Pyrazole compositions useful as inhibitors of erk

Field of the Invention

The present invention relates to the field of drug chemistry and relates to pyrazole compounds which are protein kinase inhibitors, in particular ERK inhibitors, to compositions containing such compounds and their uses. The compounds are useful in the treatment of cancer and other conditions facilitated by protein kinase inhibitors.

State of the art

Mammalian mitogen-activated protein (MAP) 1 kinases are serine / threonine kinases that mediate intracellular signal transduction (Cobb and Goldsmith, 1995, J. Biol. Chem. 270, 14843; Davis, 1995, Mot. Reprod. Dev. 42, 459). The MAP kinase family shares sequence similarity and conserved structural domains and includes ERK (extracellular signal regulatory kinase), JNK (Jun N-terminal kinase) and p38 kinase. JNK and p38 kinases are activated in response to pro-inflammatory cytokines TNF-alpha and interleukin-1, and cells are shocked, such as heat shock, increased osmotic pressure, UV light, aids, liposaccharides, and protein synthesis inhibitors (Deijard et al., 1994, chte / / 76, 1025; Han et al., 1994, Science 265, 808; Raingeaud et al., 1995, J. Biol. Chem. 270, 7420; Shapiro and Dinarello, 1995, Proc. Natl Acad. Sci. USA 92, 12230). . In contrast, ERKs are activated by mitogens and growth factors (Bokemeyer et al., 1996, Kidneyint. 49,1187).

ERK2 is a widespread protein kinase that reaches maximal activity when both Thr183 and Thr185 are phosphorylated by the upstream kinase MEK1 (Anderson et al., 1990, Nature 343, 651; Crevvs et al., 1992, Science 258, 478). During activation, ERK2 phosphorylates many regulatory proteins, including the protein kinase Rsk90 (Bjorbaek et al., 1995, J. Biol. Chem. 270, 18848) and MAPKAP2 (Rouse et al., 1994, Cell 78, 1027), and transcription factors. , such as ATF2 (Raingeaud et al., 1996, Mol. Cell Biol. 16, 1247), Elk-1 (Raingeaud et al. 1996), c-Fos (Chen et al., 1993 Proc Natl Acad Sci. USA 90, 10952) and cMyc (Oliver et al., 1995, Proc. Soc. Exp. Biol. Med. . 210, 162). ERK2 is also a downstream target of Ras / Raf-dependent processes (Moodie et al., 1993, Science 260, 1658) and may assist in the signaling of these potentially oncogenic proteins. ERK2 has been shown to play a role in the negative control of breast cancer cell growth (Frey and Mulder, 1997, Cancer Pes. 57, 628), reported over-expression of ERK2 in human breast cancer (Sivaraman et al., 1997, J. CUn. Invest. 99, 1478). Activated ERK2 is also involved in the proliferation of endothelin-stimulated airway smooth muscle cells, suggesting a role for this kinase in asthma (Welchel et al., 1997, Am. J. Pespir. Cell Moli. Biol. 16, 589).

(MAP) The JNK family of 1 kinases is involved in mediating cellular responses to a variety of disorders including cancer (Oncogene 1996, 13, 135-42), liver disorders (Hepatology 1998, 28, 1022-30), and cardiovascular disease (Circ. Pes. 1998, 83, 167-78; Circulation 1998, 97: 1731-7; J. Biol. Chem. 1997, 272, 28050-6; Circ. Res. 1996, 79, 162-73; Circ. , 947-53; J.Din Invest 1996, 97, 508-14) and immunological disorders (J. Immunol. 1999, 162, 3176-87; Eur. J. Immunol. 1998, 28, 3867-77; J. Exp. med. 1997, 186, 941-53; Eur. J. Immunol. 1996, 26, 989-94, among others).

Aurora2 is a serine / threonine protein kinase that is involved in human cancers such as colon, breast and other solid tumors. This kinase is thought to be involved in protein phosphorylation reactions that regulate the cell cycle. Specifically, aurora2 may play a role in controlling the precise division of chromosomes during mitosis. Incorrect cell cycle regulation can lead to cell proliferation and other abnormalities. Aurora2 protein was found to be overexpressed in human colon tumor tissues; see Bischoff et al., EMBO J., 1998, 17, 30523065; Schumacher et al., J. Cell Biol., 143, 1635-1646, 1998; Kimura et al., J. Bioi Chem. 272: 13766-13771 (1997).

Glycogen synthase kinase-3 (GSK-3) is a serine / threonine protein kinase composed of a and β isoforms, each encoded by a separate gene [Coghlan et al., Chemistry & Biology, 7, 793-803 (2000); Kim and Kimmel, Curr. Opinion Genetics Dev. 10: 508-514 (2000). GSK-3 is involved in mechanisms of various diseases including diabetes, Alzheimer's disease, CNS disorders such as manic depressive disorder and neurodegenerative disorders, and cardiac myocyte hypertrophy [WO 99/65897; WO 00/38675; and Haq et al., J. Cell Biol. (2000) 151, 117]. Abnormal cellular signaling pathways where GSK-3 plays a role may be the cause of these diseases.

KDR is a tyrosine kinase receptor that binds VEGF (Vascular Endothelial Growth Factor) (Neufeld et al., 1999, FASEB J., 13, 9). Binding of VEGF to the DRC receptor causes angiogenesis, which means the separation of capillaries from pre-existing blood vessels. High levels of VEGF are found in a variety of cancers, leading to tumor angiogenesis and allowing rapid growth of cancer cells. Therefore, inhibition of VEGF activity is a way to inhibit tumor growth; it is shown that this can be achieved by inhibiting the KDR receptor tyrosine kinase.

AKT, also known as protein kinase B, is a serine / threonine kinase that plays a key role in the survival of a variety of cell types [Khvvaja, A., Nature, pp. 33-34 (1990). Zang et al. Human ovarian cancer cells have been shown to have elevated levels of AKT-1 and AKT-2. Inhibition of AKT induces the loss of these human ovarian cancer cells, suggesting that AKT may be an important factor in the treatment of ovarian cancer [Zang, QY, et al., Oncogene, 19 (2000)] and other proliferative disorders. AKT involvement is also accounted for in motor neuron survival and nerve regeneration [Kazuhiko, N., et al., The Journal of Neuroscience, 20 (2000)].

There is a great unmet medical need for the development of protein kinase inhibitors, particularly ERKs, suitable for the treatment of various conditions associated with ERK activation, particularly in view of the current relatively inadequate choice of treatments for these conditions.

Accordingly, there is still a great need for the development of potent inhibitors of protein kinases, including ERKs, for the treatment of various conditions associated with protein kinase activation.

Description of the Invention

The compounds of the present invention have been found to be potent inhibitors of protein kinase, particularly ERK. The following compounds, or pharmaceutically acceptable derivatives thereof, have the general formula I:

<img file="LT4981B_D0001.tif" />

I, in which:

R¹ is selected from R, halogen, N (R⁸) 2, OR, NRCOR, NRCON (R⁸) 2, CON (R⁸) 2, SO2R, NRSO2R or SO2N (R⁸)2;

T is selected from valence bond or bridge group;

each R is independently selected from hydrogen or an optionally substituted aliphatic group having up to six carbon atoms;

R² is selected from hydrogen, CN, halogen, aryl, arylalkyl, heteroaryl, heterocyclyl, an optionally substituted acyclic aliphatic chain group containing up to six carbon atoms, or an optionally substituted cyclic aliphatic group containing from four to ten carbon atoms;

R³ is selected from R, OH, OR, N (R⁸)₂, halogen or CN;

Q is a valence bond, J or an optionally substituted C_b6 an alkylidene chain in which up to two adjacent carbon atoms of the alkylidene chain are each optionally and independently substituted with J;

J is selected from -C (= 0) -, -C0₂-, -C (O) C (O) -, NRCONR⁸-, -N (R) N (R⁸) -, -C (= O) NR⁸-, -NRC (= O) -, -O-, -S-, -SO-, -SO2-, -N (R) O-, -ON (R⁸) -, -OC (= O) N (R⁸) -, -N (R) COO-, -SO2N (R⁸) -, -N (R) SO₂-, or -N {R⁸)-;

R⁴ is selected from -R⁸, -R⁵, -NH 2, -NHR⁵, -N (R⁵) 2 or -NR⁵(CH 2) y N (R⁵)2;

each R⁵is independently selected from R⁶, R⁷, - (CH 2) y CH (R⁶) (R⁷), - (CH2)_yR⁶, - (CH 2) y CH (R⁶) 2, - (CH₂)_yCH (R⁷)₂or- (CH₂)_yR⁷;

y is 0-6;

each R⁶ is an optionally substituted group selected from an aliphatic, aryl, arylalkyl, arylalkoxy, heteroaryl, heteroarylalkyl, heteroarylalkoxy, heterocyclyl, heterocyclylalkyl or heterocyclylalkoxy group;

each R⁷ is independently selected from optionally substituted hydroxyalkyl, alkoxyalkyl, aryloxyalkyl or alkoxycarbonyl;

each R⁸ is independently selected from R, or two R⁸ optionally, on the same nitrogen atom, taken together with that nitrogen, forms a 4- to 8-membered, saturated or unsaturated heterocyclic ring containing from one to three heteroatoms;

and each ring nitrogen which may be substituted is optionally substituted with R, NR², COR, CO2 (C 1 -C 6 optionally substituted alkyl), SO 2 (CrC 6 optionally substituted alkyl), CONR² and SO2NR².

Unless otherwise stated, the following descriptions apply. In addition, combinations of substituents or variables are permissible only if such combination results in a stable compound.

As used herein, the term aliphatic refers to straight, branched, or cyclic C 1 -C 8₂ a hydrocarbon which is either saturated or containing one or more unsaturated units. For example, suitable aliphatic groups include substituted or unsubstituted linear, branched or cyclic alkyl, alkenyl, alkynyl groups and their hybrids such as (cycloalkyl) alkyl, (cycloalkenyl) alkyl or (cycloalkyl) alkenyl. The term alkyl and alkoxy, used alone or as part of a larger moiety, includes straight and branched chains containing from 2 to 12 carbon atoms. The terms haloalkyl, haloalkenyl, and haloalkoxyl means alkyl, alkenyl, or alkoxy, as the case may be, substituted with one or more halogen atoms. The term halogen means F, Cl,

Br or I. The term heteroatom denotes N, O or S and includes any oxidized form of nitrogen and sulfur and quaternized form of any basic nitrogen.

The term aryl, used alone or as part of a larger moiety, refers to aromatic ring groups having five to fourteen members such as phenyl, benzyl, 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl, and heterocyclic aromatic groups, or heteroaryl groups such as 2furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl, 2-oxazole 4-oxazolyl, 5-oxazolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-pyrimidyl, 3pyridazinyl, 2-triazolyl, 5-triazolyl, 2-thienyl or 3-thienyl. The term "aryl ring" also means a ring which is optionally substituted.

Aryl groups also include fused aromatic ring systems wherein the carbocyclic aromatic ring or heteroaryl ring is fused to one or more other rings. Examples include tetrahydronaphthyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisoxazolyl, and the like. The term "aryl" as used herein also includes a group in which one or more carbocyclic aromatic rings and / or heteroaryl rings are fused to a cycloalkyl or non-aromatic heterocyclic ring, such as indanyl or tetrahydrobenzopyranyl.

Non-aromatic heterocyclic rings are non-aromatic carbocyclic rings in which one or more ring carbon atoms are replaced by heteroatoms such as nitrogen, oxygen, or sulfur. The ring may be five, six, seven or eight membered and / or fused to another ring such as a cycloalkyl or aromatic ring. Examples include 3-1H-benzimidazol-2-one, 3- (1-alkyl) benzimidazol-2-one, 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholine, 3-morpholine, 4-morpholine. , 2-thiomorpholine, 3-thiomoroline, 4-thiomortoline, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl, 1-piperidinyl, 2-piperidinyl, 37-piperidinyl, 4-piperidinyl, 4-piperidinyl, 4-piperidinyl, N-substituted diazolonyl, 1-phthalimidinyl, benzoxane, benzotriazol-1-yl, benzopyrrolidine, benzopiperidine, benzoxolane, benzothiolane and benzothianane. The term "heterocyclic ring", whether saturated or unsaturated, also refers to optionally substituted rings.

