REFERENCE TO PRIORITY APPLICATION

This application claims the benefit of priority from U.S. Provisional Patent Application Ser. No. 60/735,982, filed on Nov. 10, 2005.

FIELD OF THE INVENTION

The present invention relates to imidazo[1,2-a]pyrazine compounds useful as protein kinase inhibitors, regulators or modulators, pharmaceutical compositions containing the compounds, and methods of treatment using the compounds and compositions to treat diseases such as, for example, cancer, inflammation, arthritis, viral diseases, neurodegenerative diseases such as Alzheimer's disease, cardiovascular diseases, and fungal diseases.

BACKGROUND OF THE INVENTION

Protein kinases are a family of enzymes that catalyze phosphorylation of proteins, in particular the hydroxyl group of specific tyrosine, serine, or threonine residues in proteins. Protein kinases are pivotal in the regulation of a wide variety of cellular processes, including metabolism, cell proliferation, cell differentiation, and cell survival. Uncontrolled proliferation is a hallmark of cancer cells, and can be manifested by a deregulation of the cell division cycle in one of two waysâ€”making stimulatory genes hyperactive or inhibitory genes inactive. Protein kinase inhibitors, regulators or modulators, alter the function of kinases such as cyclin-dependent kinases (CDKs), mitogen activated protein kinase (MAPK/ERK), glycogen synthase kinase 3 (GSK3beta), Checkpoint (Chk) (e.g., CHK-1, CHK-2 etc.) kinases, AKT kinases, JNK, Aurora kinases (Aurora A, Aurora B, Aurora C etc), and the like. Examples of protein kinase inhibitors are described in WO02/22610 A1 and by Y. Mettey et al in J. Med. Chem., (2003) 46 222-236.

The cyclin-dependent kinases are serine/threonine protein kinases, which are the driving force behind the cell cycle and cell proliferation. Misregulation of CDK function occurs with high frequency in many important solid tumors. Individual CDK's, such as, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6 and CDK7, CDK8 and the like, perform distinct roles in cell cycle progression and can be classified as either G1, S, or G2M phase enzymes. CDK2 and CDK4 are of particular interest because their activities are frequently misregulated in a wide variety of human cancers. CDK2 activity is required for progression through G1 to the S phase of the cell cycle, and CDK2 is one of the key components of the G1 checkpoint. Checkpoints serve to maintain the proper sequence of cell cycle events and allow the cell to respond to insults or to proliferative signals, while the loss of proper checkpoint control in cancer cells contributes to tumorgenesis. The CDK2 pathway influences tumorgenesis at the level of tumor suppressor function (e.g. p52, RB, and p27) and oncogene activation (cyclin E). Many reports have demonstrated that both the coactivator, cyclin E, and the inhibitor, p27, of CDK2 are either over- or underexpressed, respectively, in breast, colon, nonsmall cell lung, gastric, prostate, bladder, non-Hodgkin's lymphoma, ovarian, and other cancers. Their altered expression has been shown to correlate with increased CDK2 activity levels and poor overall survival. This observation makes CDK2 and its regulatory pathways compelling targets for the development of cancer treatments.

A number of adenosine 5â€²-triphosphate (ATP) competitive small organic molecules as well as peptides have been reported in the literature as CDK inhibitors for the potential treatment of cancers. U.S. Pat. No. 6,413,974, col. 1, line 23â€”col. 15, line 10 offers a good description of the various CDKs and their relationship to various types of cancer. Flavopiridol (shown below) is a nonselective CDK inhibitor that is currently undergoing human clinical trials, A. M. Sanderowicz et al, J. Clin. Oncol. (1998) 16, 2986-2999. [Image Omitted]

Other known inhibitors of CDKs include, for example, olomoucine (J. Vesely et al, Eur. J. Biochem., (1994) 224, 771-786) and roscovitine (I. Meijer et al, Eur. J. Biochem., (1997) 243, 527-536). U.S. Pat. No. 6,107,305 describes certain pyrazolo[3,4-b]pyridine compounds as CDK inhibitors. An illustrative compound from the '305 patent is: [Image Omitted]

K. S. Kim et al., J. Med. Chem. 45 (2002) 3905-3927 and WO 02/10162 disclose certain aminothiazole compounds as CDK inhibitors. Imidazopyrazines are known. For example, U.S. Pat. No. 6,919,341 (the disclosure of which is incorporated herein by reference) and US2005/0009832 disclose various imidazopyrazines. Also being mentioned are the following: WO2005/047290; US2005/095616; WO2005/039393; WO2005/019220; WO2004/072081; WO2005/014599; WO2005/009354; WO2005/005429; WO2005/085252; US2005/009832; US2004/220189; WO2004/074289; WO2004/026877; WO2004/026310; WO2004/022562; WO2003/089434; WO2003/084959; WO2003/051346; US2003/022898; WO2002/060492; WO2002/060386; WO2002/028860; JP (1986) 61-057587; J. Burke et al., J. Biological Chem., Vol. 278(3), 1450-1456 (2003); and F. Bondavalli et al., J. Med. Chem., Vol. 45 (22), 4875-4887 (2002).

Another series of protein kinases are those that play an important role as a checkpoint in cell cycle progression. Checkpoints prevent cell cycle progression at inappropriate times, such as in response to DNA damage, and maintain the metabolic balance of cells while the cell is arrested, and in some instances can induce apoptosis (programmed cell death) when the requirements of the checkpoint have not been met. Checkpoint control can occur in the G1 phase (prior to DNA synthesis) and in G2, prior to entry into mitosis.

One series of checkpoints monitors the integrity of the genome and, upon sensing DNA damage, these â€œDNA damage checkpointsâ€ block cell cycle progression in G₁& G₂phases, and slow progression through S phase. This action enables DNA repair processes to complete their tasks before replication of the genome and subsequent separation of this genetic material into new daughter cells takes place. Inactivation of CHK1 has been shown to transduce signals from the DNA-damage sensory complex to inhibit activation of the cyclin B/Cdc2 kinase, which promotes mitotic entry, and abrogate G.sub.2 arrest induced by DNA damage inflicted by either anticancer agents or endogenous DNA damage, as well as result in preferential killing of the resulting checkpoint defective cells. See, e.g., Peng et al., Science, 277, 1501-1505 (1997); Sanchez et al., Science, 277, 1497-1501 (1997), Nurse, Cell, 91, 865-867 (1997); Weinert, Science, 277, 1450-1451 (1997); Walworth et al., Nature, 363, 368-371 (1993); and Al-Khodairy et al., Molec. Biol. Cell., 5, 147-160 (1994).

Selective manipulation of checkpoint control in cancer cells could afford broad utilization in cancer chemotherapeutic and radiotherapy regimens and may, in addition, offer a common hallmark of human cancer â€œgenomic instabilityâ€ to be exploited as the selective basis for the destruction of cancer cells. A number of factors place CHK1 as a pivotal target in DNA-damage checkpoint control. The elucidation of inhibitors of this and functionally related kinases such as CDS1/CHK2, a kinase recently discovered to cooperate with CHK1 in regulating S phase progression (see Zeng et al., Nature, 395, 507-510 (1998); Matsuoka, Science, 282, 1893-1897 (1998)), could provide valuable new therapeutic entities for the treatment of cancer.

Another group of kinases are the tyrosine kinases. Tyrosine kinases can be of the receptor type (having extracellular, transmembrane and intracellular domains) or the non-receptor type (being wholly intracellular). Receptor-type tyrosine kinases are comprised of a large number of transmembrane receptors with diverse biological activity. In fact, about 20 different subfamilies of receptor-type tyrosine kinases have been identified. One tyrosine kinase subfamily, designated the HER subfamily, is comprised of EGFR (HER1), HER2, HER3 and HER4. Ligands of this subfamily of receptors identified so far include epithelial growth factor, TGF-alpha, amphiregulin, HB-EGF, betacellulin and heregulin. Another subfamily of these receptor-type tyrosine kinases is the insulin subfamily, which includes INS-R, IGF-IR, IR, and IR-R. The PDGF subfamily includes the PDGF-alpha and beta receptors, CSFIR, c-kit and FLK-II. The FLK family is comprised of the kinase insert domain receptor (KDR), fetal liver kinase-1 (FLK-1), fetal liver kinase-4 (FLK-4) and the fms-like tyrosine kinase-1 (flt-1). For detailed discussion of the receptor-type tyrosine kinases, see Plowman et al., DN&P 7(6): 334-339, 1994.

At least one of the non-receptor protein tyrosine kinases, namely, LCK, is believed to mediate the transduction in T-cells of a signal from the interaction of a cell-surface protein (Cd4) with a cross-linked anti-Cd4 antibody. A more detailed discussion of non-receptor tyrosine kinases is provided in Bolen, Oncogene, 8, 2025-2031 (1993). The non-receptor type of tyrosine kinases is also comprised of numerous subfamilies, including Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack, and LIMK. Each of these subfamilies is further sub-divided into varying receptors. For example, the Src subfamily is one of the largest and includes Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr, and Yrk. The Src subfamily of enzymes has been linked to oncogenesis. For a more detailed discussion of the non-receptor type of tyrosine kinases, see Bolen, Oncogene, 8:2025-2031 (1993).

In addition to its role in cell-cycle control, protein kinases also play a crucial role in angiogenesis, which is the mechanism by which new capillaries are formed from existing vessels. When required, the vascular system has the potential to generate new capillary networks in order to maintain the proper functioning of tissues and organs. In the adult, however, angiogenesis is fairly limited, occurring only in the process of wound healing and neovascularization of the endometrium during menstruation. On the other hand, unwanted angiogenesis is a hallmark of several diseases, such as retinopathies, psoriasis, rheumatoid arthritis, age-related macular degeneration, and cancer (solid tumors). Protein kinases which have been shown to be involved in the angiogenic process include three members of the growth factor receptor tyrosine kinase family; VEGF-R2 (vascular endothelial growth factor receptor 2, also known as KDR (kinase insert domain receptor) and as FLK 1); FGF-R (fibroblast growth factor receptor); and TEK (also known as Tie-2).

VEGF-R2, which is expressed only on endothelial cells, binds the potent angiogenic growth factor VEGF and mediates the subsequent signal transduction through activation of its intracellular kinase activity. Thus, it is expected that direct inhibition of the kinase activity of VEGF-R2 will result in the reduction of angiogenesis even in the presence of exogenous VEGF (see Strawn et al, Cancer Research, 56, 3540-3545 (1996)), as has been shown with mutants of VEGF-R2 which fail to mediate signal transduction. Millauer et al, Cancer Research, 56, 1615-1620 (1996). Furthermore, VEGF-R2 appears to have no function in the adult beyond that of mediating the angiogenic activity of VEGF. Therefore, a selective inhibitor of the kinase activity of VEGF-R2 would be expected to exhibit little toxicity.

Similarly, FGFR binds the angiogenic growth factors aFGF and bFGF and mediates subsequent intracellular signal transduction. Recently, it has been suggested that growth factors such as bFGF may play a critical role in inducing angiogenesis in solid tumors that have reached a certain size. Yoshiji et al., Cancer Research, 57, 3924-3928 (1997). Unlike VEGF-R2, however, FGF-R is expressed in a number of different cell types throughout the body and may or may not play important roles in other normal physiological processes in the adult. Nonetheless, systemic administration of a small molecule inhibitor of the kinase activity of FGF-R has been reported to block bFGF-induced angiogenesis in mice without apparent toxicity. Mohammad et al., EMBO Journal, 17, 5996-5904 (1998).

TEK (also known as Tie-2) is another receptor tyrosine kinase expressed only on endothelial cells which has been shown to play a role in angiogenesis. The binding of the factor angiopoietin-1 results in autophosphorylation of the kinase domain of TEK and results in a signal transduction process which appears to mediate the interaction of endothelial cells with peri-endothelial support cells, thereby facilitating the maturation of newly formed blood vessels. The factor angiopoietin-2, on the other hand, appears to antagonize the action of angiopoietin-1 on TEK and disrupts angiogenesis. Maisonpierre et al., Science, 277, 55-60 (1997). The kinase, JNK, belongs to the mitogen-activated protein kinase (MAPK) superfamily. JNK plays a crucial role in inflammatory responses, stress responses, cell proliferation, apoptosis, and tumorigenesis. JNK kinase activity can be activated by various stimuli, including the proinflammatory cytokines (TNF-alpha and interleukin-1), lymphocyte costimulatory receptors (CD28 and CD40), DNA-damaging chemicals, radiation, and Fas signaling. Results from the JNK knockout mice indicate that JNK is involved in apoptosis induction and T helper cell differentiation.

Pim-1 is a small serine/threonine kinase. Elevated expression levels of Pim-1 have been detected in lymphoid and myeloid malignancies, and recently Pim-1 was identified as a prognostic marker in prostate cancer. K. Peltola, â€œSignaling in Cancer: Pim-1 Kinase and its Partnersâ€ , Annales Universitatis Turkuensis, Sarjaâ€”Ser. D Osaâ€”Tom. 616, (Aug. 30, 2005), http://kiriasto.utu.fi/iulkaisupalvelut/annaalitV2004/D616.html. Pim-1 acts as a cell survival factor and may prevent apoptosis in malignant cells. K. Petersen Shay et al., Molecular Cancer Research 3:170-181 (2005).

There is a need for effective inhibitors of protein kinases in order to treat or prevent disease states associated with abnormal cell proliferation. Moreover, it is desirable for kinase inhibitors to possess both high affinity for the target kinase as well as high selectivity versus other protein kinases. Small-molecule compounds that may be readily synthesized and are potent inhibitors of cell proliferation are those, for example, that are inhibitors of one or more protein kinases, such as CHK1, CHK2, VEGF (VEGF-R2), Pim-1, CDKs or CDK/cyclin complexes and both receptor and non-receptor tyrosine kinases.

SUMMARY OF THE INVENTION

In its many embodiments, the present invention provides a novel class of imidazo[1,2-a]pyrazine compounds, methods of preparing such compounds, pharmaceutical compositions comprising one or more such compounds, methods of preparing pharmaceutical formulations comprising one or more such compounds, and methods of treatment, prevention, inhibition or amelioration of one or more diseases associated with protein kinases using such compounds or pharmaceutical compositions.

In one aspect, the present invention provides compounds represented by Formula I: [Image Omitted]

or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof, wherein:

R is H, CN, â€”NR⁵R⁶, cycloalkyl, cycloalkenyl, heterocyclenyl, heteroaryl, â€”C(O)NR⁵R⁶, â€”N(R⁵)C(O)R⁶, heterocyclyl, heteroaryl substituted with (CH₂)_1-3NR⁵R⁶, unsubstituted alkyl, or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶;
R¹is H, halo, aryl or heteroaryl, wherein each of said aryl and heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, â€”CH₂OR⁵, â€”C(O)NR⁵R⁶, â€”C(O)OH, â€”C(O)NH₂, â€”NR⁵R⁵(wherein the R⁵and R⁶, together with the N of said â€”NR⁵R⁶, form a heterocyclyl ring), â€”S(O)R⁵, â€”S(O₂)R⁵, â€”CN, â€”CHO, â€”SR⁵, â€”C(O)OR⁵, â€”C(O)R⁵and â€”OR⁵;
R²is H, halo, aryl, arylalkyl or heteroaryl, wherein each of said aryl, arylalkyl and heteroaryl can be unsubstituted or optionally independently be substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amide, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, â€”C(O)OH, â€”C(O)NH₂, â€”NR⁵R⁶(wherein the R⁵and R⁶, together with the N of said â€”NR⁵R⁶, form a heterocyclyl ring), â€”CN, arylalkyl, â€”CH₂OR⁵, â€”S(O)R⁵, â€”S(O₂)R⁵, â€”CN, â€”CHO, â€”SR⁵, â€”C(O)OR⁵, â€”C(O)R⁵, heteroaryl and heterocyclyl;
R³is H, alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein:
- said alkyl shown above for R³can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, alkoxy, heteroaryl, and â€”NR⁵R⁶;
- said aryl shown above for R³is unsubstituted, or optionally substituted, or optionally fused, with halo, heteroaryl, heterocyclyl, cycloalkyl or heteroarylalkyl, wherein each of said heteroaryl, heterocyclyl, cycloalkyl and heteroarylalkyl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵; and
- said heteroaryl shown above for R³can be unsubstituted or optionally substituted, or optionally fused, with one or more moieties which can be the same or different with each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl;
R⁵is H, alkyl, aminoalkyl, aryl, heteroaryl, heterocyclyl or cycloalkyl; and
R⁶is H, alkyl, aryl, arylalkyl, heteroaryl, heterocyclyl or cycloalkyl;
further wherein in any â€”NR⁵R⁶in Formula I, said R⁵and R⁶can optionally be joined together with the N of said â€”NR⁵R⁶to form a heterocyclyl ring.

The compounds of Formula I can be useful as protein kinase inhibitors and can be useful in the treatment and prevention of proliferative diseases, for example, cancer, inflammation and arthritis, neurodegenerative diseases such Alzheimer's disease, cardiovascular diseases, viral diseases and fungal diseases.

DETAILED DESCRIPTION

In an embodiment, the present invention provides imidazopyrazine compounds, especially imidazo[1,2-a]pyrazine compounds which are represented by structural Formula I, or pharmaceutically acceptable salts, solvates, esters or prodrug thereof, wherein the various moieties are as described above.

In another embodiment, the present invention provides compounds represented by Formula I: [Image Omitted]
or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof, wherein:

R is H, CN, â€”NR⁵R⁶, cycloalkenyl, heterocyclenyl, â€”C(O)NR⁵R⁶, â€”N(R⁵)C(O)R⁶, or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵and â€”NR⁵R⁶;
R¹is H, halo, aryl or heteroaryl, wherein each of said aryl and heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, â€”C(O)NR⁵R⁶and â€”OR⁵;
R²is H, halo, or heteroaryl, wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl;
R³is H, alkyl, aryl or heteroaryl, wherein:
- said alkyl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, alkoxy and â€”NR⁵R⁶;
- said aryl is substituted with heteroaryl which heteroaryl can be unsubstituted or substituted with alkyl; and
- said heteroaryl shown above for R³can be unsubstituted or substituted with one or more moieties which can be the same or different with each moiety being independently selected from the group consisting of halo, â€”OR⁵, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heterocyclyl;
R⁵is H, alkyl, aryl, heteroaryl, heterocyclyl or cycloalkyl; and
R⁶is H, alkyl, aryl, heteroaryl, heterocyclyl or cycloalkyl.

In an embodiment, R, R¹, R²and R³are not all H simultaneously.

In another embodiment, in Formula I, R²is unsubstituted heteroaryl or heteroaryl substituted with alkyl.

In another embodiment, in Formula I, R²is heteroaryl substituted with alkyl.

In another embodiment, in Formula I, R²is pyrazolyl.

In another embodiment, in Formula I, R²is pyrazolyl substituted with alkyl.

In another embodiment, in Formula I, R²is 1-methyl-pyrazol-4-yl.

In another embodiment, in Formula I, R is H.

In another embodiment, in Formula I, R is CN.

In another embodiment, in Formula I, R is â€”C(O)NR⁵R⁶.

In another embodiment, in Formula I, R is â€”C(O)NH₂.

In another embodiment, in Formula I, R is heterocyclenyl.

In another embodiment, in Formula I, R is tetrahydropyridinyl.

In another embodiment, in Formula I, R is 1,2,3,6-tetrahydropyridinyl.

In another embodiment, in Formula I, R is alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR¹and â€”NR⁵R⁶.

In another embodiment, in Formula I, R is alkyl substituted with one or more â€”NR⁵R⁶.

In another embodiment, in Formula I, R is alkyl substituted with â€”NH₂.

In another embodiment, in Formula I, R is alkyl substituted with â€”NH(methyl).

In another embodiment, R is unsubstituted alkyl.

In some embodiments, both R and R¹are not H simultaneously.

In another embodiment, in Formula I, R³is H.

In another embodiment, in Formula I, R³is unsubstituted alkyl.

In another embodiment, in Formula I, R³is alkyl substituted with one or more moieties which can be the same or different, each moiety being independently selected from the group consisting of halo, â€”OR¹, alkoxy and â€”NR⁵R⁶.

In another embodiment, in Formula I, R³is unsubstituted heteroaryl.

In another embodiment, in Formula I, R³is heteroaryl substituted with alkyl.

In another embodiment, in Formula I, R³is heteroaryl substituted with methyl.

In another embodiment, in Formula I, R³is unsubstituted isothiazolyl.

In another embodiment, in Formula I, R³is isothiazolyl substituted with alkyl.

In another embodiment, in Formula I, R³is isothiazolyl substituted with methyl.

In another embodiment, in Formula I, R³is 5-methyl-isothiazol-3-yl.

In another embodiment, R³is aryl substituted with heteroaryl.

In another embodiment, R³is aryl substituted with imidazolyl.

In another embodiment, R³is phenyl substituted with imidazolyl.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is heteroaryl, Râ• R¹â• H and R³is unsubstituted alkyl, wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amide, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, â€”C(O)OH, â€”C(O)NH₂, â€”NR⁵R⁶(where R⁵and R⁶form a cyclic amine together with the N of said â€”NR⁵R⁶), â€”CN, arylalkyl, â€”CH₂OR⁵, â€”S(O)R⁵, â€”S(O₂)R⁵, â€”CN, â€”CHO, â€”SR⁵, â€”C(O)OR⁵, â€”C(O)R⁵, heteroaryl and heterocyclyl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is heteroaryl, wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amide, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, â€”C(O)OH, â€”C(O)NH₂, â€”NR⁵R⁶(where R⁵and R⁶form a cyclic amine together with the N of said â€”NR⁵R⁶), â€”CN, arylalkyl, â€”CH₂OR⁵, â€”S(O)R⁵, â€”S(O₂)R⁵, â€”CN, â€”CHO, â€”SR⁵, â€”C(O)OR⁵, â€”C(O)R⁵, heteroaryl and heterocyclyl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)₁₃â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is heteroaryl wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is heteroaryl, wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amide, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, â€”C(O)OH, â€”C(O)NH₂, â€”NR⁵R⁶(where R⁵and R⁶form a cyclic amine together with the N of said â€”NR⁵R⁶), â€”CN, arylalkyl, â€”CH₂OR⁵, â€”S(O)R⁵, â€”S(O₂)R⁵, â€”CN, â€”CHO, â€”SR⁵, â€”C(O)OR⁵, â€”C(O)R⁵, heteroaryl and heterocyclyl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is heteroaryl wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is pyrazolyl, Râ• R¹â• H and R³is unsubstituted alkyl, wherein said pyrazolyl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amide, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, â€”C(O)OH, â€”C(O)NH₂, â€”NR⁵R⁶(where R⁵and R⁶form a cyclic amine together with the N of said â€”NR⁵R⁶), â€”CN, arylalkyl, â€”CH₂OR⁵, â€”S(O)R⁵, â€”S(O₂)R⁵, â€”CN, â€”CHO, â€”SR⁵, â€”C(O)OR⁵, â€”C(O)R⁵, heteroaryl and heterocyclyl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl, Râ• R¹â• H and R³is unsubstituted alkyl.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is pyrazolyl, wherein said pyrazolyl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amide, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, â€”C(O)OH, â€”C(O)NH₂, â€”NR⁵R⁶(where R⁵and R⁶form a cyclic amine together with the N of said â€”NR⁵R⁶), â€”CN, arylalkyl, â€”CH₂OR⁵, â€”S(O)R⁵, â€”S(O₂)R⁵, â€”CN, â€”CHO, â€”SR⁵, â€”C(O)R⁵, â€”C(O)R⁵, heteroaryl and heterocyclyl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)₁₃â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is heteroaryl wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is heteroaryl wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is heteroaryl wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is isothiazolyl wherein said isothiaozlyl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is isothiazolyl wherein said isothiazolyl is substituted with one or more alkyl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is 5-methyl-isothiazol-3-yl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is pyrazolyl, wherein said pyrazolyl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, â€”C(O)NR⁵R⁶and â€”OR⁵; R is heterocyclenyl; R¹is H and R³is heteroaryl wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is heterocyclenyl; R¹is H and R³is heteroaryl wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is tetrahydropyridinyl; R¹is H and R³is heteroaryl wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is 1,2,3,6-tetrahydropyridinyl; R¹is H and R³is heteroaryl wherein said heteroaryl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is 1,2,3,6-tetrahydropyridinyl; R¹is H and R³is isothiaozlyl wherein said isothiazolyl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is 1,2,3,6-tetrahydropyridinyl; R¹is H and R³is 5-methyl-isothiazol-3-yl.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is isothiazolyl wherein said isothiaozlyl can be unsubstituted or substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of halo, amino, alkoxycarbonyl, â€”OR⁵, alkyl, â€”CHO, â€”NR⁵R⁶, â€”S(O₂)N(R⁵R⁶), â€”C(O)N(R⁵R⁶), â€”SR⁵, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclenyl, and heterocyclyl, wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is unsubstituted heteroaryl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is aryl wherein said aryl is substituted with a heteroaryl, wherein said heteroaryl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵and wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is heteroaryl substituted with alkyl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is aryl wherein said aryl is substituted with a heteroaryl, wherein said heteroaryl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵and wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is heteroaryl substituted with alkyl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is aryl wherein said aryl is substituted with a heteroaryl, wherein said heteroaryl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵and wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)₁₃â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is aryl wherein said aryl is substituted with a heteroaryl, wherein said heteroaryl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵and wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is unsubstituted alkyl or alkyl substituted with one or more moieties which can be the same or different each moiety being independently selected from the group consisting of â€”OR⁵, heterocyclyl, â€”N(R⁵)C(O)N(R⁵R⁶), â€”N(R⁵)â€”C(O)OR⁶, â€”(CH₂)_1-3â€”N(R⁵R⁶) and â€”NR⁵R⁶; R¹is H and R³is aryl wherein said aryl is substituted with imidazolyl, wherein said imidazolyl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵and wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is unsubstituted heteroaryl; R is â€”C(O)NR⁵R⁶; R¹is H and R³is aryl wherein said aryl is substituted with a heteroaryl, wherein said heteroaryl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵and wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is heteroaryl substituted with alkyl; R is â€”C(O)NR⁵R⁶; R¹is H and R³is aryl wherein said aryl is substituted with a heteroaryl, wherein said heteroaryl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵and wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is heteroaryl substituted with alkyl; R is â€”C(O)NR⁵R⁶; R¹is H and R³is aryl wherein said aryl is substituted with a heteroaryl, wherein said heteroaryl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵and wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is â€”C(O)NR⁵R⁶; R¹is H and R³is aryl wherein said aryl is substituted with a heteroaryl, wherein said heteroaryl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵and wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is â€”C(O)NR⁵R⁶; R¹is H and R³is aryl wherein said aryl is substituted with imidazolyl, wherein said imidazolyl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵, and wherein R⁵and R⁶are as defined above.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is unsubstituted heteroaryl; R is heterocyclenyl; R¹is H and R³is aryl wherein said aryl is substituted with a heteroaryl, wherein said heteroaryl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁵) and â€”S(O₂)R⁵.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is heteroaryl substituted with alkyl; R is heterocyclenyl; R¹is H and R³is aryl wherein said aryl is substituted with a heteroaryl, wherein said heteroaryl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is heterocyclenyl; R¹is H and R³is aryl wherein said aryl is substituted with a heteroaryl, wherein said heteroaryl can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is heterocyclenyl; R¹is H and R³is aryl wherein said aryl is substituted with imidazolyl, wherein said imidazolyl can be can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁵) and â€”S(O₂)R⁵.

In another embodiment, this invention discloses a compound of the formula: [Image Omitted]
or a pharmaceutically acceptable salt, solvate or ester thereof, wherein R²is 1-methyl-pyrazol-4-yl; R is 1,2,3,6-tetrahydropyridinyl; R¹is H and R³is aryl wherein said aryl is substituted with imidazolyl, wherein said imidazolyl can be can be unsubstituted or optionally independently substituted with one or more moieties which can be the same or different each moiety being independently selected from alkyl, â€”OR⁵, â€”N(R⁵R⁶) and â€”S(O₂)R⁵.

Non-limiting examples of compounds of Formula I include: [Image Omitted]

As used above, and throughout this disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings, including any possible substitutions of the stated groups or moieties:

â€œPatientâ€ includes both human and animals.

â€œMammalâ€ means humans and other mammalian animals.

â€œAlkylâ€ means an aliphatic hydrocarbon group which may be straight or branched and comprising about 1 to about 20 carbon atoms in the chain. Preferred alkyl groups contain about 1 to about 12 carbon atoms in the chain. More preferred alkyl groups contain about 1 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkyl chain. â€œLower alkylâ€ means a group having about 1 to about 6 carbon atoms in the chain which may be straight or branched. â€œAlkylâ€ may be unsubstituted or optionally substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkyl, aryl, cycloalkyl, cyano, hydroxy, alkoxy, alkylthio, amino, oxime (e.g., â• Nâ€”OH), â€”NH(alkyl), â€”NH(cycloalkyl), â€”N(alkyl)₂, â€”Oâ€”C(O)-alkyl, â€”Oâ€”C(O)-aryl, â€”Oâ€”C(O)-cycloalkyl, carboxy and â€”C(O)O-alkyl. Non-limiting examples of suitable alkyl groups include methyl, ethyl, n-propyl, isopropyl and t-butyl.

â€œAlkenylâ€ means an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched and comprising about 2 to about 15 carbon atoms in the chain. Preferred alkenyl groups have about 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkenyl chain. â€œLower alkenylâ€ means about 2 to about 6 carbon atoms in the chain which may be straight or branched. â€œAlkenylâ€ may be unsubstituted or optionally substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkyl. aryl, cycloalkyl, cyano, alkoxy and â€”S(alkyl). Non-limiting examples of suitable alkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl, octenyl and decenyl.

â€œAlkyleneâ€ means a difunctional group obtained by removal of a hydrogen atom from an alkyl group that is defined above. Non-limiting examples of alkylene include methylene, ethylene and propylene.

â€œAlkynylâ€ means an aliphatic hydrocarbon group containing at least one carbon-carbon triple bond and which may be straight or branched and comprising about 2 to about 15 carbon atoms in the chain. Preferred alkynyl groups have about 2 to about 12 carbon atoms in the chain; and more preferably about 2 to about 4 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl, are attached to a linear alkynyl chain. â€œLower alkynylâ€ means about 2 to about 6 carbon atoms in the chain which may be straight or branched. Non-limiting examples of suitable alkynyl groups include ethynyl, propynyl, 2-butynyl and 3-methylbutynyl. â€œAlkynylâ€ may be unsubstituted or optionally substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of alkyl, aryl and cycloalkyl.

