.407 mL, 5.25 mmol) and stirred for 3 h. The reaction was then diluted with DCM, washed with brine, dried over MgSO4, filtered and concentrated. The crude product was then dissolved in minimal DCM (˜20 mL) and then hexane (200 mL) was added to give a yellow suspension. The yellow suspension was then filtered and the filtered solid was rinsed with hexane and air-dried to obtain a yellow solid as the desired product (1.51 g, 94%). MS(ESI) m/z: 322.9 (M+H)+.

22C. Methyl 4-acetyl-3-nitrophenylcarbamate: A solution of 22B (0.5 g, 1.553 mmol), tributyl(1-ethoxyvinyl)stannane (1.049 mL, 3.11 mmol), and bis(triphenylphosphine)palladium(II) chloride (0.109 g, 0.155 mmol) in toluene (3 mL) in a sealed tube was heated at 110° C. After 3 h, the reaction was cooled to rt and concentrated to yield a residue. The residue was dissolved in THF (3 mL), followed by addition of 1 N HCl solution (5 mmol). The above mixture was then stirred at rt for 1 h and then diluted with EtOAc. The EtOAc mixture was then washed with brine, dried over Na2SO4, filtered and concentrated to give the crude product which was purified by silica gel chromatography to obtain the desired product (0.254 g, 69%) as a yellow solid. MS(ESI) m/z: 239.3 (M+H)+.

22D. 2-(4-((Methoxycarbonyl)amino)-2-nitrophenyl)-2-oxoacetic acid: To a solution of 22C (11.5 g, 48.3 mmol) in pyridine (48.3 mL) was added selenium dioxide (8.04 g, 72.4 mmol) in portions. The reaction mixture was then stirred under argon at 60° C. overnight and concentrated. The residue was pumped for several hours to make sure most pyridine was removed. To the solid was then added 1.0 N HCl (80 mL) and filtered to obtain a grayish solid which was dried in a vacuum-oven at 45° C. overnight. The grayish solid was then mixed with MeOH (200 mL) to yield a suspension which was then filtered and the filtrate was concentrated to give brownish foam (11.8 g, 79%) with still some residual pyridine in it. MS(ESI) m/z: 269.0 (M+H)+.

22E. Methyl 2-(4-((methoxycarbonyl)amino)-2-nitrophenyl)-2-oxoacetate: To a red oil of 22D (11.8 g, 38.3 mmol) in DCM (150 mL) at 0° C. was added TEA (7.47 mL, 53.6 mmol) and the mixture was subjected to sonication to form a red-colored solution. To this mixture was then added methyl carbonochloridate (4.15 mL, 53.6 mmol) at 0° C. After 20 min, the reaction mixture was diluted with DCM (300 mL), washed with 1 M HCl, saturated NaHCO3solution and brine. The organic phase was dried over MgSO4, filtered and concentrated to give a red colored solid. The crude product was then purified by silica gel chromatography to yield the desired product (8.6 g, 80%) as light grayish powder. MS(ESI) m/z: 283.0 (M+H)+.

22F. Methyl (4-(6-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-3-oxo-2,3-dihydropyridazin-4-yl)-3-nitrophenyl)carbamate: To a clear solution of 22A (1.16 g, 3.61 mmol) in EtOH (38.4 mL) at rt was added K2CO3(0.748 g, 5.42 mmol). The reaction mixture was stirred for 2 h and then concentrated to yield a residue which was dried under vacuum for 1 h. To the residue was then added THF (30 mL) followed by addition of a suspension of 22E (1.121 g, 3.97 mmol) in 8 mL of THF dropwise via an addition funnel. After 3 h, hydrazine (0.567 mL, 18.05 mmol) was added and the reaction was stirred at rt for 4 days. The reaction mixture was then diluted with EtOAc, washed with 1 N HCl and brine. The organic layer was then dried over MgSO4, filtered, and concentrated to give the crude product that was purified by silica gel chromatography to give the desired product (0.48 g, 29%) as a light orange solid. MS(ESI) m/z: 460.0 (M+H)+.

22G. (S)-Methyl (4-(6-(1-aminobut-3-en-1-yl)-3-chloropyridazin-4-yl)-3-nitrophenyl)carbmate: To a solution of 22F (2.2 g, 4.79 mmol) in MeOH (23.94 mL) was added HCl (4 M in Dioxane) (5.186 mL, 20.74 mmol) and stirred at rt for 6 h. The reaction mixture was then concentrated to yield a brownish solid. To the brownish solid was added ACN (23.94 mL) and phosphoryl trichloride (13.39 mL, 144 mmol), and the reaction mixture was heated at 80° C. overnight. After overnight stirring, the reaction mixture was concentrated and dried under vacuum overnight. The crude mixture was then cooled down to 0° C., followed by addition of 1 N HCl (20 mL) to quench the reaction. Neutralized the mixture with 1 N NaOH and extracted with EtOAc (2×). The organic layers were then combined, washed with brine, dried and concentrated to give a brownish solid as the desired product (1.03 g, 57%). MS(ESI) m/z: 377.9 (M+H)+.

22H. Methyl (4-(6-(1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-3-chloropyridazin-4-yl)-3-nitrophenyl)carbamate: To a solution of 22G (1.03 g, 2.73 mmol) in DCM (27.3 mL) at 0° C. was added TEA (1.140 mL, 8.18 mmol) and Boc2O (0.760 mL, 3.27 mmol). The reaction was then stirred at 0° C. for 10 min and then was slowly allowed to warm to rt and stirred overnight. The crude product was concentrated and purified by silica gel chromatography to isolate the desired product (414 mg, 36%) as orange colored foam. MS(ESI) m/z: 477.9 (M+H)+.

22I. Methyl (3-amino-4-(6-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-3-chloropyridazin-4-yl)phenyl)carbamate: To a mixture of 22H (472 mg, 0.988 mmol) and iron powder (276 mg, 4.94 mmol) in acetic acid (7.407 mL) was added H2O (2.469 mL) and heated at 70° C. for 1 h. The reaction mixture was then cooled down using an ice-H2O bath, followed by neutralization with 10 N NaOH (aq), and at final stage concentrated NaHCO3solution was used to adjusted pH to 7-8. The reaction mixture was then extracted with EtOAc (3×) and the combined EtOAc layers were further washed with brine, dried over MgSO4, filtered, concentrated and purified by silica gel chromatography. The purified product was then subjected to chiral HPLC separation using CHIRALPAK® AD column and 40% isopropanol/60% heptane) mixture as mobile phase. Two peaks were seen eluting and peak 1 was designated as Diastereomer A (22Ia) and peak 2 was designated as Diastereomer B (221B) (144 mg, 32%). MS(ESI) m/z: 447.8 (M+H)+.

24A. Methyl (4-(2-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)pyridin-4-yl)-3-((2-ethylbut-3-enoyl)amino)phenyl)carbamate: Compound 24A was prepared following the procedure described in 21 by replacing 2-methylbut-3-enoic acid with 2-ethylbut-3-enoic acid. Purification by normal phase chromatography gave 24A (0.412 g, 74%) as a yellow foam. MS(ESI) m/z: 509.3 (M+H)+.

26A. Ethyl 2-((2-(2-((S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)pyridin-4-yl)-5-((methoxycarbonyl)amino)phenyl)amino)pent-4-enoate: A mixture of 2G (954 mg, 2.313 mmol) and maleic acid (537 mg, 4.63 mmol) in ACN (20 mL) was stirred at rt under argon. To this mixture was then added ethyl 2-oxoacetate (0.458 mL, 2.313 mmol) (50% in toluene) and stirring was continued for 5 min. Next, allyltributylstannane (0.860 mL, 2.78 mmol) was added to the above mixture and stirring was continued for overnight. After overnight stirring, the reaction mixture was concentrated in vacuo and diluted with EtOAc. The organic layer was then washed with 1 N NaOH (2×) solution and dried over MgSO4. The organic layer was then concentrated in vacuo to give the crude product which was purified using silica gel chromatography to give the desired product (344 mg, 28%) as pale yellow oil. MS(ESI) m/z: 539.0 (M+H)+.

27A. 2-(1-(3-Chlorophenyl)-4-(((7S)-9-fluoro-15-((methoxycarbonyl)amino)-3-methyl-2-oxo-2,3,4,5,6,7-hexahydro-1H-8,12-(metheno)-1,10-benzodiazacyclotetradecin-7-yl)carbamoyl)-1H-pyrazol-3-yl)ethyl methanesulfonate: To a solution of Example 1 (0.01 g, 0.016 mmol) in pyridine (0.274 mL, 3.39 mmol) and DCM (0.5 mL) was added methane sulfonylchloride (1.23 μL, 0.016 mmol) and the reaction mixture was stirred at rt overnight. The reaction mixture was then concentrated and taken to the next step without further workup or purification. MS(ESI) m/z: 713.5 (M+H)+.

27B. Methyl ((7S)-7-(((3-(2-chloroethyl)-1-(3-chlorophenyl)-1H-pyrazol-4-yl)carbonyl)amino)-9-fluoro-3-methyl-2-oxo-2,3,4,5,6,7-hexahydro-1H-8,12-(metheno)-1,10-benzodiazacyclotetradecin-15-yl)carbamate: The crude product from step 27A (0.014 g, 0.020 mmol) was dissolved in DCM (1 mL) and transferred to a sealed tube where the DCM was evaporated. To the above solid was then added DIEA (0.2 mL) and toluene (1 mL). The reaction flask was sealed and heated to 110° C. for 18 h. Aliquot LCMS shows no ring closure product but only the chloroethyl product was observed. MS(ESI) m/z: 653.4 (M+H)+.

29C. Methyl (4-(1,3-dioxolan-2-yl)-3-nitrophenyl)carbamate: To a solution of 29B (6.77 g, 28.3 mmol) in THF (100 mL) was added TEA (7.89 mL, 56.6 mmol) dropwise in THF (25 mL) at −5° C. in a ice-salt bath. The temperature was maintained at −5° C., and a solution of ethyl chloroformate (3.25 mL, 34.0 mmol) in THF (30 mL) was added dropwise over 10 minutes. After stirring for an additional 30 minutes, a cold solution of sodium azide (3.68 g, 56.6 mmol) in H2O (12.5 mL) was added dropwise. After stirring for additional 1 hour, the reaction mixture was concentrated in vacuo (without heating). The oily residue was dissolved in the Et2O (100 mL), washed with H2O, brine, and dried over sodium sulfate, filtered, and concentrated (without heating) to give the acyl azide. This material was dissolved in toluene (100 mL) and heated to 110° C. After 1 h, the temperature was lowered to 80° C., MeOH (60 mL) was added, and heating was continued for overnight. The reaction mixture was concentrated and purified by silica gel chromatography to yield the desired product (5.01 g, 66%) as amber solid.1H NMR (400 MHz, DMSO-d6) δ 10.21 (s, 1H), 8.10 (d, J=1.6 Hz, 1H), 7.74-7.62 (m, 2H), 6.22 (s, 1H), 3.95-3.90 (m, 4H), 3.69 (s, 3H) ppm.