The aryl group, the carbocyclic and heterocyclic group, or the arylalkyl group such as benzyl or phenethyl may be substituted with one or more substituents. Examples of suitable substituents on the unsaturated carbon atom of the aryl group include halogen, -R, -OR, -SR, protected OH (such as acyloxy), phenyl (Ph), substituted Ph, -OPh, substituted -OPh, NO₂, CN, -N (R)_2i -NRN (R)_2i -NRCON (R)_2i -NRCOR, -NRCO₂(aliphatic), -CO₂R, -COR, -C (O) C (O) R, -CON (R)₂, -CONRN (R)₂, -S (O)₂R, -SON (R)₂, -S (O) aliphatic, -SO₂N (R)₂ or -NRS (O)₂R, wherein each R is independently selected from hydrogen, an aliphatic group, or a substituted aliphatic group.

An aliphatic group or a non-aromatic heterocyclic ring may be substituted with one or more substituents. Examples of suitable substituents on an aliphatic group or a non-aromatic heterocyclic ring include the above substituents on an unsaturated carbon as well as the following: = O, = S, = NNHR, = NNR₂, = N-, OR, = NNHCOR, = NNHCO₂(aliphatic), = NNHSO₂(aliphatic or = NR, wherein each R is independently selected from hydrogen, an aliphatic group or a substituted aliphatic group.

The term "alkylidene chain" means an optionally substituted, straight or branched carbon chain which may be fully saturated or contain one or more unsaturated moieties. Optional substituents are as described above for the aliphatic group. Optional substituents on the alkyl moiety of the Q moiety include the substituents described above for the aliphatic group.

The nitrogen of the aromatic or non-aromatic heterocyclic ring may be optionally substituted. Suitable nitrogen substitutions include R, COR, N (R)₂, CON (R)₂, CONRN (R)_2i S (O)₂R and CO₂R, wherein R is independently selected from hydrogen, optionally substituted aryl, or aliphatic.

b

The term "bridge group" refers to an organic moiety that connects two parts of a compound. Bridges usually consist of an atom such as oxygen or sulfur, groups such as -NH- or -CH₂-, or atom chains such as an alkylidene chain. The molecular weight of the bridge is typically in the range of 14 to 200. Examples of bridges include a saturated or unsaturated C 1-4 alkylidene chain which is optionally substituted and wherein up to two saturated carbon atoms are optionally substituted with -C (= O) -, -CONH- , CONHNH, -CO₂, -NHCO2-, -O-, -NHCONH-, -OC (= O) -, -OC (= O) NH-, -NHNH-, -NHC0-, -O-, -S-, -SO-, -S0₂-, -NH-, -SO₂NH- or NHSO₂-.

It will be apparent to those skilled in the art that some of the compounds of the present invention may exist in tautomeric forms and all such tautomeric forms are encompassed by the invention.

Unless otherwise stated, the structures depicted herein also encompass all stereochemical forms, i.e., R and S. configurations of each asymmetric center. Thus, the present invention encompasses both single stereochemically isomeric forms of the present compounds and mixtures of enantiomers and diastereomers. Unless otherwise stated, the structures depicted herein also include compounds that differ only in the isotopes of one or more atoms. For example, compounds having the following structure and characterized in that hydrogen is replaced by deuterium or tritium or carbon is replaced by ¹³C- or ¹⁴C- enriched in carbon is also included in the present invention.

One embodiment of the present invention relates to compounds of formula II:

<img file="LT4981B_D0002.tif" />

in which R¹, R², R³, R⁴, T and Q are as described above.

II

The preferred embodiment of the present invention pertains to compounds of formula

<img file="LT4981B_D0003.tif" />

ll-A where T, R² and R⁴ are as described above, and R¹ and R³ each is hydrogen.

Preferred compounds include those having one or more, and preferably all, of the following: (a) Q is -CO-, -CO₂- or -CONH-;

(b) T is the valence bond; (c) R¹ is hydrogen or NHR; (d) R² is an optionally substituted aryl ring, preferably an optionally substituted phenyl ring;

(c) R³ is hydrogen; (e) R⁴ is selected from R⁵, -NHR⁵, -N (R⁵) 2, -NR⁵R⁶, NHCHR⁵R⁶ or -NHCH 2 R⁵; and / or (f) R⁵ is an optionally substituted group selected from aryl, arylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, (CH 2) y R⁶, (CH2)_yR⁷or (CH₂)_yCH (R⁶) (R⁷).

Substitutions in Rs² phenyl group, examples include halogen, nitro, alkoxy and amino groups.

When R⁴ is R⁵, groups R⁵Preferred include pyrrolidin-1-yl, morpholin-1-yl, piperidin-1-yl and piperazin-1-yl, wherein each group is optionally substituted. When R⁴ is -NHR⁵ or -N (R⁵) 2 is R⁵preferred also includes (CH2)_yR⁶, (CH 2) y R⁷ and (CH2)_yCH (R⁶) (R⁷). Groups R⁶ and R⁷preferred examples include pyridin-3-yl, pyridin-4-yl, imidazolyl, furan-2-yl, tetrahydrofuran-2-yl, cyclohexyl, phenyl, -CH₂OH, - (CH₂)₂OH and isopropyl, where each group is optionally substituted.

ιυ

For Formula II structures wherein R¹ and R³ each is H, examples are given in Table I below.

table. Compounds of Formula II.

II

N

Q-R⁴

II

N /

N,

IR

II

<td>No.</td><td>TR²</td><td>QR⁴</td>

<td> 11-1</td><td>phenyl</td><td>CON (Me)₂</td>

<td>II-2</td><td>phenyl</td><td>CO₂No.</td>

<td>II-3</td><td>3-NO₂-phenyl</td><td>conhnh₂</td>

<td>II-4</td><td>phenyl</td><td>CO (pyrrolidin-1-yl)</td>

<td>II-5</td><td>phenyl</td><td>CONHCH₂(Ph)</td>

<td>II-6</td><td>3-NO₂-phenyl</td><td>CO₂No.</td>

<td>II-7</td><td>4-Cl-phenyl</td><td>CO₂No.</td>

<td>II-8</td><td>4-OMe-phenyl</td><td>CO₂No.</td>

<td>II-9</td><td>3-NH₂-phenyl</td><td>CO₂No.</td>

<td> 11-10</td><td>3-Ome-phenyl</td><td>CO₂No.</td>

<td> 11-11</td><td>4-F-phenyl</td><td>CO₂No.</td>

<td> 11-12</td><td>4-NO₂-phenyl</td><td>CO₂No.</td>

<td> 11-13</td><td>3-Cl-phenyl</td><td>CO₂No.</td>

<td> 11-14</td><td>3-F-phenyl</td><td>CO₂No.</td>

<td> 11-15</td><td>phenyl</td><td>CO₂No.</td>

<td> 11-16</td><td>4-NH₂-phenyl</td><td>CO₂No.</td>

<td> 11-17</td><td>phenyl</td><td>CONHCH₂CH₂N (Me)₂</td>

<td> 11-18</td><td>phenyl</td><td>CONHCH₂(pyridin-3-yl)</td>

<td> 11-19</td><td>phenyl</td><td>CO (morpholin-1-yl)</td>

<td>II-20</td><td>phenyl</td><td>CONH (isopropyl)</td>

<td> 11-21</td><td>phenyl</td><td>CO (4-Me-piperazin-1-yl)</td>

<td>II-22</td><td>phenyl</td><td>CONHCH₂(furan-2-yl)</td>

<td>II-23</td><td>3-OMe-phenyl</td><td>CONMe₂</td>

<td>II-24</td><td>3-OMe-phenyl</td><td>CO (pyrrolidin-1-yl)</td>

<td>II-25</td><td>3-OMe-phenyl</td><td>CONHCH₂CH₂N (Me)₂</td>

<td>II-26</td><td>3-OMe-phenyl</td><td>C0NHCH₂(pyridin-3-yl)</td>

<td>II-27</td><td>3-OMe-phenyl</td><td>CO (morpholin-1-yl)</td>

<td>II-28</td><td>3-OMe-phenyl</td><td>CONH (isopropyl)</td>

<td>II-29</td><td>3-OMe-phenyl</td><td>CO (4-Me-piperazin-1-yl)</td>

<td>II-30</td><td>3-OMe-phenyl</td><td>CONHCH₂(furan-2-yl)</td>

<td> 11-31</td><td>4-NH₂-phenyl</td><td>CO₂No.</td>

<td>II-32</td><td>H</td><td>CONMe₂</td>

<td>II-33</td><td>H</td><td>CO (pyrrolidin-1-yl)</td>

<td>II-34</td><td>3- (AcNH) -phenyl</td><td>CO₂No.</td>

<td>II-35</td><td>4- (AcNH) -phenyl</td><td>CO₂No.</td>

<td>II-36</td><td>3- (AcNH) -phenyl</td><td>CO₂No.</td>

<td>II-37</td><td>4- (AcNH) -phenyl</td><td>CO₂No.</td>

<td>II-38</td><td>3-Cl-phenyl</td><td>CON (H) Bn</td>

<td>II-39</td><td>3,5-CI₂-phenyl</td><td>O-OH</td>

<td>II-40</td><td>5-Br-phenyl</td><td>CONH (3,4-F₂-phenyl)</td>

<td> 11-41</td><td>5-Cl-phenyl</td><td>CONH (2-OH-1-Ph-ethyl)</td>

<td>II-42</td><td>4-OH, 3-1,5-nitrophenyl</td><td>CONH (2-OH-1-Ph-ethyl)</td>

<td>II-43</td><td>5-Br-phenyl</td><td>O A</td>

<td>II-44</td><td>3-NH₂, 4-OH, 5-1-phenyl</td><td>CONH (2-OH-1-Ph-ethyl)</td>

<td>II-45</td><td>5-Br-phenyl</td><td>CONH (2-OH-1-Ph-ethyl)</td>

<td>II-46</td><td>5-Br-phenyl</td><td>CONHCH₂(3-MeO-phenyl)</td>

<td>II-47</td><td>5-Br-phenyl</td><td>CONHCH₂(3-CF₃-phenyl)</td>

<td>II-48</td><td>3,5-CI₂-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td>II-49</td><td>5-CF₃-phenyl</td><td>CONH (2-OH-1-Ph-ethyl)</td>

<td>II-50</td><td>5-Cl-phenyl</td><td>CONHCH₂Ph</td>

<td> 11-51</td><td>3,5-CI₂-phenyl</td><td>CONHOCH₂Ph</td>

<td>II-52</td><td>4-OH, 3-1,5-nitrophenyl</td><td>CONHCH₂Ph</td>

<td>II-53</td><td>5-Cl-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td>II-54</td><td>4,5-CI₂-phenyl</td><td>CONHOCH₂Ph</td>

<td>II-55</td><td>5-Br-phenyl</td><td>CONHCH₂(4-SO₂Me-phenyl)</td>

<td>II-56</td><td>5-Br-phenyl</td><td>CONHNH (3-CF₃-phenyl)</td>

<td>II-57</td><td>5-Cl-phenyl</td><td>CONHOCH₂Ph</td>

<td>li-58</td><td>5-Br-phenyl</td><td> 0 ^H</td>

<td></td><td></td><td></td>

<td>II-59</td><td>5-Br-phenyl</td><td>OH</td>

<td>II-60</td><td>5-Br-phenyl</td><td>CONHCH₂(2-Me-phenyl) ·</td>

<td> 11-61</td><td>4,5-CI₂-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td> 11-62</td><td>5-Br-phenyl</td><td>CONH (1-Ph-propyl)</td>

<td> 11-63</td><td>5-F-phenyl</td><td>CONHCH₂-Ph</td>

<td> 11-64</td><td>4,5-CI₂-phenyl</td><td> 0</td>

<td> 11-65</td><td>5-Br-phenyl</td><td>0 OH</td>

<td> 11-66</td><td>3,5-CI₂-phenyl</td><td>CON (Me) (Et)</td>

<td> 11-67</td><td>5-Cl-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-68</td><td>5-Br-phenyl</td><td>CONHCH₂(3,5-OMe₂-phenyl)</td>

<td> 11-69</td><td>5-Br-phenyl</td><td>CONHCH₂(2-OMe-phenyl)</td>

<td> 11-70</td><td>4-F-5-Cl-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td> 11-71</td><td>4-F-5-Cl-phenyl</td><td>CON (Me) (Et)</td>