â€œArylâ€ means an aromatic monocyclic or multicyclic ring system comprising about 6 to about 14 carbon atoms, preferably about 6 to about 10 carbon atoms. The aryl group can be optionally substituted with one or more â€œring system substituentsâ€ which may be the same or different, and are as defined herein. Non-limiting examples of suitable aryl groups include phenyl and naphthyl.

â€œHeteroarylâ€ means an aromatic monocyclic or multicyclic ring system comprising about 5 to about 14 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the ring atoms is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. Preferred heteroaryls contain about 5 to about 6 ring atoms. The â€œheteroarylâ€ can be optionally substituted by one or more â€œring system substituentsâ€ which may be the same or different, and are as defined herein. The prefix aza, oxa or thia before the heteroaryl root name means that at least a nitrogen, oxygen or sulfur atom respectively, is present as a ring atom. A nitrogen atom of a heteroaryl can be optionally oxidized to the corresponding N-oxide. â€œHeteroarylâ€ may also include a heteroaryl as defined above fused to an aryl as defined above. Non-limiting examples of suitable heteroaryls include pyridyl, pyrazinyl, furanyl, thienyl, pyrimidinyl, pyridone (including N-substituted pyridones), isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, oxindolyl, imidazo[1,2-a]pyridinyl, imidazo[2,1-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, 1,2,4-triazinyl, benzothiazolyl and the like. The term â€œheteroarylâ€ also refers to partially saturated heteroaryl moieties such as, for example, tetrahydroisoquinolyl, tetrahydroquinolyl and the like.

â€œAralkylâ€ or â€œarylalkylâ€ means an aryl-alkyl- group in which the aryl and alkyl are as previously described. Preferred aralkyls comprise a lower alkyl group. Non-limiting examples of suitable aralkyl groups include benzyl, 2-phenethyl and naphthalenylmethyl. The bond to the parent moiety is through the alkyl.

â€œAlkylarylâ€ means an alkyl-aryl- group in which the alkyl and aryl are as previously described. Preferred alkylaryls comprise a lower alkyl group. Non-limiting example of a suitable alkylaryl group is tolyl. The bond to the parent moiety is through the aryl.

â€œCycloalkylâ€ means a non-aromatic mono- or multicyclic ring system comprising about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms. Preferred cycloalkyl rings contain about 5 to about 7 ring atoms. The cycloalkyl can be optionally substituted with one or more â€œring system substituentsâ€ which may be the same or different, and are as defined above. Non-limiting examples of suitable monocyclic cycloalkyls include cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl and the like. Non-limiting examples of suitable multicyclic cycloalkyls include 1-decalinyl, norbornyl, adamantyl and the like.

â€œCycloalkylalkylâ€ means a cycloalkyl moiety as defined above linked via an alkyl moiety (defined above) to a parent core. Non-limiting examples of suitable cycloalkylalkyls include cyclohexylmethyl, adamantylmethyl and the like.

â€œCycloalkenylâ€ means a non-aromatic mono or multicyclic ring system comprising about 3 to about 10 carbon atoms, preferably about 5 to about 10 carbon atoms which contains at least one carbon-carbon double bond. Preferred cycloalkenyl rings contain about 5 to about 7 ring atoms. The cycloalkenyl can be optionally substituted with one or more â€œring system substituentsâ€ which may be the same or different, and are as defined above. Non-limiting examples of suitable monocyclic cycloalkenyls include cyclopentenyl, cyclohexenyl, cyclohepta-1,3-dienyl, and the like. Non-limiting example of a suitable multicyclic cycloalkenyl is norbornylenyl.

â€œCycloalkenylalkylâ€ means a cycloalkenyl moiety as defined above linked via an alkyl moiety (defined above) to a parent core. Non-limiting examples of suitable cycloalkenylalkyls include cyclopentenylmethyl, cyclohexenylmethyl and the like.

â€œHalogenâ€ means fluorine, chlorine, bromine, or iodine. Preferred are fluorine, chlorine and bromine.

â€œRing system substituentâ€ means a substituent attached to an aromatic or non-aromatic ring system which, for example, replaces an available hydrogen on the ring system. Ring system substituents may be the same or different, each being independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, alkylaryl, heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl, alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, acyl, aroyl, halo, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkyl, heterocyclyl, amide, â€”CHO, â€”Oâ€”C(O)-alkyl, â€”Oâ€”C(O)-aryl, â€”Oâ€”C(O)-cycloalkyl, â€”C(â• Nâ€”CN)â€”NH₂, â€”C(â• NH)â€”NH₂, â€”C(â• NH)â€”NH(alkyl), oxime (e.g., â• Nâ€”OH), Y₁Y₂Nâ€”, Y₁Y₂N-alkyl-, Y₁Y₂NC(O)â€”, Y₁Y₂NSO₂â€” and â€”SO₂NY₁Y₂, wherein Y₁and Y₂can be the same or different and are independently selected from the group consisting of hydrogen, alkyl, aryl, cycloalkyl, and aralkyl. â€œRing system substituentâ€ may also mean a single moiety which simultaneously replaces two available hydrogens on two adjacent carbon atoms (one H on each carbon) on a ring system. Examples of such moiety are methylene dioxy, ethylenedioxy, â€”C(CH₃)₂â€” and the like which form moieties such as, for example: [Image Omitted]

â€œHeteroarylalkylâ€ means a heteroaryl moiety as defined above linked via an alkyl moiety (defined above) to a parent core. Non-limiting examples of suitable heteroaryls include 2-pyridinylmethyl, quinolinylmethyl and the like.

â€œHeterocyclylâ€ means a non-aromatic saturated monocyclic or multicyclic ring system comprising about 3 to about 10 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the atoms in the ring system is an element other than carbon, for example nitrogen, oxygen or sulfur, alone or in combination. There are no adjacent oxygen and/or sulfur atoms present in the ring system. Preferred heterocyclyls contain about 5 to about 6 ring atoms. The prefix aza, oxa or thia before the heterocyclyl root name means that at least a nitrogen, oxygen or sulfur atom respectively is present as a ring atom. Any â€”NH in a heterocyclyl ring may exist protected such as, for example, as an â€”N(Boc), â€”N(CBz), â€”N(Tos) group and the like; such protections are also considered part of this invention. The heterocyclyl can be optionally substituted by one or more â€œring system substituentsâ€ which may be the same or different, and are as defined herein. The nitrogen or sulfur atom of the heterocyclyl can be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide. Non-limiting examples of suitable monocyclic heterocyclyl rings include piperidyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothiophenyl, lactam, lactone, and the like. â€œHeterocyclylâ€ may also mean a single moiety (e.g., carbonyl) which simultaneously replaces two available hydrogens on the same carbon atom on a ring system. Example of such moiety is pyrrolidone: [Image Omitted]

â€œHeterocyclylalkylâ€ means a heterocyclyl moiety as defined above linked via an alkyl moiety (defined above) to a parent core. Non-limiting examples of suitable heterocyclylalkyls include piperidinylmethyl, piperazinylmethyl and the like.

â€œHeterocyclenylâ€ means a non-aromatic monocyclic or multicyclic ring system comprising about 3 to about 10 ring atoms, preferably about 5 to about 10 ring atoms, in which one or more of the atoms in the ring system is an element other than carbon, for example nitrogen, oxygen or sulfur atom, alone or in combination, and which contains at least one carbon-carbon double bond or carbon-nitrogen double bond. There are no adjacent oxygen and/or sulfur atoms present in the ring system. Preferred heterocyclenyl rings contain about 5 to about 6 ring atoms. The prefix aza, oxa or thia before the heterocyclenyl root name means that at least a nitrogen, oxygen or sulfur atom respectively is present as a ring atom. The heterocyclenyl can be optionally substituted by one or more ring system substituents, wherein â€œring system substituentâ€ is as defined above. The nitrogen or sulfur atom of the heterocyclenyl can be optionally oxidized to the corresponding N-oxide, S-oxide or S,S-dioxide. Non-limiting examples of suitable heterocyclenyl groups include 1,2,3,4-tetrahydropyridinyl, 1,2-dihydropyridinyl, 1,4-dihydropyridinyl, 1,2,3,6-tetrahydropyridinyl, 1,4,5,6-tetrahydropyrimidinyl, 2-pyrrolinyl, 3-pyrrolinyl, 2-imidazolinyl, 2-pyrazolinyl, dihydroimidazolyl, dihydrooxazolyl, dihydrooxadiazolyl, dihydrothiazolyl, 3,4-dihydro-2H-pyranyl, dihydrofuranyl, fluorodihydrofuranyl, 7-oxabicyclo[2.2.1]heptenyl, dihydrothiophenyl, dihydrothiopyranyl, and the like. â€œHeterocyclenylâ€ may also mean a single moiety (e.g., carbonyl) which simultaneously replaces two available hydrogens on the same carbon atom on a ring system. Example of such moiety is pyrrolidinone: [Image Omitted]

â€œHeterocyclenylalkylâ€ means a heterocyclenyl moiety as defined above linked via an alkyl moiety (defined above) to a parent core.

It should be noted that in hetero-atom containing ring systems of this invention, there are no hydroxyl groups on carbon atoms adjacent to a N, O or S, as well as there are no N or S groups on carbon adjacent to another heteroatom. Thus, for example, in the ring: [Image Omitted]
there is no â€”OH attached directly to carbons marked 2 and 5.

It should also be noted that tautomeric forms such as, for example, the moieties: [Image Omitted]
are considered equivalent in certain embodiments of this invention.

â€œAlkynylalkylâ€ means an alkynyl-alkyl- group in which the alkynyl and alkyl are as previously described. Preferred alkynylalkyls contain a lower alkynyl and a lower alkyl group. The bond to the parent moiety is through the alkyl. Non-limiting examples of suitable alkynylalkyl groups include propargylmethyl.

â€œHeteroaralkylâ€ means a heteroaryl-alkyl- group in which the heteroaryl and alkyl are as previously described. Preferred heteroaralkyls contain a lower alkyl group. Non-limiting examples of suitable aralkyl groups include pyridylmethyl, and quinolin-3-ylmethyl. The bond to the parent moiety is through the alkyl.

â€œHydroxyalkylâ€ means a HO-alkyl- group in which alkyl is as previously defined. Preferred hydroxyalkyls contain lower alkyl. Non-limiting examples of suitable hydroxyalkyl groups include hydroxymethyl and 2-hydroxyethyl.

â€œAcylâ€ means an Hâ€”C(O)â€”, alkyl-C(O)â€” or cycloalkyl-C(O)â€”, group in which the various groups are as previously described. The bond to the parent moiety is through the carbonyl. Preferred acyls contain a lower alkyl. Non-limiting examples of suitable acyl groups include formyl, acetyl and propanoyl.

â€œAroylâ€ means an aryl-C(O)â€” group in which the aryl group is as previously described. The bond to the parent moiety is through the carbonyl. Non-limiting examples of suitable groups include benzoyl and 1-naphthoyl.

â€œAlkoxyâ€ means an alkyl-Oâ€” group in which the alkyl group is as previously described. Non-limiting examples of suitable alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy and n-butoxy. The bond to the parent moiety is through the ether oxygen.

â€œAryloxyâ€ means an aryl-Oâ€” group in which the aryl group is as previously described. Non-limiting examples of suitable aryloxy groups include phenoxy and naphthoxy. The bond to the parent moiety is through the ether oxygen.

â€œAralkyloxyâ€ means an aralkyl-Oâ€” group in which the aralkyl group is as previously described. Non-limiting examples of suitable aralkyloxy groups include benzyloxy and 1- or 2-naphthalenemethoxy. The bond to the parent moiety is through the ether oxygen.

â€œAlkylthioâ€ means an alkyl-Sâ€” group in which the alkyl group is as previously described. Non-limiting examples of suitable alkylthio groups include methylthio and ethylthio. The bond to the parent moiety is through the sulfur.

â€œArylthioâ€ means an aryl-Sâ€” group in which the aryl group is as previously described. Non-limiting examples of suitable arylthio groups include phenylthio and naphthylthio. The bond to the parent moiety is through the sulfur.

â€œAralkylthioâ€ means an aralkyl-Sâ€” group in which the aralkyl group is as previously described. Non-limiting example of a suitable aralkylthio group is benzylthio. The bond to the parent moiety is through the sulfur.

â€œAlkoxycarbonylâ€ means an alkyl-Oâ€”COâ€” group. Non-limiting examples of suitable alkoxycarbonyl groups include methoxycarbonyl and ethoxycarbonyl. The bond to the parent moiety is through the carbonyl.

â€œAryloxycarbonylâ€ means an aryl-Oâ€”C(O)â€” group. Non-limiting examples of suitable aryloxycarbonyl groups include phenoxycarbonyl and naphthoxycarbonyl. The bond to the parent moiety is through the carbonyl.

â€œAralkoxycarbonylâ€ means an aralkyl-Oâ€”C(O)â€” group. Non-limiting example of a suitable aralkoxycarbonyl group is benzyloxycarbonyl. The bond to the parent moiety is through the carbonyl.

â€œAlkylsulfonylâ€ means an alkyl-S(O₂)â€” group. Preferred groups are those in which the alkyl group is lower alkyl. The bond to the parent moiety is through the sulfonyl.

â€œArylsulfonylâ€ means an aryl-S(O₂)â€” group. The bond to the parent moiety is through the sulfonyl.

The term â€œsubstitutedâ€ means that one or more hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency under the existing circumstances is not exceeded, and that the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds. By â€œstable compoundâ€™ or â€œstable structureâ€ is meant 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.

The term â€œoptionally substitutedâ€ means optional substitution with the specified groups, radicals or moieties.

The term â€œpurifiedâ€ , â€œin purified formâ€ or â€œin isolated and purified formâ€ for a compound refers to the physical state of said compound after being isolated from a synthetic process (e.g. from a reaction mixture), or natural source or combination thereof. Thus, the term â€œpurifiedâ€ , â€œin purified formâ€ or â€œin isolated and purified formâ€ for a compound refers to the physical state of said compound after being obtained from a purification process or processes described herein or well known to the skilled artisan (e.g., chromatography, recrystallization and the like), in sufficient purity to be characterizable by standard analytical techniques described herein or well known to the skilled artisan.

It should also be noted that any carbon as well as heteroatom with unsatisfied valences in the text, schemes, examples and Tables herein is assumed to have the sufficient number of hydrogen atom(s) to satisfy the valences.

When a functional group in a compound is termed â€œprotectedâ€ , this means that the group is in modified form to preclude undesired side reactions at the protected site when the compound is subjected to a reaction. Suitable protecting groups will be recognized by those with ordinary skill in the art as well as by reference to standard textbooks such as, for example, T. W. Greene et al, Protective Groups in organic Synthesis (1991), Wiley, New York.

When any variable (e.g., aryl, heterocycle, R², etc.) occurs more than one time in any constituent or in Formula I, its definition on each occurrence is independent of its definition at every other occurrence.

As used herein, the term â€œcompositionâ€ is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

Prodrugs and solvates of the compounds of the invention are also contemplated herein. A discussion of prodrugs is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems (1987) 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, (1987) Edward B. Roche, ed., American Pharmaceutical Association and Pergamon Press. The term â€œprodrugâ€ means a compound (e.g, a drug precursor) that is transformed in vivo to yield a compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (e.g., by metabolic or chemical processes), such as, for example, through hydrolysis in blood. A discussion of the use of prodrugs is provided by T. Higuchi and W. Stella, â€œPro-drugs as Novel Delivery Systems,â€ Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

For example, if a compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvate of the compound contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as, for example, (C₁-C₈)alkyl, (C₂-C₁₂)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having from 4 to 9 carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,Nâ€”(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as Î²-dimethylaminoethyl), carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)alkylcarbamoyl-(C₁-C₂)alkyl and piperidino-, pyrrolidino- or morpholino(C₂-C₃)alkyl, and the like.

Similarly, if a compound of Formula (I) contains an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as, for example, (C₁-C₆)alkanoyloxymethyl, 1-((C₁-C₆)alkanoyloxy)ethyl, 1-methyl-1-((C₁-C₆)alkanoyloxy)ethyl, (C₁-C₆)alkoxycarbonyloxymethyl, Nâ€”(C₁-C₆)alkoxycarbonylaminomethyl, succinoyl, (C₁-C₆)alkanoyl, Î±-amino(C₁-C₄)alkanyl, arylacyl and Î±-aminoacyl, or Î±-aminoacyl-Î±-aminoacyl, where each Î±-aminoacyl group is independently selected from the naturally occurring L-amino acids, P(O)(OH)₂, â€”P(O)(O(C₁-C₆)alkyl)₂or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate), and the like.

If a compound of Formula (I) incorporates an amine functional group, a prodrug can be formed by the replacement of a hydrogen atom in the amine group with a group such as, for example, R-carbonyl, RO-carbonyl, NRRâ€²-carbonyl where R and Râ€² are each independently (C₁-C₁₀)alkyl, (C₃-C₇) cycloalkyl, benzyl, or R-carbonyl is a natural Î±-aminoacyl or natural Î±-aminoacyl, â€”C(OH)C(O)OY¹wherein Y¹is H, (C₁-C₆)alkyl or benzyl, â€”C(OY²)Y³wherein Y²is (C₁-C₄) alkyl and Y³is (C₁-C₆)alkyl, carboxy(C₁-C₆)alkyl, amino(C₁-C₄)alkyl or mono-Nâ€” or di-N,Nâ€”(C₁-C₆)alkylaminoalkyl, â€”C(Y⁴)Y⁵wherein Y⁴is H or methyl and Y⁵is mono-Nâ€” or di-N,Nâ€”(C₁-C₆)alkylamino morpholino, piperidin-1-yl or pyrrolidin-1-yl, and the like.

One or more compounds of the invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embrace both solvated and unsolvated forms. â€œSolvateâ€ means a physical association of a compound of this invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including 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. â€œSolvateâ€ encompasses both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like. â€œHydrateâ€ is a solvate wherein the solvent molecule is H₂O.

One or more compounds of the invention may optionally be converted to a solvate. Preparation of solvates is generally known. Thus, for example, M. Caira et al, J. Pharmaceutical Sci., 93(3), 601-611 (2004) describe the preparation of the solvates of the antifungal fluconazole in ethyl acetate as well as from water. Similar preparations of solvates, hemisolvate, hydrates and the like are described by E. C. van Tonder et al, AAPS Pharm Sci Tech., 5(1), article 12 (2004); and A. L. Bingham et al, Chem. Commun., 603-604 (2001). A typical, non-limiting, process involves dissolving the inventive compound in desired amounts of the desired solvent (organic or water or mixtures thereof) at a higher than ambient temperature, and cooling the solution at a rate sufficient to form crystals which are then isolated by standard methods. Analytical techniques such as, for example I. R. spectroscopy, show the presence of the solvent (or water) in the crystals as a solvate (or hydrate).

â€œEffective amountâ€ or â€œtherapeutically effective amountâ€ is meant to describe an amount of compound or a composition of the present invention effective in inhibiting the above-noted diseases and thus producing the desired therapeutic, ameliorative, inhibitory or preventative effect.

The compounds of Formula I can form salts which are also within the scope of this invention. Reference to a compound of Formula I herein is understood to include reference to salts thereof, unless otherwise indicated. The term â€œsalt(s)â€ , as employed herein, denotes acidic salts formed with inorganic and/or organic acids, as well as basic salts formed with inorganic and/or organic bases. In addition, when a compound of Formula I contains both a basic moiety, such as, but not limited to a pyridine or imidazole, and an acidic moiety, such as, but not limited to a carboxylic acid, zwitterions (â€œinner saltsâ€ ) may be formed and are included within the term â€œsalt(s)â€ as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful. Salts of the compounds of the Formula I may be formed, for example, by reacting a compound of Formula I with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.

Exemplary acid addition salts include acetates, ascorbates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, fumarates, hydrochlorides, hydrobromides, hydroiodides, lactates, maleates, methanesulfonates, naphthalenesulfonates, nitrates, oxalates, phosphates, propionates, salicylates, succinates, sulfates, tartarates, thiocyanates, toluenesulfonates (also known as tosylates,) and the like. Additionally, acids which are generally considered suitable for the formation of pharmaceutically useful salts from basic pharmaceutical compounds are discussed, for example, by P. Stahl et al, Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use. (2002) Zurich: Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977) 66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33 201-217; Anderson et al, The Practice of Medicinal Chemistry (1996), Academic Press, New York; and in The Orange Book (Food & Drug Administration, Washington, D.C. on their website). These disclosures are incorporated herein by reference thereto.

Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as dicyclohexylamines, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. Basic nitrogen-containing groups may be quarternized with agents such as lower alkyl halides (e.g. methyl, ethyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, and dibutyl sulfates), long chain halides (e.g. decyl, lauryl, and stearyl chlorides, bromides and iodides), aralkyl halides (e.g. benzyl and phenethyl bromides), and others.

All such acid salts and base salts are intended to be pharmaceutically acceptable salts within the scope of the invention and all acid and base salts are considered equivalent to the free forms of the corresponding compounds for purposes of the invention.

Pharmaceutically acceptable esters of the present compounds include the following groups: (1) carboxylic acid esters obtained by esterification of the hydroxy groups, in which the non-carbonyl moiety of the carboxylic acid portion of the ester grouping is selected from straight or branched chain alkyl (for example, acetyl, n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted with, for example, halogen, C_1-4alkyl, or C_1-4alkoxy or amino); (2) sulfonate esters, such as alkyl- or aralkylsulfonyl (for example, methanesulfonyl); (3) amino acid esters (for example, L-valyl or L-isoleucyl); (4) phosphonate esters and (5) mono-, di- or triphosphate esters. The phosphate esters may be further esterified by, for example, a C_1-20alcohol or reactive derivative thereof, or by a 2,3-di(C_6-24)acyl glycerol.

Compounds of Formula I, and salts, solvates, esters and prodrugs thereof, may exist in their tautomeric form (for example, as an amide or imino ether). All such tautomeric forms are contemplated herein as part of the present invention.

The compounds of Formula (I) may contain asymmetric or chiral centers, and, therefore, exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of Formula (I) as well as mixtures thereof, including racemic mixtures, form part of the present invention. In addition, the present invention embraces all geometric and positional isomers. For example, if a compound of Formula (I) incorporates a double bond or a fused ring, both the cis- and trans-forms, as well as mixtures, are embraced within the scope of the invention.

Diastereomeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. Also, some of the compounds of Formula (I) may be atropisomers (e.g., substituted biaryls) and are considered as part of this invention. Enantiomers can also be separated by use of chiral HPLC column.

It is also possible that the compounds of Formula (I) may exist in different tautomeric forms, and all such forms are embraced within the scope of the invention. Also, for example, all keto-enol and imine-enamine forms of the compounds are included in the invention.

All stereoisomers (for example, geometric isomers, optical isomers and the like) of the present compounds (including those of the salts, solvates, esters and prodrugs of the compounds as well as the salts, solvates and esters of the prodrugs), such as those which may exist due to asymmetric carbons on various substituents, including enantiomeric forms (which may exist even in the absence of asymmetric carbons), rotameric forms, atropisomers, and diastereomeric forms, are contemplated within the scope of this invention, as are positional isomers (such as, for example, 4-pyridyl and 3-pyridyl). (For example, if a compound of Formula (I) incorporates a double bond or a fused ring, both the cis- and trans-forms, as well as mixtures, are embraced within the scope of the invention. Also, for example, all keto-enol and imine-enamine forms of the compounds are included in the invention.) Individual stereoisomers of the compounds of the invention may, for example, be substantially free of other isomers, or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers. The chiral centers of the present invention can have the S or R configuration as defined by the IUPAC 1974 Recommendations. The use of the terms â€œsaltâ€ , â€œsolvateâ€ , â€œesterâ€ , â€œprodrugâ€ and the like, is intended to equally apply to the salt, solvate, ester and prodrug of enantiomers, stereoisomers, rotamers, tautomers, positional isomers, racemates or prodrugs of the inventive compounds.

The present invention also embraces isotopically-labelled compounds of the present invention which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively.

Certain isotopically-labelled compounds of Formula (I) (e.g., those labeled with ³H and ¹⁴C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., ²H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically labelled compounds of Formula (I) can generally be prepared by following procedures analogous to those disclosed in the Schemes and/or in the Examples hereinbelow, by substituting an appropriate isotopically labelled reagent for a non-isotopically labelled reagent.

Polymorphic forms of the compounds of Formula I, and of the salts, solvates, esters and prodrugs of the compounds of Formula I, are intended to be included in the present invention.

The compounds according to the invention have pharmacological properties; in particular, the compounds of Formula I can be inhibitors, regulators or modulators of protein kinases. Non-limiting examples of protein kinases that can be inhibited, regulated or modulated include cyclin-dependent kinases (CDKs), such as, CDK1, CDK2, CDK3, CDK4, CDK5, CDK6 and CDK7, CDK8, mitogen activated protein kinase (MAPK/ERK), glycogen synthase kinase 3 (GSK3beta), Pim-1 kinases, Chk kinases, such as Chk1 and Chk2, tyrosine kinases, such as the HER subfamily (including, for example, EGFR (HER1), HER2, HER3 and HER4), the insulin subfamily (including, for example, INS-R, IGF-IR, IR, and IR-R), the PDGF subfamily (including, for example, PDGF-alpha and beta receptors, CSFIR, c-kit and FLK-II), the FLK family (including, for example, kinase insert domain receptor (KDR), fetal liver kinase-1 (FLK-1), fetal liver kinase-4 (FLK-4) and the fms-like tyrosine kinase-1 (fit-1)), non-receptor protein tyrosine kinases, for example LCK, Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack, and LIMK, growth factor receptor tyrosine kinases such as VEGF-R2, FGF-R, TEK, Akt kinases and the like.

The compounds of Formula (I) can be inhibitors of protein kinases such as, for example, the inhibitors of the checkpoint kinases such as Chk1, Chk2 and the like. Preferred compounds can exhibit IC₅₀values of less than about 5 Î¼m, preferably about 0.001 to about 1.0 Î¼m, and more preferably about 0.001 to about 0.1 Î¼m. The assay methods are described in the Examples set forth below.

The compounds of Formula I can be useful in the therapy of proliferative diseases such as cancer, autoimmune diseases, viral diseases, fungal diseases, neurological/neurodegenerative disorders, arthritis, inflammation, anti-proliferative (e.g., ocular retinopathy), neuronal, alopecia and cardiovascular disease. Many of these diseases and disorders are listed in U.S. Pat. No. 6,413,974 cited earlier, incorporated by reference herein.

More specifically, the compounds of Formula I can be useful in the treatment of a variety of cancers, including (but not limited to) the following:

carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, including small cell lung cancer, non-small cell lung cancer, head and neck, esophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin, including squamous cell carcinoma;

hematopoietic tumors of lymphoid lineage, including leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, mantle cell lymphoma, myeloma, and Burkett's lymphoma;

hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias, myelodysplastic syndrome and promyelocytic leukemia;

tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma;

tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannomas; and

other tumors, including melanoma, seminoma, teratocarcinoma, osteosarcoma, xenoderoma pigmentosum, keratoctanthoma, thyroid follicular cancer and Kaposi's sarcoma.

Due to the key role of CDKs in the regulation of cellular proliferation in general, inhibitors could act as reversible cytostatic agents which may be useful in the treatment of any disease process which features abnormal cellular proliferation, e.g., benign prostate hyperplasia, familial adenomatosis polyposis, neuro-fibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, hypertrophic scar formation, inflammatory bowel disease, transplantation rejection, endotoxic shock, and fungal infections. Compounds of Formula I may also be useful in the treatment of Alzheimer's disease, as suggested by the recent finding that CDK5 is involved in the phosphorylation of tau protein (J. Biochem, (1995) 117, 741-749). Compounds of Formula I may induce or inhibit apoptosis. The apoptotic response is aberrant in a variety of human diseases. Compounds of Formula I, as modulators of apoptosis, will be useful in the treatment of cancer (including but not limited to those types mentioned hereinabove), viral infections (including but not limited to herpevirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus), prevention of AIDS development in HIV-infected individuals, autoimmune diseases (including but not limited to systemic lupus, erythematosus, autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes mellitus), neurodegenerative disorders (including but not limited to Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellar degeneration), myelodysplastic syndromes, aplastic anemia, ischemic injury associated with myocardial infarctions, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol related liver diseases, hematological diseases (including but not limited to chronic anemia and aplastic anemia), degenerative diseases of the musculoskeletal system (including but not limited to osteoporosis and arthritis) aspirin-sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis, kidney diseases and cancer pain.

Compounds of Formula I, as inhibitors of the CDKs, can modulate the level of cellular RNA and DNA synthesis. These agents would therefore be useful in the treatment of viral infections (including but not limited to HIV, human papilloma virus, herpesvirus, poxvirus, Epstein-Barr virus, Sindbis virus and adenovirus).

Compounds of Formula I may also be useful in the chemoprevention of cancer. Chemoprevention is defined as inhibiting the development of invasive cancer by either blocking the initiating mutagenic event or by blocking the progression of pre-malignant cells that have already suffered an insult or inhibiting tumor relapse.

Compounds of Formula I may also be useful in inhibiting tumor angiogenesis and metastasis.

Compounds of Formula I may also act as inhibitors of other protein kinases, e.g., protein kinase C, her2, raf 1, MEK1, MAP kinase, EGF receptor, PDGF receptor, IGF receptor, PI3 kinase, wee1 kinase, Src, Abl and thus be effective in the treatment of diseases associated with other protein kinases. Another aspect of this invention is a method of treating a mammal (e.g., human) having a disease or condition associated with the CDKs by administering a therapeutically effective amount of at least one compound of Formula I, or a pharmaceutically acceptable salt, solvate, ester or prodrug of said compound to the mammal.

A preferred dosage is about 0.001 to 1000 mg/kg of body weight/day of the compound of Formula I. An especially preferred dosage is about 0.01 to 25 mg/kg of body weight/day of a compound of Formula I, or a pharmaceutically acceptable salt, solvate, ester or prodrug of said compound. The compounds of this invention may also be useful in combination (administered together or sequentially) with one or more of anti-cancer treatments such as radiation therapy, and/or one or more anti-cancer agents different from the compound of Formula I. The compounds of the present invention can be present in the same dosage unit as the anti-cancer agent or in separate dosage units.