29G. Methyl (4-(6-(1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-2-oxo-1,2-dihydropyridin-4-yl)-3-nitrophenyl)carbamate: To a solution of 29F (3.0 g, 7.15 mmol) and 1-(2-ethoxy-2-oxoethyl)pyridinium bromide (1.189 g, 7.15 mmol) in EtOH (130 mL), was added ammonium acetate (11.03 g, 143 mmol) portionwise. After 15 min, the mixture was stirred at 75° C. The reaction mixture was then concentrated and dissolved in EtOAc. The organic layer was then washed with 1.0 N HCl, H2O, saturated sodium bicarbonate solution and finally by brine. The organic phase was dried over sodium sulfate, filtered and concentrated to yield a residue which was purified by normal phase chromatography to isolate the desired product (2.2 g, 67%) as a brown solid. MS(ESI) m/z: 459.3 (M+H)+.

29H. Methyl (3-amino-4-(6-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-2-oxo-1,2-dihydropyridin-4-yl)phenyl)carbamate: To a solution of 29G (2.9 g, 6.33 mmol) in MeOH (120 mL) was added ammonium chloride (0.677 g, 12.65 mmol) and zinc (4.14 g, 63.3 mmol). The suspension was stirred for 1 hour at rt and then at 65° C. for overnight. The suspension was filtered hot through a plug of CELITE® and the filter cake was washed with hot MeOH. The filtrate was concentrated and dried under vacuum to give a yellowish brown solid. This residue was re-dissolved in EtOAc (with 10% MeOH), washed with saturated sodium bicarbonate solution and brine. The organic layer was then dried over sodium sulfate, filtered and concentrated. The crude product was then subjected to chiral separation using chiral AD-H 21×250 mm, using a mixture of 35% (50/50 EtOH, i-PrOH and 0.1% DEA) and 65% CO2with a flow rate of 70 mL/min and 150 bar at 40° C. Each separated enantiomer was concentrated separately and the resulting solid placed under vacuum overnight. Analytical data corresponds to the desired product (1.12 g, 41%, 29 Ha). MS(ESI) m/z: 429.2 (M+H)+.1H NMR: (400 MHz, MeOD) δ 7.03 (d, J=8.6 Hz, 2H), 6.79 (dd, J=8.3, 2.0 Hz, 1H), 6.48 (d, J=5.6 Hz, 2H), 5.91-5.74 (m, 1H), 5.22-5.09 (m, 2H), 4.58-4.48 (m, 1H), 3.75 (s, 3H), 2.55 (t, J=5.9 Hz, 1H), 2.53-2.43 (m, 1H), 1.45 (br. s., 9H) ppm. The other isomer (29Hb) is also separately isolated.

29I. Methyl (4-(6-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-2-oxo-1,2-dihydropyridin-4-yl)-3-((2-methylbut-3-enoyl)amino)phenyl)carbamate: Isobutyl chloroformate (0.956 g, 7.00 mmol) was added to 2-methylbut-3-enoic acid (0.701 g, 7.00 mmol) and 4-methylmorpholine (0.770 mL, 7.00 mmol) in THF (33.3 mL) at 0° C. under a nitrogen atmosphere and stirred for 3 h. The resulting solids were filtered off and the filtrate was used directly for next step. To a round bottom flask containing the mixed anhydride, 29H (0.200 g, 0.467 mmol) and 4-methylmorpholine (0.770 ml, 7.00 mmol) in DMF (6 mL) was added portionwise (1 mL) every ten minutes over 1 h. The reaction mixture was then stirred at rt. After 3 d, the reaction mixture was partitioned between EtOAc and 1.0 NaOH (20 mL). The organic layer was washed with 1.0 N NaOH, H2O, 1.0 N HCl solution, H2O, and brine. The organic layer was dried, filtered and concentrated. The crude product was again dissolved in THF (20 mL) and treated with NaOH (10 mL, 10.00 mmol). After stirring for 1 h, the reaction mixture was concentrated and purified using reverse phase HPLC to give the desired product (0.09 g, 38%). MS(ESI) m/z: 511.4 (M+H)+.

29J. tert-Butyl methyl ((7S)-3-methyl-2,10-dioxo-2,3,4,5,6,7,9,10-octahydro-1H-12,8-(metheno)-1,9-benzodiazacyclotetradecine-7,15-diyl)biscarbamate: A solution of 29I (90 mg, 0.176 mmol) in DCE (anhydrous) (9793 μL) in a microwave vial was degassed for 15 minutes. To this solution was then added tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene][benzylidine]ruthenium(IV)dichloride (60.0 mg, 0.071 mmol) and the mixture was heated to 120° C. for 30 min under microwave conditions. The reaction mixture was then concentrated and purified by reverse phase HPLC. Fractions for diastereomer 29J1 (minor; Peak 1; more polar RT (by ACN PREP)=3.776 minutes) and diastereomer 29J2 (major; Peak 2; less polar RT (by ACN PREP)=3.978 minutes) were concentrated. Recovered 29J1 (8.2 mg, 19%) and 29J2 (14.8 mg, 35%) after Grubbs macrocyclization. Each diastereomer was taken on to PtO2reduction by dissolution with EtOH (10 mL) in two separate hydrogenation vessels, treatment to each with equal amount of platinum(IV) oxide (12.01 mg, 0.053 mmol), and exposed to hydrogen gas (55 psi) overnight. The reactions were filtered, concentrated, and carried forward to the next reaction without further purification. Final saturated analogs 29J3 (8.4 mg, 20%) and 29J4 (13.8 mg, 32%) were recovered as brown films. MS(ESI) m/z: 485.3 (M+H)+for both diastereomers.

33A. Methyl (4-(6-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-2-oxo-1,2-dihydropyridin-4-yl)-3-(pent-4-enoylamino)phenyl)carbamate: To a solution of 29H (115.4 mg, 0.269 mmol) in DCM (40 mL) was added pyridine (0.109 mL, 1.347 mmol). The flask was placed under nitrogen and the mixture cooled to 0° C. To this mixture was then added pent-4-enoyl chloride (0.104 mL, 0.943 mmol) and the mixture was stirred for 10 minutes at the same temperature and the reaction mixture was slowly allowed to warm to rt and stirred at rt. After stirring for overnight, the reaction mixture was concentrated under reduced pressure and the resulting yellow residue was taken up in THF and 1 N NaOH (5:2, 7 mL) and stirred at 30° C. for 1.5 h to effect hydrolysis of o-acylated intermediate to desired product. The reaction mixture was diluted with EtOAc and 1 N HCl was added to adjust the pH to 5-6 and the two phases were separated. The aqueous layer was further extracted with EtOAc (2×) and the combined organics washed with brine, dried (Na2SO4), filtered and evaporated to a residue. The crude product was then purified using silica gel chromatography to yield the desired product (97 mg, 70%) as a tan solid. MS(ESI) m/z: 511.1 (M+H)+.

33B. tert-Butyl methyl ((5E,8S)-2,11-dioxo-1,2,3,4,7,8,10,11-octahydro-13,9-(metheno)-1,10-benzodiazacyclopentadecine-8,16-diyl)biscarbamate: To a microwave vial was charged with 33A (96.6 mg, 0.189 mmol) and tricyclohexylphosphine [1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydro imidazol-2-ylidene][benzylidine]ruthenium(IV)dichloride (64.4 mg, 0.076 mmol). The vial was then capped, purged with argon and DCE (anhydrous—degassed) (10 mL) was added. The reaction mixture was then heated to 120° C. for 30 min in the microwave. The mixture was then cooled to rt and washed with saturated NaHCO3followed by brine. The organic layers were then dried over Na2SO4, filtered and evaporated to give a dark solid. The crude product was then purified using reverse phase HPLC to yield the desired product (24 mg, 26%) as a brown solid. MS(ESI) m/z: 483.0 (M+H)+.

34A. (S)-2-(4-(Methoxycarbonylamino)-2-nitrophenyl)-2-oxoethyl 2-(tert-butoxycarbonylamino)pent-4-enoate: To a clear, colorless solution of (S)-2-(tert-butoxycarbonylamino)pent-4-enoic acid (2.91 g, 13.50 mmol) in DMF (33.7 mL) was added potassium hydrogen carbonate (1.622 g, 16.20 mmol). The reaction mixture was stirred for 20 min at rt and then cooled to 0° C. To the above reaction mixture was added a solution of Intermediate 16 (4.28 g, 13.50 mmol) in DMF (33.7 mL) dropwise and the reaction mixture was allowed to warm to rt and then stirred at rt. After 18 h, the reaction was cooled to 0° C. and poured into ice-cold water. The aqueous layer was then extracted with EtOAc (3×) and the combined organic layers were washed with H2O and brine. The organic layer was then dried over Na2SO4, filtered and concentrated to yield a yellow foam as (S)-2-(4-(methoxycarbonylamino)-2-nitrophenyl)-2-oxoethyl 2-(tert-butoxycarbonylamino)pent-4-enoate (6.09 g, 100%). MS(ESI) m/z: 450.5 (M−H)+.

34B. Methyl (4-(2-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-1H-imidazol-5-yl)-3-nitrophenyl)carbamate: To a 1000-mL RBF containing 34A (6.09 g, 13.49 mmol) was added xylene (135 mL) and the above mixture was sonicated to obtain a clear yellow solution. To this solution was then added ammonium acetate (10.40 g, 135 mmol) and the flask was equipped with a dean-stark trap and reflux condenser. The reaction was warmed to 110° C. for 2 h and then raised to 140° C. for 2 h. After a total of 4 h stirring, the reaction was stopped and cooled to rt. The reaction mixture was then diluted with EtOAc and washed with saturated NaHCO3(2×) solution and brine. The organic layer was then dried over Na2SO4, filtered and concentrated to yield a brown gum which was purified using silica gel chromatography to isolate a brown foam as the desired product (0.91 g, 16%). MS(ESI) m/z: 432.5 (M+H)+.

34C. Methyl (4-(2-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-3-nitrophenyl)carbamate: A flame-dried 25 mL RBF was charged with NaH (0.092 g, 2.295 mmol) and then THF (4.17 mL) was added to give a gray suspension. The above suspension was then cooled to 0° C. and then a clear, yellow solution of 34B (0.9 g, 2.086 mmol) in THF (4.17 mL) was added dropwise. The reaction mixture was then stirred at 0° C. for 30 min and then allowed to warm to rt and stirred for 0.5 h. The yellow suspension was again cooled to 0° C. and then SEM-Cl (0.370 mL, 2.086 mmol) was added dropwise. The resulting cloudy reaction mixture was stirred at 0° C. After 1 h, the reaction was stopped by quenching with saturated NH4Cl followed by dilution with EtOAc. The layers were then separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with saturated NaHCO3, brine, dried over Na2SO4, filtered, and concentrated to obtain a yellow oil which was purified by silica gel chromatography to yield the desired product as yellow foam (0.424 g, 36%). MS(ESI) m/z: 562.0 (M+H)+. 1D NOE confirmed the regioisomeric position of SEM on the imidazole ring.