<td>II-72</td><td>5-Br-phenyl</td><td>CONH (2-OH-1-Ph-ethyl)</td>

<td>II-73</td><td>5-NH₂-phenyl</td><td>CONHCH₂Ph</td>

<td>II-74</td><td>4,5-CI₂-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td>II-75</td><td>5-Me-phenyl</td><td>CONH (2-OH-1-Ph-ethyl)</td>

<td>II-76</td><td>3,5-CI₂-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td>U-77</td><td>4-F-5-Cl-phenyl</td><td>CONHOCH₂Ph</td>

<td>II-78</td><td>3,5-CI₂-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td>II-79</td><td>5-NO₂-phenyl</td><td>CONHCH₂Ph</td>

<td>II-80</td><td>5-F-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td> 11-81</td><td>5-Cl-6-F-phenyl</td><td>CON (Me) (Et)</td>

<td>II-82</td><td>2-F-3-Cl-phenyl</td><td>CONHOCH₂Ph</td>

<td>II-83</td><td>5-Br-phenyl</td><td>Λίο</td>

<td>II-84</td><td>5-Cl-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl) __</td>

<td>II-85</td><td>4.5-F₂-phenyl</td><td>CONHOCH₂Ph</td>

<td>il-86</td><td>5-Br-phenyl</td><td>CONH (3-OH-1-Ph-propyl)</td>

<td>il-87</td><td>5-Br-phenyl</td><td>ArP</td>

<td>il-88</td><td>4.5-F₂-phenyl</td><td>CONHCH₂(pyrid-'4-yl)</td>

<td>li-89</td><td>5-F-phenyl</td><td>CONHOCH₂Ph</td>

<td>il-90</td><td>5-Me-phenyl</td><td>CONHCH₂Ph</td>

<td> 11-91</td><td>5-Br-phenyl</td><td>O_X® H</td>

<td>II-92</td><td>4-Cl-phenyl</td><td>CONHCH₂Ph</td>

<td>II-93</td><td>5-Cl-phenyl</td><td>CON (Me) (Et)</td>

<td>II-94</td><td>5-Br-phenyl</td><td>CONHCH₂(4-SO₂NH₂-phenyl)</td>

<td>II-95</td><td>5-OH-phenyl</td><td>CONHCH₂Ph</td>

<td>II-96</td><td>5-Me-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td>II-97</td><td>Phenyl</td><td>CONHCH₂Ph</td>

<td>II-98</td><td>2.5-F₂-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td>il-99</td><td>4-Cl-phenyl</td><td>CONHOCH₂Ph</td>

<td> 11-100</td><td>4-F-5-Cl-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td> 11-101</td><td>4-F-5-Cl-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-102</td><td>5-Br-phenyl</td><td>CO (4-OH-4-Ph-piperidin-1-yl)</td>

<td> 11-103</td><td>5.6-F₂-phenyl</td><td>CONHOCH₂Ph</td>

<td> 11-104</td><td>5-Cl-phenyl</td><td>CO (morpholin-1-yl)</td>

<td> 11-105</td><td>5-Br-phenyl</td><td>aBf</td>

<td> 11-106</td><td>2-F-3-Cl-phenyl</td><td>CO NHCH₂ (tetrahydrofuran -2-i 1)</td>

<td> 11-107</td><td>4-F-5-Cl-phenyl</td><td>CO (morpholin-1-yl)</td>

<td> 11-108</td><td>4-F-5-Cl-phenyl</td><td>CON (Me) (Et)</td>

<td> 11-109</td><td>5-Br-phenyl</td><td>CONHCH₂(4-NH₂-phenyl)</td>

<td> 11-110</td><td>5-Br-phenyl</td><td>vVys^HT; n</td>

ι η

<td></td><td></td><td></td>

<td> 11-111</td><td>4-F-phenyl</td><td>CONHCH₂Ph</td>

<td> 11-112</td><td>3,5-CI₂-phenyl</td><td>CO (morpholin-1-yl)</td>

<td> 11-113</td><td>2.5-F₂-phenyl</td><td>CONHOCH₂Ph</td>

<td> 11-114</td><td>2-F-3-Cl-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-115</td><td>2-F-3-Cl-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td> 11-116</td><td>4.5-F₂-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-117</td><td>4-OMe-phenyl</td><td>CONHCH₂Ph</td>

<td> 11-118</td><td>5-Br-phenyl</td><td>CONHCH₂(2,4₁6-OMe₃-phenyl)</td>

<td> 11-119</td><td>5-F-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-120</td><td>₄ - * 4,5-F₂-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td> 11-121</td><td>5-Cl-6-F-phenyl</td><td>S ° kX</td>

<td> 11-122</td><td>5-Br-phenyl</td><td></td>

<td> 11-123</td><td>5-Br-phenyl</td><td></td>

<td> 11-124</td><td>5-Br-phenyl</td><td>CONHCH₂(2,5-OMe₂-phenyl)</td>

<td> 11-125</td><td>3,5-CI₂-phenyl</td><td></td>

<td> 11-126</td><td>5-Br-phenyl</td><td></td>

<td> 11-127</td><td>4,5-CI₂-phenyl</td><td>CO (morpholin-1-yl)</td>

<td> 11-128</td><td>5-Br-phenyl</td><td></td>

<td> 11-129</td><td>2-F-3-Cl-phenyl</td><td>CO (morpholin-1-yl)</td>

<td> 11-130</td><td>5-Br-phenyl</td><td>CONHCH₂CH₂OH</td>

<td> 11-131</td><td>5-NH₂-phenyl</td><td>CONHCH₂Ph</td>

<td> 11-132</td><td>5-MeOC (O) -phenyl</td><td>CONHCH₂Ph</td>

<td> 11-133</td><td>4-MeO-phenyl</td><td>CONHOCH₂Ph</td>

<td> 11-134</td><td>phenyl</td><td>CO (pyrrolidin-1-yl)</td>

<td> 11-135</td><td>5-MeO-phenyl</td><td>CO (morpholin-1-yl)</td>

<td> 11-136</td><td>5-Cl-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td> 11-137</td><td>5-NO₂-phenyl</td><td>CONH₂NH₂</td>

<td> 11-138</td><td>5-Br-phenyl</td><td></td>

<td> 11-139</td><td>5-Br-phenyl</td><td>ήηο OH</td>

<td> 11-140</td><td>5-Cl-phenyl</td><td>CONHPh</td>

<td> 11-141</td><td>5.6-F₂-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td> 11-142</td><td>5-Cl-phenyl</td><td>o hn- \. H</td>

<td> 11-143</td><td>phenyl</td><td>CON (Me)₂</td>

<td> 11-144</td><td>5-OMe-phenyl</td><td>CO (pyrrolidin-1-yl)</td>

<td> 11-145</td><td>5-OMe-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-146</td><td>4-F-phenyl</td><td>CONHOCH₂Ph</td>

<td> 11-147</td><td>5-OMe-phenyl</td><td>CONHCH₂(furan-2-yl)</td>

<td> 11-148</td><td>5-NO₂-phenyl</td><td>COOEt</td>

<td> 11-149</td><td>phenyl</td><td>CONHCH₂(furan-2-yl)</td>

<td> 11-150</td><td>phenyl</td><td>CO (morpholin-1-yl)</td>

<td> 11-151</td><td>5-Cl-phenyl</td><td>COOEt</td>

<td> 11-152</td><td>5-Br-phenyl</td><td>CONHMe</td>

<td> 11-153</td><td>phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-154</td><td>5-OMe-phenyl</td><td>CON (Me)₂</td>

<td> 11-155</td><td>5-Cl-phenyl</td><td>F H OH</td>

<td> 11-156</td><td>5-Br-phenyl I</td><td>ύρο OH</td>

<td></td><td></td><td></td>

<td> 11-157</td><td>5-Br-phenyl</td><td>COOEt</td>

<td> 11-158</td><td>phenyl</td><td>CONH (iPr)</td>

<td> 11-159</td><td>5-OMe-phenyl</td><td>CONH (iPr)</td>

<td> 11-160</td><td>5-COOH-phenyl</td><td>CONH (iPr)</td>

<td> 11-161</td><td>5-Br-phenyl</td><td>CONHO (iPr)</td>

<td> 11-162</td><td>5-F-phenyl</td><td>COOEt</td>

<td> 11-163</td><td>5-OMe-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td> 11-164</td><td>4-NH₂-phenyl</td><td>COOEt</td>

<td> 11-165</td><td>4-NO₂-phenyl</td><td>COOEt</td>

<td> 11-166</td><td>phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td> 11-167</td><td>4-Cl-phenyl</td><td>COOEt</td>

<td> 11-168</td><td>4-OMe-phenyl</td><td>COOEt</td>

<td> 11-169</td><td>phenyl</td><td>COOEt</td>

<td> 11-170</td><td>5-OMe-phenyl</td><td>COOEt</td>

<td> 11-171</td><td>4-F-phenyl</td><td>COOEt</td>

<td> 11-172</td><td>5-NH₂-phenyl</td><td>COOEt</td>

<td> 11-173</td><td>5-Cl-phenyl</td><td>COOEt</td>

<td> 11-174</td><td>5-Cl-phenyl</td><td>χόο H</td>

<td></td><td></td><td></td>

<td> 11-175</td><td>5-O-phenyl</td><td> 0</td>

<td> 11-176</td><td>5-OMe-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td> 11-177</td><td>3.5- (OMe)₂-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td> 11-178</td><td>4-F-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-179</td><td>4-OMe-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-180</td><td>2.5- (OMe)₂-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-181</td><td>2.5-F₂-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-182</td><td>4-F-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td> 11-183</td><td>4-OMe-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td> 11-184</td><td>5-F-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td> 11-185</td><td>5-OMe-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td> 11-186</td><td>2.5- (OMe)₂-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td> 11-187</td><td>5.6-F₂-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td> 11-188</td><td>2.5-F₂-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td> 11-189</td><td>4-F-phenyl</td><td>CONHCH₂(1-Et-pyrrole idi η-2-i)</td>

<td> 11-190</td><td>4-OMe-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td> 11-191</td><td>5-F-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td> 11-192</td><td>5-OMe-phenyl</td><td>CONHCH₂(1-Et-pyrrole id i η-2-i I)</td>

<td> 11-193</td><td>3.6- (OMe)₂-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td> 11-194</td><td>4.5-F₂-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td> 11-195</td><td>5.6-F₂-phenyl</td><td>CONHCH₂(1-Et-pyroJ idin-2-i)</td>

<td> 11-196</td><td>3.6-F₂-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td> 11-197</td><td>4-F-phenyl</td><td>CO (morpholin-1-yl)</td>

<td> 11-198</td><td>4-OMe-phenyl</td><td>CO (morpholin-1-yl)</td>

<td> 11-199</td><td>5-F-phenyl</td><td>CO (morpholin-1-yl)</td>

<td>II-200</td><td>2.5- (OMe)₂-phenyl</td><td>CO (morpholin-1-yl)</td>

<td> 11-201</td><td>4.5-F₂-phenyl</td><td>CO (morpholin-1-yl)</td>

<td>II-202</td><td>5.6-F₂-phenyl</td><td>CO (morpholin-1-yl)</td>

<td>II-203</td><td>2.5-F₂-phenyl</td><td>CO (morpholin-1-yl)</td>

<td>II-204</td><td>4-F-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td>II-205</td><td>4-OMe-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td>II-206</td><td>5-F-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td>II-207</td><td>2.5- (OMe)₂-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td>II-208</td><td>4.5-F₂-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td>II-209</td><td>5.6-F₂-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td> 11-210</td><td>3.6-F₂-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td> 11-211</td><td>4-Cl-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td> 11-212</td><td>4,5- (OMe)₂-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td> 11-213</td><td>4-benzo [1,3] dioxo-5-yl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td> 11-214</td><td>4-Cl-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-215</td><td>4,5- (OMe)₂-phenyl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-216</td><td>4-benzo [1,3] dioxo-5-yl</td><td>CONHCH₂(pyrid-3-yl)</td>

<td> 11-217</td><td>4-Cl-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td> 11-218</td><td>4,5- (OMe)₂-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td> 11-219</td><td>4-benzo [1,3] dioxo-5-yl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td>II-220</td><td>4-Cl-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td> 11-221</td><td>4,5-CI₂-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td>II-222</td><td>5-Cl-6-F-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td>II-223</td><td>4-F-5-Cl-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td>II-224</td><td>4,5- (OMe)₂-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td>II-225</td><td>4-benzo [1,3] dioxo-5-yl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td>I1-226</td><td>3,5-CI₂-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td>II-227</td><td>4-Cl-phenyl</td><td>CO (morpholin-1-yl)</td>