Another aspect of the present invention is a method of treating one or more diseases associated with cyclin dependent kinase, comprising administering to a mammal in need of such treatment an amount of a first compound, which is a compound of claim 1, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof; and an amount of at least one second compound, the second compound being an anti-cancer agent different from the compound of claim 1, wherein the amounts of the first compound and the second compound result in a therapeutic effect.

Non-limiting examples of suitable anti-cancer agents include cytostatic agents, cytotoxic agents (such as for example, but not limited to, DNA interactive agents (such as cisplatin or doxorubicin)); taxanes (e.g. taxotere, taxol); topoisomerase II inhibitors (such as etoposide); topoisomerase I inhibitors (such as irinotecan (or CPT-11), camptostar, or topotecan); tubulin interacting agents (such as paclitaxel, docetaxel or the epothilones); hormonal agents (such as tamoxifen); thymidilate synthase inhibitors (such as 5-fluorouracil); anti-metabolites (such as methoxtrexate); alkylating agents (such as temozolomide (TEMODARâ„¢ from Schering-Plough Corporation, Kenilworth, N.J.), cyclophosphamide); Farnesyl protein transferase inhibitors (such as, SARASARâ„¢ (4-[2-[4-[(11R)-3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl-]-1-piperidinyl]-2-oxoehtyl]-1-piperidinecarboxamide, or SCH 66336 from Schering-Plough Corporation, Kenilworth, New Jersey), tipifarnib (ZarnestraÂ® or R115777 from Janssen Pharmaceuticals), L778,123 (a farnesyl protein transferase inhibitor from Merck & Company, Whitehouse Station, New Jersey), BMS 214662 (a farnesyl protein transferase inhibitor from Bristol-Myers Squibb Pharmaceuticals, Princeton, N.J.); signal transduction inhibitors (such as, Iressa (from Astra Zeneca Pharmaceuticals, England), Tarceva (EGFR kinase inhibitors), antibodies to EGFR (e.g., C225), GLEEVECâ„¢ (C-abl kinase inhibitor from Novartis Pharmaceuticals, East Hanover, N.J.); interferons such as, for example, intron (from Schering-Plough Corporation), Peg-Intron (from Schering-Plough Corporation); hormonal therapy combinations; aromatase combinations; ara-C, adriamycin, cytoxan, and gemcitabine.

Other anti-cancer (also known as anti-neoplastic) agents include but are not limited to Uracil mustard, Chlormethine, Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, cytostatic agents, cytotoxic agents (such as for example, but not limited to, DNA interactive agents (such as cisplatin or doxorubicin)); taxanes (e.g. taxotere, taxol); topoisomerase II inhibitors (such as etoposide); topoisomerase I inhibitors (such as irinotecan (or CPT-11), camptostar, or topotecan); tubulin interacting agents (such as paclitaxel, docetaxel or the epothilones); hormonal agents (such as tamoxifen); thymidilate synthase inhibitors (such as 5-fluorouracil); anti-metabolites (such as methoxtrexate); alkylating agents (such as temozolomide (TEMODARâ„¢ from Schering-Plough Corporation, Kenilworth, New Jersey), cyclophosphamide); Farnesyl protein transferase inhibitors (such as, SARASARâ„¢ (4-[2-[4-[(11R)-3,10-dibromo-8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl-]-1-piperidinyl]-2-oxoehtyl]-1-piperidinecarboxamide, or SCH 66336 from Schering-Plough Corporation, Kenilworth, New Jersey), tipifarnib (ZarnestraÂ® or R115777 from Janssen Pharmaceuticals), L778,123 (a farnesyl protein transferase inhibitor from Merck & Company, Whitehouse Station, New Jersey), BMS 214662 (a farnesyl protein transferase inhibitor from Bristol-Myers Squibb Pharmaceuticals, Princeton, N.J.); signal transduction inhibitors (such as, Iressa (from Astra Zeneca Pharmaceuticals, England), Tarceva (EGFR kinase inhibitors), antibodies to EGFR (e.g., C225), GLEEVECâ„¢ (C-abl kinase inhibitor from Novartis Pharmaceuticals, East Hanover, N.J.); interferons such as, for example, intron (from Schering-Plough Corporation), Peg-Intron (from Schering-Plough Corporation); hormonal therapy combinations; aromatase combinations; ara-C, adriamycin, cytoxan, Clofarabine (ClolarÂ® from Genzyme Oncology, Cambridge, Mass.), cladribine (LeustatÂ® from Janssen-Cilag Ltd.), aphidicolon, rituxan (from Genentech/Biogen Idec), sunitinib (SutentÂ® from Pfizer), dasatinib (or BMS-354825 from Bristol-Myers Squibb), tezacitabine (from Aventis Pharma), Sml1, fludarabine (from Trigan Oncology Associates), pentostatin (from BC Cancer Agency), triapine (from Vion Pharmaceuticals), didox (from Bioseeker Group), trimidox (from ALS Therapy Development Foundation), amidox, 3-AP (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), MDL-101,731 ((E)-2â€²-deoxy-2â€²-(fluoromethylene)cytidine) and gemcitabine.

Other anti-cancer (also known as anti-neoplastic) agents include but are not limited to Uracil mustard, Chlormethine, Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine, Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine, Streptozocin, Dacarbazine, Floxuridine, Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate, oxaliplatin, leucovirin, oxaliplatin (ELOXATINâ„¢ from Sanofi-Synthelabo Pharmaceuticals, France), Pentostatine, Vinblastine, Vincristine, Vindesine, Bleomycin, Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase, Teniposide 17Î±-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone, Megestrolacetate, Methylprednisolone, Methyltestosterone, Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide, Toremifene, goserelin, Cisplatin, Carboplatin, Hydroxyurea, Amsacrine, Procarbazine, Mitotane, Mitoxantrone, Levamisole, Navelbene, Anastrazole, Letrazole, Capecitabine, Reloxafine, Droloxafine, Hexamethylmelamine, Avastin, Herceptin, Bexxar, Velcade, Zevalin, Trisenox, Xeloda, Vinorelbine, Profimer, Erbitux, Liposomal, Thiotepa, Altretamine, Melphalan, Trastuzumab, Lerozole, Fulvestrant, Exemestane, Fulvestrant, Ifosfomide, Rituximab, C225 and Campath.

If formulated as a fixed dose, such combination products employ the compounds of this invention within the dosage range described herein and the other pharmaceutically active agent or treatment within its dosage range. For example, the CDC2 inhibitor olomucine has been found to act synergistically with known cytotoxic agents in inducing apoptosis (J. Cell Sci., (1995) 108, 2897. Compounds of Formula I may also be administered sequentially with known anticancer or cytotoxic agents when a combination formulation is inappropriate. The invention is not limited in the sequence of administration; compounds of Formula I may be administered either prior to or after administration of the known anticancer or cytotoxic agent. For example, the cytotoxic activity of the cyclin-dependent kinase inhibitor flavopiridol is affected by the sequence of administration with anticancer agents. Cancer Research, (1997) 57, 3375. Such techniques are within the skills of persons skilled in the art as well as attending physicians.

Accordingly, in an aspect, this invention includes combinations comprising an amount of at least one compound of Formula I, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof, and an amount of one or more anti-cancer treatments and anti-cancer agents listed above wherein the amounts of the compounds/treatments result in desired therapeutic effect.

Another aspect of the present invention is a method of inhibiting one or more Checkpoint kinases in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of at least one compound of claim 1 or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

Another aspect of the present invention is a method of treating, or slowing the progression of, a disease associated with one or more Checkpoint kinases in a patient in need thereof, comprising administering a therapeutically effective amount of at least one compound of claim 1 or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

Yet another aspect of the present invention is a method of treating one or more diseases associated with Checkpoint kinase, comprising administering to a mammal in need of such treatment an amount of a first compound, which is a compound of claim 1, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof; and an amount of at least one second compound, the second compound being an anti-cancer agent, wherein the amounts of the first compound and the second compound result in a therapeutic effect.

Another aspect of the present invention is a method of treating, or slowing the progression of, a disease associated with one or more Checkpoint kinases in a patient in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising in combination at least one pharmaceutically acceptable carrier and at least one compound according to claim 1, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

In the above methods, the checkpoint kinase to be inhibited can be Chk1 and/or Chk2.

Another aspect of the present invention is a method of inhibiting one or more tyrosine kinases in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of at least one compound of claim 1 or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

Yet another aspect of the present invention is a method of treating, or slowing the progression of, a disease associated with one or more tyrosine kinases in a patient in need thereof, comprising administering a therapeutically effective amount of at least one compound of claim 1 or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

Another aspect of the present invention is a method of treating one or more diseases associated with tyrosine kinase, comprising administering to a mammal in need of such treatment an amount of a first compound, which is a compound of claim 1, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof; and an amount of at least one second compound, the second compound being an anti-cancer agent, wherein the amounts of the first compound and the second compound result in a therapeutic effect.

Another aspect of the present invention is a method of treating, or slowing the progression of, a disease associated with one or more tyrosine kinases in a patient in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising in combination at least one pharmaceutically acceptable carrier and at least one compound according to claim 1 or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

In the above methods, the tyrosine kinase can be VEGFR (VEGF-R2), EGFR, HER2, SRC, JAK and/or TEK.

Another aspect of the present invention is a method of inhibiting one or more Pim-1 kinases in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of at least one compound of claim 1 or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

Yet another aspect of the present invention is a method of treating, or slowing the progression of, a disease associated with one or more Pim-1 kinases in a patient in need thereof, comprising administering a therapeutically effective amount of at least one compound of claim 1 or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

Another aspect of the present invention is a method of treating one or more diseases associated with Pim-1 kinase, comprising administering to a mammal in need of such treatment an amount of a first compound, which is a compound of claim 1, or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof; and an amount of at least one second compound, the second compound being an anti-cancer agent, wherein the amounts of the first compound and the second compound result in a therapeutic effect.

Another aspect of the present invention is a method of treating, or slowing the progression of, a disease associated with one or more Pim-1 kinases in a patient in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising in combination at least one pharmaceutically acceptable carrier and at least one compound according to claim 1 or a pharmaceutically acceptable salt, solvate, ester or prodrug thereof.

The pharmacological properties of the compounds of this invention may be confirmed by a number of pharmacological assays. The exemplified pharmacological assays which are described herein below have been carried out with compounds according to the invention and their salts, solvates, esters or prodrugs.

This invention is also directed to pharmaceutical compositions which comprise at least one compound of Formula I, or a pharmaceutically acceptable salt, solvate, ester or prodrug of said compound and at least one pharmaceutically acceptable carrier.

For preparing pharmaceutical compositions from the compounds described by this invention, inert, pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, dispersible granules, capsules, cachets and suppositories. The powders and tablets may be comprised of from about 5 to about 95 percent active ingredient. Suitable solid carriers are known in the art, e.g., magnesium carbonate, magnesium stearate, talc, sugar or lactose. Tablets, powders, cachets and capsules can be used as solid dosage forms suitable for oral administration. Examples of pharmaceutically acceptable carriers and methods of manufacture for various compositions may be found in A. Gennaro (ed.), Remington's Pharmaceutical Sciences, 18^thEdition, (1990), Mack Publishing Co., Easton, Pa.

Liquid form preparations include solutions, suspensions and emulsions. As an example may be mentioned water or water-propylene glycol solutions for parenteral injection or addition of sweeteners and opacifiers for oral solutions, suspensions and emulsions. Liquid form preparations may also include solutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions and solids in powder form, which may be in combination with a pharmaceutically acceptable carrier, such as an inert compressed gas, e.g. nitrogen.

Also included are solid form preparations that are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration. Such liquid forms include solutions, suspensions and emulsions.

The compounds of the invention may also be deliverable transdermally. The transdermal compositions can take the form of creams, lotions, aerosols and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.

The compounds of this invention may also be delivered subcutaneously.

Preferably the compound is administered orally or intravenously.

Also contemplated are delivery methods that are combinations of the above-noted delivery methods. Such methods are within the skill of, or typically decided, by, those skilled in the art.

Preferably, the pharmaceutical preparation is in a unit dosage form. In such form, the preparation is subdivided into suitably sized unit doses containing appropriate quantities of the active component, e.g., an effective amount to achieve the desired purpose.

The quantity of active compound in a unit dose of preparation may be varied or adjusted from about 1 mg to about 100 mg, preferably from about 1 mg to about 50 mg, more preferably from about 1 mg to about 25 mg, according to the particular application.

The actual dosage employed may be varied depending upon the requirements of the patient and the severity of the condition being treated. Determination of the proper dosage regimen for a particular situation is within the skill of the art. For convenience, the total daily dosage may be divided and administered in portions during the day as required.

The amount and frequency of administration of the compounds of the invention and/or the pharmaceutically acceptable salts thereof will be regulated according to the judgment of the attending clinician considering such factors as age, condition and size of the patient as well as severity of the symptoms being treated. A typical recommended daily dosage regimen for oral administration can range from about 1 mg/day to about 500 mg/day, preferably 1 mg/day to 200 mg/day, in two to four divided doses.

Another aspect of this invention is a kit comprising a therapeutically effective amount of at least one compound of Formula I, or a pharmaceutically acceptable salt, solvate, ester or prodrug of said compound and a pharmaceutically acceptable carrier, vehicle or diluent.

Yet another aspect of this invention is a kit comprising an amount of at least one compound of Formula I, or a pharmaceutically acceptable salt, solvate, ester or prodrug of said compound and an amount of at least one anticancer therapy and/or anti-cancer agent listed above, wherein the amounts of the two or more ingredients result in desired therapeutic effect.

The invention disclosed herein is exemplified by the following preparations and examples which should not be construed to limit the scope of the disclosure. Alternative mechanistic pathways and analogous structures will be apparent to those skilled in the art.

Where NMR data are presented, ¹H spectra were obtained on either a Varian VXR-200 (200 MHz, ¹H), Varian Gemini-300 (300 MHz) or XL-400 (400 MHz) and are reported as ppm down field from Me₄Si with number of protons, multiplicities, and coupling constants in Hertz indicated parenthetically. Where LC/MS data are presented, analyses was performed using an Applied Biosystems API-100 mass spectrometer and Shimadzu SCL-10A L column: Altech platinum C18, 3 micron, 33 mmÃ—7 mm ID; gradient flow: 0 minâ€”10% CH₃CN, 5 minâ€”95% CH₃CN, 7 minâ€”95% CH₃CN, 7.5 minâ€”10% CH₃CN, 9 minâ€”stop. The retention time and observed parent ion are given.

The following solvents and reagents may be referred to by their abbreviations in parenthesis:

Thin layer chromatography: TLC

dichloromethane: CH₂Cl₂

ethyl acetate: AcOEt or EtOAc

methanol: MeOH

trifluoroacetate: TFA

triethylamine: Et₃N or TEA

butoxycarbonyl: n-Boc or Boc

nuclear magnetic resonance spectroscopy: NMR

liquid chromatography mass spectrometry: LCMS

high resolution mass spectrometry: HRMS

milliliters: mL

millimoles: mmol

microliters: Î¼l

grams: g

milligrams: mg

room temperature or rt (ambient): about 25Â° C.

dimethoxyethane: DME

The synthesis of the inventive compounds is illustrated below. Also, it should be noted that the disclosure of commonly-owned U.S. Pat. No. 6,919,341 is incorporated herein by reference.

SYNTHESIS Example 100

[Image Omitted]

A mixture 2,3-dichloropyrazine (50 g, 0.34 mmol) and concentrated aqueous ammonium hydroxide (200 mL) was stirred at 85Â° C. in a closed pressure vessel for 4 days. The mixture was cooled to 25Â° C., water (200 mL) was added, and the mixture was filtered. The solid was washed with water (400 mL), then with dichloromethane (400 mL) and dried under vacuum. Compound 100 was isolated as a white solid 32.5 g (73%). ¹H NMR (400 MHz, DMSO-d₆Î´ 7.93 (d, 1H), 7.55 (d, 1H), 6.79 (bs, 2H).

Example 101

[Image Omitted]

Î±-Bromo diethyl acetal (51.6 mL, 332.7 mmol, 2.5 eq) was added to a solution of 7.7 mL HBr (conc.) and 80 mL of H₂O. The reaction was heated at reflux for 1 h. The reaction was cooled and extracted 2Ã— with Et₂O (200 mL). The Et₂O extracts were combined, washed with brine, and dried over Na₂SO₄before being concentrated. The material was not left on the rotavap for an extended time or put under high vacuum. The oily residue was mixed with DME (200 mL) and the 2-amino-3-chloropyrazine (2, 17.240 g, 133.1 mmol) was added. HBr conc. (1-1.5 mL) was added and the reaction was heated at reflux. The reaction is heterogeneous reaction mixture, becomes homogenous after 10-15 minutes. After approximately 30 minutes a precipitate begins to form. After 1 hour at reflux the black reaction was cooled to room temperature, filtered, and washed with Et₂O (4Ã—, 75 mL) to give compound 101 ¹H NMR (DMSO-d₆, 400 MHz) â–¡ 8.70 (d, J=2.0 Hz, 1H), 8.32 (s, 1H), 7.93 (s, 1H), 7.79 (d, J=3.0 Hz, 1H). LC/MS shows a mixture of two products (one product by LC and two by MS). By MS there is a mass for X=Cl (major) MH⁺=154 (m/z) and one for X=Br (minor) MH⁺198 (m/z). This mixture gave the product in approximately 90% yield as the HBr salt.

Example 102

[Image Omitted]

The 7-halo compound 101 (4.92 g, 20.2 mmol) was mixed with Br₂(1.54 mL, 30.0 mmol) in AcOH (100 mL) at room temperature. After 5-10 minutes the reaction became homogeneous. After 1.5 hours a precipitate began to form. The reaction stirred at room temperature for 3 days. The reaction was concentrated in vacuo. The residue was taken up in 10% iso-PrOH in CH₂Cl₂(300 mL) and washed with sat. NaHCO₃(2Ã—, 100 mL), 1M Na₂S₂O₃(100 mL), and brine (100 mL). The organic layer was dried with Na₂SO₄and concentrated in vacuo to give 4.460 g of the product, compound 102 (91% yield). ¹H NMR (DMSO-d₆, 400 MHz) â–¡ 8.47 (d, J=4.8 Hz, 1H), 8.02 (s, 1H), 7.84 (d, J=4.4 Hz, 1H).

Example 103

[Image Omitted]

To a solution of compound 102 (13.0 g, 55.9 mmol) in DMSO (150 mL) was added sodium methanethiolate (4.70 g, 67.08 mmol) as a DMSO solution (100 mL) at room temperature. The reaction mixture was stirred at 100Â° C. for 16 hours. The mixture was cooled to 25Â° C. and added to a brine solution (300 mL), and extracted with 10% IPA/dichloromethane (300 mL, 3Ã—). The combined organic layer was dried over anhydrous sodium sulfate and concentrated. Purification by column chromatography (SiO₂, ethyl acetate/hexanes (1:1)) afforded compound 103 as a yellow solid 10 g (70%). ¹H-NMR (400 MHz, DMSO-d₆Î´ 8.15 (d, 1H), 7.88 (d, 1H), 7.83 (s, 1H), 2.6 (s, 3H).

Example 104

[Image Omitted]

A mixture of compound 103 (5.0 g, 17.8 mmol), 1-methyl-4-(4,4,5,5-teramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (7.44 g, 35.7 mmol), Pd(dppf)Cl₂(1.46 g, 10 mol %), sodium carbonate (9.50 g, 89.5 mmol) in 1,2-dimethoxyethane (150 mL) and water (37 mL) was stirred at 70Â° C. under Argon for 16 hours. The solvents were evaporated and the residue was purified by column chromatography (SiO₂, ethyl acetate to 5% methanol/ethyl acetate) to afford compound 104 as a beige solid 3.80 g (86%). ¹H NMR (400 MHz, DMSO-d₆Î´ 8.35 (s, 1H), 8.27 (d, 1H), 7.96 (d, 1H), 7.82 (s, 1H), 7.81 (d, 1H), 3.93 (s, 3H), 2.59 (s, 3H).

Example 105

[Image Omitted]

To a solution of compound 104 (3.0 g, 12.2 mmol) in dichloromethane (100 mL) at room temperature was added m-CPBA (5.75 g, 25.6 mmol) in one portion. The mixture was stirred at room temperature for 1 hour at which time thin layer chromatography (10% MeOH/ethyl acetate) indicated that the reaction was complete. The reaction mixture was poured into saturated aqueous sodium bicarbonate (100 mL). The layers were separated and the aqueous layer was extracted with dichloromethane (2Ã—100 mL). The organic layers were combined and washed with brine (150 mL). The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to yield a dark yellow oil. Purification by column chromatography (SiO₂, 10% methanol/ethyl acetate) afforded compound 105 as a yellow solid 2.10 g (62%). ¹H NMR (400 MHz, DMSO-d₆Î´ 8.83 (d, 2H), 8.45 (s, 1H), 8.21 (s, 1H), 8.11 (d, 1H), 8.06 (d, 1H), 3.96 (s, 3H), 3.61 (s, 3H). HPLC-MS t_R=0.75 min (UV_254nm). Mass calculated for formula C₁₁H₁₁N₅O₂S 277.06; observed MH⁺ (LCMS) 278.1 (m/z).

Example 106

[Image Omitted]

A solution of the respective aromatic amine (2 equivalents) in DMSO (1 mL) was treated with NaH (60% dispersion in oil, 2 equivalents) for 15 minutes at room temperature. Compound 105 (1 equivalent) was then added to this solution at room temperature and this solution was stirred at room temperature for 1 hour at which time thin layer chromatography (10% methanol/ethyl acetate) indicate the reaction was complete. The reaction mixture was diluted with sat. ammonium chloride (0.5 mL) and acetonitrile (0.5 mL). Purification by Prep-LC and conversion to a hydrochloric salt afforded compound 106.

Examples 106-1-106-83

By essentially the same procedure given in Preparative Example 106, compounds given in Column 2 of Table 8 can be prepared from compound 105.

TABLE 8LCMSMH⁺HPLCExampleColumn 2MWm/zMS t_R106-1 [Image Omitted]

368.4369.12.73 106-2 [Image Omitted]

290.3291.12.47 106-3 [Image Omitted]

320.3321.12.34 106-4 [Image Omitted]

382.4383.13.84 106-5 [Image Omitted]

382.4383.14.24 106-6 [Image Omitted]

368.4369.12.91 106-7 [Image Omitted]

329.3330.12.44 106-8 [Image Omitted]

341.3342.12.45 106-9 [Image Omitted]

297.3298.12.46 106-10 [Image Omitted]

355.4356.22.57 106-11 [Image Omitted]

340.3341.23.54 106-12 [Image Omitted]

342.3343.12.96 106-13 [Image Omitted]

331.3332.21.93 106-14 [Image Omitted]

356.3357.22.89 106-15 [Image Omitted]

291.3292.12.10 106-16 [Image Omitted]

298.3299.22.45 106-17 [Image Omitted]

292.3293.22.00 106-18 [Image Omitted]

357.3358.12.98 106-19 [Image Omitted]

356.3357.22.18 106-20 [Image Omitted]

324.7325.13.36 106-21 [Image Omitted]

344.3345.22.35 106-22 [Image Omitted]

334.3335.22.40 106-23 [Image Omitted]

320.3321.22.35 106-24 [Image Omitted]

291.3292.12.20 106-25 [Image Omitted]

291.3292.12.15 106-26 [Image Omitted]

292.3293.22.05 106-27 [Image Omitted]

315.3316.12.82 106-28 [Image Omitted]

397.4398.23.49 106-29 [Image Omitted]

430.4431.24.05 106-30 [Image Omitted]

402.8403.13.67 106-31 [Image Omitted]

357.3358.11.94 106-32 [Image Omitted]

320.3321.22.70 106-33 [Image Omitted]

338.3339.13.24 106-34 [Image Omitted]

347.4348.12.34 106-35 [Image Omitted]

356.3357.22.96 106-36 [Image Omitted]

358.4359.13.75 106-37 [Image Omitted]

373.4374.24.30 106-38 [Image Omitted]

295.3296.22.05 106-39 [Image Omitted]

308.3309.22.32 106-40 [Image Omitted]

341.3342.32.96 106-41 [Image Omitted]

295.3296.23.04 106-42 [Image Omitted]

311.3312.12.52 106-43 [Image Omitted]

294.3295.12.19 106-44 [Image Omitted]

341.3342.32.09 106-45 [Image Omitted]

347.4348.12.75 106-46 [Image Omitted]

341.3342.33.83 106-47 [Image Omitted]

374.5375.21.78 106-48 [Image Omitted]

377.4378.32.07 106-49 [Image Omitted]

377.4378.31.81 106-50 [Image Omitted]

356.3357.22.46 106-51 [Image Omitted]

409.4410.22.55 106-52 [Image Omitted]

331.3332.22.87 106-53 [Image Omitted]

346.4347.23.12 106-54 [Image Omitted]

344.3345.22.02 106-55 [Image Omitted]

357.3358.12.97 106-56 [Image Omitted]

375.3376.13.21 106-57 [Image Omitted]

370.4371.22.71 106-58 [Image Omitted]

427.4428.23.50 106-59 [Image Omitted]

439.4440.22.33 106-60 [Image Omitted]

373.4374.22.19 106-61 [Image Omitted]

373.4374.22.10 106-62 [Image Omitted]

373.4374.22.10 106-63 [Image Omitted]

373.4374.21.99 106-64 [Image Omitted]

375.4376.12.21 106-65 [Image Omitted]

388.4389.22.51 106-66 [Image Omitted]

361.4362.12.51 106-67 [Image Omitted]

341.3342.12.10 106-68 [Image Omitted]

341.3342.22.35 106-69 [Image Omitted]

384.4385.13.49 106-69 [Image Omitted]

312.3313.12.97 106-70 [Image Omitted]

340.4341.23.80 106-71 [Image Omitted]

348.2349.23.49 106-72 [Image Omitted]

311.1312.12.87 106-73 [Image Omitted]

403.1404.15.16 106-74 [Image Omitted]

297.07298.12.71 106-75 [Image Omitted]

296.08297.13.03 106-76 [Image Omitted]

310.10311.13.55 106-77 [Image Omitted]

389.00390.04.41 106-78 [Image Omitted]

389.5390.31.80 106-79 [Image Omitted]

345.17346.20.85 106-80 [Image Omitted]

407.44408.42.15 106-81 [Image Omitted]

424.44425.42.30 106-82 [Image Omitted]

407.44408.41.85 106-83 [Image Omitted]

372.29373.11.05

Example 107

The compounds shown in column 2 of Table 9 were prepared as follows. [Image Omitted]

To a solution of compound 105 (1 equivalent) in NMP (0.5 mL) was added DIEA (10 equivalents), and the respective aliphatic amine (2 equivalents) at room temperature. The reaction was heated to 50Â° C. overnight. LC-MS analysis of the reaction indicates the reaction is complete. The crude reaction mixture was concentrated. Purification by Prep-LC and conversion to a hydrochloric salt afforded compound 107-1 to 107-13 as a white solid.

TABLE 9LCMSExam-MH⁺HPLCpleColumn 2MWm/zMS t_R107-1 [Image Omitted]

256.3257.31.60 107-2 [Image Omitted]

298.3299.31.90 107-3 [Image Omitted]

228.2229.21.49 107-4 [Image Omitted]

242.3243.21.81 107-5 [Image Omitted]

254.3255.11.82 107-6 [Image Omitted]

297.4298.21.41 107-7 [Image Omitted]

272.3273.21.85 107-8 [Image Omitted]

258.3259.21.47 107-9 [Image Omitted]

297.4298.21.39 107-10 [Image Omitted]

311.4312.31.42 107-11 [Image Omitted]

327.4328.21.55 107-12 [Image Omitted]

296.4297.32.70 107-13 [Image Omitted]

345.17346.20.85

Example 108

[Image Omitted]

A mixture of compound 102 (2.00 g, 8.6 mmol), conc. aqueous NH₄OH (60 mL) and 2-propanol (6 mL) was stirred in a closed pressure vessel at 85Â° C. for 3 days. The reaction mixture was cooled to 25Â° C., diluted with water (120 mL) and stirred at 25Â° C. for 10 minutes. The resulting heterogeneous solution was filtered, the solid was washed with water (3Ã—) and air dried overnight. This gave compound 108 as a beige solid 1.50 g (82%). ¹H-NMR (400 MHz, DMSO-d₆) Î´ 7.66 (s, 1H), 7.56 (d, 1H), 7.35 (d, 1H), 7.1 (bs, 2H).

Example 109

[Image Omitted]

A mixture of compound 108 (1.50 g, 7.10 mmol), 1-methyl-4-(4,4,5,5-teramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (2.94 g, 14.2 mmol), Pd (dppf)Cl₂(0.58 g, 10 mol %), sodium carbonate (3.75 g, 35.4 mmol) in 1,2-dimethoxyethane (60 mL) and water (15 mL) was stirred at 80Â° C. under Argon for 16 hours. The solvents were evaporated and the residue purified by column chromatography (SiO₂5% methanol/ethyl acetateâ†’15% methanol/ethyl acetate) to afford compound 109 as a grey solid 1.50 g (99%). ¹H NMR (400 MHz, DMSO-d₆Î´ 8.27 (s, 1H), 7.88 (s, 1H), 7.72 (d, 1H), 7.64 (s, 1H), 7.26 (d, 1H), 6.91 (bs, 2H), 3.92(s, 1H)HPLC-MS t_R=0.3 nm (UV_254nm). Mass calculated for formula C₁₀H₁₀N₆, 214.1; observed MH⁺ (LC/MS) 215.2 (m/z).

Example 110

[Image Omitted]

A solution of compound 109 (1 equivalent) in DMF (1 mL) was treated with NaH (60% dispersion in oil, 1.2 equivalents) for 15 minutes at room temperature. The respective isocyanate (1 equivalent) was then added to this solution at room temperature and the resultant solution was stirred overnight. When LC-MS analysis indicated the reaction was complete, the reaction mixture was concentrated. Purification by Prep-LC and conversion to a hydrochloric salt afforded compounds 110-1 to 110-4.