34D. (S)-Methyl 4-(2-(1-Boc-aminobut-3-enyl)-1-(2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-3-aminophenylcarbamate: To a solution of 34C (0.424 g, 0.755 mmol) in MeOH (5 mL) was added zinc (0.494 g, 7.55 mmol) and ammonium chloride (0.404 g, 7.55 mmol). The mixture was then stirred at 60° C. in a sealed tube. After 4 h, the reaction mixture was cooled to rt and the yellow suspension was diluted with DCM and washed with H2O. The aqueous layer was extracted with 15% IPA in CHCl3. The combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated. The crude product was then purified using silica gel chromatography to give an orange solid as the desired product (0.31 g, 77%). MS(ESI) m/z: 532.4 (M+H)+.

34E. Methyl (4-(2-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-3-((trifluoroacetyl)amino)phenyl)carbamate: A solution of 34D (10.2 g, 19.18 mmol) and TEA (3.19 mL, 23.02 mmol) in EtOAc (50 mL) was cooled down to 0° C. under argon. To this solution was added 2,2,2-trifluoroacetic anhydride (2.97 mL, 21.10 mmol) dropwise via a syringe pump. After completion of addition, the reaction mixture was stirred for another 30 min at 0° C. The reaction mixture was then diluted with EtOAc, washed with H2O, brine and dried over MgSO4. The crude product was then filtered to remove the solids and the organic layer was concentrated and purified by silica gel chromatography to yield the desired product (10.69 g, 89%) as a yellow solid. MS(ESI) m/z: 627.9 (M+H)+.

34F. (6S,E)-Benzyl 6-((tert-butoxycarbonyl)amino)-6-(4-(4-((methoxycarbonyl)amino)-2-(2,2,2-trifluoroacetamido)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)-2-methylhex-3-enoate: To a solution of 34E (3.3 g, 5.26 mmol) and Intermediate 17 (5.91 g, 31.1 mmol) in DCM (80 mL) was added pTsOH (0.905 g, 5.26 mmol). The above solution was then bubbled with argon for 30 min. The reaction mixture was sealed, heated to 40° C. under argon for 10 min, and added Grubbs II (1.5 g, 1.767 mmol) in 20 mL argon degassed DCM dropwise via syringe pump over 3 h while maintaining the reaction temperature at 40° C. After overnight stirring, the reaction mixture was washed with concentrated NaHCO3(aq) and brine. The organic layer was dried over MgSO4, filtered and concentrated. The crude product was then purified by silica gel chromatography to yield the desired product (1.93 g, 46%) as a yellow solid. MS(ESI) m/z: 790.4 (M+H)+.

34G. (6S)-6-((tert-Butoxycarbonyl)amino)-6-(4-(4-((methoxycarbonyl)amino)-2-(2,2,2-trifluoroacetamido)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)-2-methylhexanoic acid: A solution of 34F (1.76 g, 2.228 mmol) in MeOH (40 mL) was vacuumed and refilled with argon. To this solution under argon was added palladium on carbon (10%) (500 mg, 0.470 mmol), vacuumed and refilled with H2gas (3×). The reaction mixture was then stirred at rt under H2balloon. After overnight stirring, the reaction mixture was filtered through CELITE®. The filtrate was concentrated and purified by silica gel chromatography to isolate the desired product (1.23 g, 79%) as a beige solid. MS(ESI) m/z: 702.1 (M+H)+.

34H. (6S)-6-(4-(2-Amino-4-((methoxycarbonyl)amino)phenyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-2-yl)-6-((tert-butoxycarbonyl)amino)-2-methylhexanoic acid: To a solution of 34G (1.656 g, 2.360 mmol) in MeOH (14 mL) was added LiOH (2 N aq) (7 mL, 14.00 mmol). The reaction mixture was sealed and heated at 60° C. After 1 h, the reaction mixture was cooled down on an ice H2O bath, 1 N HCl (aq) was added to adjust pH to 6. The aqueous layer was extracted with EtOAc (2×60 mL). The combined EtOAc layers were washed with brine, dried over MgSO4, filtered, and concentrated to yield the desired product (1.43 g, 100%) as a grayish solid. MS(ESI) m/z: 606.3 (M+H)+.

34I. tert-Butyl methyl ((3R,7S)-3-methyl-2-oxo-9-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,4,5,6,7,9-octahydro-11,8-(azeno)-1,9-benzodiazacyclotridecine-7,14-diyl)biscarbamate: To a mixture of BOP (1141 mg, 2.58 mmol), DMAP (529 mg, 4.33 mmol) and DIEA (1.261 mL, 7.22 mmol) in DCM (300 mL) was added 34H (625 mg, 1.032 mmol) in DMF (5 mL) dropwise via syringe pump. The reaction mixture was stirred at rt for 2 days before transferred to a sealed vessel. The reaction was heated at 50° C. for 48 h before cooling down to rt. The reaction mixture was concentrated to small volume and to the residue was added EtOAc. The EtOAc layer was washed with 10% LiCl solution to remove DMF and the organic layers were dried over MgSO4. The organic layer was then concentrated and purified by silica gel chromatography followed by reverse phase HPLC. Two major peaks were seen on HPLC and the first peak was identified as the desired product (second peak is the other isomer) based on previous X-ray studies and stereochemistry is assigned based on previous compounds. Isolated 132 mg (22%) of the desired product as a white solid. MS(ESI) m/z: 588.1 (M+H)+.

34J. Methyl ((3R,7S)-7-amino-3-methyl-2-oxo-9-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,4,5,6,7,9-octahydro-11,8-(azeno)-1,9-benzodiazacyclotridecin-14-yl)carbamate: To a solution of 34I (120 mg, 0.204 mmol) in DCM (4 mL) was added TFA (0.8 mL, 10.38 mmol) and stirred at rt for 1 h. The reaction was quenched with concentrated Na2CO3aqueous solution followed by extraction with DCM and EtOAc. The organic layers were washed with brine, dried over MgSO4and concentrated under vacuo to yield the desired product (71 mg, 71%) as a yellow gum. MS(ESI) m/z: 488.3 (M+H)+.

The following Examples in Table 3 were made by using the same procedure as shown in Example 34. The acids used in the final step are as indicated in the below table in the Intermediate section. Various coupling reagents could be used other than the one described in Example 34 like BOP, PyBop, EDC/HOBt, HATU or T3P. Boc and SEM deprotection was achieved prior to the final coupling unlike with Example 34 where the Boc group alone was removed in step 34J.

The following Examples in Table 4 were made by using the same procedure as shown in Example 34 using the second isomer at step 341. The acids used in the final step are as indicated in the below table in the Intermediate section. Various coupling reagents could be used other than the one described in Example 34 such as BOP, PyBop, EDC/HOBt, HATU or T3P. Boc and SEM deprotection was achieved prior to the final coupling unlike with Example 34 where the Boc group alone was removed in step 34J.

45A. Methyl (3-bromo-4-(2-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)phenyl)carbamate: 45A was prepared following the procedures described in step 34A, by replacing Intermediate 16 with Intermediate 18; followed by step 34B and 34C. MS(ESI) m/z: 597.1 (M+2+H)+.

45B: (R)-2-(2-(2-((S)-1-(tert-Butoxycarbonylamino)but-3-enyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-5-(methoxycarbonylamino)phenylamino)pent-4-enoic acid: To a mixture of 45A (2 g, 3.36 mmol), copper(I) iodide (0.064 g, 0.336 mmol) and K2CO3(1.160 g, 8.39 mmol) in a sealable tube were added (R)-2-aminopent-4-enoic acid (0.464 g, 4.03 mmol) and DMSO (6.72 mL). The reaction mixture was vacuumed and back-filled with argon for three times, then capped and heated at 90° C. for 18 h. The reaction mixture was then cooled to rt and then diluted with EtOAc and H2O. The aqueous layer was extracted with EtOAc (2×). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to give the crude residue. A small amount of DCM (˜5 mL) was added to give a brown solution followed by addition of hexanes (˜300 mL) to result in a yellow suspension which was filtered. The solid was rinsed with hexane and air-dried to yield the desired product as a yellow solid (1.8 g, 85%). MS(ESI) m/z: 630.4 (M+H)+.

45C. (R)-Methyl 2-(2-(2-((S)-1-(tert-butoxycarbonylamino)but-3-enyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-5-(methoxycarbonylamino)phenylamino)pent-4-enoate: To the solution of 45B (1.8 g, 2.86 mmol) in DMF (25 mL) was added K2CO3(0.395 g, 2.86 mmol) and MeI (0.179 mL, 2.86 mmol). The reaction mixture was stirred at rt. After 20 h, the reaction mixture was diluted with EtOAc and H2O. The aqueous layer was extracted with EtOAc and the combined organic layers were washed with H2O and brine. The organic layer was then dried over Na2SO4, filtered and concentrated. The crude product was then purified by silica gel chromatography to give brown foam as the desired product (0.58 g, 32%). MS(ESI) m/z: 644.3 (M+H)+.

45D. Methyl (2R,7S)-7-((tert-butoxycarbonyl)amino)-14-((methoxycarbonyl)amino)-9-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,4,5,6,7,9-octahydro-11,8-(azeno)-1,9-benzodiazacyclotridecine-2-carboxylate: A solution of 45C (0.58 g, 0.901 mmol) and Grubbs (II) (0.306 g, 0.360 mmol) in DCE (22.52 mL) was heated at 120° C. under microwave conditions for 20 min and then cooled to rt. The reaction mixture was diluted with EtOAc and then washed with saturated NaHCO3solution and brine. The organic layer was then dried over MgSO4, filtered, and concentrated. The crude product was then purified using silica gel chromatography to give a yellow solid as the desired product (0.128 g, 23%). MS(ESI) m/z: 616.4 (M+H)+.

45E. Methyl (2R,7S)-7-((tert-butoxycarbonyl)amino)-14-((methoxycarbonyl)amino)-9-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,4,5,6,7,9-octahydro-11,8-(azeno)-1,9-benzodiazacyclotridecine-2-carboxylate: To a solution of 45D (0.128 g, 0.208 mmol) in EtOAc (5 mL) was added TFA (0.032 mL, 0.416 mmol) and 10% palladium on carbon (0.022 g, 0.021 mmol). Hydrogen was bubbled through the reaction mixture for 5 min, and the reaction was stirred under H2-balloon. After 17 h, EtOH (1 mL) was added to the reaction mixture, and the reaction was filtered through a 0.45 μM GMF rinsing with MeOH (filtered twice) and concentrated. The crude product was then purified by reverse phase HPLC and isolated the desired product as a solid (0.113 g, 64%). MS(ESI) m/z: 618.4 (M+H)+.