<td>II-228</td><td>4,5- (OMe)₂-phenyl</td><td>CO (morpholin-1-yl)</td>

<td>II-229</td><td>4-benzo [1,3] dioxo-5-yl</td><td>CO (morpholin-1-yl)</td>

<td>II-230</td><td>4-Cl-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td> 11-231</td><td>4,5-CI₂-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td>II-232</td><td>5-Cl-6-F-phenyl</td><td>C0 (4-Me-piperidin-1-yl)</td>

<td>II-233</td><td>4-F-5-Cl-phenyl</td><td>C0 (4-Me-piperidin-1-yl)</td>

<td>II-234</td><td>4,5- (OMe)₂-phenyl</td><td>C0 (4-Me-piperidin-1-yl)</td>

<td>II-235</td><td>4-benzo [1,3] dioxo-5-yl</td><td>C0 (4-Me-piperidin-1-yl)</td>

<td>II-236</td><td>3,5-CI₂-phenyl</td><td>C0 (4-Me-piperidin-1-yl)</td>

<td>II-237</td><td>5.6-F₂-phenyl</td><td>CON (Me) (Et)</td>

<td>II-238</td><td>4-F-phenyl</td><td>A-JP⁴</td>

<td>II-239</td><td>5-OMe-phenyl</td><td>--JP⁴</td>

<td>II-240</td><td>2.5- (OMe)₂-phenyl</td><td>° v</td>

<td> 241</td><td>4.5-F₂-phenyl</td><td>o O, V</td>

<td>II-242</td><td>5.6-F₂-phenyl</td><td>while O. \ Z</td>

<td>II-243</td><td>3.6-F₂-phenyl</td><td>⁰ ° κ \ V</td>

<td>II-244</td><td>5-MeO-phenyl</td><td>CONHOCH₂Ph</td>

<td>II-245</td><td>2.5- (OMe)₂-phenyl</td><td>CONHOCH₂Ph</td>

<td>II-246</td><td>5-F-phenyl</td><td>⁰ hn \, H</td>

<td>ΙΙ-247</td><td>5-MeO-phenyl</td><td>H</td>

<td>II-248</td><td>4.5-F₂-phenyl</td><td>0 HN \, H</td>

<td></td><td></td><td></td>

<td>II-249</td><td>5.6-F₂-phenyl</td><td>⁰ ΗΝ'Χ. H</td>

<td>II-250</td><td>5-Cl-phenyl</td><td></td>

<td> 11-251</td><td>4-Cl-phenyl</td><td></td>

<td>II-252</td><td>4-Cl-phenyl</td><td>O HN-% H</td>

<td>II-253</td><td>4,5-CI₂-phenyl</td><td>° RV</td>

<td>II-254</td><td>4,5-Clp-phenyl</td><td>Αχχ H</td>

<td>II-255</td><td>2-F-3-Cl-phenyl</td><td>0 HN%. H</td>

<td>II-256</td><td>4-F-5-Cl-phenyl</td><td></td>

<td>II-257</td><td>4-F-5-Cl-phenyl</td><td>n ^HfP^NH</td>

<td></td><td></td><td></td>

<td>li-258</td><td>4,5- (OMe)₂-phenyl</td><td>CON (Me) (Et)</td>

<td>II-259</td><td>4,5- (OMe)₂-phenyl</td><td></td>

<td>II-260</td><td>4,5- (OMe)₂-phenyl</td><td>CONHOCH₂Ph</td>

<td> 11-261</td><td>4,5- (OMe)₂-phenyl</td><td>H^NVNH 0</td>

<td>II-262</td><td>4-benzo [1,3] dioxo-5-yl</td><td>CON (Me) (Et)</td>

<td></td><td></td><td></td>

<td>II-263</td><td>4-benzo [1,3] dioxo-5-yl</td><td>o C) \ V</td>

<td>li-264</td><td>4-benzo [1,3] dioxo-5-yl</td><td>CONHOCH₂Ph</td>

<td>H-265</td><td>4-benzo [1,3] dioxo-5-yl</td><td>o hnX H</td>

<td>II-266</td><td>3,5-Cl 2 -phenyl</td><td>o ηνΎ., vvv- '¹H</td>

<td>II-267</td><td>5-Br-phenyl</td><td></td>

<td>II-268</td><td>5-Br-phenyl</td><td>tlVjH 0</td>

<td>II-269</td><td>5-Br-phenyl</td><td>Άο-b</td>

<td></td><td></td><td></td>

<td>II-270</td><td>5-Br-phenyl</td><td></td>

<td> 11-271</td><td>5-Br-phenyl</td><td></td>

<td>II-272</td><td>5-Br-phenyl</td><td>^ ob</td>

<td>II-273</td><td>5-Br-phenyl</td><td>^ ob *</td>

<td>II-274</td><td>5-Br-phenyl</td><td>O NHj</td>

<td>II-275</td><td>5-Br-phenyl</td><td>etc.</td>

<td>II-276</td><td>5-Br-phenyl</td><td></td>

<td>II-277</td><td>5-Br-phenyl</td><td></td>

<td>II-278</td><td>5-Br-phenyl</td><td>o</td>

<td></td><td></td><td></td>

<td>II-279</td><td>5-Br-phenyl</td><td>H</td>

<td>II-280</td><td>5-Br-phenyl</td><td>CONH (CH₂)₂COOH</td>

<td> 11-281</td><td>5-Br-phenyl</td><td></td>

<td>II-282</td><td>5-Br-phenyl</td><td>CONHCH₂(4-COOH-phenyl)</td>

<td></td><td></td><td></td>

<td>II-283</td><td>5-Br-phenyl</td><td>O OH DU HO</td>

<td>II-284</td><td>5-Br-phenyl</td><td>O r _</td>

<td>II-285</td><td>3-NO₂-phenyl</td><td>CONHCH₂phenyl</td>

<td>II-286</td><td>5-Cl-phenyl</td><td>CONHCH₂(1-Et-pyrrolidin-2-yl)</td>

<td>II-287</td><td>5 - (N-Et-NHC0) -phenyl</td><td>CONHCH₂phenyl</td>

<td>II-288</td><td>5-Br-phenyl</td><td> .0 75</td>

<td>II-289</td><td>5-NO₂-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td>II-290</td><td>5-Br-phenyl</td><td>0 O In- °^vVn M</td>

<td> 11-291</td><td>5-F-phenyl</td><td>CON (Me) (Et)</td>

<td>li-292</td><td>5-MeO-phenyl</td><td>CON (Me) (Et)</td>

<td>II-293</td><td>5-Br-phenyl</td><td>"/ R H</td>

<td>il-294</td><td>5-Br-phenyl</td><td>hn ^_n0 -X H</td>

<td></td><td></td><td></td>

<td>II-295</td><td>5-Br-phenyl</td><td>o r-^OH</td>

<td>li-296</td><td>5-Br-phenyl</td><td>OMe M H</td>

<td>li-297</td><td>phenyl</td><td>CONH (CH₂)₂NMe₂</td>

<td>li-298</td><td>5-MeO-phenyl</td><td>CONH (CH₂)₂NMe₂</td>

<td>li-299</td><td>5-Br-phenyl</td><td>CONHCH₂phenyl</td>

<td>II-300</td><td>3-Cl-phenyl</td><td>O r - OH X</td>

<td> 11-301</td><td>3-Cl-phenyl</td><td>O Γ- °^Η</td>

<td>II-302</td><td>3-Cl-phenyl</td><td>0 r-OH^H U-cf,</td>

<td></td><td></td><td></td>

<td>II-303</td><td>3-Cl-phenyl</td><td></td><td>OH Oh</td>

<td>II-304</td><td>3-Cl-phenyl</td><td></td><td>-OH cc</td>

<td>II-305</td><td>3-Cl-phenyl</td><td colspan="2">0, ΤθΗ</td>

<td>II-306</td><td>3-Cl-phenyl</td><td>or yV H</td><td>OH</td>

<td>II-307</td><td>3-Cl-phenyl</td><td colspan="2">• 9 --- ° γ^Ζ^H O</td>

<td>II-308</td><td>3-Cl-phenyl</td><td colspan="2"> .0 ^H 0</td>

<td>II-309</td><td>3-Cl-phenyl</td><td></td><td>OH cx</td>

<td> 11-310</td><td>3,5-CI₂-phenyl</td><td colspan="2"> 0</td>

<td> 11-311</td><td>3-Br-5-CF₃-phenyl</td><td>θ Λ ' H</td><td>OH υ</td>

<td> 11-312</td><td>3-Cl-phenyl</td><td>Oh, OH^h LJ Cl</td>

<td> 11-313</td><td>3,5-CI₂-phenyl</td><td>O r- °^H</td>

<td></td><td></td><td></td>

<td> 11-314</td><td>3-Cl-4-CN-phenyl</td><td>H</td>

<td> 11-315</td><td>3-CI-4-CH₂OH-phenyl</td><td>.0 VW « H</td>

<td> 11-316</td><td>3-CI-4-CH₂NH₂-phenyl</td><td>• 9 V ^ n ^ oh H</td>

<td> 11-317</td><td>0 Cl</td><td>»9 vL_nA &gt;&gt; h H</td>

<td> 11-318</td><td>CA. Cl</td><td>»9 H</td>

<td> 11-319</td><td>^HH0 no</td><td>9th 9 '»A H</td>

<td>I1-320</td><td>0 Cl</td><td>• 9 HI</td>

<td> 11-321</td><td> o C * i</td><td>• 9 VYl-Y '⁰¹¹H</td>

<td>II-322</td><td>l / V¹a</td><td>• 9 H</td>

<td>II-323</td><td>CH₂Ph</td><td>CON (Me)₂</td>

<td>II-324</td><td>cyclopentylmethyl</td><td>CO₂NHCH₂Ph</td>

<td>II-325</td><td>isopropyl</td><td>CN</td>

<td>II-326</td><td>3-Cl-phenyl</td><td>NHCOCH₂Ph</td>

<td>II-327</td><td>3-Cl-phenyl</td><td>NHSO₂-morpholin-1-yl</td>

<td>II-328</td><td>3-Cl-phenyl</td><td>NHCONHCH₂Ph</td>

<td>II-329</td><td>3-Cl-phenyl</td><td>NHCO₂-tetrahydrofuran-2-yl</td>

<td>II-330</td><td>CH₂Ph</td><td>CONHCH₂Ph</td>

<td> 11-331</td><td>Me</td><td>CONHCH₂Ph</td>

<td>II-332</td><td>isopropyl</td><td>CONHCH₂Ph</td>

<td>II-333</td><td>H</td><td>CON (Me)₂</td>

Another preferred embodiment of the invention relates to compounds of formula II-B:

<img file="LT4981B_D0004.tif" />

ll-B where T, R, R² and R⁴ are as described above.

Preferred compounds of formula II-B include those having one or more, and preferably all, of the following: (a) T is a valence bond; (b) R³ is hydrogen; and / or (c) R² is an optionally substituted aryl ring, preferably an optionally substituted phenyl ring.

Structures of Formula II-B wherein R³ is H, examples are given in Table 2 below.

table. Compounds of Formula II-B.