TABLE 10LCMSMH⁺HPLCExampleColumn 2MWm/zMS t_R110-1 [Image Omitted]

333.4334.14.10 110-2 [Image Omitted]

285.3286.22.30 110-3 [Image Omitted]

367.8368.23.60 110-4 [Image Omitted]

397.8398.23.60

Example 111

[Image Omitted]

To a solution of nicotinic acid (25.0 mg, 0.203 mmol) in DMF (1.5 mL) was added compound 109 (65.2 mg, 0.304 mmol) and diisopropylethylamine (0.159 mL, 0.91 mmol). The reaction mixture was stirred at room temperature for 10 minutes, cooled to 0Â° C. (ice-bath) and then added HATU (115.6 mg, 0.304 mmol) and catalytic DMAP. The reaction mixture was allowed to warm to room temperature and then heated to 70Â° C., stirred overnight. LC-MS analysis indicated the reaction was complete. The reaction mixture was concentrated. Purification by Prep-LC and conversion to a hydrochloric salt afforded compound 111. HPLC-MS t_R=1.78 min (UV_254nm). Mass calculated for formula C₁₆H₁₃N₇O, 319.12; observed MH⁺ (LC/MS) 320.2 (m/z).

Example 112

[Image Omitted]

5-Amino-3-methyl isothiazole hydrochloride (5.00 g, 33.2 mmol) was added to water (35 mL). The insolubles were filtered and the filtrate's pH was adjusted to 10 with the addition of 2N NaOH. The mixture was stirred for five minutes and extracted with ethyl ether. The organic layer was separated and the aqueous layer was saturated with NaCl, extracted with ethyl ether (10 mL, 2Ã—). The combined ether extracts were washed with brine, dried over sodium sulfate and then concentrated to afford compound 112 as dark orange oil, 3.12 g (82%). ¹H-NMR (400 MHz, DMSO-d₆Î´ 6.5 (bs, 2H), 5.9 (s, 1H), 2.1 (s, 3H).

5-amino-3-methyl isothiazole (1.00 g, 8.75 mmol) was slurried in CCl₄(30 mL) under an atmosphere of argon. N-Bromosuccinimide (1.56 g, 8.75 mmol) was added portion-wise to the amine slurry over a 10 minute period at room temperature. The reaction stirred at 65Â° C. for 1.5 hours. Thin layer chromatography (DCM/Hexanes 1:1) indicates the reaction is complete. The reaction mixture was cooled to room temperature and diluted with ethyl ether (40 mL). The resulting mixture was cooled to 5Â° C. for 30 minutes and filtered to remove any solid material. The filtrate was concentrated to yield a dark red solid that was dissolved in ethyl acetate and washed with water (100 mL, 2Ã—). The organic layer was separated, washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to afford compound 112 as a dark red solid (1.49 g, 88%). This was used without further purification. ¹H-NMR (400 MHz, DMSO-d₆) Î´ 6.7 (bs, 2H), 2.2 (s, 3H).

Example 113

[Image Omitted]

A solution of thiophene2-carboxylic acid (1.00 g, 7.8 mmol), diphenylphosphoryl azide (2.15 g, 7.80 mmol) and triethylamine (1.1 mL, 7.8 mmol) in tert-butanol (20 mL) was heated at reflux for 5 hours, at which time thin layer chromatography (DCM/Hexanes) indicates the reaction is complete. The reaction mixture was cooled to room temperature, poured into water and extracted with diethyl ether (3Ã—). The combined ether extracts were washed with brine, dried over anhydrous sodium sulfate, and then concentrated to afford a beige solid. Purification by column chromatography (SiO₂, DCM/Hexanes) afforded compound 113 as a white solid 1.07 g (69%). ¹H-NMR (400 MHz, DMSO-d₆)Î´ 6.87 (dd, 1H), 6.77 (m, 1H), 6.5 (dd, 1H), 1.46 (s, 9H).

Example 114

[Image Omitted]

A solution of compound 113 (0.20 g, 1.00 mmol) was stirred in 4 M HCl solution in 1,4-dioxane (3 mL) at 50Â° C. for 2 hrs at which time thin layer chromatography (DCM/Hexanes) indicated the reaction was complete. The reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was diluted with acetonitrile, sonicated, and concentrated to afford compound 114 as a grey solid 0.13 g (96%). ¹H-NMR (400 MHz, DMSO-d₆) Î´ 7.38 (m, 1H), 7.02 (m, 1H), 6.97 (m, 2H).

Example 115

[Image Omitted]

A solution of 4-methyl thiophene-2carboxylic acid (1.00 g, 7.03 mmol), diphenylphosphoryl azide (1.94 g, 7.03 mmol) and triethylamine (0.98 mL, 7.03 mmol) in tert-butanol (20 mL) was heated at reflux for 5 hours, at which time thin layer chromatography (DCM/Hexanes) indicates the reaction is complete. The reaction mixture was cooled to room temperature, poured into water and extracted with diethyl ether (3Ã—). The combined ether extracts were washed with brine, dried over anhydrous sodium sulfate and then concentrated to afford a beige solid. Purification by column chromatography (SiO₂DCM/Hexanes) afforded compound 115 as a white solid 0.96 g (64%). ¹H-NMR (400 MHz, DMSO-d₆) Î´ 6.42(s, 1H), 6.35 (d, 1H), 1.46 (s, 9H).

Example 116

[Image Omitted]

A solution of compound 115 (0.21 g, 1.00 mmol) was stirred in 4 M HCl solution in 1,4-dioxane (3 mL) at 50Â° C. for 2 hrs at which time thin layer chromatography (DCM/Hexanes) indicated the reaction was complete. The reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was diluted with acetonitrile, sonicated, and concentrated to afford compound 116 as a grey solid 0.14 g (91%). ¹H-NMR (400 MHz, DMSO-d₆) Î´ 11.6 (bs, 2H) 6.83 (d, 1H), 6.7 (d, 1H), 4.55 (s, 3H).

Example 117

[Image Omitted]

To a solution of isothiazole-5-carboxylic acid methyl ester (0.50 g, 3.49 mmol) in THF/MeOH (20 mL/5 mL) was added 1N NaOH (5.24 mL, 5.24 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 hours at which time thin layer chromatography indicated the reaction was complete. The reaction mixture was acidified to pH 2 with 1N HCl resulting in the formation of a precipitate, this was filtered and dried to afford compound 2 as a beige solid 0.35 g (76%). ¹H-NMR (400 MHz, DMSO-d₆) Î´ 8.69 (d, 1H), 7.85 (d, 1H).

Example-118

[Image Omitted]

A solution of compound 117 (0.35 g, 2.67 mmol), diphenylphosphoryl azide (0.57 mL, 2.67 mmol) and triethylamine (0.37 mL, 2.67 mmol) in tert-butanol (10 mL) was heated at reflux for 5 hours, at which time thin layer chromatography (DCM/Hexanes) indicates the reaction is complete. The reaction mixture was cooled to room temperature, poured into water and extracted with diethyl ether (3Ã—). The combined ether extracts were washed with brine, dried over sodium sulfate, and concentrated to afford a beige solid. Purification by column chromatography (SiO₂, 40% ethyl acetate/hexanes) afforded compound 121 as a white solid 0.245 g (46%). ¹H-NMR (400 MHz, DMSO-d₆) Î´ 8.15(d, 1H), 6.72 (d, 1H), 1.48 (s, 9H).

Example 119

[Image Omitted]

A solution of compound 118 (0.25 g, 1.22 mmol) was stirred in 4 M HCl solution in 1,4-dioxane (3 mL) at 50Â° C. for 2 hrs at which time thin layer chromatography (DCM/Hexanes) indicated the reaction was complete. The reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was diluted with acetonitrile, sonicated, and concentrated to afford compound 119 as a grey solid 0.15 g (93%). ¹H-NMR (400 MHz, DMSO-d₆) Î´ 8.09 (d, 1H), 6.26 (d, 1H).

Example 120

[Image Omitted]

To a solution of 3-nitrophenol (0.35 g, 2.48 mmol, 1.00 equiv), triphenyl phosphine (0.68 g, 2.61 mmol, 1.05 equiv) and Boc-L-prolinol (0.53 g, 2.61 mmol, 1.05 equiv) in THF (10 mL) at rt was added drop wise diisopropyl azodicarboxylate (0.51 mL, 2.61 mmol, 1.05 equiv). The resulting solution was allowed to stir overnight at rt. Concentration and purification by chromatography (30% ethyl acetate in hexanes) afforded the title compound as a viscous oil (0.39 g, 48%).

Example 121

[Image Omitted]

A suspension of (S)-2-(3-nitro-phenoxymethyl)-pyrrolidine-1-carboxylic acid tert-butyl ester (0.39 g) and 10% Pd/C (0.20 g) in ethanol was stirred under an hydrogen atmosphere (1 atm at balloon pressure) for 3.5 hr. The reaction mixture was filtered through a bed of Celite using ethyl acetate as solvent. Concentration afforded the title compound as a clear oil (0.316 g, 90%). ¹H NMR (400 MHz, DMSO-d₆) â–¡ 6.85 (t, 1H), 6.10 (appt, 3H), 5.00 (br s, 2H), 3.91 (app t, 1H), 3.71 (app t, 1H), 3.28-3.19 (m, 2H), 1.95-1.75 (m, 4H), 1.38 (s, 9H). LCMS: (MHâˆ’C₄H₈)⁺=237.3.

Example 122

[Image Omitted]

To a suspension of NaH (0.17 g, 4.4 mmol, 1.1 equiv) in DMSO (4 mL) at rt was added (3S)-1-Boc-3-pyrrolidinol (0.75 g, 4.0 mmol, 1.00 equiv) in one portion. After stirring for 20 min, 3-fluoronitrobenzene (0.51 g, 3.6 mmol, 0.90 equiv) was added drop wise and the resulting suspension was stirred an additional 1.5 hours at rt. The reaction mixture was quenched with the addition of saturated, aqueous NH₄Cl and extracted with ethyl acetate (3Ã—). The combined organic layers were washed with brine, dried (Na₂SO₄), and concentrated. Purification of the crude residue by chromatography (30% ethyl acetate in hexanes) afforded 3-(3-nitro-phenoxy)-pyrrolidine-1-carboxylic acid tert-butyl ester as a bright yellow oil (0.676 g, 60%).

Example 123

[Image Omitted]

A suspension of 3-(3-nitro-phenoxy)-pyrrolidine-1-carboxylic acid tert-butyl ester (0.676 g) and 10% Pd/C (0.200 g) in ethanol was stirred under an hydrogen atmosphere (1 atm at balloon pressure) for 16 hr. The reaction mixture was filtered through a bed of Celite using ethyl acetate as solvent. Concentration afforded the title compound as a clear oil (0.529 g, 87%). ¹H NMR (400 MHz, DMSO-d₆) â–¡ 6.87 (t, 1H), 6.14-6.03 (m, 3H), 5.04 (br s, 2H), 4.81 (br s, 1H), 3.52-3.23 (m, 4H), 2.10-1.95 (m, 2H), 1.38 (d, 9H). LCMS: (MHâˆ’C₄H₈)⁺=223.1.

Example 124

[Image Omitted]

To a suspension of NaH (0.165 g, 4.14 mmol, 1.1 equiv) in DMSO (4 mL) at rt was added 1-BOC-4-hydroxypiperidine (0.794 g, 3.94 mmol, 1.00 equiv) in one portion. After stirring for 20 min, 3-fluoronitrobenzene (0.62 g, 4.34 mmol, 1.10 equiv) was added dropwise and the resulting suspension was stirred an additional 16 hours at rt. The reaction mixture was quenched with the addition of saturated, aqueous NH₄Cl and extracted with ethyl acetate (50 mL, 3Ã—). The combined organic layers were washed with brine, dried with sodium sulfate and concentrated. Purification of the crude residue by chromatography (30% ethyl acetate in hexanes) afforded 4-(3-nitro-phenoxy)-piperidine-1-carboxylic acid tert-butyl ester as a dark orange oil (0.390 g, 31%).

Example 125

[Image Omitted]

A suspension of 4-(3-nitro-phenoxy)-piperidine-1-carboxylic acid tert-butyl ester (0.390 g) and 10% Pd/C (0.100 g) in ethanol was stirred under an hydrogen atmosphere (1 atm at balloon pressure) for 16 hr. The reaction mixture was filtered through a bed of Celite using ethyl acetate as solvent. Concentration afforded 4-(3-amino-phenoxy)-piperidine-1-carboxylic acid tert-butyl ester as a clear oil (0.353 g, 90%). ¹H NMR (400 MHz, DMSO-d₆) E16.85 (t, 1H), 6.15-6.05 (m, 3H), 4.99 (br s, 2H), 4.43-4.30 (m, 1H), 3.67-3.53 (m, 2H), 3.20-3.06 (m, 2H), 1.89-1.80 (m, 2H), 1.53-1.4 (m, 2H), 1.38 (s, 9H).

Example 126

[Image Omitted]
Part A:

A solution of 3-amino-4-methyl-pent-2-enenitrile (Hackler, R. E., et. al. J. Heterocyclic Chem. 1989, 1575-1578) (0.700 g, 6.35 mmol, 1.00 equiv) in 1/1 THF/ethanol (5 mL) was cooled to 0Â° C. and treated with hydrogen sulfide gas for ca. 5 min. The tube was sealed and heated at 90Â° C. (16 hr). The reaction vessel was cooled in an ice-bath, carefully vented and the reaction mixture was concentrated. The crude residue was used in Part B without further purification.

Part B:

A suspension of the crude residue from Part A and potassium carbonate (1.34 g, 9.71 mmol, 2.0 equiv) in diethyl ether (7 mL) was heated at reflux. To the reaction mixture was added drop wise a solution of iodine (1.2 g, 4.85 mmol, 1.00 equiv) in ether (7 mL). The mixture was heated at reflux for an additional 2 hr. Water and ethyl acetate were added. The aqueous phase was washed with ethyl acetate and the combined organic phases were washed with water, brine, and dried with sodium sulfate. Purification of the residue by chromatography (30% ethyl acetate in hexanes) afforded 449 mg (50% yield based on 3-amino-4-methyl-pent-2-enenitrile) of 3-isopropyl-isothiazol-5-ylamine as a waxy, orange solid. ¹H NMR (400 MHz, DMSO-d₆) â–¡ 6.46 (br s, 2H), 5.97 (s, 1H), 3.31 (dq, 1H), 1.12 (d, 6H), (MH)⁺ (LCMS) 143.1 (m/z)

Example 127

[Image Omitted]

The compound of example 127 was prepared by the same procedure set forth in the above example 126, MH⁺ (LCMS) 141.1 (m/z).

Example 128

[Image Omitted]

4-(1-Amino-2-cyano-vinyl)-piperidine-1-carboxylic acid tert-butyl ester was prepared from 4-cyano-piperidine-1-carboxylic acid tert-butyl ester (10.0 mmol) according to the procedure described in WO 2004/014910 A1 (p. 32). The crude residue was used in the next step without purification.

Example 129

[Image Omitted]

A solution of crude 4-(1-amino-2-cyano-vinyl)-piperidine-1-carboxylic acid tert-butyl ester (compound 128) in 1:1 THF/Ethanol (10 mL) was cooled to 0Â° C. and treated with hydrogen sulfide gas for ca. 5 min. The tube was sealed and heated at 85Â° C. for 4 hr. The reaction vessel was cooled in an ice-bath, carefully vented and the reaction mixture was concentrated. The crude residue was used in the next step without purification.

Example 130

[Image Omitted]

To the crude product from example 129 and potassium carbonate (2.1 g, 15.0 mmol) in diethyl ether (15 mL) at rt was added drop wise a solution of iodine (1.02 g, 4.0 mmol) in ether (6 mL). The mixture was stirred at rt for an additional 2 hr. Water and ethyl acetate were added. The aqueous phase was washed with ethyl acetate and the combined organic extracts were washed with water, brine and dried with sodium sulfate. Purification of the residue by chromatography (40% ethyl acetate in hexanes) afforded 250 mg of 4-(5-amino-isothiazol-3-yl)-piperidine-1-carboxylic acid tert-butyl ester (9% yield based on 4-cyano-piperidine-1-carboxylic acid tert-butyl ester). ¹H NMR (400 MHz, DMSO-d₆) â–¡ 6.51 (br s, 2H), 5.98 (s, 1H), 4.02-3.88 (m, 2H), 2.82-2.68 (m, 2H), 2.68-2.58 (m, 2H), 2.82-2.75 (m, 2H), 2.60-2.51 (m, 1H), 1.38 (s, 9H). LCMS: (Mâˆ’C₄H₈)⁺=228.1.

Example 131

[Image Omitted]

To a suspension of benzyl 4-(amino carbonyl)tetrahydro-1(2H)-pyridinecarboxylate (2.79 g, 10.6 mmol, 1.00 equiv) in toluene (50 mL) was added chlorocarbonylsulfonyl chloride (0.97 mL, 11.7 mmol, 1.1 equiv) drop wise. The resulting suspension was refluxed for one hour, allowed to cool and then concentrated. The residue was dissolved in ethyl acetate and washed with saturated sodium bicarbonate, water, brine and dried with sodium sulfate. Concentration afforded 3-(2-oxo-[1,3,4]oxathiazol-5-yl)-piperidine-1-carboxylic acid benzyl ester as a clear, pale yellow oil, MH⁺ (LCMS) 321.1 (m/z).

Example 132

[Image Omitted]

A solution of the crude residue from example 131 and ethyl propiolate (2 mL) in xylenes (15 mL) was heated in a sealed tube at 150Â° C. for 4 hr. Concentration and chromatographic purification (25% ethyl acetate and hexanes) afforded 3-(5-ethoxycarbonyl-isothiazol-3-yl)-piperidine-1-carboxylic acid benzyl ester and 3-(4-ethoxycarbonyl-isothiazol-3-yl)-piperidine-1-carboxylic acid benzyl ester as a 1:1 mixture (1.24 g), MH⁺ (LCMS) 375.1 (m/z).

Example 133

[Image Omitted]

A solution of the residue from example 132 in THF (20 mL) and 1 N LiOH (6.7 mL) was heated at 50Â° C. for 4 hr. The reaction mixture was poured into ethyl acetate and acidified to pH 3 with 1 N HCl. The aqueous phase was extracted with ethyl acetate and the combined organic extracts were washed with water, brine, and dried with sodium sulfate. Concentration afforded 3-(5-carboxy-isothiazol-3-yl)-piperidine-1-carboxylic acid benzyl ester and 3-(4-carboxy-isothiazol-3-yl)-piperidine-1-carboxylic acid benzyl ester as a 1:1 mixture (1.02 g), MH⁺ (LCMS) 347.1 (m/z).

Example 134 and 134-1

[Image Omitted]

To a solution of crude residue from example 133 (1.02 g, 2.94 mmol, 1.00 equiv), N,N-diisopropylethylamine (0.56 mL, 3.23 mmol, 1.1 equiv) in tert-BuOH (25 mL) at rt was added diphenylphosphoryl azide (0.7 mL, 3.2 mmol, 1.1 equiv) drop wise. The resulting solution was refluxed for one hour and concentrated. The regioisomers were separated chromatographically (15% ethyl acetate in hexanes) affording 3-(5-tert-butoxycarbonylamino-isothiazol-3-yl)-piperidine-1-carboxylic acid benzyl ester (134; R_f=0.50 (15% ethyl acetate in hexanes), LCMS: (MH)⁺=418.1 m/z) and 3-(4-tert-butoxycarbonylamino-isothiazol-3-yl)-piperidine-1-carboxylic acid benzyl ester (134-1; R_f=0.31 (15% ethyl acetate in hexanes), MH⁺ (LCMS) 418.1 (m/z).

Example 135

[Image Omitted]

The crude residue from 134-1 was treated with 4 N HCl in dioxane at rt for 4 hours and then was concentrated. The residue was freeze-dried from a solution of acetonitrile and water. 3-(5-Amino-isothiazol-3-yl)-piperidine-1-carboxylic acid benzyl ester was used without further purification, MH⁺ (LCMS) 318.2 (m/z). 3-(4-Amino-isothiazol-3-yl)-piperidine-1-carboxylic acid benzyl ester was prepared using the same method, MH⁺ (LCMS) 318.2 (m/z).

Example 135-1

[Image Omitted]

The crude residue from 134-1 was treated with 4 N HCl in dioxane at rt for 4 hours and then was concentrated. The residue was freeze-dried from a solution of acetonitrile and water. 3-(5-Amino-isothiazol-3-yl)-piperidine-1-carboxylic acid benzyl ester was used without further purification. MH⁺ (LCMS) 318.2 (m/z). 3-(4-amino-isothiazol-3-yl)-piperidine-1-carboxylic acid benzyl ester was prepared using the same method, MH⁺ (LCMS) 318.2 (m/z).

Examples 136-141

By essentially the same procedure set forth in Example 106, the compounds shown in column 3 were prepared from compounds given in column 2.

TABLE 11Ex-LCMSam-MH⁺HPLCpleColumn 2Column 3MWm/zMS t_R136 [Image Omitted]

466.1467.21.66 137 [Image Omitted]

475.2476.21.80 138 [Image Omitted]

489.2490.32.02 139 [Image Omitted]

489.2490.32.02 140 [Image Omitted]

480.2481.11.84 141 [Image Omitted]

514.1515.21.93 141-1 [Image Omitted]

514.1515.22.02

Example 142

[Image Omitted]

A solution of compound from example 121 (0.25 g,) was stirred in 4 N HCl solution in 1,4-dioxane (3 mL) at room temperature for 2 hrs at which time LC MS analysis indicated the reaction was complete. The reaction mixture concentrated under vacuum. The residue was diluted with acetonitrile, water, and lyophilized to afford compound 142; HPLC t_R=20.50 min, calculated molecular formula weight, 366.10; observed MH⁺ (LCMS) 367.2 (m/z).

By essentially the same procedure given in example 142, starting from compounds given in column 2, compounds given in column 3 in Table 12 can be prepared:

TABLE 12LCMSExam-MH⁺HPLCpleColumn 2Column 3MWm/zMS t_R143 [Image Omitted]

375.2376.22.18 144 [Image Omitted]

389.2390.22.27 145 [Image Omitted]

389.2390.22.26 146 [Image Omitted]

380.2381.22.23 147 [Image Omitted]

345.2346.20.85

Example 148

[Image Omitted]

A suspension of compound from example 141 (0.05 g) and 4 N HCl in dioxane was stirred at 60Â° C. for 1 hr. The reaction mixture evaporated to dryness, dissolved in acetonitrile-water (1:1), and lyophilized to give the product 148. HPLC t_R=2.49 min, calculated molecular formula weight 380.2, observed MH⁺ (LCMS) 381.2 (m/z).

Example 148-1

[Image Omitted]

By essentially the process in example 148-1 can be prepared from the procedure described in example 148. HPLC t_R=2.66 min, calculated molecular weight, 380.2, observed MH⁺ (LCMS) 381.2 (m/z).

Example 149

[Image Omitted]

The mixed halo-products (3:1 Cl:Br) from Preparative Example 102 (3.67 g, 15.0 mmol), were combined with N,N-dimethyl-m-phenylenediamine.2HCl (4.71 g, 22.5 mmol), i-Pr₂NEt (15.7 mL, 90.2 mmol), and NMP solvent (75 mL). The reaction was heated in an oil bath at 160Â° C. for 18 hours. The reaction was cooled and concentrated under vacuum. The crude material was purified by column chromatography; 2 columns using a gradient of 20% EtOAc/Hexanes increasing to 50% EtOAc/Hexanes. The product 149 was isolated in 95% purity as determined by ¹H NMR (400 MHz DMSO-d₆,) â–¡ 9.36 (s, 1H), 7.77 (s, 1H), 7.74 (d, J=4.4 Hz, 1H), 7.54 (d, J=4.8 Hz, 1H), 7.47 (m, 1H), 7.42 (t, J=2.0 Hz), 7.09 (t, J=8.0 Hz, 1H), 6.40 (dd, J=8.0 Hz, 2.0 Hz, 1H), 2.87 (s, 6H). Product was isolated in 77% yield, 3.83 g.

Example 150-1 to 150-30

[Image Omitted]

A 1.5 M solution of Na₂CO₃in H₂O (0.5 mL) was added to 4 mL vials containing 10 mol % Pd(dppf)Cl₂and 1.5 eq. of the appropriate boronic acid. The product from example 149 was added last as a 0.06 M solution in DME (2.0 mL). The reactions were flushed with Argon, capped, and placed in a sand bath at 80Â° C. overnight. The reactions were cooled, concentrated, and purified via preparative HPLC to give products 150.

TABLE 13LCMSHPLCExam-MH⁺MS t_RpleProductMWm/z(min)150-1 [Image Omitted]

407.5408.31.30 150-2 [Image Omitted]

380.5381.21.50 150-3 [Image Omitted]

380.5381.21.42 150-4 [Image Omitted]

407.5408.11.29 150-5 [Image Omitted]

335.4336.23.15 150-6 [Image Omitted]

354.4355.23.23 150-7 [Image Omitted]

330.4331.21.79 150-8 [Image Omitted]

346.4347.21.98 150-9 [Image Omitted]

354.4355.23.25 150-10 [Image Omitted]

359.4360.33.41 150-11 [Image Omitted]

365.4366.33.65 150-12 [Image Omitted]

375.5376.23.86 150-13 [Image Omitted]

401.5402.23.93 150-14 [Image Omitted]

398.5399.34.23 150-15 [Image Omitted]

414.5415.33.52 150-16 [Image Omitted]

371.4372.23.42 150-17 [Image Omitted]

391.5392.22.55 150-18 [Image Omitted]

349.5350.23.85 150-19 [Image Omitted]

372.4373.22.39 150-20 [Image Omitted]

377.5378.23.29 150-21 [Image Omitted]

369.4370.24.23 150-22 [Image Omitted]

385.5386.24.36 150-23 [Image Omitted]

360.4361.23.05 150-24 [Image Omitted]

373.5374.22.83 150-25 [Image Omitted]

373.4374.32.02 150-26 [Image Omitted]

428.5429.32.10 150-27 [Image Omitted]

333.4334.20.72 150-28 [Image Omitted]

361.5362.22.68 150-29 [Image Omitted]

364.5365.23.05 150-30 [Image Omitted]

375.2376.31.51 150-31 [Image Omitted]

409.2410.21.53

Example 151

[Image Omitted]

To the mixture of 3-(4-bromo-1-methyl-1H-pyrazol-3-yl-)phenyl amine (1.78 g, 7.1 mmol), imidazole (1.36 g, 20 mmol), and catalytic amount DMAP in DMF (12 mL), (BOC)₂O (1.7 g, 7.8 mmol) was added at room temperature. The mixture was stirred overnight and diluted with EtOAc (200 mL), the organics were washed with H₂O, brine and dried over Na₂SO₄. After concentration, the residue was purified with column chromatography (silica gel, hexane/EtOAc=70/30) to give the product 151 (2.52 g) as white solid. HPLC-MS t_R=2.00 min (UV_254nm). Mass calculated for formula C₁₅H₁₈BrN₃O₂, 351.1; observed MH⁺ LC/MS 352.1 (m/z).

Example 152

[Image Omitted]

To a 25 mL round bottom flask charged with bis(pinacolato)diboron (1.0 g, 4.0 mmol), KOAC (960 mg, 10 mmol), Pd(dppf)Cl₂(240 mg, 0.30 mmol) and product from example 151 (1.16 g, 3.30 mmol) was added DMSO (6 mL) under argon. The mixture was degassed thoroughly. This resulting mixture was then heated at 80Â° C. overnight, diluted by EtOAc (40 mL) and filtered through celite. After concentration, the residue was purified with column chromatography (silica gel, hexane/EtOAc=80/20) to give the product 152 (997 mg) as an oil. HPLC-MS t_R=2.11 min (UV_{254 nm}); mass calculated for formula C₂₁H₃₀BN₃O₄, 399.2; observed MH⁺ LCMS 400.3 (m/z).

Example 153

[Image Omitted]

Under argon, the boronate compound 152 (120 mg, 0.3 mmol) in THF (3.0 mL, 5% H₂O) was added to the flask which was charged with Pd(dppf)Cl₂(8.0 mg, 10 mol %), K₂CO₃(138 mg, 1.0 mmol), and 3-bromoimidazopyrazine 149 (51 mg, 0.15 mmol). The mixture was degassed thoroughly with argon. The resulting solution was heated up to 80Â° C. and stirred overnight. After cooling to room temperature, the mixture was diluted with EtOAc (50 mL) and the solid was removed by filter through Celite and washed with some EtOAc. Concentration resulted in a residue 153 and was used in the next step directly without further purification. HPLC-MS t_R=2.05 min (UV_{254 nm}); mass calculated for formula C₂₉H₃₂N₈O₂; 524.3, observed MH⁺ (LCMS) 525.2.1 (m/z).

Example 154

[Image Omitted]

To the product from example 153 was added HCl (6 N, 3 mL), and the mixture was stirred at room temperature for 10 min. The reaction was concentrated, and the residue purified with HPLC to give the compound 154 (48 mg). HPLC-MS t_R=1.16 min (UV_{254 nm}); mass calculated for formula C₂₄H₂₄N₈, 424.2; Observed MH⁺ (LCMS) 425.2 (m/z).

Example 155

[Image Omitted]

To a mixture of hydroxy benzotriazole (7 mg, 0.05 mmol and benzoic acid (6 mg, 0.05 mmol) in DMF (1 mL), EDC (10 mg, 0.05 mmol) was added and the mixture was stirred at room temperature for 10 min. Then product 154 (21 mg, 0.05 mmol) in DMF (1 mL) was added and the resulting mixture was heated up to 50Â° C. and stirred overnight. The mixture was diluted with EtOAc (50 mL), washed with H₂O, brine and dried over Na₂SO₄. After concentration the residue was purified by prep-LC to give the product 155. HPLC-MS t_R=1.54 min (UV_254nm); mass calculated for formula C₃₁H₂₈N₈O, 528.2; observed MH⁺ (LCMS) 529.3 (m/z).

Example 156

[Image Omitted]

Compound 156 was prepared using the boronation conditions described in Example 152. HPLC-MS t_R=1.83 min (UV_{254 nm}); mass calculated for formula C₁₁H₁₇BN₂O₃, 236.1; observed MH⁺ (LCMS) 237.3 (m/z).