The following Examples in Table 5 were made by using the same procedure as shown in Example 45. The acids used in the final step are as indicated in the below table in the Intermediate section. Various coupling reagents could be used other than the one described in Example 45 like BOP, PyBop, EDC/HOBt, HATU or T3P.

51A. (S)-2-(4-(Methoxycarbonylamino)-2-nitrophenyl)-2-oxoethyl 2-(tert-butoxycarbonylamino)pent-4-enoate: To a clear, colorless solution of (S)-2-(tert-butoxycarbonylamino)pent-4-enoic acid (2.91 g, 13.50 mmol) in DMF (33.7 mL) was added potassium hydrogen carbonate (1.622 g, 16.20 mmol). The reaction mixture was then stirred for 20 min at rt and then cooled to 0° C. To the above cooled solution was then added a solution of Intermediate 16 (4.28 g, 13.50 mmol) in DMF (33.7 mL) dropwise and the reaction mixture was allowed to warm to rt and continued to stir at rt. After 18 h, the reaction was cooled to 0° C. and poured into ice-cold H2O. The aqueous layer was then extracted with EtOAc (3×) and the combined organic layers were washed with H2O, brine, dried over Na2SO4, filtered, and concentrated to yield the desired product as a yellow foam (6.09 g, 100%). MS(ESI) m/z: 450.5 (M−H)−.

51B. Methyl (4-(2-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-1H-imidazol-5-yl)-3-nitrophenyl)carbamate: To a 1000-mL RBF containing 51A (6.09 g, 13.49 mmol) was added xylene (135 mL) and the reaction mixture was sonicated to obtain a clear yellow solution. To the clear solution was then added ammonium acetate (10.40 g, 135 mmol) and the flask was equipped with a dean-stark trap and reflux condenser. The reaction mixture was then heated 110° C. for 2 h and then at 140° C. for additional 2 h. The reaction was cooled to rt and diluted with EtOAc. The mixture was then washed with saturated NaHCO3(2×) solution followed by brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography to yield the desired product as a brown foam (0.91 g, 16%). MS(ESI) m/z: 432.5 (M+H)+.

51C. Methyl (4-(2-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-3-nitrophenyl)carbamate: A flame-dried 25 mL RBF was charged with NaH (0.092 g, 2.295 mmol) and then THF (4.17 mL) was added to give a gray suspension. The suspension was cooled to 0° C. and then a clear, yellow solution of 51B (0.9 g, 2.086 mmol) in THF (4.17 mL) was added dropwise. The reaction mixture was stirred at 0° C. for 30 min and then allowed to warm to rt and stirred for 0.5 h. The yellow suspension was cooled to 0° C. and then SEM-Cl (0.370 mL, 2.086 mmol) was added dropwise. The resulting cloudy reaction mixture was stirred at 0° C. After 1 h, the reaction was stopped and quenched with saturated NH4Cl solution followed by dilution with EtOAc. The layers were separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with saturated NaHCO3, brine, dried over Na2SO4, filtered, and concentrated. The residue was then purified by silica gel chromatography to obtain the desired product as a yellow foam (0.424 g, 36%). MS(ESI) m/z: 562.0 (M+H)+. 1D NOE confirmed the regioisomeric position of SEM on the imidazole ring.

51D. (S)-Methyl 4-(2-(1-Boc-aminobut-3-enyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-3-aminophenylcarbamate: To a solution of 51C (0.424 g, 0.755 mmol) in MeOH (5 mL) was added zinc (0.494 g, 7.55 mmol) and ammonium chloride (0.404 g, 7.55 mmol). The combined reaction mixture was stirred at 60° C. in a sealed tube for 4 h and then cooled to rt. The yellow suspension was diluted with DCM and washed with H2O. The aqueous layer was extracted with 15% IPA/CHCl3and the combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated. The crude product was then purified using silica gel chromatography to give an orange solid as the desired product (0.31 g, 77%). MS(ESI) m/z: 532.4 (M+H)+.

51E. (S)-Methyl 4-(2-(1-Boc-aminobut-3-enyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-3-(but-4-enamido)-phenylcarbamate: To a solution of but-3-enoic acid (0.024 g, 0.282 mmol) and 51D (0.15 g, 0.282 mmol) in EtOAc (8.06 mL) was added DIEA (0.148 mL, 0.846 mmol). The reaction mixture was allowed to cool to −10° C. under argon. Next, T3P (0.332 mL, 0.564 mmol) was added and the reaction was allowed to stir for 5 min. The reaction mixture was then warmed to rt while stirring under argon for 1 h. The crude product was then purified by silica gel chromatography to yield a yellow solid (0.130 g, 77%). MS(ESI) m/z: 600.4 (M+H)+.

51F. tert-Butyl methyl ((7S)-2-oxo-9-((2-(trimethylsilyl)ethoxy)methyl)-1,2,3,4,5,6,7,9-octahydro-11,8-(azeno)-1,9-benzodiazacyclotridecine-7,14-diyl)biscarbamate: 51E was subjected to the macrocyclization protocol as described previously to obtain the unsaturated macrocyclized product. The purified product was then subjected to hydrogenation using palladium on carbon (10%) (83 mg, 0.042 mmol). The flask was purged with nitrogen and to the flask was added EtOH (absolute) (10 mL) and EtOAc (10 mL). The flask was again purged with nitrogen (3×), evacuated and an atmosphere of hydrogen (approx. 55 psi) was introduced and the reaction was stirred at ambient temperature. The reaction mixture was then filtered through a pad of CELITE® with the aid of additional EtOAc and the solvent was evaporated. The desired product was obtained as a colorless solid (113 mg, 93%) which was used without further purification. MS(ESI) m/z: 574.5 (M+H)+.

55A. tert-Butyl N-[(1S)-1-(4-{4-[(methoxycarbonyl)amino]-2-[2-(propan-2-yl)but-3-enamido]phenyl}-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-imidazol-2-yl)but-3-en-1-yl]carbamate, TFA salt: 55A was prepared in the same way as 21B by substituting but-3-enoic acid with 2-isopropylbut-3-enoic acid and 21A with 51D. The desired product was isolated as a greenish oil. MS(ESI) m/z: 642.6 (M+H)+.

Example 56. Methyl N-[(14S)-14-[1-(3-chloro-2-fluorophenyl)-1H-1,2,3-triazole-4-amido]-9-oxo-10-(propan-2-yl)-8,16,18-triazatricyclo[13.2.1.02,7]octadeca-1(17),2,4,6,15(18)-pentaen-5-yl]carbamate, TFA salt: Example 56 was made in the same way as Example 55 and was isolated as a single diastereomer during Grubbs macrocyclization protocol. It was isolated as the second peak following macrocyclization and the final compound was homochiral. MS(ESI) m/z: 609.3 (M+H)+. Analytical HPLC (Method A) RT=7.34 min.

57A. (S)-2-(4-(Methoxycarbonylamino)-2-nitrophenyl)-2-oxoethyl 2-(tert-butoxycarbonylamino)pent-4-enoate: To a clear, colorless solution of (S)-2-(tert-butoxycarbonyl amino)pent-4-enoic acid (2.91 g, 13.50 mmol) in DMF (33.7 mL) was added potassium hydrogen carbonate (1.622 g, 16.20 mmol) and the reaction mixture was stirred for 20 min at rt and then cooled to 0° C. To the above mixture was then added a solution of Intermediate 16 (4.28 g, 13.50 mmol) in DMF (33.7 mL) dropwise and the reaction was allowed to warm to rt and stirring was continued. After 18 h, the reaction was stopped, cooled to 0° C. and poured into ice-cold H2O. The aqueous layer was then extracted with EtOAc (3×) and the combined organic layers were washed with H2O and brine. The organic layers were then dried over Na2SO4, filtered and concentrated to give the desired product as a yellow foam (6.09 g, 100%). MS(ESI) m/z: 450.5 (M−H)−.

57B. Methyl (4-(2-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-1H-imidazol-5-yl)-3-nitrophenyl)carbamate: To a 1000-mL RBF containing 57A (6.09 g, 13.49 mmol) was added xylene (135 mL) and sonicated to obtain a clear yellow solution. To the above clear solution was then added ammonium acetate (10.40 g, 135 mmol) and the flask was equipped with a dean-stark trap and reflux condenser. The reaction mixture was then warmed to 110° C. for 2 h, then 140° C. for additional 2 h. The reaction was cooled to rt and diluted with EtOAc. The mixture was then washed with saturated NaHCO3(2×) and brine. The organic layer was dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography to yield a brown foam as the desired product (0.91 g, 16%). MS(ESI) m/z: 432.5 (M+H)+.

57C. Methyl (4-(2-((1S)-1-((tert-butoxycarbonyl)amino)but-3-en-1-yl)-1-(2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-3-nitrophenyl)carbamate: A flame-dried 25 mL RBF was charged with NaH (0.092 g, 2.295 mmol) and then THF (4.17 mL) was added to give a gray suspension. The suspension was cooled to 0° C. and then a clear, yellow solution of 57B (0.9 g, 2.086 mmol) in THF (4.17 mL) was added dropwise. The reaction mixture was stirred at 0° C. for 30 min and then allowed to warm to rt and stirred for 0.5 h. The yellow suspension was cooled to 0° C. and then SEM-Cl (0.370 mL, 2.086 mmol) was added dropwise. The resulting cloudy reaction mixture was then stirred at 0° C. After 1 h, the reaction mixture was quenched with saturated NH4Cl solution followed by dilution with EtOAc. The layers were separated and the aqueous layer was extracted with EtOAc. The combined organic layers were washed with saturated NaHCO3, brine, dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel chromatography to yield the desired product as a yellow foam (0.424 g, 36%). MS(ESI) m/z: 562.0 (M+H)+. 1D NOE confirmed the regioisomeric position of SEM on the imidazole ring.

57D. (S)-Methyl 4-(2-(1-Boc-aminobut-3-enyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-3-aminophenylcarbamate: To a solution of 57C (0.424 g, 0.755 mmol) in MeOH (5 mL) was added zinc (0.494 g, 7.55 mmol) and ammonium chloride (0.404 g, 7.55 mmol). The mixture was stirred at 60° C. in a sealed tube for 4 h and then cooled to rt. The yellow suspension was diluted with DCM and then washed with H2O. The aqueous layer was extracted with 15% IPA/CHCl3. The combined organic layers were washed with brine, dried over MgSO4, filtered and concentrated. The crude product was purified using silica gel chromatography to give an orange solid as the desired product (0.31 g, 77%). MS(ESI) m/z: 532.4 (M+H)+.