<td>No.</td><td>R</td><td>TR²</td><td>QR⁴</td>

<td>II-B-1</td><td>H</td><td>phenyl</td><td>CON (Me)₂</td>

<td>II-B-2</td><td>H</td><td>phenyl</td><td>CO₂No.</td>

<td>II-B-3</td><td>H</td><td>3-NO₂-phenyl</td><td>CONHNH₂</td>

<td>II-B-4</td><td>H</td><td>phenyl</td><td>CO (pyrrolidin-1-yl)</td>

<td>II-B-5</td><td>Me</td><td>phenyl</td><td>CONHCH₂(Ph)</td>

<td>II-B-6</td><td>H</td><td>3-NO₂-phenyl</td><td>CO₂No.</td>

<td>II-B-7</td><td>H</td><td>4-Cl-phenyl</td><td>CO₂No.</td>

<td>II-B-8</td><td>Me</td><td>4-OMe-phenyl</td><td>CO₂No.</td>

<td>II-B-9</td><td>H</td><td>3-NH₂-phenyl</td><td>CO₂No.</td>

<td>II-B-10</td><td>H</td><td>3-OMe-phenyl</td><td>CO₂No.</td>

<td>II-B-11</td><td>H</td><td>4-F-phenyl</td><td>CO₂No.</td>

<td>II-B-12</td><td>H</td><td>4-NO₂-phenyl</td><td>CO₂No.</td>

<td>II-B-13</td><td>No.</td><td>3-Cl-phenyl</td><td>CO₂No.</td>

<td>II-B-14</td><td>H</td><td>3-F-phenyl</td><td>CO₂No.</td>

<td>II-B-15</td><td>H</td><td>phenyl</td><td>CO₂No.</td>

<td>II-B-16</td><td>Me</td><td>3-Cl-phenyl</td><td>CONHCH₂(pyridin-4-yl)</td>

<td>II-B-17</td><td>H</td><td>5-Cl-phenyl</td><td>X</td>

<td>II-B-18</td><td>H</td><td>5-F-phenyl</td><td>CONHCH₂(tetrahydrofuran-2-yl)</td>

<td>II-B-19</td><td>Me</td><td>5.6-F₂-phenyl</td><td>CO (4-Me-piperidin-1-yl)</td>

<td>II-B-20</td><td>H</td><td>4-Cl-phenyl</td><td>CONHCH₂(pyrid-4-yl)</td>

<td>II-B-21</td><td>H</td><td>4,5- (OMe)₂-phenyl</td><td>H VNH O</td>

<td>II-B-22</td><td>Me</td><td>4,5-CI₂-phenyl</td><td></td>

<td></td><td></td><td></td><td></td>

<td>II-B-23</td><td>H</td><td>3-Cl-phenyl</td><td>^H . 0</td>

<td>II-B-24</td><td>H</td><td>3-Cl-phenyl</td><td>0 Γ- °^Η^H LJ Cl</td>

<td>II-B-25</td><td>Me</td><td>3,5-CI₂-phenyl</td><td>0 r-OH</td>

<td>II-B-26</td><td>H</td><td></td><td>H</td>

<td>II-B-27</td><td>H</td><td>H</td><td>CON (Me)₂</td>

The following compounds may be synthesized according to procedures known to those of ordinary skill in the art from analogous compounds, as illustrated in Schemes I and II and the following synthesis examples.

<img file="LT4981B_D0005.tif" />

<img file="LT4981B_D0006.tif" />

<img file="LT4981B_D0007.tif" />

Reagents and Conditions: (a) PhCH₂COCI, AICI₃, CH₂CI₂, 2 hours, room temperature (RT); (b) DMF, 24 or., RT; (c) (Me₂N)₂-Ot-Bu, THF, 24h or RT; (d) H₂NNH₂, EtOH, 12 hours, refluxing.

Scheme I above illustrates the general synthesis route by which the compounds of the present invention were synthesized when R² is an optionally substituted phenyl group. In step (a), the optionally substituted benzoyl chloride is mixed in dichloromethane with compound i and aluminum chloride to give compound 2. This reaction is responsible for various phenyl ring substituents. For eligible Rs² Examples of groups include but are not limited to Table 1 above.

Amide 4 is formed by reacting compound 2 with amine 3 in DMF solution. When amine 3 is primary, the reaction proceeds at room temperature. When amine 3 is a secondary amine, the reaction mixture is heated at 50 ° C to complete the reaction to afford amide 4.

Enamine 5 is formed in step (c) by treatment of amide 4 (Me₂N)₂-Ot-Bu at room temperature. Alternatively, the reaction whereby the enamine 5 is formed in step (c) also takes place using dimethylformamide-dimethylacetal (DMF-DMA). The reaction using DMF-DMA requires higher temperatures to yield the enamine 5, whereas {Me₂N)₂The use of -Ot-Bu has the advantage that the enamine 5 is purer at room temperature.

The pyrazole compound 6 is formed in step (d) by treatment of the enamine 5 with hydrazine hydrate at elevated temperature. Compounds of formula II synthesized by this method as shown in Table 1 were isolated by HPLC (reverse phase, 10-&gt; 90% MeCN in water over 15 minutes). Details of how to obtain these devices are given in the Examples.

Scheme II

<img file="LT4981B_D0008.tif" />

II-B-16

Reagents and Conditions: (a) 3-CI-PhCH₂COCI, AICI₃, CH₂CI_2| 2 or. RT; (b) DMF, 24 or., RT; (c) NBS, CCI₄, reflux welding; (e) Formic acid, refluxing, 2 hours.

Scheme II above demonstrates a general synthesis procedure for the preparation of compounds of formula II-B based on the example of compound II-B-16. These are modified by Jira T. et al., Pharmazie, pp. 401-406 (1994). Compounds of formula II-B may also be prepared according to procedures similar to those described in Woller, J., et al., Pharmazie, p. 937-940 (1996), Rychmans T. et al., Tetrahedron, p. 1729-1734 (1997) and Tupper DE et al., Synthesis, p. 337-341 (1997).

In another embodiment, the invention provides a method of inhibiting kinase activity in a biological sample. The method comprises the step of contacting a biological sample with a compound of the present invention.

As used herein, the term biological sample includes cell culture or extract thereof, mammalian biopsy material or extract thereof, and blood, saliva, urine, faeces, semen, tears or other body fluids or extracts thereof. The term biological sample also includes living organisms, and in this case contacting a compound of the present invention with a biological sample is synonymous with introducing the term said compound (or composition containing said compound) to a mammal.

Another aspect of the present invention relates to a method of treating a mammalian disease condition wherein the condition is ameliorated by a protein kinase inhibitor, the method comprising administering to the mammal in need of such treatment a therapeutically effective amount of a compound of formula

II or a pharmaceutically acceptable derivative thereof, wherein

R¹ is selected from R, halogen, N (R⁸) 2, OR, NRCOR, NRCON (R⁸)2,

CON (R⁸) 2, SO2R, NRSO2R or SO2N (R⁸)2;

T is selected from valence bond or bridge group;

each R is independently selected from hydrogen or an optionally substituted aliphatic group having up to six carbon atoms;

R² is selected from hydrogen, CN, halogen, aryl, arylalkyl, heteroaryl, heterocyclyl, an optionally substituted aliphatic chain group containing up to six carbon atoms, or an optionally substituted cyclic aliphatic group having four to ten carbon atoms;

R³ is selected from R, OH, OR, N (R⁸)₂, halogen or CN;

Q is a valence bond, J or an optionally substituted Ci-₆ an alkylidene chain in which up to two adjacent carbon atoms of the alkylene chain are each optionally and independently substituted with J;

J is selected from -C (= O) -, -CO₂-, -C (O) C (O) -, -NRCONR⁸-, -N (R) N (R⁸) -, -C (= O) NR⁸-, -NRC (= O) -, -O-, -S-, -SO-, -SO2-, -N (R) O-, -ON (R⁸) -, -OC (= O) N (R⁸) -, -N (R) COO-, -SO2N (R⁸) -, -N (R) SO₂-, or -N {R⁸)-;

R⁴ is selected from -R⁸, -R⁵, -NH 2, -NHR⁵, -N (R⁵) 2 or -NR⁵(CH 2) y N (R⁵) 2; each R⁵is independently selected from R⁶, R⁷, - (CH₂)_yCH (R⁶) (R⁷),

- (CH₂)_yR⁶, - (CH 2) y CH (R⁶) 2, - (CH₂)_yCH (R⁷)₂ or - (CH₂)_yR⁷; y is 0-6;

each R⁶ is an optionally substituted group selected from an aliphatic, aryl, arylalkyl, arylalkoxy, heteroaryl, heteroarylalkyl, heteroarylalkoxy, heterocyclyl, heterocyclylalkyl or heterocyclylalkoxy group;

each R⁷ is independently selected from optionally substituted hydroxyalkyl, alkoxyalkyl, aryloxyalkyl or alkoxycarbonyl;

each R⁸ is independently selected from R, or two R⁸ optionally, on the same nitrogen atom, taken together with that nitrogen, forms a 4- to 8-membered, saturated or unsaturated heterocyclic ring containing from one to three heteroatoms;

and each ring nitrogen which may be substituted is optionally substituted with R, NR², COR, CO2 (CrC6 optionally substituted alkyl), SO2 (CrC6 optionally substituted alkyl), CONR² and SO2NR².

One embodiment involves the administration of compounds of formula II. Preferred embodiments include the administration of compounds of formula II-A, and most preferably, the compounds listed in Table 1. Another preferred embodiment involves the administration of compounds of formula II-B, and most preferably the compounds listed in Table 2. Pharmaceutical compositions suitable for such a process are described below.

This method is particularly suitable for treating disease states facilitated by the use of ERK, JAK, JNK, Aurora, GSK, DRC or AKT inhibitors. The terms ERK, JAK, JNK, Aurora, GSK, DRC and AKT as used herein refer to all isoforms of the respective enzymes, including but not limited to ERK1, ERK2, ERK3, ERK4, ERK5,

ERK6, ERK7, JAK1, JAK2, JAK3, JAK4, JNK1, JNK2, JNK3, Aurora, Aurora2, GSK3-alpha, GSK3-beta, KDR, AKT-1, AKT-2 and AKT-3.

The activity of the compounds as protein kinase inhibitors, such as ERK inhibitors, can be determined in vitro, in vivo or in a cell line. Using ERK as an example, in. The in vitro assay involves a method that detects either inhibition of kinase activity or inhibition of activated ERK ATPase activity. Alternatively, in vitro assays quantitatively inhibit the ability of the inhibitor to bind to ERK, and can be measured either by radiolabelling the inhibitor, isolating the inhibitor / ERK complex and determining the amount of bound radioactivity, or by a competitive experiment incubating the inhibitor with ERK. known radioligands. Any ERK isoform or type can be used, depending on which ERK isoform or type will be inhibited.

The compounds of the present invention were found to be potent ERK inhibitors by an enzymatic assay. These compounds have also been shown to inhibit ERK in cell proliferation assays. Details of both enzymatic and cell proliferation assays are provided in the Examples below.

The compounds of the present invention are also inhibitors of JNK, Aurora, GSK, DRC and AKT, as demonstrated by enzymatic assays. Details of these studies are provided in the Examples below. The compounds of the present invention are likely to inhibit other protein kinases.

Protein kinase inhibitors of the present invention, or pharmaceutical salts thereof, may be included in pharmaceutical compositions for administration to animals or humans. These pharmaceutical compositions effective for the treatment or prophylaxis of protein kinase dependent conditions and comprising an amount of a protein kinase inhibitor sufficient to detectably inhibit protein kinase activity and a pharmaceutically acceptable carrier are another embodiment of the present invention. As used herein, the term "inhibitory detectable" refers to a measurable change in activity between a sample containing an inhibitor and a sample containing only protein kinase.

As used herein, the term ERK-dependent condition refers to a disease state or other deleterious condition known to play a role in ERK. Such conditions include, but are not limited to, cancer, stroke, diabetes, hepatomegaly, cardiovascular disease including cardiac dilatation, Alzheimer's disease, mucoviscidosis, viral diseases, autoimmune diseases, atherosclerosis, restenosis, psoriasis, allergic diseases, disorders and hormone related disorders. The term cancer includes, but is not limited to, the following types of cancer: breast, ovarian, uterine, prostate, testicular, urinary and genital, esophageal, larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung, epidermoid carcinoma, small cell carcinoma, small cell carcinoma, adenocarcinoma, , thyroid, follicular carcinoma, undifferentiated carcinoma, nipple carcinoma, seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma, bile duct, renal carcinoma, myeloid disorders, lymphoid disorders, Hodgkins disease, hair cells, oral and pharyngeal, lip, tongue, oral, pharyngeal, small intestine, large intestine, and colon, cancer of the brain and central nervous system, and leukemia.

The compounds of the present invention are also useful as inhibitors of related kinases. The term related kinases refers to protein kinases that have residues similar to those surrounding the ERK binding site. Without claiming any theoretical generalization, the Applicants believe that this inhibitory activity is due to the strong similarity between the active sites of ERKs and related kinases. Comparison of ERK sequences with other kinases is possible through a computer program such as the Genetics Computer Group Best Match program. This program uses the local homology algorithm described by Smith and Waterman, A dvances in Applied Mathematics £ 482 (1981).