Example 157

[Image Omitted]

Compound 157 was prepared using the coupling conditions described in example 153.HPLC-MS t_R=1.18 min (UV_{254 nm}); mass calculated for formula C₁₉H₁₉N₇O, 361.2; observed MH⁺ (LCMS) 362.1 (m/z).

Example 158

[Image Omitted]

Product from example 157 (50 mg, 0.14 mmol) was dissolved in MeOH (5 mL) and the mixture cooled to 0Â° C. NaBH₄(38 mg, 1.0 mmol) was added and the resulting mixture was stirred at 0Â° C. for 30 min. After concentration, the residue was purified with prep-LC gave the product 158. HPLC-MS t_R=0.92 min. (UV_254nm); mass calculated for formula C₁₉H₂₁N₇O, 363.2; observed MH⁺ (LCMS) 364.3 (m/z).

Example 159

[Image Omitted]

Product of example 159 was prepared using the coupling condition described in 153. HPLC-MS t_R=0.94 min (UV_{254 nm}); mass calculated for formula C₁₆H₁₄N₆290.1, observed MH⁺ (LCMS) 291.3 (m/z).

Example 160

[Image Omitted]

By essentially the same procedure given in example 106, combining the product from example 105 and 2-chloro-4-amino pyridine to give the product 160. HPLC t_R=1.45 min. Calculated molecular weight, 325.1, observed MH⁺ (LCMS) 326.0 (m/z).

Example 161

[Image Omitted]

A mixture of the product from example 160, 1-methyl piperazine (excess) is stirred and heated at 100Â° C. for 72 hrs. The mixture poured in to 10% aqueous Na₂CO₃and extracted with ethyl acetate. The extracts dried over sodium sulfate, filtered and evaporated. Preparative HPLC purification afford the product, HPLC t_R=1.92 min. Calculated molecular weight=389.5, observed MH⁺ (LCMS) 390.30 (m/z).

By essentially the same procedure given in example 161, combining intermediates from preparative example 160 with the amines given in column 1, compounds given in column 2 were prepared. The compounds obtained were purified by preparative HPLC. The purified products were treated with 4 N HCl in dioxane to remove the BOC protecting group. The volatiles were removed under vacuum. The product was dissolved in acetonitrile-water and lyophilized to give the product(s).

TABLE 14LCMSExam-MH⁺HPLCpleColumn 1Column 2MWm/zMS t_R163 [Image Omitted]

375.1376.10.75 164 [Image Omitted]

389.2390.20.75 164-1 [Image Omitted]

375.1376.01.94

Example 165

[Image Omitted]

By essentially the same procedure given in example 106, combining the product from example 105 and 2-chloro-4-amino pyridine to give the product 165.

HPLC t_R=1.48 min. Calculated molecular weight, 325.1; observed MH⁺ (LCMS), 326.0 (m/z).

Example 166

[Image Omitted]

A mixture of the product from example 165, 1-methyl piperazine (excess) is stirred and heated at 100Â° C. for 72 hrs. The mixture poured into 10% aqueous Na₂CO₃and extracted with ethyl acetate. The extracts were dried over sodium sulfate, filtered and evaporated. Preparative HPLC purification afforded the product. HPLC t_R=1.80 min. Calculated molecular weight, 389.5.1; observed MH⁺ (LCMS) 390.23 (m/z).

By essentially the same procedure given in example 161, combining intermediates from preparative example 160 with the amines given in column 1, the compounds given in column 2 were prepared. The compounds obtained were purified by preparative HPLC. The purified products obtained were treated with 4 N HCl dioxane to remove the BOC protecting group and volatiles were removed under vacuum. The product was dissolved in acetonitrile-water and lyophilized to give the product(s).

TABLE 15LCMSExam-MH⁺HPLCpleColumn 1Column 2MWm/zMS t_R167 [Image Omitted]

349.1350.10.50 168 [Image Omitted]

375.4376.20.80 169 [Image Omitted]

403.4404.20.85

Example 170

[Image Omitted]

To a solution of 2-amino-3-chloropyrazine (0.20 g, 1.5 mmol, 1.00 equiv) and 3-methoxyphenacyl bromide (0.71 g, 3.1 mmol, 2.0 equiv) in dioxane (10 mL) was heated at 90Â° C. for 3 hr. The resulting mixture was cooled to rt and filtered. The filtrate was partitioned between 10% IPA/DCM and 1 N NaOH. The aqueous extract was washed with 10% IPA/DCM (2Ã—) and the combined organic extracts were washed with brine and dried with sodium sulfate. Concentration afforded 8-chloro-2-(3-methoxy-phenyl)-imidazo[1,2-a]pyrazine (76 mg, 19%). MH⁺ (LCMS) 260.1 (m/z).

Example 171

[Image Omitted]

To the product from example 170 in acetic acid (10 mL) was added a solution of bromine in acetic acid (0.25 mmol, 1 mL). Concentration of the reaction mixture afforded crude 3-bromo-8-chloro-2-(3-methoxy-phenyl)-imidazo[1,2-a]pyrazine. MH⁺ (LCMS) 338.0 (m/z).

Example 172

[Image Omitted]

A solution of 3-bromo-8-chloro-2-(3-methoxy-phenyl)-imidazo[1,2-a]pyrazine (0.13 g, 0.38 mmol, 1.00 equiv) product from example 171, N,N-dimethyl-m-phenylenediamine hydrochloride (0.15 g, 0.71 mmol, 1.9 equiv) and N,N-diisopropylethylamine (0.33 mL, 1.9 mmol, 5.0 equiv) in NMP (2 mL) was heated at 140Â° C. for 20 h. Concentration and purification by chromatography (25% ethyl acetate in hexanes) afforded the title compound. MH⁺ (LCMS) 438.1 (m/z).

Example 173

[Image Omitted]

A suspension of 3-bromo-8-chloro-2-(3-methoxy-phenyl)-imidazo[1,2-a]pyrazine (38.2 mg, 0.0871 mmol, 1.00 equiv), [1,1â€²-bis(diphenylphosphino) ferrocene]dichloropalladium(II) (3 mg, 0.004 mmol, 5 mol %), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole (0.036 g, 0.17 mmol, 2.0 equiv) and sodium carbonate (0.028 g, 0.26 mmol, 3.0 equiv) in 1,2-dimethoxy ethane/water (0.4 mL/0.1 mL) was heated at 90Â° C. for 2.5 hr. The mixture was allowed to cool, filtered, concentrated and purified using chromatography (25% ethyl acetate in hexanes). The title compound was obtained as a colorless solid. HPLC t_R=1.68 min), MH⁺ (LCMS) 440.2 (m/z).

Example 174

[Image Omitted]

The title compound, example 174 was prepared by the same procedure set forth in the above example 173 HPLC (t_R=0.64 min). Calculated M.Wt. 228.1, observed MH⁺ (LCMS) 229.1 (m/z).

Example 175

[Image Omitted]

The title compound, example 175 was prepared by the same procedure set forth in the above example 173. HPLC (t_R=0.75 min). Calculated M.Wt. 286.2, observed MH⁺ (LCMS) 287.2 (m/z).

Example 176

[Image Omitted]

The mixture of bromoacetaldehyde diethyl acetal (5.2 mL, 33.3 mmol) and HBr (0.8 mL, 48% in H₂O) in H₂O (8 mL) was heated at reflux and stirred for 1 hour. After cooling to room temperature. The mixture was extracted with ethyl ether (100 mL, 5Ã—). The ether was dried over Na₂SO₄and concentrated to give the crude bromoacetaldehyde. To the crude acetaldehyde, 2-amino-3,5-dibromopyrazine (4.30 g, 17 mmol) and DME (120 mL) were added followed by the addition of HBr (1 mL, 48% in H₂O). The mixture was heated at reflux with stirring overnight. After cooling to room temperature the solid was collected with filtration and washed with DME. After drying under vacuum, the product 176 (4.50 g) obtained as HBr salt, a black solid. HPLC-MS t_R=1.13 min (UV_{254 nm}); mass calculated for formula C₆H₃Br₂N₃, 274.9; observed MH⁺ (LCMS) 276.0 (m/z).

Example 177

[Image Omitted]

The dibromo compound 176 (2.16 g, 6.0 mmol) was dissolved in MeOH (20 mL). NaSMe (840 mg, 12 mmol) was added. The mixture was stirred for 2 hours at room temperature and concentrated. The residue was taken up in H₂O (20 mL) and extracted with DCM/iso-PrOH (9/1) (50 mL, 3Ã—). The combined organic layers were dried over Na₂SO₄and concentrated. The crude compound was purified with column chromatography (silica gel, EtOAc/hexane=40/60 to 100% EtOAc) to give the pure compound 177 (1.12 g) as yellowish solid. ¹H NMR (400 MHz, CDCl₃) Î´7.97 (s, 1H), 7.68 (d, 1H), 7.57 (d, 1H), 2.66 (s, 3H). HPLC-MS t_R=1.40 min (UV_{254 nm}); mass calculated for formula C₇H₆BrN₃S, 242.9; observed MH⁺ (LCMS) 244.1 (m/z).

Example 178

[Image Omitted]

Under Ar, a solution of 9-BBN (10 mL, 0.5 M in THF) was added drop wise to the solution of benzyl N-vinylcarbamate (875 mg, 5.00 mmol) in THF (10 mL) at room temperature and stirred for 2 hours. The resulting mixture was transferred to another flask that was charged with product from example 177 (610 mg, 2.5 mmol), K₃PO₄(850 mg, 4.0 mmol) and Pd(dppf)Cl₂(160 mg, 0.2 mmol) in THF (20 mL, together with 1 mL of water) under Argon. The resulting mixture was heated to 60Â° C. and stirred overnight under Argon. The reaction was cooled to room temperature. EtOAc (200 mL) was added to the reaction mixture and filtered through celite. After concentration the residue was purified with column (silica gel, EtOAc/hexane=50/50) to give the product 178 (457 mg) and 178 A (150 mg) as oil.

178: ¹H NMR (400 MHz, CDCl₃) Î´ 7.65 (s, 1H), 7.63 (d, 1H), 7.51 (d, 1H), 7.34 (m, 5H), 5.43 (s, 1H), 5.10(s, 2H), 3.64 (m, 2H), 2.89 (t, 2H), 2.62 (s, 3H). HPLC-MS t_R=1.59 min (UV_{254 nm}); mass calculated for formula C₁₇H₁₈N₄O₂S 342.1; observed MH⁺ (LCMS) 343.1 (m/z).

178 A: HPLC-MS t_R=1.50 min (UV_{254 nm}); mass calculated for formula C₁₇H₁₈N₄O₂S, 342.1; observed MH⁺ (LCMS) 343.1 (m/z).

Example 179

[Image Omitted]

NBS (104 mg, 0.59 mmol) was added to a solution of compound 178 (200 mg, 0.59 mmol) in EtOH (10 mL), at room temperature. The mixture was stirred for 30 min and concentrated. The residue was diluted with EtOAc and washed with saturated aq.NaHCO₃(30 mL, 2Ã—), brine and dried over Na₂SO₄. After concentrating, the crude product 179 was used in the next step directly without further purification. HPLC-MS t_R=1.88 min (UV_{254 nm}); mass calculated for formula C₁₇H₁₇BrN₄O₂S, 420.0; observed MH⁺ (LCMS) 421.0 (m/z).

Example 180

[Image Omitted]

The boronate (122 mg, 0.585 mmol), was mixed with Pd(dppf)Cl₂(50 mg, 0.06 mmol), K₃PO₄(318 mg, 1.5 mmol), and the product from example 179 (246 mg, 0.585 mmol) in dioxane (5 mL) was added. The mixture was degassed thoroughly and kept under argon blanket. The resulting solution was heated at 80Â° C. and stirred overnight. After cooling to room temperature the mixture was diluted with EtOAc (50 mL). The solid was removed by filter through Celite and washed with EtOAc. The solvent was removed under reduced pressure and the resulting residue was purified with column chromatography (silica gel, EtOAc to MeOH/EtOAc=5/95) gave the product 180 (212 mg) as oil. HPLC-MS t_R=1.62 min (UV_{254 nm}); mass calculated for formula C₂₁H₂₂N₆O₂S, 422.2; observed MH⁺ (LCMS) 423.3 (m/z).

Example 181

[Image Omitted]

A mixture of compound 180 (212 mg, 0.5 mmol) and m-CPBA (224 mg, 77%, 1.0 mmol) in DCM (10 mL) was stirred at room temperature for 30 min then diluted with EtOAc (100 mL). The organics were washed with NaHCO₃(sat. aq., 10 mlÃ—2), brine and dried over Na₂SO₄. After concentration, the crude product 181 was used in the next step directly without further purification. HPLC-MS t_R=1.36 min (UV_{254 nm}); mass calculated for formula C₂₁H₂₂N₆O₄S, 454.1; observed MH⁺ (LCMS) 455.2 (m/z).

Example 182

[Image Omitted]

The aniline (16 mg, 0.21 mmol) was dissolved in dry DMSO (2 mL) with NaH (60% in oil, 4 mg, 0.1 mmol) under argon. The mixture was stirred for 10 min at room temperature and sulfone 181 (25 mg, 0.05 mmol) in dry DMSO (0.5 mL) was added. The reaction mixture was heated at 80Â° C. and stirred for 10 min. After cooling to room temperature, the mixture was purified by prep-LC to give the product 182 as a TFA salt. HPLC-MS t_R=1.15 min (UV_{254 nm}); mass calculated for formula C₂₉H₂₇N₉O₂, 533.2; observed MH⁺ (LCMS) 534.2 (m/z).

Example 183

[Image Omitted]

The TFA salt of compound 182 (20 mg, 0.038 mmol) was treated with 4 N HCl (2 mL) and the mixture was stirred at room temperature for 30 min. After concentration the residue was dried by lyophilization gave the final compound 183. HPLC-MS t_R=0.75 min (UV_{254 nm}); mass calculated for formula C₂₁H₂₁N₉, 399.2; observed MH⁺ (LCMS) 400.1 (m/z).

By essentially the same procedures given in examples 178-183 to give compound 184 and 185.

TABLE 16LCMSExam-MH⁺HPLCpleColumn 2MWm/zMS t_R184 [Image Omitted]

354.1355.10.87 185 [Image Omitted]

354.1355.10.90

Example 186

[Image Omitted]

To a solution of NaH (24 mg, 60% in oil, 0.6 mmol), compound 178 (200 mg, 0.585 mmol) in dry DMF (5 mL) was added carefully. The mixture was stirred at room temperature for 10 min. Iodomethane (100 Î¼L) was added to the above reaction mixture. The resulting mixture was stirred overnight, cooled to 0Â° C. and water was added carefully to quench the reaction. The aqueous was extracted with EtOAc and the organics was dried over Na₂SO₄. After concentration, the crude product was purified with column chromatography (silica gel, hexane/EtOAc=70/30) to give the product 186 (201 mg). HPLC-MS t_R=1.65 min (UV_{254 nm}), mass calculated for formula C₁₈H₂₀N₄O₂S, 356.1; observed MH⁺ (LCMS) 357.2 (m/z).

Example 187

[Image Omitted]

Compound 187 was prepared using the brominating conditions described in example 179. HPLC-MS t_R=2.01 min (UV_{254 nm}); mass calculated for formula C₁₈H₁₉BrN₄O₂S, 434.0; observed MH⁺ (LCMS) 435.1 (m/z).

Example 188

[Image Omitted]

Compound 188 was synthesized using the same coupling condition described in example 180. HPLC-MS t_R=1.73 min (UV_{254 nm}); mass calculated for formula C₂₂H₂₄N₆O₂S, 436.2; observed MH⁺ (LCMS) 437.2 (m/z).

Example 189

[Image Omitted]

Compound 189 was prepared using the oxidation conditions described in example 181. HPLC-MS t_R=1.43 min (UV_{254 nm}); mass calculated for formula C₂₂H₂₄N₆O₄S, 468.2; observed MH⁺ (LCMS) 469.1 (m/z).

Example 190

[Image Omitted]

Compound 190 was prepared using the amination conditions described in example 182. HPLC-MS t_R=1.25 min (UV_{254 nm}); mass calculated for formula C₃₀H₂₉N₉O₂, 547.2; observed MH⁺ (LCMS) 548.2 (m/z).

Example 191

[Image Omitted]

Compound 190 was synthesized using the deprotecting conditions described in example 183. HPLC-MS t_R=0.75 min (UV_{254 nm}); mass calculated for formula C₂₂H₂₃N₉, 413.2; observed MH⁺ (LCMS) 414.2 (m/z). By essentially the same procedure given in Preparative Example 186-191, compounds given in Column 2 can be prepared from 183 and 185.

TABLE 17LCMSExam-MH⁺HPLCpleColumn 2MWm/zMS t_R192 [Image Omitted]

368.2355.10.87 193 [Image Omitted]

368.2369.10.90 194 [Image Omitted]

413.2414.20.78

Example 195

[Image Omitted]

A solution of LDA (28.6 mmol) was prepared from iso-Pr₂NH (4.03 mL, 28.6 mmol) and n-BuLi (11.40 mL, 2.5 M in hexane, 28.6 mmol) in THF (50 mL). The solution was cooled at âˆ’78Â° C. and N-Boc-3-piperidone (4.0 g, 20 mmol) in THF (10 mL) was added with a syringe. After 15 min, N-phenyltriflimide (8.60 g, 24.0 mmol) in THF (20 mL) was added. The reaction mixture was then warmed up to room temperature slowly and stirred overnight. After evaporation, of the solvent under vacuum, the residue was dissolved in DCM (120 mL). The solution was then filtered on neutral alumina and evaporated. Flash chromatography (hexane/EtOAc 80/20) of the crude oil on silica gel gave products 195 and 196.

Product 195: HPLC-MS t_R=1.65 min (UV_{254 nm}); mass calculated for formula C₁₁H₁₆F₃NO₅S, 231.1; observed MH⁺ (LCMS) 232.1 (m/z).

Product 196: HPLC-MS t_R=1.68 min (UV_{254 nm}); mass calculated for formula C₁₁H₁₆F₃NO₅S, 231.1; observed MH⁺ (LCMS) 232.1 (m/z).

Example 197

[Image Omitted]

To a 25 mL round bottom flask charged with bis(pinacolato)diboron (1.50 g, 6 mmol), potassium acetate (1.5 g, 15 mmol), Pd(dppf)Cl₂(408 mg, 0.5 mmol) and DPPF (277 mg, 0.5 mmol). Compound 195 (1.55 g, 5.0 mmol) in dioxane 20 mL) was added to the above mixture. The mixture was degassed thoroughly and placed under argon. This resulting mixture was then heated at 80Â° C. for overnight, diluted with EtOAc (40 mL) and filtered through celite. After concentration, the residue was purified with column chromatography (silica gel, Hexane/EtOAc=60/40) to give the product (832 mg) as an oil. HPLC-MS t_R=2.41 min (UV_{254 nm}), mass calculated for formula C₁₆H₂₈BNO₄, 309.2; observed MH⁺; -t-Bu (LCMS) 254.2 (m/z).

Example 198

[Image Omitted]

To a 25 mL round bottom flask charged with boronate 197 (456 mg, 1.5 mmol), K₂CO₃(800 mg, 6 mmol), and Pd(dppf)Cl₂(160 mg, 0.2 mmol) was added a solution of product from example 177 (360 mg, 1.5 mmol) in DMF (10 mL). The mixture was degassed thoroughly and placed under argon. This resulting mixture was then heated at 80Â° C. overnight. The reaction mixture was diluted with EtOAc (40 mL) and filtered through Celite. After concentration, the residue was purified by column chromatography (silica gel, Hexane/EtOAc=60/40) to give the product 198 (258 mg) as an oil. HPLC-MS t_R=1.91 min (UV_{254 nm}); mass calculated for formula C₁₇H₂₂N₄O₂S, 346.1; observed MH⁺ (LCMS) 347.2 (m/z).

Example 199

[Image Omitted]

Compound 199 was prepared using brominating conditions described in example 179. HPLC-MS t_R=2.26 min (UV_{254 nm}); mass calculated for formula C₁₇H₂₁BrN₄O₂S, 424.1; observed MH⁺ (LCMS) 425.0 (m/z).

Example 200

[Image Omitted]

By essentially, example product 200 was synthesized using the same coupling conditions described in example 180. HPLC-MS t_R=1.96 min (UV_{254 nm}); mass calculated for formula C₂₁H₂₆N₆O₂S, 426.2; observed MH⁺ (LCMS) 427.1 (m/z).

Example 201

[Image Omitted]

The mixture of compound 200 (130 mg, 0.305 mmol) and m-CPBA (68 mg, 77%, 0.305 mmol) in DCM (5 mL) was stirred at 0Â° C. for 30 min and then diluted with EtOAc (100 mL). The organics were washed with saturated aqueous NaHCO₃(10 mL, 2Ã—), brine, and dried over Na₂SO₄. After concentration the crude product 201 was used in the next step directly without further purification. HPLC-MS t_R=1.48 min (UV_{254 nm}); mass calculated for formula C₂₁H₂₆N₆O₃S, 442.2; observed MH⁺ (LCMS) 443.2 (m/z).

Example 202

[Image Omitted]
The product example 202 was prepared using the similar experimental conditions described in product example 182. HPLC-MS t_R=1.44 min (UV_{254 nm}); mass calculated for formula C₂₉H₃₁N₉O₂, 537.3; observed MH⁺ (LCMS) 538.3 m/z).

Example 203

[Image Omitted]

The product from example 202 (20 mg) was treated with 4 N HCl in dioxane (4 mL) and stirred at room temperature for 10 min. After concentration, the residue was dried by lyophilization gave compound 203. HPLC-MS t_R=0.75 min (UV_{254 nm}); mass calculated for formula C₂₄H₂₃N₉, 437.2; observed MH⁺ (LCMS) 438.3 (m/z).

By essentially the same procedures given in Preparative Example 203, compounds given in Column 2 of Table 18 can be prepared from example 195 through 203.

TABLE 18LCMSExam-MH⁺HPLCpleColumn 2MWm/zMS t_R204 [Image Omitted]

437.2438.30.74 205 [Image Omitted]

392.2393.10.97 206 [Image Omitted]

392.2393.20.95

Example 207

[Image Omitted]

The product from example 202 (20 mg, TFA salt) was dissolved in THF (5 mL), and DIEA (500 Î¼L) was added. To this mixture, 10% Pd/C (5 mg) was added and the resulting mixture was hydrogenated under H₂atm. while stirring for overnight. After filtration and concentration the residue was purified by prep-LC to give the product 207. HPLC-MS t_R=1.45 min (UV_{254 nm}); mass calculated for formula C₂₉H₃₃N₉O₂, 539.3; observed MH⁺ (LCMS) m/z 540.3 (m/z).

Example 208

[Image Omitted]

Product from example 207 was treated with was treated with 4 N HCl in dioxane (4 mL) and stirred at room temperature for 10 min. After concentration, the residue was dried with lyophilization to give 208. HPLC-MS t_R=0.80 min (UV_{254 nm}); mass calculated for formula C₂₄H₂₅N₉, 439.2; observed MH⁺ (LCMS) 440.2 (m/z).

By essentially the same procedure given in Preparative Example 208, compounds given in Column 2 of Table 19 can be prepared.

TABLE 19LCMSExam-MH⁺HPLCpleColumn 2MWm/zMS t_R209 [Image Omitted]

394.2395.20.95

Example 210

[Image Omitted]

The product from example 198 (175 mg, 0.50 mmol) was dissolved in 20 mL of DME and 4 mL of water. To the mixture was added p-toluenesulfonyl hydrazide (1.86 g, 10 mmol). The mixture was heated up to 90Â° C. following the addition of NaOAc (1.64 g, 20.0 mmol) to the reaction. After stirring at reflux for 4 hours, additional p-toluenesulfonyl hydrazide (1.86 g, 10.0 mmol) and NaOAc (1.64 g, 20 mmol) were added. The mixture was at reflux overnight. After cooling to room temperature, the mixture was diluted with EtOAc (200 mL) and washed with H₂O, and brine. The organics were dried over Na₂SO₄and concentrated. The resulting residue was purified by prep-LC to give the product 210. HPLC-MS t_R=1.92 min (UV_{254 nm}); mass calculated for formula C₁₇H₂₄N₄O₂S, 348.2; observed MH⁺ (LCMS) 349.2 (m/z).

Example 211

[Image Omitted]

Product from example 211 was prepared using brominating conditions described in example 179. HPLC-MS t_R=5.89 min (UV_{254 nm}); mass calculated formula C₁₇H₂₃BrN₄O₂S, 426.1; observed MH⁺ (LCMS) 427.0 (m/z).

Example 212

[Image Omitted]

Compound 212 was synthesized using coupling conditions described in example 180. HPLC-MS t_R=1.99 min (UV_{254 nm}); mass calculated for formula C₂₁H₂₈N₆O₂S, 428.2; observed MH⁺ (LCMS) 429.2 (m/z).

Example 213

[Image Omitted]

Compound 213 was synthesized using oxidation conditions described in example 181. HPLC-MS t_R=1.64 min (UV_{254 nm}); mass calculated for formula C₂₁H₂₈N₆O₄S; 460.2, observed MH⁺ (LCMS) 461.2 (m/z).

Example 214

[Image Omitted]

Compound 214 was prepared using the experimental condition described in example 182. HPLC-MS t_R=1.84 min (UV_{254 nm}); mass calculated for formula C₂₄H₃₀N₈O₂S; 494.2, observed MH⁺ (LCMS) 495.2 (m/z).

Example 215

[Image Omitted]

The compound 214 (20 mg) was treated with HCl (4 N in dioxane, 4 mL) and stirred at room temperature for 10 min. After concentrating, the residue was dried by lyophilization to give compound 215. HPLC-MS t_R=0.98 min (UV_{254 nm}); mass calculated for formula C₁₉H₂₂N₈S, 394.2; observed MH⁺ (LCMS) 395.2 (m/z).

Example 216

[Image Omitted]

To a 25 mL round bottom flask charged with product from example 177 (486 mg, 2.0 mmol), Pd₂(dba)₃(180 mg, 0.2 mmol), dppf (235 mg, 0.4 mmol), and Zn(CN)₂(500 mg, 4.2 mmol) was added DME (10 ml) as solvent. The mixture was degassed thoroughly and placed under argon. This resulting mixture was then heated at 80Â° C. overnight. The reaction was diluted with EtOAc (100 mL) and filtered through Celite. After concentrating, the residue was purified with column chromatography (silica gel, Hexane/EtOAc=60/40) to give the product 216 (399 mg) as yellowish solid. ¹H NMR (400 MHz, CDCl₃) Î´ 8.31 (s, 1H), 7.80 (d, 1H), 7.69 (d, 1H), 2.66 (s, 3H). HPLC-MS t_R=1.15 min (UV_{254 nm}); mass calculated for formula C₈H₆N₄S; 190.0, observed MH⁺ (LCMS) 191.1 (m/z).

Example 217

[Image Omitted]

Product of the example 217 was prepared using brominating conditions described in example 179. HPLC-MS t_R=1.53 min (UV_{254 nm}); mass calculated for formula C₈H₅BrN₄S, 267.9; observed MH⁺ (LCMS) 269.0 (m/z).

Example 218

[Image Omitted]

Compound 218 was synthesized using the coupling condition described in example 180. HPLC-MS t_R=1.36 min (UV_{254 nm}); mass calculated for formula C₁₂H₁₀N₆S, 270.1; observed MH⁺ (LCMS) 271.0 (m/z).

Example 219, 220

[Image Omitted]

The aniline (32 mg, 0.42 mmol) was dissolved in dry DMSO (2 mL) and NaH (60% in oil, 8 mg, 0.2 mmol) was added under argon. The mixture was stirred for 10 min at room temperature then, sulfide 219 (27 mg, 0.1 mmol) in dry DMSO (0.5 mL) was added. The resulting mixture was heated up to 80Â° C. and stirred for 10 min. After cooling and LCMS analysis shown the formation of two products. The mixture was purified with Prep-LC to give the product 219 and 220 as TFA salt.

219: HPLC-MS t_R=0.77 min (UV_{254 nm}); mass calculated for formula C₂₀H₁₅N₉, 381.1; observed MH⁺ (LCMS) 382.1 (m/z).

220: HPLC-MS t_R=0.63 min (UV_{254 nm}); mass calculated for formula C₂₀H₁₇N₉O 399.2; observed MH⁺ (LCMS) 400.1 (m/z).

Example 221

[Image Omitted]

Compound 105 was synthesized via the synthetic method described in Preparative Example 105 described above. Also disclosed on page 71 in US20060 0106023 (A1).

3-(5-aminoisothiazol-3-yl) pyrrolidine-1-carboxylic-tert-butyl ester was prepared similar to the procedures described above for the synthesis in Examples 128-130.

A solution of the 3-(5-aminoisothiazol-3-yl)pyrrolidine-1-carboxylic-tert-butyl ester, (2 equivalents) in DMSO (10 mL) was treated with NaH (60% dispersion in oil, 2 equivalents) for 15 min at room temperature. Compound 105 (1 equivalent, 300 mg, 1.08 mmol) was then added to this solution at rt and the resultant solution was stirred at room temperature for 1 hr at which time LC-MS analysis indicated the reaction was complete. The reaction mixture was diluted with sat. ammonium chloride (10 mL) and extracted with 10% i-propylalcohol/dichloromethane (Ã—3). The combined organic layers were washed with water, brine, dried over anhydrous sodium sulfate and concentrated. Purification by column chromatography ((SiO₂10% methanol/ethyl acetate) afforded compound 221 as a red solid 0.46 g (91%).

Example 222

[Image Omitted]

To compound 221 in THF (8 mL) was added 4N HCl in dioxane (2 mL). The resulting solution was stirred at room temperature for 16 hr at which time LC-MS analysis indicated that the reaction was complete. The solvent was evaporated. Purification by Prep-LC and conversion to a hydrochloric salt afforded compound 222. HPLC-MS t_R=2.55 Min (UV_254nm). Mass calculated for formula C₁₇H₁₈N₈S 366.1, observed LC/MS m/z 367.1 (M+H).

Example 223

[Image Omitted]

To compound 222 (50 mg, 0.14 mmol) in DCM (2 mL) was added DIEA (2.5 equivalents) at room temperature and the resulting heterogeneous solution was stirred at room temperature, then added methanesulfonyl chloride (1.5 equivalents). The resulting solution was stirred at room temperature for 15 min at which time LC-MS analysis indicated that the reaction was complete. After concentration the residue was purified by Prep-LC and conversion to a hydrochloric salt afforded compound 223. HPLC-MS t_R=3.34 Min (UV_254nm). Mass calculated for formula C₁₈H₂₀N₈O₂S₂444.12, observed LC/MS m/z 445.1 (M+H).