57E. (S)-Methyl 4-(2-(1-Boc-aminobut-3-enyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-imidazol-4-yl)-3-(pent-4-enamido)-phenylcarbamate: To a solution of pent-4-enoic acid (0.028 g, 0.282 mmol) and 57D (0.15 g, 0.282 mmol) in EtOAc (8.06 mL) was added DIEA (0.148 mL, 0.846 mmol). The reaction mixture was allowed to cool to −10° C. under argon. To the above mixture was then added 1-propanephosphonic acid cyclic anhydride in EtOAc (0.332 mL, 0.564 mmol) and the reaction was allowed to stir for 5 min. The reaction mixture was then warmed to rt while stirring under argon for additional 1 h and then it was concentrated. The crude product was purified by silica gel chromatography to obtain a yellow solid (0.092 g, 53%). MS(ESI) m/z: 614.1 (M+H)+.

57F. tert-Butyl methyl ((5E,8S)-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)-1,3,4,7,8,10-hexahydro-2H-12,9-(azeno)-1,10-benzodiazacyclotetradecine-8,15-diyl)biscarbamate: To a round bottom flask equipped with an argon bubbler was charged with finely powdered 57E (1.0165 g, 1.656 mmol) and p-TsOH monohydrate (0.299 g, 1.739 mmol). The flask was then purged with argon and DCM (anhydrous—degassed) (78 mL) was added followed by heating of the colorless mixture at 40° C. The mixture was rapidly stirred at this temperature until the reactants went into solution (˜5 min) after which a solution of tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene][benzylidine]ruthenium(IV)dichloride (0.070 g, 0.083 mmol) in DCM (anhydrous—degassed) (5.0 mL) was added at the rate of ˜1 drop per second. Stirring was continued at 40° C. for 90 minutes at which time an aliquot was removed. The mixture was then cooled to rt and washed with saturated NaHCO3solution and brine. The organic layer was then dried over Na2SO4, filtered, and concentrated to give a dark solid. The residue was purified using silica gel chromatography to give the desired product, as a mixture of the cis- and trans-olefin isomers. The crude product was purified by reverse phase HPLC to give two fractions, fraction 1 (trans-olefin isomer) and fraction 2 (cis-olefin isomer). Appropriate trans fractions were evaporated to obtain the desired product as a colorless solid (404 mg, 42%). MS(ESI) m/z: 586.5 (M+H)+.

57H. tert-Butyl methyl ((5E,8S)-11-cyano-2-oxo-10-((2-(trimethylsilyl)ethoxy)methyl)-1,3,4,7,8,10-hexahydro-2H-12,9-(azeno)-1,10-benzodiazacyclotetradecine-8,15-diyl)biscarbamate: A solution of 57G (0.18 g, 0.271 mmol), zinc cyanide (0.019 g, 0.162 mmol), DPPF (0.018 g, 0.032 mmol) and Pd2(dba)3-CHCl3(0.012 g, 0.014 mmol) in DMF (2 mL) was degassed for 0.5 h under argon bubbling. The solution was then stirred at 130° C. for 0.5 h under microwave conditions. The reaction mixture was then diluted with EtOAc and washed with NaHCO3solution followed by brine. The organic layer was then dried over MgSO4and concentrated to give the crude product which was purified using reverse phase HPLC to isolate the desired product (0.145 g, 88%). MS(ESI) m/z: 611.3 (M+H)+.

57I. Methyl ((5E,8S)-8-amino-11-cyano-2-oxo-1,3,4,7,8,10-hexahydro-2H-12,9-(azeno)-1,10-benzodiazacyclotetradecin-15-yl)carbamate: To a solution of 57H (145 mg, 0.237 mmol) in DCM (3 mL) was added TFA (0.500 mL) and the reaction was stirred at rt. After 2 h, the reaction mixture was concentrated to dryness. To the solid was added EtOAc and enough saturated NaHCO3(to make it basic). The aqueous layer was then extracted with EtOAc (3×) and the combined organic layers were washed with brine, dried over MgSO4, filtered, and concentrated to give 421 (90 mg, 100%) as a reddish solid. MS(ESI) m/z: 381.1 (M+H)+.

The following Examples in Table 6 were made by using the same procedure as shown in Example 63. The acids used in the final step are as indicated in the below table in the Intermediate section. Various coupling reagents could be used other than the one described in Example 63 such as BOP, PyBop, EDC/HOBt or HATU.

The following Examples in Table 7 were made by using the same procedure as shown in Example 31. The acids used in the final step are as indicated in the below table in the Intermediate section. Various coupling reagents could be used other than the one described in Example 2 like BOP, PyBop, EDC/HOBt or HATU. In step 2F methyl chloroformate can be replaced with 3-methoxypropanoyl chloride.

87A. Methyl N-{3-amino-4-[2-(1-{[(tert-butoxy)carbonyl]amino}but-3-en-1-yl)pyridin-4-yl]-2-fluorophenyl}carbamate: To a solution of 2H (50 mg, 0.121 mmol) in DMF (0.5 mL) was added Na2CO3(22 mg, 0.208 mmol), followed by Accufluor (50% in alumina) (143 mg, 0.222 mmol). The reaction was stirred at rt for 40 min and concentrated. The residue was purified by reverse phase HPLC to isolate the desired product (10 mg, 19%). MS(ESI) m/z: 431.1 (M+H)+.

88A. (S,E)-N-((4-Chloropyridin-2-yl)methylene)-2-methylpropane-2-sulfinamide: Angew. Chem. Int. Ed., 48:914-917 (2009). To a stirred suspension of (S)-2-methylpropane-2-sulfinamide (5 g, 41.3 mmol) and Cs2CO3(20.16 g, 61.9 mmol) in DCM (100 mL) was added a solution of 4-chloropicolinaldehyde (5.84 g, 41.3 mmol) in DCM (50 mL) dropwise over a period of 10 min. The solution was then stirred for 2 h at rt. The reaction mixture was diluted with EtOAc (50 mL) and washed with brine (20 mL×3). The organic layer was dried over MgSO4and concentrated to give the desired product (9.56 g, 95%) as brown thick oil. MS(ESI) m/z: 246.9 (M+H)+.

88B. (S)—N-((1S,2R)-1-(4-Chloropyridin-2-yl)-2-methylbut-3-enyl)-2-methylpropane-2-sulfinamide: To a solution of 88A (1.7 g, 6.95 mmol) in THF (50 mL) at −78° C. was added 1-methyl-2-propenylmagnesium chloride (0.5 M in THF) (13.89 mL, 6.95 mmol) dropwise over a period of 1 h. The resulting solution was stirred at −78° C. for 0.5 h and at rt overnight. The reaction was cooled to 0° C. and quenched with saturated NH4Cl. The mixture was extracted with EtOAc (3×). The combined organic layer was concentrated and purified by silica gel chromatography to give the desired product (1.27 g, 61%) as a crude beige oil.1H NMR indicated a ˜4:1 mixture of diastereomers whereby the major diastereomer corresponds to the title compound. MS(ESI) m/z: 301.1 (M+H)+.

88C. tert-Butyl (1S,2R)-1-(4-chloropyridin-2-yl)-2-methylbut-3-enylcarbamate: To a solution of 88B (1.27 g, 4.22 mmol) in MeOH (20 mL) at 0° C. was added HCl (5.28 mL, 21.11 mmol) (4 M in dioxane). The reaction was warmed to rt and stirred for 1 h. The mixture was concentrated and added Et2O. The yellow suspension was filtered, washed Et2O and dried. The above solid was dissolved in DCM (20 mL) and Et3N (2.354 mL, 16.89 mmol) and cooled to 0° C. BOC2O (0.980 mL, 4.22 mmol) was added and the reaction was stirred at rt for 2 h. The reaction was diluted with saturated NaHCO3and extracted with DCM (2×). The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel chromatography to give the desired product (1 g, 80% yield) as a white solid. MS(ESI) m/z: 297.1 (M+H)+.

88D. tert-Butyl (1S,2R)-1-(4-(2-amino-4-nitrophenyl)pyridin-2-yl)-2-methylbut-3-enylcarbamate: To a 20 mL microwave vial was added 88C (0.25 g, 0.842 mmol), 2-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-5-nitroaniline (0.253 g, 1.011 mmol), potassium phosphate, tribasic (0.358 g, 1.685 mmol), water (0.076 mL, 4.21 mmol) and DMSO (4.21 mL). The mixture was bubbled with N2for 5 min and added PdCl2(dppf)-CH2Cl2adduct (0.069 g, 0.084 mmol). The vial was sealed and heated at 90° C. for 3 h and then stirred at rt for 2 d. The mixture was partitioned between EtOAc and brine. The aqueous layer was extracted with EtOAc. The combined organic layer was concentrated and purified by silica gel chromatography to yield the desired product (0.27 g, 80%) as a yellow foam. MS(ESI) m/z: 399.1 (M+H)+.

88E. tert-Butyl ((1S,2R)-1-(4-(2,4-diaminophenyl)pyridin-2-yl)-2-methylbut-3-en-1-yl)carbamate: To a clear orange solution of 88D (0.27 g, 0.678 mmol) in methanol (6.78 mL) was added zinc (0.443 g, 6.78 mmol) and NH4Cl (0.362 g, 6.78 mmol). The resulting yellow-orange suspension turned clear after a few minutes and was stirred at rt for 1 h. The reaction was filtered and washed with MeOH. The filtrate was concentrated. The residue was diluted with EtOAc and washed with saturated aq. NaHCO3, brine, dried over MgSO4, filtered, and concentrated to give the desired product (0.25, 100%) as a brownish foam. MS(ESI) m/z: 369.2 (M+H)+.

88F. tert-Butyl N-[(1S)-1-(4-{2-amino-4-[(methoxycarbonyl)amino]phenyl}pyridin-2-yl)-2-methylbut-3-en-1-yl]carbamate: To a clear orange solution of 88E (0.25 g, 0.678 mmol) and pyridine (0.055 mL, 0.678 mmol) in DCM (6.78 ml) at −78° C. was added methyl chlorocarbonate (0.047 mL, 0.611 mmol) dropwise. The reaction was stirred at −78° C. for 1 h and quenched with saturated NH4Cl. The reaction was diluted with EtOAc and water. The aqueous layer was extracted with EtOAc. The combined organic layer was washed with brine, dried over MgSO4, filtered and concentrated to give the desired product (0.3 g, 100%) as a brown glass. MS(ESI) m/z: 427.1 (M+H)+.

88G. tert-Butyl N-[(1S)-1-{4-[2-(but-3-enamido)-4-[(methoxycarbonyl)amino]phenyl]pyridin-2-yl}-2-methylbut-3-en-1-yl]carbamate: To a solution of 88F (100 mg, 0.234 mmol) and but-3-enoic acid (0.020 mL, 0.234 mmol) in pyridine (1 mL) at 0° C. was added POCl3(0.044 mL, 0.469 mmol). The resulting orange solution was stirred at 0° C. for 10 min and diluted with DCM. The mixture was washed with aq. NaHCO3, brine, and concentrated. The residue was purified by silica gel chromatography to give the desired product (52 mg, 45%) as a beige foam. MS(ESI) m/z: 495.1 (M+H)+.