The kinases inhibited by the compounds of the present invention should have sequences identified by the above standard protein sequence comparison program corresponding to ERK residues 131, E33, G34, A35, Y36, G37, M38, V39, A52, K54, R67, T68, E71, L75, I84, I86, 1103, Q105, D106, L107, M108, E109, D111, K114, D149, K151, S153, N154, L156, C166, and D167 at 80% or more overlap. The degree of overlap can be determined using standard amino acid replacement tables such as those described by Dayhoff (Dayhoff MO, et al., Atlas of Protein Sequence and Structure, 1979) and BlosomHenikoff (Blosom-Henikoff S. and Henikoff JG, PNAS, 1992, 89). : 10915-10919). The term related kinases also includes kinases containing residues 80% or more overlapping with the following ERK residues 131, G37, A52, 1103, E109 and N154.

The compounds of the present invention are also suitable inhibitors of the JAK family kinases. Without claiming theoretical conclusions, the Applicants believe that this inhibitory activity is due to the close similarity of the ERK and JAK active centers as determined by standard methods described below.

X-ray crystalline studies of ERK-bound inhibitor crystals have shown that the three amino acid residues at the active site of ERK form the main hydrogen bonding interaction with this type of inhibitor. Those three amino acid residues are M108, D106 and Q105. The following amino acid numbering corresponds to Swiss-Prot Database Use Number # P28442. The Swiss-Prot database is an international database of protein sequences distributed by the European Institute of Bioinformatics (EBI) in Geneva, Switzerland. The database is available at www.ebi.ac.uk/swissprot.

The skeletal atoms of M108 and D106 and related interactions are common to all kinases. M108 gives both a hydrogen bond donor and acceptor, and D106 gives a hydrogen bond acceptor through its skeletal CO. An inhibitor that can form a hydrogen bond to one or more of these hydrogen bonding groups inside the active center is likely to bind to the enzyme and therefore inhibit it.

The glutamine residue of Q105 belongs to a subset of kinases that include ERK and JAK, as determined by analysis of comparison data obtained with the aforementioned computer program. Q105 forms the basic hydrogen bond by bonding the side chain CO. Model experiments revealed that for both ERK and JAK, the Ht-ring hydrogen bond donor is located at the hydrogen bond length from the Q105 residue. Because of such similar interactions in the active site, the ERK inhibitors of the present invention also inhibit JAK. Accordingly, these compounds are useful for treating JAK dependent conditions.

The term JAK-dependent condition as used herein refers to a disease state or other deleterious condition known to play a role in JAK. Such conditions include, but are not limited to, allergic conditions such as asthma and atopic dermatitis, autoimmune diseases such as SLE lupus and psoriasis, and conditions associated with organ transplants.

The compounds of the present invention are also suitable inhibitors of the JNK family kinases. Accordingly, these compounds are useful in the treatment of JNK dependent conditions.

As used herein, the term JNK dependent condition refers to a disease state or other deleterious condition known to play a role in JNK. Such conditions include, but are not limited to, neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, lateral amyotrophic sclerosis (ALS), epilepsy and seizures, Huntington's disease, traumatic brain injury, ischemic and hemorrhagic stroke, heart disease, , inflammatory diseases, allergic diseases, autoimmune diseases, bone destructive diseases such as osteoporosis, proliferative diseases, infections, viral disorders, cell death and hyperplasia, including reperfusion / ischemia in stroke, heart attack, organ hypoxia, thrombin-induced platelet aggregation, chronic myelogenous leukemia (CML), rheumatoid arthritis, asthma, osteoarthritis , liver problems including liver ischaemia, heart problems such as myocardial infarction and hyperemic heart failure, pathological immune conditions, including T cell activation, and neurodegenerative disorders.

The compounds of the present invention are also suitable as Aurora inhibitors. Accordingly, these compounds are useful for treating Aurora-dependent conditions.

As used herein, the term Aurora-dependent condition refers to a disease state or other deleterious condition known to play a role in Aurora. Such conditions include, but are not limited to, cancer. Cancer refers to, but is not limited to, cancer of the colon and ovaries.

The compounds of the present invention are also suitable inhibitors of GSK family kinases. Accordingly, these compounds are useful in the treatment of GSK-dependent conditions. As used herein, the term GSK-dependent condition refers to a disease state or other deleterious condition known to play a role in GSK. Such conditions include, but are not limited to, diabetes, Alzheimer's disease, neurodegenerative disorders, and CNS disorders such as manic depressive psychosis and schizophrenia.

The compounds of the present invention are also suitable inhibitors of the DRC family kinases. Accordingly, these compounds are useful in the treatment of DRC dependent conditions. As used herein, the term DRC-dependent condition refers to a disease state or other deleterious condition known to play a role in the DRC. Such conditions include, but are not limited to, cancers such as brain cancer, urinary and genital cancer, lymphatic system cancer, stomach cancer, laryngeal cancer, lung cancer, pancreatic cancer, breast cancer, Kaposi's sarcoma and leukemia; endometriosis, benign prostatic hyperplasia, vascular disorders such as restenosis and atherosclerosis; autoimmune diseases such as rheumatoid arthritis and psoriasis; ocular conditions such as proliferative or angiogenic retinopathy and degeneration; and inflammatory conditions such as contact dermatitis, asthma, and delayed hypersensitivity reactions.

The compounds of the present invention are also suitable inhibitors of AKT family kinases. Accordingly, these compounds are useful for treating AKT-dependent conditions. As used herein, the term AKT-dependent condition refers to a disease state or other deleterious condition known to play a role in AKT.

Such conditions include, but are not limited to, proliferative disorders, cancer, and neurodegenerative disorders.

In addition to the compounds of the present invention, pharmaceutically acceptable derivatives or prodrugs thereof may also be used in compositions for the treatment or prophylaxis of the above disorders.

A pharmaceutically acceptable derivative or prodrug means any pharmaceutically acceptable salt, ester, ester salt or other derivative of a compound of the invention that, when administered to a patient, can deliver, directly or indirectly, a compound of the present invention, or an inhibitory metabolite or residue. . Particularly desirable are derivatives or prodrugs that enhance the bioavailability of the compounds of the present invention when administered to a mammal (e.g., by making the oral compound more easily absorbed into the blood), or which accelerate delivery of the parent compound to a biological entity (e.g., brain or lymphatic system). .

Pharmaceutically acceptable prodrugs of the compounds of this invention include, but are not limited to, esters, amino acid esters, phosphate esters, metal salts, and sulfonate esters.

Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetates, adipates, alginates, aspartates, benzoates, benzenesulfonates, bisulfates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentane propionates, diglyuconates, dodecyl sulfates, ethanesulfonates, formates, fumarates, gliofumans, , hydrochlorides, hydrobromides, hydroiodides, 2-hydroxy ethanesulfonates, lactates, maleate, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinate, nitrate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate, and undecanoate. Other acids, such as oxalic, although not pharmaceutically acceptable per se, may be used in the preparation of salts used as intermediates in the preparation of the compounds of the present invention and their pharmaceutically acceptable acid addition salts.

Salts derived from suitable bases include alkali metals (such as sodium and potassium), alkaline earth metals (such as magnesium), ammonium, and N.⁺(C₁. ₄alkyl)₄ salts. The present invention also provides for the quaternization of any of the basic nitrogen-containing groups of the compounds disclosed herein. Such quaternization may result in water or oil soluble or dispersible products.

Pharmaceutically acceptable carriers that may be used in these pharmaceutical compositions include, but are not limited to, ionites, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate. , partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon oxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene ene-propylene block copolymers, polyethylene glycol.

The compositions of the present invention may be administered orally, parenterally, by aerosol inhalation, topically, rectally, nasally, orally, vaginally, or via an implanted container. The term parenteral as used herein includes methods of subcutaneous, intravenous, intramuscular, intra-articular, intra-articular, sternal, spinal, hepatic, and intracranial. Preferably, the compositions are administered orally, intraperitoneally, or intravenously.

The sterile injectable form of the compositions of the present invention may be an aqueous or oily suspension. Such suspensions may be formulated according to methods known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,340 butanediol. Suitable carriers and solvents that may be used include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any neutral fixed oil may be employed, including synthetic mono- or di-glycerides. Injectable preparations use fatty acids such as oleic acid and its glycerides, as well as natural pharmaceutically acceptable oils such as olive oil or castor oil, in particular in their polyoxyethylated forms. Solutions or suspensions of such oils may also contain a long-chain alcohol diluent or dispersant such as carboxymethylcellulose or a similar dispersant commonly used in the preparation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other widely used surfactants such as Tveen, Span and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms may also be used to formulate the formulation.

The compositions of the present invention may be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and maize starch. Typically, also tapping agents such as magnesium stearate are added. Suitable diluents for oral administration in capsule form include lactose and dry corn starch. When an aqueous suspension is required for oral administration, the active ingredient is mixed with emulsifying and suspending agents. If desired, sweetening, perfuming or coloring agents are sometimes added.

Alternatively, the compositions of the present invention may be administered in the form of suppositories for rectal administration. They may be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and will therefore melt in the outlet to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of the present invention may also be administered topically, especially when the treatment sought includes areas or organs readily accessible locally, including diseases of the eye, skin or lower gastrointestinal tract. Suitable topical formulations can readily be prepared for each of these areas or organs.

Topical administration to the lower gastrointestinal tract may be effected rectally in the form of suppositories (see above), or in the form of a suitable enema formulation. Topical transdermal patches may also be used.

For topical use, the pharmaceutical compositions may be in the form of a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum jelly, white petroleum jelly, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the pharmaceutical compositions may be in the form of a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, ceteraryl alcohol, 2-octyldodecanol, benzyl alcohol, and water.

For use in ophthalmology, pharmaceutical compositions may take the form of micronized suspensions in isotonic, pH-adjusted, sterile saline, with or without preservatives such as benzylalkonium chloride. Alternatively, for ophthalmic use, the pharmaceutical compositions may take the form of an ointment such as petroleum jelly.

The pharmaceutical compositions of the present invention may also be administered in the form of a nasal aerosol or by inhalation. Such compositions are prepared by methods well known in the art of dosage forms and may be prepared in the form of solutions containing sodium chloride, benzyl alcohol or other suitable preservative, absorption enhancers to improve bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

The amount of the protein kinase inhibitor of the present invention that can be mixed with the carriers to produce a unit dosage form will depend upon the subject being treated, the form of administration. Preferably, the compositions are in such a dosage form that the patient will receive about 0.01-100 mg / kg body weight / day of the inhibitor.

It will also be appreciated that the specific dosage and treatment regimen for each individual patient will depend upon a number of factors including patient activity, patient age, body weight, overall health, sex, diet, administration time, rate of elimination, drug combination and physician judgment. and the severity of the particular disease being treated. The amount of inhibitor will also depend on the particular compound in the composition.

The kinase inhibitors of the present invention may also include compositions for coating implantable medical devices, such as prostheses, artificial valves, vascular grafts, stents, and catheters. Vascular stents, for example, are used to treat restenosis (dilate the blood vessel after injury). However, patients using stents or other implantable devices are at risk of clot formation or platelet activation. These undesirable effects can be avoided or mitigated by pre-coating the device with a kinase inhibitor-containing composition. Suitable coatings and a common method for manufacturing coated implantable devices are described in U.S. Pat. 6099562; 5886026; and 5304121. Coatings are typical biocompatible polymeric materials such as hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic acid, ethylene vinyl acetate and mixtures thereof. The coatings may optionally be further coated with a suitable outer coating of fluorosilicone, polysaccharides, polyethylene glycol, phospholipids, or combinations thereof to provide controlled release properties. Implantable devices coated with a kinase inhibitor of the present invention are another embodiment of the present invention.

In another embodiment, the invention provides methods of treating or preventing ERK, JAK, JNK, Aurora, GSK, DRC or AKT-dependent conditions or diseases comprising administering to a patient one of the pharmaceutical compositions described above. As used herein, the term patient refers to a mammal, preferably a human.

Preferably, the method is used for the treatment or prophylaxis of a condition or disease selected from cancer such as breast, colon, prostate, skin, pancreas, brain, urinary tract, lymphatic system, stomach, larynx and lung, including lung adenocarcinoma and small cell lung cancer, stroke, diabetes, hepatomegaly, cardiomegaly, cardiovascular disease, Alzheimer's disease, mucoviscidosis and viral disease, or any specific disease or disorder, described above.