Example 224

[Image Omitted]

To compound 222 (50 mg, 0.14 mmol) in DCM (2 mL) was added trimethylsilyl isocyanate (2.1 equivalents) at room temperature. The resulting solution was stirred at room temperature for 15 min at which time LC-MS analysis indicated that the reaction was completed. After concentration the residue was purified by Prep-LC and conversion to a hydrochloric salt afforded compound 223. HPLC-MS t_R2.72=Min (UV_254nm). Mass calculated for formula C₁₈H₁₉N₉OS 409.1, observed LC/MS m/z 410.1 (M+H).

Example 225

[Image Omitted]

To compound 222 (50 mg, 0.14 mmol) in DCM (2 mL) was added DIEA (2.5 equivalents) at room temperature and the resulting heterogeneous solution was stirred at room temperature for 10 min. Then added ethyl chloroformate (1.5 equivalents) at room temperature. The resulting solution was stirred at room temperature for 15 min at which time LC-MS analysis indicated that the reaction was complete. After concentration the residue was purified by Prep-LC and conversion to a hydrochloric salt afforded compound 225. HPLC-MS t_R=3.88 Min (UV_254nm). Mass calculated for formula C₂₀H₂₂N₈O₂S 438.16, observed LC/MS m/z 439.1 (M+H).

The compounds 226-1 through 226-8 in Table 20 were prepared from the free amine and the appropriate reagents.

TABLE 20MSExam-Exactm/zHPLCpleColumn 2mass(MH)⁺MS t_R226-1 [Image Omitted]

4584593.49 226-2 [Image Omitted]

4724733.75 226-3 [Image Omitted]

5245254.25 226-4 [Image Omitted]

4584593.44 226-5 [Image Omitted]

4524534.10 226-6 [Image Omitted]

4584593.59 226-7 [Image Omitted]

4524534.22 226-8 [Image Omitted]

4234242.97

Example 227

[Image Omitted]

Compound 227 was synthesized from compound 1 via the synthetic method described by Hackler et al., Journal of Heterocyclic Chemistry (1989), 26 (6), 1575-8.

Example 228

[Image Omitted]

A 2.5M n-BuLi solution (20.4 mL, 50.9 mmol) was slowly added to a solution of diisopropylamine (7.2 mL, 50.9 mmol) in anhydrous THF (75 mL) under argon at âˆ’78Â° C. After stirring at âˆ’78Â° C., the solution was treated with acetonitrile (2.5 mL, 48.5 mmol) dissolved in anhydrous THF (10 mL). After 10 minutes, Benzonitrile was added dropwise to the above solution at âˆ’78Â° C. The resulting suspension was allowed to warm to room temperature. The reaction mixture was stirred at room temperature overnight at which time thin layer chromatography (40% ethyl acetate/hexanes) indicated that the reaction was complete. The reaction mixture was poured into ice water (200 mL), and then concentrated to remove the organic solvent. The resulting emulsion was extracted twice with diethyl ether. The combined organic layers were dried over anhydrous sodium sulfate and concentration afforded the title compound 228 that was used directly in the next step.

Example 229

[Image Omitted]

A solution of compound 228 (1 g, 6.9 mmol) in THF/ethanol (1:1, 10 mL) in a high pressure vessel was cooled to 0Â° C. (ice-bath) and treated with hydrogen sulfide gas for 5 minutes. The tube was sealed and heated to 90Â° C. for 2 hr. LC-MS analysis indicated the reaction was complete; concentration afforded the title compound 229 that was used directly in the next step.

Example 230

[Image Omitted]

To compound 229 (1.15 g, 3.47 mmol) and potassium carbonate (2 equivalents) in diethyl ether (20 mL) was added an ethereal solution of iodine (1 equivalent) dropwise at reflux. The resulting solution was heated at reflux for 2 hr at which time LC-MS analysis indicated that the reaction was complete. The mixture was cooled to 25Â° C. and concentrated. Purification by column chromatography (SiO₂, 40% ethyl acetate/hexanes) afforded compound 230 as a red/orange solid 0.29 g (48%). HPLC-MS t_R=1.38 Min (UV_254nm). Mass calculated for formula C₉H₈N₂S 176.0, observed LC/MS m/z 177.1 (M+H).

Example 231 & 232

[Image Omitted]

A 2.5M n-BuLi solution (20.4 mL, 50.9 mmol) was slowly added to a solution of diisopropylamine (7.2 mL, 50.9 mmol) in anhydrous THF (75 mL) under argon at âˆ’78Â° C. After stirring at âˆ’78Â° C., the solution was treated with acetonitrile (2.5 mL, 48.5 mmol) dissolved in anhydrous THF (10 mL). After 10 minutes, a solution of 3-methyl butyronitrile (5.1 mL, 40 mmol) in anhydrous THF (75 mL), under argon at âˆ’78 C, was added drop wise to the above solution. The resulting suspension was allowed to warm to room temperature. The reaction mixture was stirred at room temperature overnight at which time thin layer chromatography (40% ethyl acetate/hexanes) indicated that the reaction was complete. The reaction mixture was poured into ice water (200 mL), and then concentrated to remove the organic solvent. The resulting emulsion was extracted twice with diethyl ether. The combined organic layers were dried over anhydrous sodium sulfate and concentration afforded the a mixture of two compounds 231 and 232 in 1:3 ratio. These two compounds separated by column chromatography and the compound 231, HPLC-MS t_R=Min (UV_254nm). Mass calculated for formula C₇H₁₂N₂, M+124.18, observed LC/MS m/z 125.20.10 (M+H), is used in the next step. Undesired compound, 232 HPLC-MS t_R=Min (UV_254nm). Mass calculated for formula C₁₀H₁₈N₂, M+ 166.26, observed LC/MS m/z 167.40 (M+H). was discarded.

Example 233

[Image Omitted]

A solution of compound 231 (1 g, mmol) in THF/ethanol (1:1, 10 mL) in a high pressure vessel was cooled to 0Â° C. (ice-bath) and treated with hydrogen sulfide gas for 5 minutes. The tube was sealed and heated to 90Â° C. for 2 h. LC-MS analysis indicated the reaction was complete, concentration afforded the title compound 233 that was used directly in the next step. HPLC-MS t_R=Min (UV_254nm). Mass calculated for formula C₇H₁₄N₂S, M+ 158.26, observed LC/MS m/z 159.30 (M+H).

Example 234

[Image Omitted]

To compound 233 (1.15 g, mmol) and potassium carbonate (2 equivalents) in diethyl ether (20 mL) was added an ethereal solution of iodine (1 equivalent) dropwise at reflux. The resulting solution was heated at reflux for 2 hr. at which time LC-MS analysis indicated that the reaction was complete. The mixture was cooled to 25Â° C. and concentrated. Purification by column chromatography (SiO₂, 40% ethyl acetate/hexanes) afforded compound 234 as a viscous liquid 0.29 g (48%). HPLC-MS t_R=Min (UV_254nm). Mass calculated for formula C₇H₁₂N₂S, M+ 156.25, observed LC/MS m/z 157.40 (M+H).

Example 235

[Image Omitted]

A solution of benzo[b]thiophene-2 carboxylic acid (1.25 g, 7.03 mmol), diphenylphosphoryl azide (1.94 g, 7.03 mmol) and triethylamine (0.98 mL, 7.03 mmol) in tert-butanol (20 mL) was heated at reflux for 5 hours, at which time thin layer chromatography (DCM/Hexanes) indicates the reaction is complete. The reaction mixture was cooled to room temperature, poured into water and extracted with diethyl ether (3Ã—). The combined ether extracts were washed with brine, dried over anhydrous sodium sulfate and then concentrated to afford a beige solid. Purification by column chromatography (SiO₂DCM/Hexanes) afforded compound 235 as a white solid 0.96 g (64%). HPLC-MS t_R=2.7 Min (UV_254nm). Mass calculated for formula C₁₃H₁₅NO₂S, M+ 249.33, observed LC/MS m/z 250.40 (M+H).

Example 236

[Image Omitted]

A solution of compound 235 (0.250 g, 1.00 mmol) was stirred in 4 M HCl solution in 1,4-dioxane (3 mL) at room temperature for 2 hrs at which time thin layer chromatography (DCM/Hexanes) indicated the reaction was complete. The reaction mixture was cooled to room temperature and concentrated under vacuum. The residue was diluted with acetonitrile, sonicated, and concentrated to afford compound 236 as a grey solid 0.24 g (91%). HPLC-MS t_R=1.5 Min (UV_254nm). Mass calculated for formula C₈H₇NS, M+ 149.21, observed LC/MS m/z 150.40 (M+H).

Example 237

[Image Omitted]

By essentially the same procedure given in Preparative Example 235, 237 can be prepared from compound, 5-pyridin-2yl-thiophene-2carboxylic acid.

Example 238

[Image Omitted]

By essentially the same procedure given in Preparative Example 236, 238 can be prepared from compound 237.

Example 239

[Image Omitted]

Compound 2-methylpyridine-3-carboxaldehyde (2.5 g, 17.7 mmol) was dissolved in DMF (25 mL) and water (2.5 mL). Potassium carbonate (1.1 equivalents) and methyl thioglycolate (1.1 equivalents) are added portion wise resulting in a bright orange solution which was heated at 40Â° C. for 16 hr. LC-MS analysis indicated that the reaction was complete. The reaction mixture was allowed to cool to room temperature and then quenched with ice-cold water (150 mL) and placed in an ice-bath to enhance precipitation. The precipitate was isolated by filtration, affording compound 242 as an off-white solid 1.87 g (55%).

Example 240

[Image Omitted]

By essentially following the same procedure given in Preparative Example 133, compound 240 can be prepared from compound 239.

Example 241

[Image Omitted]

By essentially following the same procedure given in Preparative Example 237, compound 241 can be prepared from compound 240.

Example 242

[Image Omitted]

By essentially following the same procedure given in Preparative Example 238, compound 242 can be prepared from compound 241.

Example 243

[Image Omitted]

By essentially the same procedure given in Preparative Example 106, the compounds given in Column 2 of Table 21 can be prepared from compound 105.

TABLE 21MSExam-Exactm/zHPLCpleColumn 2mass(M + H)MS t_R243-1 [Image Omitted]

353.1354.14.37 243-2 [Image Omitted]

353.14354.104.50 243-3 [Image Omitted]

373.1374.14.76 243-4 [Image Omitted]

346.1347.14.60 243-5 [Image Omitted]

373.1374.02.96 243-6 [Image Omitted]

347.1348.03.05 243-7 [Image Omitted]

353.1354.14.20 243-8 [Image Omitted]

309.1310.22.19 243-9 [Image Omitted]

3823831.97

Example 244

[Image Omitted]

5-Chlorosulfonyl-4-methyl-thiophene-2-carboxylic acid methyl ester (1.76 g, 6.92 mmol) was dissolved in 1,4-dioxane (40 mL) and cooled in an ice-bath. Ammonia gas was bubbled into the reaction mixture until thin layer chromatography indicated the reaction was complete (ca Ëœ10 minutes). The reaction mixture was filtered, the solids were rinsed with dichloromethane and the filtrate was concentrated to afford the title compound 231 as a white solid 1.53 g (94%).

Example 245

[Image Omitted]

To a solution of compound 231 (1.50 g, 6.37 mmol) in THF/water (80 mL/20 mL) was added 1N LiOH (12.8 mL, 12.8 mmol) at room temperature. The reaction mixture was stirred at room temperature for 16 hr at which time thin layer chromatography indicated the reaction was complete. The reaction mixture was concentrated, the residue acidified to pH 4 with 1N HCl and extracted with ethyl acetate (Ã—4). The combined organic layer was dried over anhydrous Na₂SO₄and concentrated to afford compound 232 as a white solid 1.29 g (92%).

Example 246

[Image Omitted]

A solution of compound 232 (0.59 g, 2.69 mmol), diphenylphosphoryl azide (0.58 mL, 2.69 mmol) and triethylamine (0.37 mL, 2.69 mmol) in t-butanol (20 mL) was heated at reflux for 5 hr, at which time thin layer chromatography (DCM/Hexanes) indicated that the reaction is complete. The reaction mixture was cooled to room temperature, poured into water and extracted with diethyl ether (Ã—3). The combined ether extracts were washed with brine, dried over anh. sodium sulfate and then concentrated to afford a beige solid. Purification by column chromatography (SiO₂40% ethyl acetate/hexanes) afforded compound 233 as a white solid 0.36 g (46%).

Example 247

[Image Omitted]

A solution of compound 233 (0.20 g, 0.68 mmol) was stirred in 4M HCl solution in 1,4-dioxane (3 mL) at room temperature for 2 h at which time thin layer chromatography (DCM/Hexanes) indicated that the reaction was complete. The reaction mixture was concentrated under vacuum. The residue was diluted with acetonitrile, sonicated and concentrated to afford compound 234 as a grey solid 0.15 g (96%).

Preparative Examples 248-1-10

By essentially using the same procedures set forth in Preparative Example 244 through 247 by using amines listed in column 1 compounds in column 2 of the table 22, are prepared.

TABLE 22LCMS MH⁺Serial No.Column 1Column 2MWm/z248-1 [Image Omitted]

262.04263.1 248-2 [Image Omitted]

246.05247.1 248-3 [Image Omitted]

246.05247.1 248-4 [Image Omitted]

232.03233.1 248-5 [Image Omitted]

246.05247.1 248-6 [Image Omitted]

236.03237.1 248-7 [Image Omitted]

232.03233.1 248-8 [Image Omitted]

220.03221.1 248-9 [Image Omitted]

220.03221.10 248-10 [Image Omitted]

248.07249.20

Example 249

[Image Omitted]

5-(cyclopropylmethyl-sulfamoyl)-4-methyl-thiophene-2-carboxylic acid methyl ester prepared as in example 244.

Example 250

[Image Omitted]

Compound of preparative 249 (0.275 g, 1.0 mmol) in THF (5 mL) was added to the suspension of NaH (60% dispersion in oil) (0.040 g, 1.5 mmol) in THF (5 mL) at 0Â° C. and stirred for 10 minutes. Then the Iodomethane 0.284 g, 2 mmol) in THF (1 mL) was added the reaction mixture. The reaction was stirred for 2 hours at room temperature. After the completion of the reaction (LCMS analysis), reaction is quenched with NH₄Cl soln. and extracted with ethyl acetate. The organic layer was washed with brine and dried over anhydrous Na₂SO₄. Filtered and concentrated to obtain crude product 250 (0.250 g. 86%). HPLC-MS t_R=1.826 min (UV_{254 nm}); mass calculated for formula C₁₁H₁₅NO₄S₂, 289.04; observed MH⁺ (LCMS) 290.0 (m/z).

Example 251

[Image Omitted]

By essentially the same procedure given in Preparative Example 245, the compound 251 can be prepared from compound 250.

Example 252

[Image Omitted]

By essentially the same procedure given in Preparative Example 246, the compound 252 can be prepared from compound 251

Example 253

[Image Omitted]

By essentially the same procedure given in Preparative Example 247, the compound 253 can be prepared from compound 252

Compounds listed in column 2 (254-1 through 254-7) of Table-23 were essentially prepared from the amines ranging from 247 and 248-1 through 10 following the procedure described in preparation of compound 106.

TABLE 23MSEx-Exactm/zHPLCampleColumn 2Mass(M + H)MS t_R254-1 [Image Omitted]

389.1390.02.87 254-2 [Image Omitted]

417.1418.13.82 254-3 [Image Omitted]

445.16446.204.10 254-4 [Image Omitted]

459.11460.234.02 254-5 [Image Omitted]

443.12444.234.38 254-6 [Image Omitted]

429.10430.203.91 254-7 [Image Omitted]

443.12444.204.19 254-8 [Image Omitted]

374.1375.13.09

Example 255

[Image Omitted]

Acetoacetate (45.4 g, 458 mmol), cyanoacetic acid (39 g, 458 mmol), NH₄OAc (7.3 g, 94.7 mmol), AcOH (13.0 mL), and benzene (130 mL) was stirred for 24 hr at reflux with a Dean-Stark trap. The mixture was cooled to room temperature, washed with sat. NaHCO₃, brine, dried with Na₂SO₄, and conc. in vacuo. The crude product was distilled at 65Â° C. at 0.5 Torr: to give compound, methyl 4-Cyano-3-methylbut-3-enoate (44.27 g, 70%) as a mixture E/Z isomers. ¹H NMR DMSO_d6: 5.69 (q, J=0.6 Hz, 1H), 5.62 (q, J=0.6 Hz, 1H), 3.61 (s, 3H), 3.60 (s, 3H), 3.42 (s, 2H), 3.35 (d, J=1.2 Hz, 2H), 2.01 (d, J=1.2 Hz, 3H), 1.93 (d, J=1.2 Hz, 3H).

Example 256

[Image Omitted]

Et₂NH (36.2 mL, 350 mmol) was added dropwise to a mixture of compound methyl 4-Cyano-3-methylbut-3-enoate (44.27 g, 318 mmol) and S-flakes (10.20 g, 318 mmol) in EtOH (250 mL). The reaction stirred at room temperature for 3 hr. The mixture was concentrated to a minimal volume and placed in an ice bath. HCl (conc.) was slowly added to the mixture to give a yellow/orange solid. The precipitate was collected by vacuum filtration and washed with Et₂O to give compound (256) Methyl 5-Amino-3-methylthiophene-2-carboxylate Hydrochloride (41.22 g, 62%). ¹H NMR DMSO_d6: 6.91 (s, 2H), 5.76(s, 1H), 3.61 (s, 3H), 2.62 (s, 3H).

Example 257

[Image Omitted]

Compound (256) Methyl 5-Amino-3-methylthiophene-2-carboxylate Hydrochloride (1.25 g, 6.75) was mixed with tert-BOC anhydride (1.62 g, 7.42 mmol), diisopropyl ethyl amine (1.29 mL, 7.42 mmol), and a catalytic amount of dimethylaminopyridine (10 mg) in DMF (50 mL). The reaction was heated at 60Â° C. for 3 hr. The reaction was concentrated and the residue dissolved in EtOAc (100 mL). This solution was washed with water followed by brine. The organic layer was then dried over Na₂SO₄and conc. in vacuo. The crude material was purified via column chromatography using a gradient of 5% EtOAc/Hexanes to 40% EtOAc/Hexanes. Compound, 5-tert-Butoxycarbonylamino-3-methyl-thiophene-2-carboxylic acid ethyl ester, was isolated in 32% yield (0.612 g). 0.304 g of the starting material was also recovered. ¹H NMR CDCl₃: 7.29, (bs, 1H), 6.30, (s, 1H), 4.26 (q, J=6.8 Hz, 2H) 2.46 (s, 3H), 1.52 (s, 9H), 1.32 (t, J=6.8 Hz, 3H).

Example 258

[Image Omitted]

5-tert-Butoxycarbonylamino-3-methyl-thiophene-2-carboxylic acid ethyl ester (0.600 g, 2.10 mmol) was mixed with 1M NaOH (2.3 mL) in MeOH (15 mL) and H₂O (5 mL). The solution was heated to reflux for 48 h. The reaction was cooled to 0Â° C. and 1M HCl was added until the solution had a pH between 4 to 5. The reaction was washed with EtOAc (3Ã—, 50 mL). The organic layer was dried with Na₂SO₄and conc. in vacuo. This material was used without further purification.

Example 259

[Image Omitted]

5-tert-Butoxycarbonylamino-3-methyl thiophene-2-carboxylic acid (258,1 mmol, 257 mg) was dissolved in dichloromethane and added with, 1.5 eq of EDCl, and 4.0 eq. of DIEA in CH₂Cl₂at room temperature. After 10 minutes, the NN-dimethylamine.HCl salt (3 eq.) was added. The reaction stirred at room temperature for 3 hrs. Then the crude reaction material was concentrated, was dissolved in EtOAc (25 mL), and washed with H₂O (2Ã—, 25 mL), followed by brine (25 mL). The organic layer was dried over Na₂SO₄, filtered, and concentrated to give the crude product which was chromatographed to give the product 259. HPLC-MS t_R=2.4 Min (UV_254nm). Mass calculated for formula C₁₃H₂₀N₂O₃S, M+ 284.37, observed LC/MS m/z 285.40 (M+H).

Example 260

[Image Omitted]

The compound 259 from the above step was dissolved in dichloromethane (2 mL) and cooled to 0Â° C. To this solution, a 50% TFA-DCM (2 mL) was added and the reaction mixture stirred for 30 minutes at room temperature. The reaction was concentrated and dried under vacuum to give the TFA salt of the 5-amino 3-methyl thiophene-2-carboxylic acid dimethyl amide, HPLC-MS t_R=0.6 Min (UV_254nm). Mass calculated for formula C₈H₁₂N₂OS, M+ 184.26, observed LC/MS m/z 185.40 (M+H).

Example 261

[Image Omitted]

By essentially the same procedure given in Preparative Example, 259 compound 261 can be prepared from compound 258.

Example 262

[Image Omitted]

By essentially the same procedure given in Preparative Example, 260 compound 262 can be prepared from compound 261.

Example 263

[Image Omitted]

By essentially the same procedure given in Preparative Example, 259 compound 261 can be prepared from compound 258.

Example 264

[Image Omitted]

By essentially the same procedure given in Preparative Example, 260 compound 264 can be prepared from compound 263.

Example 265

[Image Omitted]

By essentially following the procedure in the example 255, the compound, 265 can be prepared.

Example 266

[Image Omitted]

By essentially following the procedure in the example 256, the compound, 266 can be prepared.

Example 267

[Image Omitted]

By essentially following the procedure in the example 255, the compound, 267 can be prepared.

Example 268

[Image Omitted]

By essentially following the procedure in the example 256, the compound, 268 can be prepared.

Compounds (269-1 through 269-7) listed in column 2 of Table-24 were essentially prepared from the amines ranging fromâ€”following the procedure described in preparation of compound 106.

TABLE 24Ex-MSam-Exactm/zHPLCpleColumn 2Mass(M + H)MS t_R269-1 [Image Omitted]

382.1383.14.68 269-2 [Image Omitted]

381.14382.204.35 269-3 [Image Omitted]

381.14382.204.50 269-4 [Image Omitted]

353.11354.203.25 269-5 [Image Omitted]

410.15411.305.10 269-6 [Image Omitted]

451.18452.204.30 269-7 [Image Omitted]

529.16530.203.50

Example 270

[Image Omitted]

To a suspension of potassium carbonate (5.85 g, 1.5 equiv) and 1H-pyrazole-4-boronate (5.48 g, 1.0 equiv) in NMP (50 mL) at room temperature was added SEMCl (5.2 mL, 1.05 equiv) dropwise (mildly exothermic). The resulting mixture was allowed to stir for an additional 45 min at room temperature. The reaction was diluted with ethyl acetate, rinsed with water (Ã—2), brine and dried (sodium sulfate). Filtration and concentration afforded the title compound (270) that used directly in the next step.

Example 271

[Image Omitted]

A flask was charged with compound 103 (1.83 g, 1.00 equiv), Bpin-compound 270 (2.08 g, 1.3 equiv), PdCl2(dppf) (0.4 g, 0.1 equiv) and potassium phosphate monohydrate (3.4 g, 3.0 equiv). After purging the flask with argon, 1,4-dioxane (50 mL) and water (5 mL) were added and the resulting mixture was heated at 40Â° C. overnight (23 hr). The reaction was cooled to room temperature. EtOAc was added to the reaction mixture and filtered through Celite. After concentration the residue was purified by column chromatography (silica gel, 25% EtOAc/hexane) to give the title compound 271 (46%).

Example 272

[Image Omitted]

To a solution of compound 271 (1.02 g, 1.0 equiv) in DCM (10 mL) was added m-CPBA (1.1 g, 77%, 2.05 equiv) in one portion. The resulting mixture was stirred at room temperature for 30 min. The mixture was concentrated and then partitioned between EtOAc and water. The organic layer was washed with NaHCO₃(sat. aq., Ã—2), brine and dried (Na₂SO₄). After concentration, the crude product compound 272 was used in the next step directly without further purification.

Example 273

[Image Omitted]

To a solution of compound 177 (2.00 g, 8.19 mmol) in DMF (50 mL) was added N-iodosuccinimide (1.84 g, 8.19 mmol). The reaction mixture was stirred at 60Â° C. for 16 hr. The mixture was cooled to 25Â° C. and concentrated. Purification by column chromatography (SiO₂, 40% ethyl acetate/hexanes) afforded compound 273 as a white solid 2.30 g (76%). ¹H-NMR (400 MHz, DMSO-d₆) Î´ 8.3 (s, 1H), 7.8 (s, 1H), 2.6 (s, 3H). HPLC-MS t_R=1.87 Min (UV_{254 nm}). Mass calculated for formula C₇H₅BrIN₃S, 370.01, observed LC/MS m/z 370.9 (M+H).

Example 274

[Image Omitted]

A flask was charged with iodo-compound 273 (1.83 g, 1.00 equiv), Bpin-compound 270 (2.08 g, 1.3 equiv), PdCl2(dppf) (0.4 g, 0.1 equiv) and potassium phosphate monohydrate (3.4 g, 3.0 equiv). After purging the flask with argon, 1,4-dioxane (50 mL) and water (5 mL) were added and the resulting mixture was heated at 40Â° C. overnight (23 hr). The reaction was cooled to rt. EtOAc was added to the reaction mixture and filtered through Celite. After concentration the residue was purified by column chromatography (silica gel, 25% EtOAc/hexane) to give the title compound 274 (46%).

Example 275

[Image Omitted]

To a solution of compound 274 (1.02 g, 1.0 equiv) in DCM (10 mL) was added m-CPBA (1.1 g, 77%, 2.05 equiv) in one portion. The resulting mixture was stirred at room temperature for 30 min. The mixture was concentrated and then partitioned between EtOAc and water. The organic layer was washed with NaHCO₃(sat. aq., Ã—2), brine and dried (Na₂SO₄). After concentration, the crude product compound 275 was used in the next step directly without further purification.

Example 276

[Image Omitted]

To a solution of aminoisothiazole hydrochloride (0.135 g, 1.4 equiv.) in DMSO (9 mL) at room temperature was added NaH (0.11 g of 60% dispersion in oil, 3.0 equiv) in one portion. After ca. 10 min, compound 273 (0.30 g, 1.00 equiv) was added in one portion. After 15 min at room temperature, the reaction was quenched with sat. aq. ammonium chloride and then extracted with ethyl acetate (Ã—2). The combined organic layers were washed with water (Ã—2), brine and dried (sodium sulfate). Evaporation of solvent afforded the title compound 276 (0.18 g. 56%).

Example 277

[Image Omitted]

A solution of crude compound 276 in THF (1 mL) was treated with 4N HCl in dioxane solution (1 mL) at 60Â° C. for 10 min at which time HPLC-MS indicated that the reaction was complete. The solvent was removed and the residue was purified by Prep-LC. Conversion to a hydrochloric salt afforded compound 277. ¹H-NMR (400 MHz, DMSO-d₆) Î´ 12.35 (bs, 1H), 8.27 (bs, 2H), 8.18 (s, 1H), 7.92 (s, 1H), 7.03 (s, 1H) and 3.24 (s, 3H). HPLC-MS t_R=2.93 Min (UV_254nm). Mass calculated for formula C₁₃H₁₀BrN₇S, 374.99, observed LC/MS m/z 376.0 (M+H).

Example 278

[Image Omitted]

By essentially following the experimental procedure given in example 274 and 275, using appropriate amine (4-amino N,N-dimethyl benzenesulfonamide) compound 278 can be made. HPLC-MS t_R=4.06 Min (UV_254nm). Mass calculated for formula C₁₇H₁₆BrN₇O₂S, 461.03, observed LC/MS m/z 462.10 (M+H).

Example 279

[Image Omitted]

By essentially the same procedure given in Preparative Example 274 & 275, compounds (279, 1-7) given in Column 2 of Table 25 can be prepared.

TABLE 25MSExactm/zHPLCExampleColumn 2Mass(M + H)MS t_R279-1 [Image Omitted]

283.1284.02.33 279-2 [Image Omitted]

425.1426.13.16 279-3 [Image Omitted]

425.1426.13.06 279-4 [Image Omitted]

2972982.37 279-5 [Image Omitted]

3663670.86 279-6 [Image Omitted]

4444452.89 279-7 [Image Omitted]

2912921.33

Example 280

[Image Omitted]

A mixture of compound 276 (30 mgs, 0.059 mmol, 1 equivalent), sodium methanethiolate (1.4 equivalent), PdCl₂(dppf) (0.07 equivalents), sodium tert-butoxide (1.1 equivalents) in 1,2-dimethoxyethane (1 ml) was stirred at 85 C under Ar for 16 h. The reaction mixture was cooled to room temperature, filtered through Celite and the filtrate concentrated. The residue was taken back up in ethyl acetate and washed with water, brine, dried over anhydrous sodium sulfate and concentrated to afford crude compound 280. HPLC-MS t_R=2.26 Min (UV_254nm). Mass calculated for formula C₂₁H₂₉N₇OS₂Si 487.16, observed LC/MS m/z 488.1.

Example 281

[Image Omitted]

By essentially the same procedure used in the preparative example 275 to give the product 281. ¹H-NMR (400 MHz, DMSO-d₆) Î´ 8.27 (s, 2H), 7.96 (s, 1H), 7.84 (s, 1H), 7.07 (s, 1H), 2.66 (3.43) and 2.42 (s, 3H). HPLC-MS t_R=Min (UV_254nm). Mass calculated for formula C₁₄H₁₃N₇S 343.07, observed LC/MS m/z 344.1.

Examples 282

By essentially the same procedure given in Preparative 278 & 279 or by metal catalyzed reactions, the compounds 282 (1-11) given in Column 2 of Table 26 can be prepared from compound 274.