Example 89 (Alternative, 2HCl salt): Example 89 (0.067 g, 0.086 mmol) was dissolved in 1.25 M HCl in MeOH (1 mL, 1.250 mmol) and then concentrated. The process was repeated one more time to give the desired product (55 mg, 100%) as a yellow solid.

The following Examples in Table 8 were made in the same way as shown in Example 95. Carbonochloridates can be either from commercial source or generated by corresponding alcohol with various reagents such as phosgene, triphosgene. Carbonochloridates can also be replaced with activated alcohols by treating alcohols with 4-nitrophenyl carbonochloridate.

139A. Fluoromethyl carbonofluoridate: A mixture of chloromethyl carbonochloridate (0.16 g, 1.241 mmol), potassium fluoride (0.29 g, 4.99 mmol), and 18-crown-6 (0.1 g, 0.378 mmol) in acetonitrile (2.5 mL) in a sealed tube was stirred at rt overnight. The mixture was used in the next step without further purification as a 0.5 M solution.

145A. 2-Oxopropyl N-[(10R,14S)-14-[1-(3-chloro-2-fluorophenyl)-5-methyl-1H-1,2,3-triazole-4-amido]-10-methyl-9-oxo-8,16-diazatricyclo[13.3.1.02,7]nonadeca-1(19),2(7),3,5,15,17-hexaen-5-yl]carbamate: To a solution of Example 89 (0.02 g, 0.032 mmol) in DCM (1 mL) and acetonitrile (1 mL) was added sodium bicarbonate (8.12 mg, 0.097 mmol). The mixture was cooled to 0° C. under argon, and then added phosgene solution (20% in toluene) (0.051 mL, 0.097 mmol). After another 2 h, the reaction was concentrated. The residue was dissolved in acetonitrile (1 mL) and DCM (1 mL) and cooled to 0° C. under argon. 1-Hydroxypropan-2-one (7.2 mg, 0.097 mmol) and TEA (9 μL, 0.064 mmol) were added and the resulting cloudy mixture was stirred at 0° C. for 30 min, then at rt overnight. The reaction was concentrated and purified by reverse phase HPLC to isolate the desired product as a yellow solid (11 mg, 18%). MS(ESI) m/z: 648.2 (M+H)+.

The following Examples in Table 9 were synthesized using methods similar as those described in Example 149.

The following Examples in Table 10 were synthesized using procedures similar to those shown in Example 164. Example 171 was a common by-product of the coupling reaction.

173A. tert-Butyl N-[(10R,14S)-1′-hydroxy-5-[(methoxycarbonyl)amino]-10-methyl-9-oxo-8,16-diazatricyclo[13.3.1.02,7]nonadeca-1(18),2,4,6,15(19),16-hexaen-14-yl]carbamate and tert-butyl N-[(10R,14S)-12-hydroxy-5-[(methoxycarbonyl)amino]-10-methyl-9-oxo-8,16-diazatricyclo[13.3.1.02,7]nonadeca-1(18),2,4,6,15(19),16-hexaen-14-yl]carbamate (1:1 mixture): To a solution of 2J (634 mg, 1.36 mmol) in THF (13.6 mL) at 0° C. was added borane tetrahydrofuran complex (4.08 mL, 4.08 mmol) dropwise. The reaction was allowed to warm up to rt and stirred for 2.5 h. The reaction mixture was cooled to 0° C. and added sodium acetate (9.06 ml, 27.2 mmol), followed by hydrogen peroxide (4.16 mL, 40.8 mmol) dropwise. The reaction was warmed up to rt and stirred at for 8 h. The mixture was diluted with H2O and extracted with EtOAc (2×). The combined organic layer was washed with brine, dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel chromatography (0-10% MeOH/DCM) to yield a mixture of two products (323 mg, 49%) as a light grey solid. MS(ESI) m/z: 485.1 (M+H)+.

173C. Methyl N-[(10R,14,5)-14-amino-10-methyl-9,11-dioxo-8,16-diazatricyclo[13.3.1.02,7]nonadeca-1(18),2,4,6,15(19),16-hexaen-5-yl]carbamate and 173D, methyl N-[10R,14S)-14-amino-10-methyl-9,12-dioxo-8,16-diazatricyclo[13.3.1.02,7]nonadeca-1(18),2,4,6,15(19),16-hexaen-5-yl]carbamate: 173B (1:1 mixture of regioisomers) (78 mg, 0.162 mmol) was suspended in DCM (3 mL) and added TFA (0.623 mL, 8.08 mmol). The reaction became a clear light brownish solution and was stirred at rt for 1 h. The reaction was concentrated and purified by reverse phase HPLC to yield 173C, early eluting regioisomer (40 mg, 38%) as brownish oil and 173D, late eluting regioisomer (27 mg, 26%) as brownish oil. MS(ESI) m/z: 383.1 (M+H)+.

175A. To a solution of 2J (68.9 mg, 0.148 mmol) in THF (1477 μL) at 0° C. was added borane tetrahydrofuran complex (443 μL, 0.443 mmol) dropwise. The reaction was allowed to warm up to rt and stirred for 4 h. 3 M NaOAc (985 μL, 2.95 mmol) and H2O2(453 μL, 4.43 mmol) were added dropwise. The reaction was stirred at rt for 2 h and diluted with H2O. The mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated. The residue was purified by silica gel chromatography. Further purification was carried out by using Chiral OD column (mobile phase: 50% MeOH/EtOH: 50% Heptane) to give 175A (the second peak) as diastereomer mixture (7 mg, 10%). The other regioisomers, 175B (the first peak) (5 mg, 6%) and 175C (the third peak) (3 mg, 4%), were separated as methyl N-[(10R,14S)-14-{[(tert-butoxy)carbonyl]amino}-11-hydroxy-10-methyl-9-oxo-8,16-diazatricyclo[13.3.1.02,7]nonadeca-1(19),2(7),3,5,15,17-hexaen-5-yl]carbamate as homochiral compounds. MS(ESI) m/z: 485.1 (M+H)+.

197A. (14S)-14-C-1-(3-Chloro-2-fluorophenyl)-5-methyl-1H-pyrazole-4-10-methyl-9-oxo-8,16-diazatricyclo[13.3.1.02,7]nonadeca-1(19),2(7),3,5,15,17-hexaene-5,14-diamido, TFA salt: 197A was made in the same way as Example 189 by replacing (2-amino-4-(methoxycarbonyl)phenyl)boronic acid with 2-amino-5-(methoxycarbonyl)phenylboronic acid in step 2C. The diastereomer mixture was not separated and carried on through the syntheses. MS(ESI) m/z: 575.6 (M+H)+.

199D. tert-Butyl N-[(14S)-17-methoxy-5-[(methoxycarbonyl)amino]-10-methyl-9-oxo-8,16-diazatricyclo[13.3.1.02,7]nonadeca-1(19),2,4,6,15,17-hexaen-14-yl]carbamate: A solution of 199C (1.50 g, 2.86 mmol) and Ts-OH (0.598 g, 3.15 mmol) in DCM (337 mL) was heated for 0.5 h. The solution was cooled down to room temperature and bubbled with argon for 0.5 h. To the solution was added tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene][benzylidine]ruthenium(IV)dichloride (0.728 g, 0.858 mmol) and the resulting solution bubbled with argon for additional 0.5 h before heating at 45° C. for 12 hours. The reaction mixture was washed with aqueous saturated NaHCO3solution. Aqueous layer was further extracted with DCM (2×30 mL). The combined organic extract was dried over Na2SO4, concentrated, and purified by normal phase chromatography. The olefin double bond was reduced by dissolution in EtOH (50 mL), treatment with platinum oxide (0.065 gram, 0.286 mmol), and subjected to a hydrogen atmosphere (55 psi) overnight. The catalyst was filtered off through a plug of CELITE® and the filtrate concentrated to give 199D (720 mg, 51%) as a diastereomer mixture.

201A. Methyl N-(4-{6-[(1S)-1-{[(tert-butoxy)carbonyl]amino}but-3-en-1-yl]-1-methyl-2-oxo-1,2-dihydropyridin-4-yl}-3-nitrophenyl)carbamate: To a stirred solution of 29G (1.0 g, 2.181 mmol; enriched by SFC separation method similar to that used for 29H) in chloroform (43.6 mL) under an argon atmosphere was added Cs2CO3(0.711 g, 2.181 mmol) and iodomethane (0.929 g, 6.54 mmol). The reaction mixture was heated at 65° C. After 14 hours, the reaction shows a 1:1 ratio of the desired N-methylated product (more polar by LC) and the O-methoxy (less polar by LC). The reaction mixture was filtered, concentrated, and purified by normal phase column chromatography. Both products were isolated with desired product (0.542 g, 53%) being carried forward to subsequent reaction and the O-methylated side-product (382 mg, 37%, analytical data corresponds that from an earlier alternative synthesis) being set aside. MS(ESI) m/z: 473 (M+H)+.

201B. Methyl N-(4-{6-[(1S)-1-{[(tert-butoxy)carbonyl]amino}but-3-en-1-yl]-1-methyl-2-oxo-1,2-dihydropyridin-4-yl}-3-(2-methylbut-3-enamido)phenyl)carbamate: Ammonium chloride (0.122 g, 2.286 mmol) was added to a suspension of 201A (0.540 g, 1.143 mmol) and zinc (0.747 g, 11.43 mmol) in MeOH (11.43 mL). The reaction mixture was heated at 65° C. overnight. The reaction mixture was through a plug of CELITE® and concentrated. This residue was re-dissolved in EtOAc, washed with saturated sodium bicarbonate solution, brine, dried over sodium sulfate, filtered, and concentrated. 1-Propanephosphonic acid cyclic anhydride (1.455 g, 2.286 mmol; 50% in EtOAc) was added to a solution of aniline intermediate, 2-methylbut-3-enoic acid (0.460 g, 4.58 mmol), and DIPEA (1.2 ml, 6.86 mmol) in EtOAc (30 mL). After stirring for 48 hours, the reaction mixture was washed with saturated sodium bicarbonate solution, brine, dried over sodium sulfate, filtered, and concentrated to give the desired product. MS(ESI) m/z: 525.2 (M+H)+.