Depending on the particular conditions or condition of the disease to be treated or prevented, additional therapeutic agents, which are usually administered for the treatment or prevention of the disease, may be administered in addition to the inhibitors of the present invention. For example, chemotherapeutic agents or other antiproliferative agents may be combined with inhibitors of the present invention for the treatment of proliferative diseases and cancer. Examples of known chemotherapeutic agents include, but are not limited to, adriamycin, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, taxol, interferons, and platinum derivatives.

Other examples of agents with which the inhibitors of this invention may be combined include, but are not limited to, anti-inflammatory agents such as corticosteroids, TNF-blockers, IL-1 RA, azathioprine, cyclophosphamide, and sulfasalazine; immunomodulatory and immunosuppressive agents such as cyclosporine, tacrolimus, rapamycin, mofetil mycophenolate, interferons, corticosteroids, cyclophosphamide, azathioprine and sulfasalazine; neurotropic factors such as acetylcholinesterase inhibitor, MAO inhibitor, interferons, anticonvulsants, ion channel blockers, riluzole and antiparkinsonian agents; agents for the treatment of cardiovascular diseases such as beta-blockers, ACE inhibitors, diuretics, nitrates, calcium channel blockers and statins; agents for treating liver diseases such as corticosteroids, cholestyramine, interferons, and antiviral agents; agents for treating blood disorders such as corticosteroids, antileukemic agents and growth factors; agents for the treatment of diabetes, such as insulin, insulin analogues, alpha-glucosidase inhibitors, biguanides, and insulin sensitizers; and agents for the treatment of immunodeficiency disorders, such as gamma-globulin.

These additional agents may be administered separately from the inhibitor-containing composition as part of a multi-dose regimen. Alternatively, these agents may be part of a single dosage form mixed together with an inhibitor in a single composition.

In order that the invention described herein may be more fully understood, the following are examples. It is to be understood that these examples are merely illustrative of the invention and are not intended to limit it in any way.

EXAMPLES example

<img file="LT4981B_D0009.tif" />

2,2,2-T-Richloro-1- (4-phenylacetyl-1H-pyrrol-2-yl) -ethanone (1): In a dry flask, mix a minimum amount of dichloromethane (DCM) with phenylacetyl chloride (1 equivalent) and 2-trichloroacetylpyrrole (1). 1 equivalent). To the resulting solution was added aluminum trichloride (1 equivalent) at room temperature. After 2 or. the reaction mixture is passed directly through a silica gel column. Gradient elution from 10% ethyl acetate to 50% ethyl acetate in hexane gave compound 1 in 60% yield.¹1 H NMR (CDCl 3)₃δ 4.0 (s, 2H), 7.1-7.35 (m, 7H), 9.7 (m.s, NH). HPLC using Method B (as described in Example 5 below) shows a retention time of 4.9 min. LC / MS (M + 1) 330.2, (M-1) 328.1.

<img file="LT4981B_D0010.tif" />

4-Phenylacetyl-1H-pyrrole-2-carboxylic acid benzylamide (2): To a solution of compound (1) (1 equivalent) in DMF was added benzylamine (1.2 equivalent) at room temperature. After 24 or. the solvent is evaporated off and the crude product 2 is used without further purification. HPLC according to Method B (as described in Example 5 below) shows a retention time of 3.8 minutes. FIA / MS (M + 1) 319.3, (M-1) 317.2.

<img file="LT4981B_D0011.tif" />

4 - (- Dimethylamino-2-phenyl-acryloyl) -1H-pyrrole-2-carboxylic acid benzylamide (3); A solution of compound 2 (1 equivalent) in THF at room temperature is added (Me₂N) 2CHO / -Bu (3 equivalents). After 24 hours the solvent is evaporated off and the crude product 3 is used without further purification.¹1 H NMR (CDCl 3) δ 4.4 (s, 2H), 4.8 (s, NH), 6.8-7.4 (m, 13H).

example

<img file="LT4981B_D0012.tif" />

4- (Phenyl-1H-pyrazol-3-yl) -1H-pyrrole-2-carboxylic acid benzylamide (II ·

5): To a solution of compound 3 (1 equivalent) in ethanol at room temperature is added hydrazine hydrate (3 equivalents) and the resulting mixture is refluxed. After 12 or. the solvent is evaporated and the crude product is purified by preparative HPLC (reverse phase, 10-&gt; 90% MeCN in water; 15 min) to give the desired compound li-5. LC / MS (M + 1) 343.3, (M-1) 341.2.

example

According to the procedures described in Examples 1-4 above and illustrated in Scheme I, we prepared other compounds of Formula II. The characteristics of these compounds are given in Table 3 below and include LC / MS, HPLC and¹H NMR data.

For compounds marked by HPLC as A, the following procedure was used: A gradient of water: MeCN with 0.1% TFA (95: 5 → 0: 100) was run for 22 minutes at 1 ml / min and 214 nm. For those compounds where HPLC is designated B, the following procedure was used: gradient of water: MeCN with 0.1% TFA (90: 0 -&gt; 0: 100) for 8 min. At 1 ml / min and at 214 nm. Both Methods A and B use a YMC ODS-AQ 55 12A column, measuring 3.0 x 150 mm. The term T_ret (min) ¹¹ refers to the retention time in minutes associated with the compound using the indicated HPLC procedure.

Where was the use, ¹H NMR data are also reported in Table 3, where the notation Y indicates that ¹H NMR data were obtained and agree with the structure of the compound. The numbering of the compounds corresponds to that used in Table 1.

table. Characteristics of selected compounds

<td>The compound No.</td><td>M + 1</td><td>M-1</td><td>HPLC method</td><td>T_rel (min)</td><td>NMR</td>