TABLE 26MSExactm/zHPLCExampleColumn 2Mass(M + H)MS t_R282-1â€‚ [Image Omitted]

357.08358.13.17 282-2â€‚ [Image Omitted]

371.1372.13.41 282-3â€‚ [Image Omitted]

385.1386.13.48 282-4â€‚ [Image Omitted]

3373381.10 282-5â€‚ [Image Omitted]

4624631.45 282-6â€‚ [Image Omitted]

3743750.96 282-7â€‚ [Image Omitted]

4054061.38 282-8â€‚ [Image Omitted]

3433441.12 282-9â€‚ [Image Omitted]

3223231.09 282-10 [Image Omitted]

3253261.12 282-11 [Image Omitted]

3113120.97

The compound 283 in Table 27 was prepared by essentially the same procedure as in Preparative examples starting from compound 271.

TABLE 27MSExactm/zHPLCExampleColumn 2Mass(MH)+MS t_R283 [Image Omitted]

3403410.82

Example 284

[Image Omitted]

To the mixture of compound, [3-(4-bromo-1-methyl-1H-pyrazol-3-yl)-phenyl]carbamic acid tert-butylester (1.78 g, 7.1 mmol), imidazole (1.36 g, 20 mmol), and catalytic amount DMAP in DMF (12 mL), Boc₂O (1.7 g, 7.8 mmol) was added at room temperature. The mixture was stirred overnight at room temperature and diluted with EtOAc (200 mL), the organics were washed with H₂O, brine and dried over Na₂SO₄. After concentration, the residue was purified with column (silica gel, hexane/EtOAc=70/30) gave the product 284 (2.52 g) as white solid. HPLC-MS t_R=2.00 Min (UV_254nm). Mass calculated for formula C₁₅H₁₈BrN₃O₂351.1, observed LC/MS m/z 352.1 (M+H).

Example 285

[Image Omitted]

To a 25 ml round bottom flask charged with bis(pinacolato)diboron (1.0 g, 4.0 mmol), KOAc (960 mg, 10 mmol), PdCl₂(dppf) (240 mg, 0.3 mmol) and compound 284 (1.16 g, 3.3 mmol) was added DMSO (6 ml) under Argon. The mixture was thoroughly degassed by alternately connected the flask to vacuum and Argon. This resulting mixture was then heated at 80Â° C. overnight, diluted by EtOAc (40 ml) and filtered through celite. After concentration, the residue was purified with column (silica gel, Hexane/EtOAc=80/20) to give the product 285 (997 mg) as oil. HPLC-MS t_R=2.11 min (UV_{254 nm}); mass calculated for formula C₂₁H₃₀BN₃O₄399.2, observed LCMS m/z 400.3(M+H).

Example 286

[Image Omitted]

Under Argon, the compound 285 (120 mg, 0.3 mmol) in THF (3.0 mL, 5% H₂O) was added to the flask which was charged with Pd(dppf)Cl₂(8 mg, 0.01 mmol), K₂CO₃(138 mg, 1.0 mmol), and compound 149 (51 mg, 0.15 mmol). The mixture was thoroughly degassed by alternately connected the flask to vacuum and Argon. The resulting solution was heated upto 80Â° C. and stirred overnight. After cooling to room temperature, the mixture was diluted with EtOAc (50 mL) and the solid was removed by filter through Celite and washed with some EtOAc. Concentration to remove the solvent and the resulting residue 286 was used in the next step directly without further purification. HPLC-MS t_R=2.05 min (UV_{254 nm}); mass calculated for formula C₂₉H₃₂N₈O₂524.3, observed LCMS m/z 525.2.1 (M+H).

Example 287

[Image Omitted]

To the compound 286 was added HCl (6N, 3 mL), and the mixture was stirred at room temperature for 10 min. Then, concentrated, and the residue was purified with HPLC and gave the final compound 287 (48 mg). HPLC-MS t_R=1.16 min (UV_{254 nm}); mass calculated for formula C₂₄H₂₄N₈424.2, observed LCMS m/z 425.2 (M+H).

Example 288

[Image Omitted]

The benzoic acid (6 mg, 0.05 mmol) in DMF (1 mL) was added HOBt (7 mg, 0.05 mmol), EDC (10 mg, 0.05 mmol) and the mixture was stirred at room temperature for 10 min. Then, compound 287 (21 mg, 0.05 mmol) in DMF (1 mL) was added and the resulting mixture was allowed to heated up to 50Â° C. and stirred overnight. The mixture was diluted with EtOAc (50 mL) and washed with H₂O, brine and dried over Na₂SO₄. After concentration, the residue was purified with HPLC gave the product 288. HPLC-MS t_R=1.54 min (UV_{254 nm}); mass calculated for formula C₃₁H₂₈N₈O 528.2, observed LCMS m/z 529.3 (M+H).

Example 289

[Image Omitted]

Compound 289 was prepared using the boronation conditions described in Example 285. HPLC-MS t_R=1.83 min (UV_{254 nm}); mass calculated for formula C₁₁H₁₇BN₂O₃236.1, observed LCMS m/z 237.3 (M+H).

Example 290

[Image Omitted]

Compound 290 was prepared using the coupling conditions described in Example 286. HPLC-MS t_R=1.18 min (UV_{254 nm}); mass calculated for formula C₁₉H₁₉N₇O 361.2, observed LCMS m/z 362.1 (M+H).

Example 291

[Image Omitted]

Compound 290 (50 mg, 0.14 mmol) was dissolved in MeOH (5 mL) and the mixture was cooled to 0Â° C. NaBH4 (38 mg, 1.0 mmol) was added and the resulting mixture was stirred at 0Â° C. for 30 min. After concentration, the residue was purified with HPLC gave the product 291. HPLC-MS t_R=0.92 min (UV_{254 nm}); mass calculated for formula C₁₉H₂₁N₇O 363.2, observed LCMS m/z 364.3 (M+H).

Example 292

By essentially the same procedure given in Preparative Example 290, compounds given in Column 2 of Table 28 can be prepared from compound 149 and appropriate pyrazole boronate.

TABLE 28Ex-MSam-Exactm/zHPLCpleColumn 2mass(M + H)MS t_R292-1 [Image Omitted]

375.2376.31.51 292-2 [Image Omitted]

409.2410.21.53

Example 293

[Image Omitted]

Compound 293 was prepared using the coupling condition described in example 286 starting from-3-bromo-7-amino imidazopyrazines and n-benzyl pyrazole-4-boronate. HPLC-MS t_R=0.94 min (UV_{254 nm}); mass calculated for formula C₁₆H₁₄N₆290.1, observed LCMS m/z 291.3 (M+H).

Example 294

[Image Omitted]

Compound 294 was prepared using the coupling condition described in example 198. HPLC-MS t_R=0.79 min (UV_{254 nm}); mass calculated for formula C₁₂H₁₀N₄S 242.1, observed LCMS m/z 243.1 (M+H).

Example 295

[Image Omitted]

Compound 295 was prepared using the bromination condition described in 179. HPLC-MS t_R=1.11 min (UV_{254 nm}); mass calculated for formula C₁₂H₉BrN₄S 320.0, observed LCMS m/z 321.0 (M+H).

Example 296

[Image Omitted]

Compound 296 was synthesized using the same coupling condition described in example 180. HPLC-MS t_R=1.04 min (UV_{254 nm}); mass calculated for formula C₁₆H₁₄N₆S, 322.1, observed LCMS m/z 323.2 (M+H).

Example 297

[Image Omitted]

Compound 297 was synthesized using the same oxidation condition described in example 181. HPLC-MS t_R=0.71 min (UV_{254 nm}); mass calculated for formula C₁₆H₁₄N₆O₂S 354.1, observed LCMS m/z 355.0 (M+H).

Example 298

[Image Omitted]

Compound 298 was prepared using the amination condition described in example 182. HPLC-MS t_R=0.63 min (UV_{254 nm}); mass calculated for formula C₁₉H₁₆N₈S 388.1, observed LCMS m/z 389.2 (M+H).

Example 299

[Image Omitted]

Compound 299 was synthesized with the using the procedures described in examples 177 through 183. HPLC-MS t_R=0.93 min (UV_{254 nm}); mass calculated for formula C₁₇H₂₀N₈S 368.2, observed LCMS m/z 369.1 (M+H).

Example 300

[Image Omitted]

Compound 300 was synthesized using preparative procedures described in examples 186 through 191. HPLC-MS t_R=0.99 min (UV_{254 nm}); mass calculated for formula C₁₈H₂₂N₈S 382.2, observed LCMS m/z 383.1 (M+H).

Example 301

[Image Omitted]

Compound 301 was synthesized with the same procedure using in example 178. HPLC-MS t_R=0.82 min (UV_{254 nm}); mass calculated for formula C₁₀H₁₃N₃OS 223.1, observed LCMS m/z 224.1 (M+H).

Example 302

[Image Omitted]

Compound 302 (223 mg, 1.0 mmol) was dissolved in DCM (10 mL) and DIEA (200 Î¼L) was added followed by DMAP (cat. Amount) and pivaloyl chloride (150 Î¼L). The resulting mixture was stirred at room temperature for 1 hour and diluted with EtOAc. The organics was washed with NaHCO₃(aq), water and brine, dried over Na₂SO₄. After concentration, the crude product was used in the next step directly without further purification. HPLC-MS t_R=1.82 min (UV_{254 nm}); mass calculated for formula C₁₅H₂₁N₃O₂S 307.1, observed LCMS m/z 308.2 (M+H).

Example 303

[Image Omitted]

Compound 303 was prepared using the bromination condition described in example 179. HPLC-MS t_R=2.28 min (UV_{254 nm}); mass calculated for formula C₁₅H₂₀BrN₃O₂S 385.0, observed LCMS m/z 386.0 (M+H).

Example 304

[Image Omitted]

Compound 304 was synthesized using the same coupling condition described in example 180. HPLC-MS t_R=1.89 min (UV_{254 nm}); mass calculated for formula C₁₉H₂₅N₅O₂S 387.2, observed LCMS m/z 388.2 (M+H).

Example 305

[Image Omitted]

Compound 305 was synthesized using the same oxidation condition described in example 181. HPLC-MS t_R=1.53 min (UV_{254 nm}); mass calculated for formula C₁₉H₂₅N₅O₄S 419.2, observed LCMS m/z 420.1 (M+H).

Example 306

[Image Omitted]

Compound 306 was prepared using the amination condition described in example 182 and deprotection of butyloxy carbonyl group as in example 183. HPLC-MS t_R=2.55 min (UV_{254 nm}, 10 min LC-MS); mass calculated for formula C₁₇H₁₉N₇OS 369.1, observed LCMS m/z 370.1 (M+H).

Example 307

By essentially the same procedure given in Preparative Example 306 starting from compound 305, compound given in Column 2 of Table 29 can be prepared.

TABLE 29MSExactm/zHPLCExampleColumn 2Mass(M + H)MS t_R301 [Image Omitted]

424.2425.10.85

Example 308

[Image Omitted]

Compound 308 was synthesized using the same condition as described in preparative example 186. HPLC-MS t_R=1.03 min (UV_{254 nm}); mass calculated for formula C₁₁H₁₅N₃OS 237.1, observed LCMS m/z 238.1 (M+H).

Example 309

[Image Omitted]

Compound 309 was prepared using the bromination condition described in example 187. HPLC-MS t_R=2.33 min (UV_{254 nm}); mass calculated for formula C₁₁H₁₄BrN₃OS 315.0, observed LCMS m/z 316.0 (M+H).

Example 310

[Image Omitted]

Compound 310 was synthesized using the same coupling condition described in example 188. HPLC-MS t_R=1.43 min (UV_{254 nm}); mass calculated for formula C₁₅H₁₉N₅OS 317.1, observed LCMS m/z 318.1 (M+H).

Example 311

[Image Omitted]

Compound 311 was synthesized using the same oxidation condition described in example 189. HPLC-MS t_R=1.06 min (UV_{254 nm}); mass calculated for formula C₁₅H₁₉N₅O₃S 349.1, observed LCMS m/z 350.2 (M+H).

Example 312

[Image Omitted]

Compound 312 was prepared using the amination condition described in example 190. HPLC-MS t_R=1.26 min (UV_{254 nm}); mass calculated for formula C₁₈H₂₁N₇OS 383.2, observed LCMS m/z 384.1 (M+H).

Example 313

[Image Omitted]

Compound 313 (596 mg, 2.0 mmol) was dissolved in THF (20 mL) and cooled to âˆ’78Â° C. n-BuLi (1.6 ml, 2.5 M in hexane, 4.0 mmol) was added dropwise and the resulting mixture was stirred at âˆ’78Â° C. for 30 min. Triisopropyl borate (752 mg, 4.0 mmol) was added and the mixture was stirred for 30 min at âˆ’78Â° C., then warmed to room temperature slowly. 1N HCl (10 mL) was added and the mixture was extracted with EtOAc. The organics was dried over Na₂SO₄and concentrated. The crude product 2 was used in the next step without further purification. HPLC-MS t_R=1.49 min (UV_{254 nm}); mass calculated for formula C₁₀H₁₆BNO₄S 257.1, observed LCMS m/z 202.1 (M+Hâ€”t-Bu).

Example 314

[Image Omitted]

Compound 314 was synthesized using the same coupling condition described in example 178. HPLC-MS t_R=1.89 min (UV_{254 nm}); mass calculated for formula C₁₇H₂₀N₄O₂S₂376.1, observed LCMS m/z 377.1 (M+H).

Example 315

[Image Omitted]

Compound 315 was prepared using the bromination condition described in example 179. HPLC-MS t_R=2.20 min (UV_{254 nm}); mass calculated for formula C₁₇H₁₉BrN₄O₂S₂, 454.0, observed LCMS m/z 455.0 (M+H).

Example 316

[Image Omitted]

Compound 316 was synthesized using the same coupling condition described in example 180. HPLC-MS t_R=1.96 min (UV_{254 nm}); mass calculated for formula C₂, H₂₄N₆O₂S₂456.1, observed LCMS m/z 427.1 (M+H).

Example 317

[Image Omitted]

Compound 317 was synthesized using the same oxidation condition described in example 201. HPLC-MS t_R=1.54 min (UV_{254 nm}); mass calculated for formula C₂, H₂₄N₆O₃S₂472.1, observed LCMS m/z 473.1 (M+H).

Example 318

[Image Omitted]

Compound 318 was prepared using the amination condition described in example 202. HPLC-MS t_R=1.44 min (UV_{254 nm}); mass calculated for formula C₂₉H₂₉N₉O₂S 567.2, observed LCMS m/z 568.3 (M+H).

Example 319

[Image Omitted]

Compound 319 was synthesized using the deprotecting condition described in example 203. HPLC-MS t_R=0.87 min (UV_{254 nm}); mass calculated for formula C₂₄H₂₁N₉S 467.2, observed LCMS m/z 468.1 (M+H).

Example 320

By essentially the same procedure given in Preparative Example 318 and 319 starting from compound 317, compound given in Column 2 of Table 30 can be prepared.

TABLE 30MSExactm/zHPLCExampleColumn 2Mass(M + H)MS t_R320 [Image Omitted]

422.1423.10.98 337-1 [Image Omitted]

394.2395.10.91 337-2 [Image Omitted]

500.2501.11.25 337-3 [Image Omitted]

514.2515.21.29

Example 321

[Image Omitted]

Compound 321 was synthesized using the same condition described in example 302. NMR (CDCl₃, ppm): 5.69(m, 1H), 5.25(m, 2H), 4.73(m, 1H), 4.45(m, 1H), 4.13(m, 2H), 3.68(m, 1H), 2.07(s, 3H), 1.46(s, 9H).

Example 322

[Image Omitted]

Compound 322 was synthesized with the same procedure using in example 178. HPLC-MS t_R=1.62 min (UV_{254 nm}); mass calculated for formula C₁₈H₂₆N₄O₄S 394.2, observed LCMS m/z 395.1 (M+H).

Example 323

[Image Omitted]

Compound 323 was prepared using the bromination condition described in example 179. HPLC-MS t_R=1.97 min (UV_{254 nm}); mass calculated for formula C₁₈H₂₅BrN₄O₄S 472.1, observed LCMS m/z 473.0 (M+H).

Example 324

[Image Omitted]

Compound 324 was synthesized using the same coupling condition described in example 180. HPLC-MS t_R=1.70 min (UV_{254 nm}); mass calculated for formula C₂₂H₃₀N₆O₄S 474.2, observed LCMS m/z 475.1 (M+H).

Example 325

[Image Omitted]

Compound 325 was synthesized using the same oxidation condition described in example 181. HPLC-MS t_R=1.41 min (UV_{254 nm}); mass calculated for formula C₂₂H₃₀N₆O₆S 506.2, observed LCMS m/z 507.1 (M+H).

Example 326

[Image Omitted]

Compound 326 was prepared using the amination condition described in example 182. HPLC-MS t_R=1.52 min (UV_{254 nm}); mass calculated for formula C₂₅H₃₂N₈O₄S 540.2, observed LCMS m/z 541.2 (M+H).

Example 327

[Image Omitted]

Compound 326 (150 mg) was dissolved in the mixture of THF (10 mL) and methanol (5 mL). LiOH (1N, 4 mL) was added and the resulting mixture was stirred at 50Â° C. for 2 hours. After cooling to room temperature, the mixture was concentrated followed by taking up with EtOAc. The organics was washed with water, brine and dried over Na₂SO₄. After concentration, the crude product 327 (122 mg) was used in the next step without further purification. HPLC-MS t_R=1.29 min (UV_{254 nm}); mass calculated for formula C₂₃H₃₀N₈O₃S 498.2, observed LCMS m/z 499.1 (M+H).

Example 328

[Image Omitted]

Compound 328 was synthesized using the deprotecting condition described in example 183. HPLC-MS t_R=0.80 min (UV_{254 nm}); mass calculated for formula C₁₈H₂₂N₈OS 398.2, observed LCMS m/z 399.0 (M+H).

Example 329

[Image Omitted]

Compound 328 (25 mg) was dissolved in DMF (5 mL) and NaH (8 mg, 0.2 mmol) was added. The resulting mixture was stirred at room temperature overnight and quenched with NH₄Cl (sat. aq.) extracted with EtOAc. After concentration, the crud product was purified by HPLC gave the compound 329. HPLC-MS t_R=1.05 min (UV_{254 nm}); mass calculated for formula C₁₉H₂₀N₈O₂S 424.1, observed LCMS m/z 425.1 (M+H).

Example 330

[Image Omitted]

A suspension of methyltriphenylphosphonium bromide (8.93 g, 25 mmol) in THF (50 mL) was placed under argon and treated with t-BuOK (25 mL, 1M in THF). The mixture quickly became bright yellow and was stirred at room temperature for 1 hour. A solution of 1-Boc-3-piperidone (1.97 g, 10 mmol) in THF (10 mL) was then added to the mixture and stirred for 3 hours. The mixture was poured into water, extracted with ether and dried over Na₂SO₄and concentrated. The crude material was purified by column (silica gel, 5% EtOAc in hexane) to afford product 330 as an oil (1.51 g).

Example 331

[Image Omitted]

Compound 331 was synthesized with the same procedure using in example 178. HPLC-MS t_R=1.90 min (UV_{254 nm}); mass calculated for formula C₁₈H₂₆N₄O₂S 362.2, observed LCMS m/z 363.3 (M+H).

Example 332

[Image Omitted]

Compound 332 was prepared using the bromination condition described in example 179. HPLC-MS t_R=2.31 min (UV_{254 nm}); mass calculated for formula C18H₂₅BrN₄O₂S 440.1, observed LCMS m/z 441.1 (M+H).

Example 333

[Image Omitted]

Compound 333 was synthesized using the same coupling condition described in example 180. HPLC-MS t_R=1.99 min (UV_{254 nm}); mass calculated for formula C₂₂H₃₀N₆O₂S 442.2, observed LCMS m/z 443.2 (M+H).

Example 334

[Image Omitted]

Compound 334 was synthesized using the same oxidation condition described in example 181. HPLC-MS t_R=1.66 min (UV_{254 nm}); mass calculated for formula C₂₂H₃₀N₆O₄S 474.2, observed LCMS m/z 475.1 (M+H).

Example 335

[Image Omitted]

Compound 335 was prepared using the amination condition described in example 182. HPLC-MS t_R=1.58 min (UV_{254 nm}); mass calculated for formula C₂₅H₃₂N₈O₂S 508.2, observed LCMS m/z 509.2 (M+H).

Example 336

[Image Omitted]

Compound 336 was synthesized using the deprotecting condition described in example 183. HPLC-MS t_R=0.95 min (UV_{254 nm}); mass calculated for formula C₂₀H₂₄N₈S 408.2, observed LCMS m/z 409.1 (M+H).

Example 337

By essentially the same procedure given in Preparative Example 335 & 336 starting from 334 and appropriate amines, compounds given in Column 2 of Table 31 can be prepared.

TABLE 31ExactMS m/zHPLCExampleColumn 2mass(M + H)MS t_R337-1 [Image Omitted]

394.2395.10.91 337-2 [Image Omitted]

500.2501.11.25 337-3 [Image Omitted]

514.2515.21.29

Example 338

[Image Omitted]

Under Argon, to the flask which charged with the boronate compound (81 mg, 0.39 mmol), Pd(dppf)Cl₂(32 mg, 0.039 mmol), and K₃PO₄(212 mg, 1.0 mmol), compound 273 (145 mg, 0.0.39 mmol) in dioxane (5 mL) was added. The mixture was thoroughly degassed by alternately connecting the flask to vacuum and Argon. The resulting solution was heated upto 40Â° C. and stirred overnight. After cooling to room temperature, the mixture was diluted with EtOAc (50 mL) and the solid was removed by filter through Celite and washed with some EtOAc. Concentration to remove the solvent and the resulting residue was purified with column (silica gel, EtOAc) gave the product 338 (98 mg) as solid. HPLC-MS t_R=1.50 min (UV_{254 nm}); mass calculated for formula C₁₁H₁₀BrN₅S 323.0, observed LCMS m/z 324.0 (M+H).

Example 339

[Image Omitted]

Compound 339 was synthesized using the same oxidation conditions described in example 181. HPLC-MS t_R=1.23 min (UV_{254 nm}); mass calculated for formula C₁₁H₁₀BrN₅O₂S 355.0, observed LCMS m/z 356 (M+H).

Example 340

[Image Omitted]

Compound 340 was prepared using the amination condition described in example 182. HPLC-MS t_R=1.44 min (UV_{254 nm}); mass calculated for formula C₁₄H₁₂BrN₇S 389.0, observed LCMS m/z 390.0 (M+H).

Example 341

[Image Omitted]

Under Argon, to the vial which charged with the compound 340 (Ëœ20 mg, 0.05 mmol), Pd(dppf)Cl₂(8 mg, 0.01 mmol), and sodium t-butoxide (15 mg, 0.15 mmol), thiol (15 mg, 0.06 mmol) in DME (2 mL) was added. The mixture was thoroughly degassed by alternately connected the flask to vacuum and Argon. The resulting solution was heated upto 80Â° C. and stirred overnight. After cooling to room temperature, the mixture was diluted with EtOAc (50 mL) and washed with NH₄Cl (sat. aq.), water, brine, and dried over Na₂SO₄. After concentration to remove the solvent and the resulting residue was purified with HPLC gave the product 341 (98 mg) as solid. HPLC-MS t_R=1.63 min (UV_{254 nm}); mass calculated for formula C₂₆H₂₆N₈O₂S₂546.2, observed LCMS m/z 547.2 (M+H).

Example 342

[Image Omitted]

Compound 342 was synthesized using the deprotecting condition described in example 183. HPLC-MS t_R=0.95 min (UV_{254 nm}); mass calculated for formula C₁₈H₂₀N₈S₂412.1, observed LCMS m/z 413.0 (M+H).

Example 343

[Image Omitted]

Compound 180 (100 mg) was dissolved in DMF (5 ml) and NaH (24 mg, 0.6 mmol) was added. After stirring 10 min at room temperature, cyclopropylmethylbromide (100 mg) was added and the resulting mixture was stirred at room temperature overnight. EtOAc (100 mL) was added and the organics was washed with water, brine and dried over Na₂SO₄. After concentration, the crud product was purified with column (silica gel, EtOAc/hexane=50:50-100:0) gave the product 343 (88 mg). HPLC-MS t_R=1.98 min (UV_{254 nm}); mass calculated for formula C₂₅H₂₈N₆O₂S 476.2, observed LCMS m/z 477.1 (M+H).

Example 344

[Image Omitted]

Compound 344 was synthesized using the same oxidation condition described in example 181. HPLC-MS t_R=1.69 min (UV_{254 nm}); mass calculated for formula C₂₅H₂₈N₆O₄S 508.2, observed LCMS m/z 509.2 (M+H).

Example 345

[Image Omitted]

Compound 345 was prepared using the amination condition described in example 182. HPLC-MS t_R=2.05 min (UV_{254 nm}); mass calculated for formula C₃₁H₃₆N₈O₄S₂648.2, observed LCMS m/z 649.1 (M+H).

Example 346

[Image Omitted]

Compound 346 was synthesized using the deprotecting condition described in example 183. HPLC-MS t_R=1.31 min (UV_{254 nm}); mass calculated for formula C₂₃H₃₀N₈O₂S₂514.2, observed LCMS m/z 515.2 (M+H).

Example 347

[Image Omitted]

Compound 347 was prepared from compound 213 using the amination condition described in example 4 part G. HPLC-MS t_R=2.00 min (UV_{254 nm}); mass calculated for formula C₂₇H₃₆N₈O₄S₂600.2, observed LCMS m/z 601.2 (M+H).

Example 348

[Image Omitted]

Compound 348 was synthesized using the deprotecting condition described in example 215. HPLC-MS t_R=1.26 min (UV_{254 nm}); mass calculated for formula C₂₂H₂₈N₈O₂S₂500.2, observed LCMS m/z 501.1 (M+H).

Example 349

[Image Omitted]

Compound 216 (342 mg, 1.8 mmol) and TMSCl (2.0 g) was dissolved in ethanol (20 mL). The mixture was heated to 70Â° C. and stirred 2 days. After concentration, the residue was purified with column (silica gel, EtOAC/hexane 10=30:70) gave the product 349 (280 mg). HPLC-MS t_R=1.27 min (UV_{254 nm}); mass calculated for formula C₁₀H₁₁N₃O₂S 237.1, observed LCMS m/z 238.1 (M+H).

Example 350

[Image Omitted]

Compound 349 (280 mg, 1.18 mmol) was dissolved in the mixture of THF/MeOH (10 mL/10 mL) and LiOH (1N, 5.0 mL) was added. The resulting mixture was stirred at room temperature overnight and the solvent was removed under vacuum. The residue was taken up with water (5 mL), and adjusted to pH 5 with 1N HCl. The solid was collected with filtration and washed with water and dried with air gave the product 350 (235 mg). HPLC-MS t_R=0.76 min (UV_{254 nm}); mass calculated for formula C₈H₇N₃O₂S 209.0, observed LCMS m/z 210.1 (M+H).

Example 351

[Image Omitted]

The acid 350 (42 mg, 0.2 mmol) was dissolved in DMF (5 mL) and HATU (76 mg, 0.2 mmol) was added followed by DIEA (300 Î¼L) and amine (40 mg, 0.2 mmol). The resulting mixture was stirred at room temperature overnight and diluted with EtOAc. The organics was washed with water, brine and dried over Na₂SO₄. After concentration, the crude was purified with column (silica gel, EtOAc/hexane=30/70) to afford the product 351 (62 mg). HPLC-MS t_R=1.68 min (UV_{254 nm}); mass calculated for formula C₁₈H₂₅N₅O₃S 391.2, observed LCMS m/z 392.2 (M+H).

Example 352

[Image Omitted]

Compound 352 was prepared using the bromination condition described in example 179. HPLC-MS t_R=1.96 min (UV_{254 nm}); mass calculated for formula C₁₈H₂₄BrN₅O₃S 469.1, observed LCMS m/z 470.0 (M+H).

Example 353

[Image Omitted]

Compound 353 was synthesized using the same coupling condition described in example 180. HPLC-MS t_R=1.75 min (UV_{254 nm}); mass calculated for formula C₂₂H₂₉N₇O₃S 471.2, observed LCMS m/z 472.2 (M+H).

Example 354

[Image Omitted]

Compound 354 was synthesized using the same oxidation condition described in example 181. HPLC-MS t_R=1.52 min (UV_{254 nm}); mass calculated for formula C₂₂H₂₉N₇O₅S, 503.2, observed LCMS m/z 504.2 (M+H).

Example 355

[Image Omitted]

Compound 355 was prepared using the amination condition described in example 182. HPLC-MS t_R=1.58 min (UV_{254 nm}); mass calculated for formula C₂₅H₃₁N₉O₃S 537.2, observed LCMS m/z 538.3 (M+H).

Example 356

[Image Omitted]

Compound 356 was synthesized using the deprotecting condition described in example 183. HPLC-MS t_R=0.84 min (UV_{254 nm}); mass calculated for formula C₂₀H₂₃N₉OS 437.2, observed LCMS m/z 438.3 (M+H).

Example 357 & 358

[Image Omitted]

The compound 214 was dissolved in CHCl₃(5 mL) and NCS (10 mg) was added, the mixture was heated to 50Â° C. and stirred for 2 hours. After concentration, the residue was purified with HPLC gave the product 357 and 358. Compound 357: HPLC-MS t_R=2.22 min (UV_{254 nm}); mass calculated for formula C₂₄H₂₉ClN₈O₂S 528.2, observed LCMS m/z 529.2 (M+H). Compound 358: HPLC-MS t_R=2.38 min (UV_{254 nm}); mass calculated for formula C₂₄H₂₈Cl₂N₈O₂S 562.1, observed LCMS m/z 563.0 (M+H).

Example 359

[Image Omitted]

Compound 359 was synthesized using the deprotecting condition described in 215. and purified by preparative HPLC. HPLC-MS t_R=1.17 min (UV_{254 nm}); mass calculated for formula C₁₉H₂₁ClN₈S 428.1, observed LCMS m/z 429.1 (M+H).

Example 360

[Image Omitted]

Compound 360 was synthesized using the deprotecting condition described in 215. and purified by preparative HPLC. Compound 360: HPLC-MS t_R=1.16 min (UV_{254 nm}); mass calculated for formula C₁₉H₂₁Cl₂N₈S 462.1, observed LCMS m/z 463.0 (M+H).