204A. tert-Butyl N-[(15S)-5-[(methoxycarbonyl)amino]-9-oxo-17-{[2-(trimethylsilyl)ethoxy]methyl}-8,17,19-triazatetracyclo[14.2.1.02,7.011,13]nonadeca-1(18),2,4,6,16(19)-pentaen-15-yl]carbamate: To a mixture of 34F (100 mg, 0.175 mmol) and diacetoxypalladium (1.963 mg, 8.75 μmol) in CH2Cl2(20 mL) at 0° C. was added diazomethane (73.5 mg, 1.749 mmol) dropwise. The reaction was stirred for 2 h and quenched with 1 mL HOAc. The solution was neutralized with aq. Na2CO3and extracted with ether. The organic layer was washed with brine, dried over MgSO4, filtered, and concentrated. The residue was purified by reverse phase HPLC yield the product (29 mg, 28%) as white solid. NMR was shown to be a mixture a diastereomers. MS(ESI) m/z: 586.4 (M+H)+.

The following Examples in Table 11 were made by using the same procedure as shown in Example 34. The acids used in the final step are as indicated in the below table in the Intermediate section. Various coupling reagents could be used other than the one described in Example 34 like BOP, PyBop, EDC/HOBt, HATU or T3P. Boc and SEM deprotection was achieved prior to the final coupling unlike with Example 34 where the Boc group alone was removed in step 34J.

215B. {3-Bromo-4-[2-((S)-1-tert-butoxycarbonylamino-but-3-enyl)-1-(2-trimethylsilanyl-ethoxymethyl)-1H-imidazol-4-yl]-phenyl}-carbamic acid methyl ester: To a cooled (0° C.) solution of 215A (15 g, 32.2 mmol) in THF (77 mL) was added N,N-dicyclohexylmethylamine (7.52 mL, 35.5 mmol) followed by the dropwise addition of SEM-Cl (6.29 mL, 35.5 mmol). The reaction was stirred at 0° C. for 2 h and then it was allowed to warm slowly to rt. After 18 h, the yellow suspension was diluted with EtOAc, washed with saturated sodium bicarbonate, brine, dried over MgSO4, filtered and concentrated. Purification by normal phase chromatography gave 12.24 g (64%) of 215B as an off-white solid. MS(ESI) m/z: 595.1 (M+H)+and 597.2 (M+2+H)+.

215D. tert-Butyl N-[(1S)-1-(4-{4-[(methoxycarbonyl)amino]-2-[(2R)-2-methylbut-3-enamido]phenyl}-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-imidazol-2-yl)but-3-en-1-yl]carbamate: To a cooled (0° C.), clear yellow orange solution of 215C (4.83 g, 9.08 mmol) in ethyl acetate (91 mL) was added Intermediate 45 (1.0 g, 9.99 mmol) and Hunig's base (6.34 mL, 36.3 mmol). Next, 1-propanephosphonic acid cyclic anhydride (T3P) (50% in EtOAc) (13.38 mL, 22.70 mmol) was added dropwise over 20 min. and the reaction was stirred at 0° C. After 3 h, the reaction was diluted with EtOAc and washed with sat. NaHCO3. The aqueous layer was extracted with EtOAc (2×). The organic layers were combined and washed with brine, dried over sodium sulfate, filtered and concentrated to give an orange foam. Purification by normal phase chromatography gave 215D (4.53 g, 81%) as a white foam. Proton NMR indicated a 3:1 mixture of diastereomers. MS(ESI) m/z: 614.4 (M+H)+.

215G. tert-Butyl N-[(10R,14S)-5-[(methoxycarbonyl)amino]-10-methyl-9-oxo-16-{[2-(trimethylsilyl)ethoxy]methyl}-8,16,18-triazatricyclo[13.2.1.02,7]octadeca-1(17),2,4,6,15(18)-pentaen-14-yl]carbamate: A dark brown solution of 215E (1.71 g, 2.92 mmol) in EtOAc (97 mL) was degassed with argon for 30 minutes. Next, platinum(IV) oxide (0.066 g, 0.292 mmol) was added and hydrogen gas from a balloon was bubbled through the reaction mixture for several minutes. The reaction was stirred under a hydrogen atmosphere. After 24 h, an additional amount of platinum(IV) oxide (0.192 g, 0.876 mmol) was added and the reaction was stirred under a hydrogen atmosphere. After 21 h, the reaction was stopped. The vessel was purged with vacuum/argon three times, then CELITE® was added, and the reaction was filtered rinsing with EtOAc. The resulting clear, yellow brown filtrate was concentrated to give an off-white solid weighing 1.66 g. Recrystallization from methanol (30 mL) gave 215G (0.575 g, 34%) as a white solid. MS(ESI) m/z: 588.4 (M+H)+.

215H. tert-Butyl N-[(10R,14S)-5-amino-10-methyl-9-oxo-16-{[2-(trimethylsilyl)ethoxy]methyl}-8,16,18-triazatricyclo[13.2.1.02,7]octadeca-1(17),2,4,6,15(18)-pentaen-14-yl]carbamate (Diastereomer A), 2 TFA and 2151. tert-butyl N-[(10S,14S)-5-amino-10-methyl-9-oxo-16-{[2-(trimethylsilyl)ethoxy]methyl}-8,16,18 triazatricyclo[13.2.1.02,7]octadeca-1(17),2,4,6,15(18)-pentaen-14-yl]carbamate (Diastereomer B), 2 TFA: A sealed tube containing a white suspension of 215G (0.100 g, 0.170 mmol) in MeOH (2.84 mL) and 1.0 M NaOH (1.021 mL, 1.021 mmol) was warmed to 75° C. After 2.5 h, additional MeOH (5.6 mL) and 1.0 M NaOH (1.021 mL, 1.021 mmol) were added and the reaction was heated at 75° C. After 16.5 h, additional 1.0 M NaOH (2 mL) was added and the reaction was heated at 75° C. After 21 h, the reaction was stopped and cooled to rt. The reaction was neutralized with 1.0 N HCl and concentrated. The solid was partitioned between EtOAc and saturated NaHCO3and the layers were separated. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated to give a white solid weighing 0.107 g. Purification by reverse phase chromatography gave 215H (Diastereomer A) (0.082 g, 63.6% yield) and 215I (Diastereomer B) (0.025 g, 19%). MS(ESI) m/z: 530.4 (M+H)+.

216A. tert-Butyl N-[(12E,15S)-18-chloro-5-[(methoxycarbonyl)amino]-9-oxo-17-{[2-(trimethylsilyl)ethoxy]methyl}-8,17,19-triazatricyclo[14.2.1.02,7]nonadeca-1(18),2,4,6,12,16(19)-hexaen-15-yl]carbamate: A white suspension of 57F (2.56 g, 4.37 mmol) and NCS (0.700 g, 5.24 mmol) in CHCl3(18.36 mL) and ACN (18.36 mL) was heated to 65° C. After 10 h, the reaction mixture was cooled to rt and partitioned between DCM and water and the layers were separated. The aqueous layer was extracted with DCM (2×). The organic layers were combined, washed with saturated NaHCO3, brine, dried over MgSO4, filtered and concentrated to give a brown foam. The E- and Z-alkene isomers were separated by reverse phase chromatography. The fractions containing the E-alkene isomer were combined, neutralized with a solution of saturated NaHCO3, and then concentrated to give a solid. The solid was partitioned between EtOAc and water and the layers were separated. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated to give the desired product (1.15 g, 42%) as yellow foam. MS(ESI) m/z: 620.1 (M+H)+.

216B. Methyl N-[(12E,15S)-15-amino-18-chloro-9-oxo-17-{[2-(trimethylsilyl)ethoxy]methyl}-8,17,19-triazatricyclo[14.2.1.02,7]nonadeca-1(18),2,4,6,12,16(19)-hexaen-5-yl]carbamate: To a solution of 216A (0.24 g, 0.387 mmol) in DCM (5 mL) was added TFA (1 mL, 12.98 mmol). The reaction was stirred at rt for 1 h and concentrated. Purification by reverse phase chromatography gave, after neutralization of the fractions with saturated NaHCO3and concentration, a solid. The solid was partitioned between EtOAc and water and the layers were separated. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated to give the desired product (0.095 g, 47%) as a white solid. MS(ESI) m/z: 520.1 (M+H)+.

217A. (S)-1-(4-Chloropyridin-2-yl)but-3-en-1-amine: To (S)-tert-butyl (1-(4-chloropyridin-2-yl)but-3-en-1-yl)carbamate (3 g, 10.61 mmol) was added HCl in dioxane (15 mL, 60.0 mmol) and the reaction was stirred at room temperature for 1 h. The reaction mixture was then concentrated and taken to the next step without further purification. MS(ESI) m/z: 183.1 (M+H)+.

217B. (S)-Benzyl (1-(4-chloropyridin-2-yl)but-3-en-1-yl)carbamate: To a solution of 217A (1.938 g, 10.61 mmol) in MeOH (40 mL) was added benzyl (2,5-dioxopyrrolidin-1-yl)carbonate (2.64 g, 10.61 mmol) followed by DIEA (3.71 mL, 21.22 mmol) and the reaction was stirred at room temperature over night. The mixture was concentrated. The residue was diluted with ethylacetate and washed with brine. The organic layer was dried over MgSO4and concentrated. The crude product was purified by silica gel chromatography to yield the desired product (3.3 g, 95%) as a yellow solid. MS(ESI) m/z: 317.0 (M+H)+.

217C. (S)-Benzyl (1-(4-chloropyridin-2-yl)-3-oxopropyl)carbamate: (Reference:J. Org. Chem.,58(4):860-866 (1993)) To a solution of 217B (1 g, 3.16 mmol) in MeOH (40 mL) and water (20 mL) was added OsO44 wt % in water (1.350 mL, 0.221 mmol). After 5 min of stirring, a clear tan yellow solution formed. To this solution was then added sodium periodate (2.026 g, 9.47 mmol) with vigorous stirring. The color discharged then gradually a white suspension formed. The reaction mixture was continued to stir at room temperature over night. The reaction mixture was diluted with water (˜100 mL) and extracted with EtOAc (3×). The combined organic layers were dried over MgSO4, filtered, and concentrated. The residue was purified by silica gel chromatography to yield the desired product (66%). MS(ESI) m/z: 319.0 (M+H)+.

217D. (S)-Benzyl (1-(4-chloropyridin-2-yl)-3-hydroxypropyl)carbamate: To a solution of 217C (2.2 g, 6.90 mmol) in ethanol (50 mL) was added sodium borohydride (0.522 g, 13.80 mmol) and the reaction was stirred at rt over night. The reaction mixture was then quenched with brine and extracted with EtOAc. The organic layer was concentrated and the residue was purified by silica gel chromatography to yield the desired product (1.58 g, 68%). MS(ESI) m/z: 321.0 (M+H)+.

217E. (S)-tert-Butyl 2-(3-(((benzyloxy)carbonyl)amino)-3-(4-chloropyridin -2-yl)propoxy)acetate: To a mixture of tert-butyl bromoacetate (0.415 mL, 2.81 mmol) and NaH (224 mg, 5.61 mmol) in THF (18 mL) at 0° C. was added a solution of 217D (900 mg, 2.81 mmol) in THF (9 mL) dropwise. The reaction mixture was stirred at 0° C. for additional 1 h and then quenched with saturated NH4Cl. The mixture was extracted with ethylacetate. The organic layer was dried over MgSO4and concentrated. The residue was purified by silica gel chromatography to isolate the desired product (300 mg, 23%). MS(ESI) m/z: 435.0 (M+H)+.

217F. (S)-tert-Butyl 2-(3-(4-(2-amino-4-nitrophenyl)pyridin-2-yl)-3-(((benzyloxy)carbonyl)amino)propoxy)acetate: Argon was bubbled through a solution of DMSO (8 mL) and water (0.062 mL, 3.45 mmol) for 30 min. Then this solvent mixture was added to a microwave vial containing 217E (300 mg, 0.690 mmol), 2-(5,5-dimethyl -1,3,2-dioxaborinan-2-yl)-5-nitroaniline (345 mg, 1.380 mmol) and phosphoric acid, potassium salt (293 mg, 1.380 mmol). Argon was again bubbled through the deep red solution for 15-20 min. Then 1,1′-bis(diphenylphosphino)ferrocenepalladium(II) dichloride, DCM (56.7 mg, 0.069 mmol) was added and the mixture was stirred at 90° C. for 5 h. The reaction mixture was diluted with ethyl acetate and washed with brine. The organic layer was dried over MgSO4and concentrated. The residue purified by silica gel chromatography to yield the desired product (170 mg, 43%). MS(ESI) m/z: 537.0 (M+H)+.

217G. (S)-tert-Butyl 2-(3-(((benzyloxy)carbonyl)amino)-3-(4-(2,4-diaminophenyl)pyridin-2-yl)propoxy)acetate: To a solution of 217F (170 mg, 0.317 mmol) in MeOH (8 mL) was added zinc (207 mg, 3.17 mmol) and ammonium chloride (169 mg, 3.17 mmol). The reaction was stirred at rt for 5 h. The reaction mixture was filtered using a 0.45 micron filter and concentrated to give the crude product which was purified by silica gel chromatography to yield the desired product (70 mg, 41%). MS(ESI) m/z: 507.0 (M+H)+.

217H. (S)-tert-Butyl 2-(3-(4-(2-amino-4-((methoxycarbonyl)amino)phenyl)pyridin-2-yl)-3-(((benzyloxy)carbonyl)amino)propoxy)acetate: To a solution of 217G (70 mg, 0.138 mmol) in DCM (5 mL) at −78° C. was added pyridine (0.011 mL, 0.138 mmol) followed by methyl chloroformate (10.70 μL, 0.138 mmol). The reaction was stirred at −78° C. for 1 h and then quenched with saturated ammonium chloride. The mixture was extracted with DCM and EtOAc. The combined organic layer was concentrated and purified by silica gel chromatography to yield the desired product (70 mg, 85%). MS(ESI) m/z: 565.1 (M+H)+.

217I. (S)-2-(3-(4-(2-Amino-4-((methoxycarbonyl)amino)phenyl)pyridin-2-yl)-3-(((benzyloxy)carbonyl)amino)propoxy)acetic acid: 217H (70 mg, 0.124 mmol) was treated with HCl in dioxane (5 mL, 20.00 mmol) at rt under argon for 1 h. The reaction mixture was then concentrated and the crude product was taken to the next step without further purification (56 mg, 84%). MS(ESI) m/z: 509.0 (M+H)+.

218B. tert-Butyl N-[(1R)-2-(benzyloxy)-1-(4-{4-[(methoxycarbonyl)amino]-2-(trifluoroacetamido)phenyl}-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-imidazol-2-yl)ethyl]carbamate: To a solution of 218A (2.035 g, 3.33 mmol) in DCM (107 mL) at 0° C. was added pyridine (0.404 mL, 4.99 mmol), followed by TFAA (0.611 mL, 4.32 mmol). After 30 min, the reaction was stopped and it was washed with saturated NaHCO3, 1 N HCl, brine, dried over magnesium sulfate, filtered and concentrated to give the desired product (2.3 g, 98% yield) as a yellow solid. The material was carried onto the next step without further purification.

218E. 3-[(2R)-2-(4-{2-Amino-4-[(methoxycarbonyl)amino]phenyl}-1-{[2-(trimethylsilyl)ethoxy]methyl}-1H-imidazol-2-yl)-2-{[(tert-butoxy)carbonyl]amino}ethoxy]propanoic acid, 2 TFA salt: To the solution of 218D (0.013 g, 0.015 mmol) in MeOH (1 mL) was added 1 N NaOH (0.1 mL, 0.100 mmol). The reaction was stirred at 75° C. in a sealed tube. After 7 h, the reaction was cooled to rt and then it was concentrated. Purification by reverse phase HPLC afforded the desired product (0.008 g, 67%) as a yellow solid. MS(ESI) m/z: 594.4 (M+H)+.

218G. Methyl N-[(14R)-14-amino-9-oxo-12-oxa-8,16,18-triazatricyclo[13.2.1.02,7]octadeca-1(17),2,4,6,15(18)-pentaen-5-yl]carbamate, 2HCl salt: The solution of 218F (0.005 g, 7.25 μmol) in 4 M HCl in 1,4-dioxane (0.5 mL, 2.000 mmol) in a sealed vial was heated at 65° C. After 1 h, the reaction was cooled to rt and then it was concentrated to give the desired product (3 mg, 100% yield) as a yellow solid. MS(ESI) m/z: 346.2 (M+H)+. The material was carried onto the next step without further purification.

222B. tert-Butyl N-[(12E,15S)-5-[(methoxycarbonyl)amino]-11-methyl-9-oxo-17-{[2-(trimethylsilyl)ethoxy]methyl}-8,17,19-triazatricyclo[14.2.1.02,7]nonadeca -1(18),2,4,6,12,16(19)-hexaen-15-yl]carbamate and Z-isomer: A flame-dried RBF, equipped with condenser, containing a solution of 222A (1.1 g, 1.752 mmol) and p-toluenesulfonic acid monohydrate (0.367 g, 1.927 mmol) in DCM (1600 mL) was degassed for 1 h with nitrogen. The reaction mixture was refluxed for 1 h under nitrogen atmosphere. Next, a solution of tricyclohexylphosphine[1,3-bis(2,4,6-trimethylphenyl) -4,5-dihydroimidazol-2-ylidene][benzylidine]ruthenium(IV)dichloride (0.596 g, 0.701 mmol) in DCM (15 mL), purged with nitrogen for 10 min, was added slowly. The reaction was stirred overnight at 45° C. The reaction was cooled to rt. The reaction mixture was washed with saturated NaHCO3(2×250 mL), brine solution (250 mL), dried by Na2SO4, filtered and concentrated to give a gummy brown solid. Purification using silica gel chromatography gave the desired product (0.88 g, 84%) as a brown solid and a mixture of E and Z isomers. MS(ESI) m/z: 600.4 (M+H)+.

223A. tert-Butyl N-(1-diazo-2-oxohex-5-en-3-yl)carbamate: To a cooled (−40° C.) solution of 2-((t-butoxycarbonyl)amino)pent-4-enoic acid (15 g, 69.7 mmol) in THF (250 mL) was added N-methylmorpholine (9.19 mL, 84 mmol) followed by the dropwise addition of isobutyl chloroformate (10.98 mL, 84 mmol). The reaction was stirred at −40° C. for 20 min, whereupon it was filtered to remove the salts. The filtrate was added to a solution of diazomethane (4.39 g, 105 mmol) in Et2O (500 mL) [Generated from 1-methyl-3-nitro-1-nitrosoguanidine]. The reaction mixture was stirred at −40° C. for 3 h and then the reaction was allowed to warm to rt. After 1 h, the reaction was purged with nitrogen for 30 min to remove the excess diazomethane. The reaction mixture was washed with a saturated solution of NaHCO3(2×100 mL), water (2×50 mL), brine solution (1×80 mL), dried by Na2SO4, filtered and concentrated to give a yellow solid (16 g). Purification by normal phase chromatography afforded the desired product (12.5 g, 75%) as a yellow solid.1H NMR (300 MHz, CDCl3) δ 5.66-5.83 (m, 1H), 5.48 (br. s., 1H), 5.19 (dd, J=3.21, 1.79 Hz, 1H), 5.03-5.16 (m, 2H), 4.24 (br. s., 1H), 2.35-2.62 (m, 2H), 1.46 (s, 9H).

223C. tert-Butyl N-[1-(1H-imidazol-4-yl)but-3-en-1-yl]carbamate: A pressure tube containing a solution of 223B (28 g, 96 mmol), formamidine acetate (19.95 g, 192 mmol) and K2CO3(53.0 g, 383 mmol) in DMF (200 mL) was heated at 100° C. overnight. The reaction mixture was cooled to rt and concentrated. The residue was partitioned between water (200 mL) and ethyl acetate (500 mL) and the layers were separated. The aqueous layer was extracted with ethyl acetate (2×200 mL). The organic layers were combined and washed with brine (1×100 mL), dried by Na2SO4, filtered and concentrated to give the desired product (25.5 g, 84%) as a gummy brown solid. This was used in the next step without purification. MS(ESI) m/z: 238.2 (M+H)+.

224A. tert-Butyl N-[(3,6-dichloropyridazin-4-yl)methyl]carbamate: A mixture of 3,6-dichloropyridazine (0.765 g, 5.14 mmol) in water (25 mL) was heated to 75° C. 2-((tert-Butoxycarbonyl)amino)acetic acid (1 g, 5.71 mmol) and ammonium formate (0.072 g, 1.142 mmol) were added. Then, a solution of silver nitrate (0.194 g, 1.142 mmol) in water (1 mL) was added dropwise over 2 min. To the resulting dark brown solution was added dropwise a solution of ammonium persulfate (5.21 g, 22.83 mmol) in water (30 mL) over 25 min. A purple precipitate formed. The reaction mixture was stirred for additional 40 min, and then cooled to rt. The reaction mixture was poured onto ice, basified with aq. ammonia, keeping the temperature below 5° C. The reaction mixture was extracted with ethyl acetate and the organic layer was dried over sodium sulfate, filtered and concentrated. Purification by normal phase chromatography afforded the desired product (0.3 g, 18%) as an off-white solid. MS(ESI) m/z: 278.1 (M+H)+.1H NMR (300 MHz, CDCl3) δ 7.51 (s, 1H), 5.15 (br. s., 1H), 4.40 (s, 2H), 1.49 (s, 9H).

The following Examples in Table 12 were made by using coupling acids with amines. The acids used are as indicated in the below table in the Intermediate section. Various coupling reagents could be used other than the one described in Example 34 like BOP, PyBop, EDC/HOBt, HATU or T3P. Boc and SEM deprotection was achieved when necessary.