Example 361

[Image Omitted]

To a stirred solution of 5-chlorosulfonyl-3-methyl-thiophene-2-carboxylic acid methyl ester (0.254 g, 1 mmol) in dioxane (4 mL.) at room temperature is treated with a solution of sodium sulphite (0.252 g, 2 mmol) and sodium bicarbonate (0.168 g, 2 mmol) in water (4 mL). The reaction mixture is heated to 90Â° C. for 30 minutes and then allowed to cool to room temperature. The solvent is removed in vacuo. The residue is dissolved in DMF (4 mL), Iodomethane (0.248 g, 2 mmol) is added and stirred for 1 hr. The reaction mixture is diluted with water and extracted with ethyl acetate. The combined organic layers were washed with water, brine, dried over anhydrous sodium sulfate and concentrated. Crude product was purified on silica column using Hexane/Ethylacetate solvents to yield compound 361 (50%).

Example 362

[Image Omitted]

By essentially the same procedure given in Preparative Example 117, compound 362 can be prepared.

Example 363

[Image Omitted]

By essentially the same procedure given in Preparative Example 118, compound 363 can be made.

Example 364

[Image Omitted]

By essentially the same procedure given in Preparative Example 119, compound 364 can be prepared.

Example 365

By essentially using the same procedures set forth in Preparative Example 361 through 364 by using isopropyl bromide, compound given in column 2 is prepared.

TABLE 32LCMSExam-MH⁺pleColumn 1Column 2MWm/z365 [Image Omitted]

262.04263.1

Example 366

[Image Omitted]

By essentially the same procedure given in Preparative Example 118, compound 366 can be prepared from 2-methyl thiazole-5-carboxylic acid HPLC-MS t_R=2.5 Min (UV_254nm). Mass calculated for formula C₉H₁₄N₂O₂S, M+214.20, observed LC/MS m/z 215.30(M+H)

Example 367

[Image Omitted]

By essentially the same procedure given in Preparative Example 119, compound 367 can be prepared from 366. HPLC-MS t_R=1.25 Min (UV_254nm). Mass calculated for formula C₄H₆N₂S, M+114.20, observed LC/MS m/z 115.30 (M+H).

Example 368

[Image Omitted]

By essentially the same procedure given in Preparative Example 182, compounds given in Column 2 of Table 33 are prepared from compound 201 and amines listed in column 1, Table 33.

TABLE 33LCMSExam-MH⁺HPLCpleColumn 1Column 2MWm/zMS t_R368-1 [Image Omitted]

626.25627.355.95 368-2 [Image Omitted]

640.23641.345.43 368-3 [Image Omitted]

624.23625.375.71 368-4 [Image Omitted]

624.23625.375.59 368-5 [Image Omitted]

610.21611.325.37 368-6 [Image Omitted]

624.23625.405.56 368-7 [Image Omitted]

670.27671.425.76 368-8 [Image Omitted]

610.21611.325.20 368-9 [Image Omitted]

598.21599.345.27 368-10 [Image Omitted]

598.21599.275.48 368-11 [Image Omitted]

520.24521.335.27 368-12 [Image Omitted]

534.25535.25.28 368-13 [Image Omitted]

527.21528.266.21 368-14 [Image Omitted]

528.21529.225.10 368-15 [Image Omitted]

522.25523.394.30 368-16 [Image Omitted]

578.24579.315.16 368-17 [Image Omitted]

624.23625.25.6 368-18 [Image Omitted]

492.21493.404.50 368-19 [Image Omitted]

569.19570.345.07 368-20 [Image Omitted]

597.22598.415.49

Example 369

[Image Omitted]

By essentially the same procedure given in Preparative Example 203, compounds given in Column 2 of Table 34 are prepared from compounds in column 1, Table 4.

TABLE 34LCMSExam-MH⁺HPLCpleColumn 1Column 2MWm/zMS t_R369-1 [Image Omitted]

526.19527.23.494 369-2 [Image Omitted]

540.17541.21.099 369-3 [Image Omitted]

524.18525.11.18 369-4 [Image Omitted]

524.18525.11.147 369-5 [Image Omitted]

510.16511.11.094 369-6 [Image Omitted]

524.18525.33.46 369-7 [Image Omitted]

514.16515.22.81 369-8 [Image Omitted]

510.165111.190 369-9 [Image Omitted]

498.16499.33.16 369-10 [Image Omitted]

498.16499.11.14 369-11 [Image Omitted]

420.18421.252.99 369-12 [Image Omitted]

434.20435.11.12 369-13 [Image Omitted]

427.16428.11.232 369-14 [Image Omitted]

428.16428.11.1 369-15 [Image Omitted]

422.20423.10.83 369-16 [Image Omitted]

478.19479.22.99 369-17 [Image Omitted]

524.18525.13.62 369-18 [Image Omitted]

392.15393.21.30 369-19 [Image Omitted]

469.19470.22.99 369-20 [Image Omitted]

497.17498.11.12

Example 370

[Image Omitted]

By essentially the same procedure given in Preparative Example 182, compounds given in Column 2 of Table 35 are prepared from compound 201 and amines listed in column 1, Table-35.

TABLE 35LCMSExam-MH⁺HPLCpleColumn 1Column 2MWm/zMS t_R370-1 [Image Omitted]

463.18464.303.50 370-2 [Image Omitted]

491.2492.31.37 370-3 [Image Omitted]

434.16435.252.50 370-4 [Image Omitted]

462.20463.302.80 370-5 [Image Omitted]

462.2463.33.03 370-6 [Image Omitted]

595.2596.33.09

Example 371

[Image Omitted]

By essentially the same procedure given in Preparative Example 203, compounds given in Column 2 of Table 36 are prepared from compounds in column 1,

TABLE 36LCMSExam-MH⁺HPLCpleColumn 1Column 2MWm/zMS t_R371-1 [Image Omitted]

363.18364.302.50 371-2 [Image Omitted]

391.2392.31.37 371-3 [Image Omitted]

334.16335.251.50 371-4 [Image Omitted]

362.20363.301.80 371-5 [Image Omitted]

362.2363.32.03 371-6 [Image Omitted]

495.2496.32.09

Example 372

[Image Omitted]

By essentially the same procedure given in Preparative 118, compound 372 can be prepared from thieno{2,3-b]pyrazine-6-carboxylic acid Compound 372: HPLC-MS t_R=2.5 Min (UV_254nm). Mass calculated for formula C₁₁H₁₃N₃O₂S, M+251.2018, observed LC/MS m/z 252.30(M+H).

Example 373

[Image Omitted]

By essentially the same procedure given in Preparative 118, compound 372 can be prepared from 371:HPLC-MS t_R=1.5 Min (UV_254nm). Mass calculated for formula C₆H₅N₃S, M+151.2018 observed LC/MS m/z 152.30(M+H)

Example 374

[Image Omitted]

By essentially the same procedure given in Preparative Example 182, compounds given in Column 2 of Table 37 are prepared from compound 181 and amines listed in column 1, Table 37.

TABLE 37LCMSExam-MH⁺HPLCpleColumn 1Column 2MWm/zMS t_R374-1 [Image Omitted]

516.21517.23.87 374-2 [Image Omitted]

530.22531.11.836 374-3 [Image Omitted]

468.20469.11.149 374-4 [Image Omitted]

474.16475.34.20 374-5 [Image Omitted]

525.17528.305.60 374-6 [Image Omitted]

621.19622.305.50 374-7 [Image Omitted]

580.17581.254.30

Example 375

[Image Omitted]

By essentially the same procedure given in Preparative Example 183, compounds given in Column 2 of Table 38 are prepared from compounds column 1, Table 38.

TABLE 38Ex-LCMSam-MH⁺HPLCpleColumn 1Column 2MWm/zMS t_R375-1 [Image Omitted]

382.17383.262.66 375-2 [Image Omitted]

396.18397.242.93 375-3 [Image Omitted]

334.17335.281.76 375-4 [Image Omitted]

340.12341.202.01 375-5 [Image Omitted]

391.13392.202.20 375-6 [Image Omitted]

4874882.59 375-7 [Image Omitted]

4464471.14

Example 376

[Image Omitted]

A solution of the isoxazole (2 equivalents) in DMSO (1 mL) was treated with NaH (60% dispersion in oil, 2 equivalents) for 15 min at room temperature. Compound 181 (1 equivalent) was then added to this solution at room temperature and the resultant solution was stirred at room temperature for 1 h at which time LC-MS analysis indicated the reaction was complete. The reaction mixture was diluted with sat. ammonium chloride (0.5 mL) and acetonitrile (0.5 mL). Purification by Prep-LC and conversion to a hydrochloric salt afforded compound 376. HPLC-MS t_R=3.33 Min (UV_254nm). Mass calculated for formula C₂₁H₂₂N₁₀O₃462.187, observed LC/MS m/z 463.24 (M+H).

Example 377

[Image Omitted]

A solution of the isothiazole (2 equivalents) in DMSO (1 mL) was treated with NaH (60% dispersion in oil, 2 equivalents) for 15 min at room temperature. Compound 181 (1 equivalent) was then added to this solution at rt and the resultant solution was stirred at room temperature for 1 hr at which time LC-MS analysis indicated the reaction was complete. The reaction mixture was diluted with sat. ammonium chloride (0.5 mL) and acetonitrile (0.5 mL). Purification by Prep-LC and conversion to a hydrochloric salt afforded compound 377. ¹H-NMR (400 MHz, DMSO-d₆) Î´ 10.45 (bs, 1H), 8.42 (s, 1H), 7.96 (d, 2H), 7.91 (s, 1H), 7.15 (s, 1H), 6.95 (bs, 1H), 6.57 (s, 1H), 3.94 (s, 3H), 3.6 (q, 3H), 3.95 (t, 2H), 1.31 (s, 9H) and 1.22 (s, 9H). HPLC-MS t_R=3.76 Min (UV_254nm). Mass calculated for formula C27H34N10OS2 578.2, observed LC/MS m/z 579.2 (M+H).

Example 378

[Image Omitted]

By essentially following the experimental procedures followed in the examples 376 & 377, the compound 378 can be prepared HPLC-MS t_R=2.15 Min (UV_{254 nm}). Mass calculated for formula C17H19N9OS 397.14, observed LC/MS m/z 398.20 (M+H).

Example 379

[Image Omitted]

By essentially the same procedure given in Preparative Example 182, compounds given in Column 2 of Table-39 are prepared from compound 181 and amines listed in column 1, Table-39.

TABLE 39LCMSExam-MH⁺HPLCpleColumn 1Column 2MWm/zMS t_R379-1 [Image Omitted]

559.20560.304.55 379-2 [Image Omitted]

530.18531.203.80 379-3 [Image Omitted]

558.22559.353.95 379-4 [Image Omitted]

558.22559.353.95

Example 380

[Image Omitted]

By essentially the same procedure given in Preparative Example 183, compounds given in Column 2 of Table 40 are prepared from compounds column 1, Table 40.

TABLE 40Ex-LCMSam-MH⁺HPLCpleColumn 1Column 2MWm/zMS t_R380-1 [Image Omitted]

425.16426.253.55 380-2 [Image Omitted]

396.15397.252.95 380-3 [Image Omitted]

424.18425.303.10 380-4 [Image Omitted]

424.18425.303.20 380-5 [Image Omitted]

391.13392.202.20

Example 381

[Image Omitted]

NBS (0.176 g, 1.0 mmol) was added to a solution of compound 176 (0.278 g, 1.0 mmol) in DCM (10 mL), at room temperature. The mixture was stirred for one hour and concentrated. The residue was diluted with EtOAc and washed with saturated aq.NaHCO₃(30 mL, 2Ã—), brine and dried over Na₂SO₄. After concentrating, the crude product 381 was used in the next step directly without further purification. HPLC-MS t_R=1.54 min (UV_{254 nm}); mass calculated for formula C₆H₂Br₃N₃, 352.78; observed MH⁺ (LCMS) 353.8 (m/z).

Example 382

[Image Omitted]

By essentially the same procedure given in Preparative Example 182, compound 382 is prepared from compound 381. HPLC-MS t_R=1.73 min (UV_{254 nm}); mass calculated for formula C₆H₂Br₃N₃, 386.88; observed MH⁺ (LCMS) 388.0 (m/z).

Example 383

[Image Omitted]

1-Methyl-4-(4,4,5,5-tetramethyl-[1,3,2]dioxoborolan-2-yl)-1H-Pyrazole (0.208 g, 1.0 mmol), was mixed with Pd(dppf)Cl₂(50 mg, 0.06 mmol), K₃PO₄(0.848 g., 4 mmol), and the product from example 382 (0.195 g, 0.50 mmol) in dioxane (5 mL) was added. The mixture was degassed thoroughly and kept under argon blanket. The resulting solution was heated at 80Â° C. and stirred overnight. After cooling to room temperature the mixture was diluted with EtOAc (50 mL). The solid was removed by filter through Celite and washed with EtOAc. The solvent was removed under reduced pressure. Purification by Prep-LC and conversion to a hydrochloric salt afforded compound 383. HPLC-MS t_R=3.08 min (UV_{254 nm}); mass calculated for formula C₁₈H₁₇N₉S, 391.13; observed MH⁺ (LCMS) 392.22 (m/z).

Example 384

[Image Omitted]

Compound 199 (0.433 g, 1.021 mmol), the 4-(4,4,5,5-tetramethyl-{1,3-2}dioxaboralan-2yl)furan-2carboxaldehyde (0.339 g, 1.52 mmol), PdCl₂dppf.CH₂Cl₂(0.081 g, 0.12 mmol), and K₃PO₄(0.865 g, 4.0 mmol) in 1,2-dimethoxyethane (10 mL) and H₂O (2 mL) was flushed with Ar and refluxed for 2 hr. The solvents were evaporated and the residue was purified by column chromatography on silica gel with 2:1 hexane/EtOAc as eluent to obtain product 384 (0.181 g,). HPLC-MS t_R=2.04 min (UV_{254 nm}); mass calculated for formula C₂₂H₂₄N₄O₄S, 440.12; observed MH⁺ (LCMS) 441.1 (m/z).

Example 385

[Image Omitted]

The product from Preparative Example 384 (0.181 g, 0.41 mmol) in CH₂Cl₂(5 mL) and MeOH (1 mL) was added NH₂OH.HCl (0.043 g, 0.616 mmol), and triethylamine (1.2 mL) and stirred in a closed flask at 25Â° C. for 4 hr. The solvent was evaporated and the residue was chromatographed on silica gel with 2:1 hexane/EtOAc as eluent to obtain pure product 385 (0.120 g.). HPLC-MS t_R=1.968 min (UV_{254 nm}); mass calculated for formula C₂₂H₂₅N₅O₄S, 455.16; observed MH⁺ (LCMS) 456.1 (m/z).

Example 386

[Image Omitted]

To the compound 385 (0.120 G., 0.263 mmol) and triethylamine (1.1 mL) in dichloromethane (5 mL) was added trifluoroacetic anhydride (0.036 mL, 0.258 mmol) was ad de d at 0Â° C. under Argon. The mixture was stirred for 2 hr, then it was poured into saturated aqueous NaHCO₃solution (50 mL), extracted with CH₂Cl₂(3Ã—40 mL), dried over Na₂SO₄, and filtered. The solvents were evaporated and the residue was purified by column chromatography on silica gel with 50:1 CH₂Cl₂/MeOH as eluent to obtain pure product 386 (0.083 g). HPLC-MS t_R=2.181 min (UV_{254 nm}); mass calculated for formula C₂₂H₂₃N₅O₃S, 437.15; observed MH⁺ (LCMS) 438.1 (m/z).

Example 387

[Image Omitted]

The mixture of compound from preparative example 386 (0.083 g, 0.183 mmol) and m-CPBA (31 mg, 77%) in DCM (5 mL) was stirred at 0Â° C. for min and then diluted with EtOAc (100 mL). The organics were washed with saturated aqueous NaHCO₃(10 mL, 2Ã—), brine, and dried over Na₂SO₄. After concentration the crude product which was used in the next step directly without further purification. HPLC-MS t_R=1.72 min (UV_{254 nm}); mass calculated for formula C₂₂H₂₃N₅O₄S, 453.15; observed MH⁺ (LCMS) 454.1 (m/z).

Example 388

[Image Omitted]

By essentially the same procedure given in Preparative Example 182, compounds 388 given in Column 2, Table 42 are prepared from compound from preparative example 387 and amines listed in column 1, Table 42

TABLE 41LCMSExam-MH⁺HPLCpleColumn 1Column 2MWm/zMS t_R388-1 [Image Omitted]

503.17504.22.07 388-2 [Image Omitted]

637.12638.22.349

Example 389

[Image Omitted]

By essentially the same procedure given in Preparative Example 183, compounds 389 series given in Column 2 of Table 43 are prepared from compounds column 1, Table 43.

TABLE 42LCMSMH³⁰HPLCExampleColumn 1Column 2MWm/zMS t_R389-1 [Image Omitted]

403.17404.22.04 389-2 [Image Omitted]

537.12538.23.81

Assays:
Aurora Enzyme Assay

An in vitro assay was developed that utilizes recombinant Aurora A or Aurora B as an enzyme source and a peptide based on PKA as the substrate.

Aurora A Assay:

Aurora A kinase assays were performed in low protein binding 384-well plates (Corning Inc). All reagents were thawed on ice. Compounds were diluted in 100% DMSO to desirable concentrations. Each reaction consisted of 8 nM enzyme (Aurora A, Upstate cat#14-511), 100 nM Tamra-PKAtide (Molecular Devices, 5TAMRA-GRTGRRNSICOOH), 25 Î¼M ATP (Roche), 1 mM DTT (Pierce), and kinase buffer (10 mM Tris, 10 mM MgCl2, 0.01% Tween 20). For each reaction, 14 Î¼l containing TAMRA-PKAtide, ATP, DTT and kianse buffer were combined with 1 Î¼l diluted compound. The kinase reaction was started by the addition of 5 Î¼l diluted enzyme. The reaction was allowed to run for 2 hours at room temperature. The reaction was stopped by adding 60 Î¼l IMAP beads (1:400 beads in progressive (94.7% buffer A: 5.3% buffer B) 1Ã— buffer, 24 mM NaCl). After an additional 2 hours, fluorescent polarization was measured using an Analyst AD (Molecular devices).

Aurora B Assay:

Aurora A kinase assays were performed in low protein binding 384-well plates (Corning Inc). All reagents were thawed on ice. Compounds were diluted in 100% DMSO to desirable concentrations. Each reaction consisted of 26 nM enzyme (Aurora B, Invitrogen cat#pv3970), 100 nM Tamra-PKAtide (Molecular Devices, 5TAMRA-GRTGRRNSICOOH), 50 Î¼M ATP (Roche), 1 mM DTT (Pierce), and kinase buffer (10 mM Tris, 10 mM MgCl2, 0.01% Tween 20). For each reaction, 14 Î¼l containing TAMRA-PKAtide, ATP, DTT and kianse buffer were combined with 1 Î¼l diluted compound. The kinase reaction was started by the addition of 5 Î¼l diluted enzyme. The reaction was allowed to run for 2 hours at room temperature. The reaction was stopped by adding 60 Î¼l IMAP beads (1:400 beads in progressive (94.7% buffer A: 5.3% buffer B) 1Ã— buffer, 24 mM NaCl). After an additional 2 hours, fluorescent polarization was measured using an Analyst AD (Molecular devices).

IC₅₀Determinations:

Dose-response curves were plotted from inhibition data generated each in duplicate, from 8 point serial dilutions of inhibitory compounds. Concentration of compound was plotted against kinase activity, calculated by degree of fluorescent polarization. To generate IC₅₀values, the dose-response curves were then fitted to a standard sigmoidal curve and IC₅₀values were derived by nonlinear regression analysis.

CHK1 SPA Assay

An in vitro assay was developed that utilizes recombinant His-CHK1 expressed in the baculovirus expression system as an enzyme source and a biotinylated peptide based on CDC25C as substrate (biotin-RSGLYRSPSMPENLNRPR).

Materials and Reagents:

1) CDC25C Ser 216 C-term Biotinylated peptide substrate (25 mg), stored at âˆ’20Â° C., Custom Synthesis by Research Genetics: biotin-RSGLYRSPSMPENLNRPR 2595.4 MW

2) His-CHK1 In House lot P976, 235 ug/mL, stored at âˆ’80Â° C.

3) D-PBS (without CaCl and MgCl): GIBCO, Cat.#14190-144

4) SPA beads: Amersham, Cat.#SPQ0032: 500 mg/vial

Add 10 mls of D-PBS to 500 mg of SPA beads to make a working concentration of 50 mg/ml. Store at 4Â° C. Use within 2 week after hydration.

5) 96-Well White Microplate with Bonded GF/B filter: Packard, Cat.#6005177

6) Top seal-A 96 well Adhesive Film: Perkin Elmer, Cat.#6005185

7) 96-well Non-Binding White Polystyrene Plate: Corning, Cat. #6005177

8) MgCl₂: Sigma, Cat.#M-8266

9) DTT: Promega, Cat.#V3155

10) ATP, stored at 4Â° C.: Sigma, Cat.#A-5394

11) Î³³³P-ATP, 1000-3000 Ci/mMol: Amersham, Cat.#AH9968

12) NaCl: Fisher Scientific, Cat.#BP358-212

13) H₃PO₄85% Fisher, Cat.#A242-500

14) Tris-HCL pH 8.0: Bio-Whittaker, Cat. #16-015V

15) Staurosporine, 100 ug: CALBIOCHEM, Cat. #569397

16) Hypure Cell Culture Grade Water, 500 mL: HyClone, Cat.#SH30529.02

Reaction Mixtures:

1) Kinase Buffer: 50 mM Tris pH 8.0; 10 mM MgCl₂; 1 mM DTT

2) His-CHK1, In House Lot P976, MW Ëœ30 KDa, stored at âˆ’80Â° C. 6 nM is required to yield positive controls of Ëœ5,000 CPM. For 1 plate (100 rxn): dilute 8 Î¼L of 235 Î¼g/mL (7.83 uM) stock in 2 mL Kinase Buffer. This makes a 31 nM mixture. Add 20 Î¼L/well. This makes a final reaction concentration of 6 nM.

3) CDC25C Biotinylated Peptide.

Dilute CDC25C to 1 mg/mL (385 uM) stock and store at âˆ’20Â° C. For 1 plate (100 rxn): dilute 10 Î¼L of 1 mg/mL peptide stock in 2 ml Kinase Buffer. This gives a 1.925 Î¼M mix. Add 20 Î¼L/rxn. This makes a final reaction concentration of 385 nM.

4) ATP Mix.

For 1 plate (100 rxn): dilute 10 Î¼L of 1 mM ATP (cold) stock and 2 uL fresh P33-ATP (20 Î¼Ci) in 5 ml Kinase Buffer. This gives a 2 Î¼M ATP (cold) solution; add 50 Î¼l/well to start the reaction. Final volume is 100 Î¼l/rxn so the final reaction concentrations will be 1 Î¼M ATP (cold) and 0.2 uCi/rxn.

5) Stop Solution:

For 1 plate add: To 10 mL Wash Buffer 2 (2M NaCl 1% H₃PO₄): 1 mL SPA bead slurry (50 mg); Add 100 Î¼L/well

6) Wash buffer 1: 2 M NaCl

7) Wash buffer 2: 2 M NaCl, 1% H₃PO₄

Assay Procedure:

AssayFinalComponentConcentrationVolumeCHK16nM20Î¼l/rxnCompoundâ€”10Î¼l/rxn(10% DMSO)CDC25C0.385Î¼M20Î¼l/rxnÎ³³³P-ATP0.2Î¼Ci/rxn50Î¼l/rxnCold ATP1Î¼MStop solution0.5mg/rxn100Î¼l/rxn*SPA beads200Î¼l/rxn**
*Total reaction volume for assay.

**Final reaction volume at termination of reaction (after addition of stop solution).

1) Dilute compounds to desired concentrations in water/10% DMSOâ€”this will give a final DMSO concentration of 1% in the rxn. Dispense 10 Î¼l/rxn to appropriate wells. Add 10 Î¼L 10% DMSO to positive (CHK1+CDC25C+ATP) and negative (CHK1+ATP only) control wells.
2) Thaw enzyme on iceâ€”dilute enzyme to proper concentration in kinase buffer (see Reaction Mixtures) and dispense 20 Î¼l to each well.
3) Thaw the Biotinylated substrate on ice and dilute in kinase buffer (see Reaction Mixtures). Add 20 Î¼L/well except to negative control wells. Instead, add 20 uL Kinase Buffer to these wells.
4) Dilute ATP (cold) and P33-ATP in kinase buffer (see Reaction Mixtures). Add 50 Î¼L/well to start the reaction.
5) Allow the reaction to run for 2 hours at room temperature.
6) Stop reaction by adding 100 uL of the SPA beads/stop solution (see Reaction Mixtures) and leave to incubate for 15 minutes before harvest
7) Place a blank Packard GF/B filter plate into the vacuum filter device (Packard plate harvester) and aspirate 200 mL water through to wet the system.
8) Take out the blank and put in the Packard GF/B filter plate.
9) Aspirate the reaction through the filter plate.
10) Wash: 200 ml each wash; 1Ã— with 2M NaCl; 1Ã— with 2M NaCl/1% H₃PO₄
11) Allow filter plate to dry 15 min.
12) Put TopSeal-A adhesive on top of filter plate.
13) Run filter plate in Top Count

Settings:

- Data mode: CPM
- Radio nuclide: Manual SPA:P33
- Scintillator: Liq/plast
- Energy Range: Low
  IC₅₀DETERMINATIONS: Dose-response curves were plotted from inhibition data generated, each in duplicate, from 8 point serial dilutions of inhibitory compounds. Concentration of compound was plotted against % kinase activity, calculated by CPM of treated samples divided by CPM of untreated samples. To generate IC₅₀values, the dose-response curves were then fitted to a standard sigmoidal curve and IC₅₀values were derived by nonlinear regression analysis. IC₅₀values for the compounds of the present invention determined according to the above method are set forth in Table 43 below. As demonstrated above by the assay values, compounds of Table A of the present invention exhibit good Chk1 inhibitory properties.
  CDK2 Assay:
  BACULOVIRUS CONSTRUCTIONS: Cyclin E was cloned into pVL1393 (Pharmingen, La Jolla, Calif.) by PCR, with the addition of 5 histidine residues at the amino-terminal end to allow purification on nickel resin. The expressed protein was approximately 45 kDa. CDK2 was cloned into pVL1393 by PCR, with the addition of a haemaglutinin epitope tag at the carboxy-terminal end (YDVPDYAS). The expressed protein was approximately 34 kDa in size.
  ENZYME PRODUCTION: Recombinant baculoviruses expressing cyclin E and CDK2 were co-infected into SF9 cells at an equal multiplicity of infection (MOI=5), for 48 hrs. Cells were harvested by centrifugation at 1000 RPM for 10 minutes, then pellets lysed on ice for 30 minutes in five times the pellet volume of lysis buffer containing 50 mM Tris pH 8.0, 150 mM NaCl, 1% NP40, 1 mM DTT and protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). Lysates were spun down at 15000 RPM for 10 minutes and the supernatant retained. 5 ml of nickel beads (for one liter of SF9 cells) were washed three times in lysis buffer (Qiagen GmbH, Germany). Imidazole was added to the baculovirus supernatant to a final concentration of 20 mM, then incubated with the nickel beads for 45 minutes at 4Â° C. Proteins were eluted with lysis buffer containing 250 mM imidazole. Eluate was dialyzed overnight in 2 liters of kinase buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 10 mM MgCl2, 100 uM sodium orthovanadate and 20% glycerol. Enzyme was stored in aliquots at âˆ’70Â° C.
  IN VITRO KINASE ASSAY: Cyclin E/CDK2 kinase assays were performed in low protein binding 96-well plates (Corning Inc, Corning, N.Y.). Enzyme was diluted to a final concentration of 50 Î¼g/ml in kinase buffer containing 50 mM Tris pH 8.0, 10 mM MgCl₂, mM DTT, and 0.1 mM sodium orthovanadate. The substrate used in these reactions was a biotinylated peptide derived from Histone H1 (from Amersham, UK). The substrate was thawed on ice and diluted to 2 Î¼M in kinase buffer. Compounds were diluted in 10% DMSO to desirable concentrations. For each kinase reaction, 20 Î¼l of the 50 Î¼g/ml enzyme solution (1 Î¼g of enzyme) and 20 Î¼l of the 2 Î¼M substrate solution were mixed, then combined with 10 Î¼l of diluted compound in each well for testing. The kinase reaction was started by addition of 50 Î¼l of 2 Î¼M ATP and 0.1 Î¼Ci of 33P-ATP (from Amersham, UK). The reaction was allowed to run for 1 hour at room temperature. The reaction was stopped by adding 200 Î¼l of stop buffer containing 0.1% Triton X-100, 1 mM ATP, 5 mM EDTA, and 5 mg/ml streptavidine coated SPA beads (from Amersham, UK) for 15 minutes. The SPA beads were then captured onto a 96-well GF/B filter plate (Packard/Perkin Elmer Life Sciences) using a Filtermate universal harvester (Packard/Perkin Elmer Life Sciences.). Non-specific signals were eliminated by washing the beads twice with 2M NaCl then twice with 2 M NaCl with 1% phosphoric acid. The radioactive signal was then measured using a TopCount 96 well liquid scintillation counter (from Packard/Perkin Elmer Life Sciences).

IC₅₀DETERMINATIONS: Dose-response curves were plotted from inhibition data generated, each in duplicate, from 8 point serial dilutions of inhibitory compounds. Concentration of compound was plotted against % kinase activity, calculated by CPM of treated samples divided by CPM of untreated samples. To generate IC₅₀values, the dose-response curves were then fitted to a standard sigmoidal curve and IC₅₀values were derived by nonlinear regression analysis. Table 43 shows the activity data for an illustrative list of compounds of the invention.

TABLE 43CDK2CHK-IC 50 =1IC50 =StructurenMnM [Image Omitted]

50000492

5000054

1251697

1137413

59427

1810031

19382181

1251697

1096614

110013

2181818

5000023

19105

57738

â€”8

51987

136380

1373116

42096

2408616

1623023

1405311

1794519

4129715

4099524

5000015

55017

828319

69497

51736

21448

15773

47925

116188

â€”10

32147

46816

458619

â€”14

â€”10

â€”12

6077

9838

â€”19

46268

â€”12

1308818

While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications and other variations thereof will be apparent to those of ordinary skill in the art. All such alternatives, modifications and variations are intended to fall within the spirit and scope of the present invention.