<td> 11-41</td><td> 407.4</td><td> 405.4</td><td>A</td><td> 8.6</td><td>Y</td>

<td>II-42</td><td> 560.2</td><td> 558.1</td><td>A</td><td> 9.5</td><td> -</td>

<td>II-43</td><td> -</td><td> -</td><td>A</td><td> 10.5</td><td> -</td>

<td>II-44</td><td> 530.3</td><td> 528.2</td><td>A</td><td> 6.3</td><td> -</td>

<td>II-45</td><td> -</td><td> -</td><td>A</td><td> 9.8</td><td> -</td>

<td>il-46</td><td> -</td><td> -</td><td>A</td><td> 10.6</td><td> -</td>

<td>li-50</td><td> 377.4</td><td> -</td><td>A</td><td> 10.1</td><td>Y</td>

<td>II-52</td><td> 530.2</td><td> 528.2</td><td>A</td><td> 10.3</td><td> -</td>

<td>II-53</td><td> 378.4</td><td> 376.3</td><td>A</td><td> 7.4</td><td>Y</td>

<td> 11-56</td><td> 490.2</td><td> 488.1</td><td>A</td><td> 10.8</td><td> -</td>

<td> 11-58</td><td> -</td><td> -</td><td>A</td><td> 10.46</td><td> -</td>

<td>il-59</td><td> -</td><td> -</td><td>A</td><td> 9.1</td><td> -</td>

<td> 11-63</td><td> 361.4</td><td> 359.3</td><td>A</td><td> 9.5</td><td>Y</td>

<td> 11-65</td><td> -</td><td> -</td><td>A</td><td> 10.0</td><td> -</td>

<td> 11-67</td><td> 378.4</td><td> 376.3</td><td>A</td><td> 7.4</td><td>Y</td>

<td> 11-72</td><td> 451.5</td><td> 449.1</td><td>A</td><td> 10.15</td><td>Y</td>

<td> 11-80</td><td> 374.4</td><td> 372.3</td><td>A</td><td> 6.6</td><td> -</td>

<td> 11-83</td><td> 435.3</td><td> 433.4</td><td>A</td><td> 10.3</td><td> -</td>

<td> 11-85</td><td> -</td><td> -</td><td>A</td><td> 10.6</td><td> -</td>

<td> 11-86</td><td> -</td><td> -</td><td>A</td><td> 9.3</td><td> -</td>

<td> 11-88</td><td> 380.4</td><td> 378.3</td><td>A</td><td> 6.9</td><td> -</td>

<td> 11-89</td><td> -</td><td> -</td><td>A</td><td> 10.5</td><td> -</td>

<td> 11-91</td><td> -</td><td> -</td><td>A</td><td> 9.6</td><td> -</td>

<td>II-92</td><td> 377.4</td><td> 375.3</td><td>A</td><td> 10.2</td><td>Y</td>

<td>li-94</td><td> -</td><td> -</td><td>A</td><td> 9.0</td><td> -</td>

<td>li-97</td><td> 342.1</td><td> -</td><td>B</td><td> 3.8</td><td>Y</td>

<td>II-98</td><td> 380.4</td><td> 378.3</td><td>A</td><td> 6.7</td><td> -</td>

<td> 11-102</td><td> -</td><td> -</td><td>A</td><td> 10.3</td><td> -</td>

<td> 11-103</td><td> -</td><td> -</td><td>A</td><td> 10.6</td><td> -</td>

<td> 11-105</td><td> -</td><td> -</td><td>A</td><td> 9.3</td><td> -</td>

<td> 11-109</td><td> -</td><td> -</td><td>A</td><td> 7.9</td><td> -</td>

<td> 11-110</td><td> -</td><td> -</td><td>A</td><td> 10.3</td><td> -</td>

<td> 11-111</td><td> 361.4</td><td> 359.3</td><td>A</td><td> 9.4</td><td>Y</td>

<td> 11-113</td><td> -</td><td> -</td><td>A</td><td> 10.6</td><td> -</td>

<td> 11-116</td><td> 380.2</td><td> 378.4</td><td>A</td><td> 6.9</td><td> -</td>

<td> 11-117</td><td> 373.4</td><td> -</td><td>A</td><td> 9.0</td><td>Y</td>

<td> 11-119</td><td> 362.4</td><td> 371.4</td><td>A</td><td> 6.5</td><td> -</td>

<td> 11-120</td><td> 373.4</td><td> 371.4</td><td>A</td><td> 8.2</td><td> -</td>

<td> 11-122</td><td> -</td><td> -</td><td>A</td><td> 10.8</td><td> -</td>

<td> 11-123</td><td> -</td><td> -</td><td>A</td><td> 11.4</td><td></td>

<td> 11-126</td><td> -</td><td> -</td><td>A</td><td> 10.2</td><td></td>

<td> 11-128</td><td> - </td><td> -</td><td>A</td><td> 10.9</td><td> -</td>

<td> 11-130</td><td> -</td><td> -</td><td>A</td><td> 7.4</td><td> -</td>

<td> 11-133</td><td> -</td><td> -</td><td>A</td><td> 9.5</td><td> -</td>

<td> 11-134</td><td> 306.1</td><td> -</td><td>B</td><td> 3.5</td><td>Y</td>

<td> 11-135</td><td> 353.4</td><td> 351.4</td><td>A</td><td> 7.7</td><td> -</td>

<td> 11-137</td><td> 313.3</td><td> 311.2</td><td>A</td><td> 6.4</td><td>Y</td>

<td> 11-141</td><td> 380.4</td><td> 378.3</td><td>A</td><td> 6.7</td><td> -</td>

<td> 11-143</td><td> 280.1</td><td> -</td><td>B</td><td> 3.3</td><td>Y</td>

<td> 11-144</td><td> 336.4</td><td> -</td><td>B</td><td> 3.5</td><td> -</td>

<td> 11-145</td><td> 373.4</td><td> -</td><td>B</td><td> 2.8</td><td> -</td>

<td> 11-146</td><td> -</td><td> -</td><td>A</td><td> 10.5</td><td> -</td>

<td> 11-147</td><td> 362.4</td><td> -</td><td>B</td><td> 3.5</td><td> -</td>

<td> 11-148</td><td> 327.3</td><td> 325.2</td><td>A</td><td> 9.2</td><td>Y</td>

<td> 11-149</td><td> 332.4</td><td> -</td><td>B</td><td> 3.5</td><td> -</td>

<td> 11-150</td><td> 322.4</td><td> -</td><td>B</td><td> 3.2</td><td> -</td>

<td> 11-151</td><td> 316.2</td><td> 314.2</td><td>A</td><td> 10.3</td><td>Y</td>

<td> 11-152</td><td> -</td><td> -</td><td>A</td><td> 6.6</td><td> -</td>

<td> 11-153</td><td> 323.4</td><td> -</td><td>B</td><td> 2.3</td><td> -</td>

<td> 11-154</td><td> 343.3</td><td> -</td><td>B</td><td> 2.8</td><td> -</td>

<td> 11-158</td><td> 294.3</td><td> -</td><td>B</td><td> 3.4</td><td> -</td>

<td> 11-159</td><td> 335.4</td><td> -</td><td>B</td><td> 2.7</td><td> -</td>

<td> 11-161</td><td> 389.3</td><td> 387.2</td><td>A</td><td> 8.9</td><td> -</td>

<td> 11-162</td><td> 300.3</td><td> 298.2</td><td>A</td><td> 9.5</td><td>Y</td>

<td> 11-163</td><td> 366.5</td><td> 364.4</td><td>B</td><td> 6.0</td><td> -</td>

<td> 11-164</td><td> 297.3</td><td> -</td><td>A</td><td> 5.1</td><td>Y</td>

<td> 11-165</td><td> 322.3</td><td> 325.2</td><td>A</td><td> 9.7</td><td>Y</td>

<td> 11-167</td><td> 316.2</td><td> 314.2</td><td>A</td><td> 10.0</td><td>Y</td>

<td> 11-168</td><td> 312.3</td><td> 310.2</td><td>A</td><td> 8.6</td><td>Y</td>

<td> 11-169</td><td> 281.1</td><td> -</td><td>B</td><td> 3.9</td><td>Y</td>

<td> 11-170</td><td> 312.3</td><td> 310.2</td><td>A</td><td> 9.1</td><td>Y</td>

<td> 11-171</td><td> 300.3</td><td> 298.2</td><td>A</td><td> 9.4</td><td>Y</td>

<td> 11-172</td><td> 297.3</td><td> 295.7</td><td>A</td><td> 5.5</td><td>Y</td>

<td> 11-174</td><td> 449.3</td><td> 447.2</td><td>A</td><td> 12.5</td><td>Y</td>

<td> 11-175</td><td> 477.3</td><td> 475.3</td><td>A</td><td> 14.0</td><td>Y</td>

<td> 11-176</td><td> 374.4</td><td> 372.4</td><td>A</td><td> 6.3</td><td> -</td>

<td> 11-178</td><td> 362.4</td><td> 360.0</td><td>A</td><td> 6.6</td><td> -</td>

<td> 11-179</td><td> 374.4</td><td> 372.4</td><td>A</td><td> 6.3</td><td> -</td>

<td> 11-180</td><td> 404.4</td><td> 402.4</td><td>A</td><td> 6.4</td><td> -</td>

<td> 11-181</td><td> 380.2</td><td> 378.3</td><td>A</td><td> 6.7</td><td> -</td>

<td> 11-182</td><td> 355.4</td><td> 353.4</td><td>A</td><td> 7.7</td><td> -</td>

<td> 11-183</td><td> 367.4</td><td> 365.4</td><td>A</td><td> 7.4</td><td> -</td>

<td> 11-184</td><td> 355.4</td><td> 353.4</td><td>A</td><td> 7.9</td><td> -</td>

<td> 11-185</td><td> 367.4</td><td> 365.3</td><td>A</td><td> 7.5</td><td> ..</td>

<td> 11-186</td><td> 397.4 .</td><td> 395.4</td><td>A</td><td> 7.1</td><td></td>

<td> 11-187</td><td> 373.4</td><td> 371.4</td><td>A</td><td> 8.0</td><td> -</td>

<td> 11-188</td><td> 373.4</td><td> 371.4</td><td>A</td><td> 7.9</td><td> -</td>

<td> 11-189</td><td> 382.4</td><td> 380.4</td><td>A</td><td> 6.9</td><td> -</td>

<td> 11-190</td><td> 394.4</td><td> 392.4</td><td>A</td><td> 6.7</td><td> -</td>

<td> 11-191</td><td> 382.4</td><td> 380.4</td><td>A</td><td> 7.0</td><td></td>

<td> 11-192</td><td> 394.5</td><td> 392.4</td><td>A</td><td> 6.7</td><td> -</td>

<td> 11-193</td><td> 424.4</td><td> 422.4</td><td>A</td><td> 6.4</td><td> -</td>

<td> 11-194</td><td> 400.4</td><td> 398.4</td><td>A</td><td> 7.3</td><td> -</td>

<td> 11-195</td><td> 400.4</td><td> 398.4</td><td>A</td><td> 7.1</td><td> -</td>

<td> 11-196</td><td> 400.4</td><td> 398.4</td><td>A</td><td> 7.2</td><td> -</td>

<td> 11-197</td><td> 341.3</td><td> 339.2</td><td>A</td><td> 7.5</td><td> -</td>

<td> 11-198</td><td> 353.4</td><td> 351.4</td><td>A</td><td> 7.1</td><td> -</td>

<td> 11-199</td><td> 341.3</td><td> 339.2</td><td>A</td><td> 7.6</td><td> -</td>

<td>II-200</td><td> 383.4</td><td> 381.4</td><td>A</td><td> 6.9</td><td> -</td>

<td> 11-201</td><td> 359.4</td><td> 357.4</td><td>A</td><td> 8.0</td><td> -</td>

<td>li-202</td><td> 359.4</td><td> 357.4</td><td>A</td><td> 7.8</td><td> -</td>

<td>II-203</td><td> 359.4</td><td> 357.4</td><td>A</td><td> 7.7</td><td> -</td>

<td>II-204</td><td> 354.4</td><td> 352.4</td><td>A</td><td> 6.2</td><td> -</td>

<td>II-205</td><td> 366.4</td><td> 364.4</td><td>A</td><td> 5.9</td><td> -</td>

<td>II-206</td><td> 354.4</td><td> 352.4</td><td>A</td><td> 5.6</td><td> -</td>

<td>II-207</td><td> 396.4</td><td> 394.4</td><td>A</td><td> 5.9</td><td> -</td>

<td>II-208</td><td> 372.4</td><td> 370.4</td><td>A</td><td> 6.7</td><td> -</td>

<td>II-209</td><td> 372.4</td><td> 370.4</td><td>A</td><td> 6.5</td><td> -</td>

<td> 11-210</td><td> 372.4</td><td> 370.4</td><td>A</td><td> 6.4</td><td> -</td>

<td>II-237</td><td> -</td><td> -</td><td>A</td><td> 9.8</td><td> -</td>

<td>II-238</td><td> -</td><td> -</td><td>A</td><td> 11.6</td><td> -</td>

<td>II-239</td><td> -</td><td> -</td><td>A</td><td> 11.3</td><td> -</td>

<td>II-240</td><td> -</td><td> -</td><td>A</td><td> 7.5</td><td> -</td>

<td> 11-241</td><td> -</td><td> -</td><td>A</td><td> 12.0</td><td> -</td>

<td>II-242</td><td> -</td><td> -</td><td>A</td><td> 11.7</td><td> -</td>

<td>II-243</td><td> -</td><td> -</td><td>A</td><td> 11.6</td><td> -</td>

<td>II-244</td><td> 389.4</td><td> 387.3</td><td>A</td><td> 10.2</td><td> -</td>

<td>II-245</td><td> -</td><td> -</td><td>A</td><td> 10.6</td><td> -</td>

<td>II-246</td><td> 365.4</td><td> 363.4</td><td>A</td><td> 7.5</td><td> -</td>

<td>II-247</td><td> -</td><td> -</td><td>A</td><td> 7.2</td><td> -</td>

<td>II-248</td><td> -</td><td> -</td><td>A</td><td> 8.0</td><td> -</td>

<td>II-249</td><td> -</td><td> -</td><td>A</td><td> 7.7</td><td> -</td>

<td>II-267</td><td> -</td><td> -</td><td>A</td><td> 10.7</td><td> -</td>

<td>II-268</td><td> -</td><td> -</td><td>A</td><td> 10.0</td><td></td>

<td>II-269</td><td> -</td><td> -</td><td>A</td><td> 12.2</td><td> -</td>

<td>II-270</td><td> -</td><td> -</td><td>A</td><td> 12.3</td><td> -</td>

<td> 11-271</td><td> -</td><td> -</td><td>A</td><td> 9.3</td><td> -</td>

<td>II-272</td><td> -</td><td> -</td><td>A</td><td> 12.7</td><td></td>

<td>II-273</td><td> -</td><td> -</td><td>A</td><td> 12.7</td><td>r</td>

<td>II-274</td><td> -</td><td> -</td><td>A</td><td> 3.8</td><td> -</td>

<td>II-275</td><td> -</td><td> -</td><td>A</td><td> 10.3</td><td> -</td>

<td>II-276</td><td> -</td><td> -</td><td>A</td><td> 8.4</td><td> -</td>

<td>II-277</td><td> -</td><td> -</td><td>A</td><td> 10.6</td><td> -</td>

<td>II-278</td><td> -</td><td> -</td><td>A</td><td> 12.8</td><td> -</td>

<td>II-279</td><td> -</td><td> -</td><td>A</td><td> 11.4</td><td> . -</td>

<td>II-280</td><td> -</td><td> -</td><td>A</td><td> 7.9</td><td> -</td>

<td> 11-281</td><td> -</td><td> -</td><td>A</td><td> 11.5</td><td> -</td>

<td>ΙΙ-282</td><td> -</td><td> -</td><td>A</td><td> 8.6</td><td> -</td>

<td>II-283</td><td> -</td><td> -</td><td>A</td><td> 8.4</td><td> -</td>

<td>II-284</td><td> -</td><td> -</td><td>A</td><td> 12.2</td><td> -</td>

<td>II-290</td><td> -</td><td> -</td><td>A</td><td> 11.4</td><td> -</td>

<td> 11-291</td><td> -</td><td> -</td><td>A</td><td> 9.7</td><td> -</td>

<td>II-292</td><td> -</td><td> -</td><td>A</td><td> 9.1</td><td> -</td>

<td>II-293</td><td> 481.3</td><td> 479.3</td><td>A</td><td> 8.3</td><td> -</td>

<td>II-294</td><td> 455.4</td><td> 453.3</td><td>A</td><td> 6.9</td><td> -</td>

<td>II-295</td><td> -</td><td> -</td><td>A</td><td> 7.5</td><td> -</td>

<td>II-296</td><td> -</td><td> -</td><td>A</td><td> 8.9</td><td> -</td>

<td>II-298</td><td> 353.4</td><td> -</td><td>B</td><td> 2.8</td><td> -</td>

<td>II-299</td><td> 421.3</td><td> 423.2</td><td>A</td><td> 10.1</td><td> -</td>

example

ERK Inhibition Assay: Compounds are assayed for ERK2 inhibition using a coupled enzyme technique (Fox et al., (1998) Protein Sci. 7, 2249). In this procedure, a constant concentration (10 nM) of activated ERK2 was incubated with various compound concentrations in DMSO (2.5%) for 10 min. At 30 ° C in 0.1 M HEPES buffer, pH 7.5 containing 10 mM MgCl 2₂, 2.5 mM phosphoenolpyruvate, 200 μΜ NADH, 150 pg / ml pyruvate kinase, 50 pg / ml lactate dehydrogenase, and 200 pM erctide peptide. The reaction is started by the addition of 65 pM ATP. A decrease in absorbance at 340 nm is observed. From the rate-dependent concentration of inhibitor, IC is calculated₅₀.

Table I shows the activity of selected compounds of the present invention in ERK2 inhibition assays. The numbering of the compounds corresponds to that used in Table 1. The activity of the compounds is designated A if K, is less than 1 micromole; activity is denoted B if K i is between 1 and 5 micromoles and activity is denoted B if K is greater than 5 micromoles.

table. Selected compounds have inhibitory activity against ERK2

<td>No.</td><td>Activity</td><td>No.</td><td>Activity</td><td>No.</td><td>Activity</td>

<td> 11-1</td><td>A</td><td>II-2</td><td>C</td><td>II-3</td><td>A</td>

<td>II-4</td><td>A</td><td>II-5</td><td>A</td><td>II-6</td><td>A</td>

<td>II-7</td><td>C</td><td>II-8</td><td>C</td><td>II-9</td><td>C</td>

<td> 11-10</td><td>C</td><td> 11-11</td><td>C</td><td> 11-12</td><td>C</td>

<td> 11-13</td><td>A</td><td> 11-14</td><td>C</td><td> 11-16</td><td>C</td>

<td> 11-17</td><td>C</td><td> 11-18</td><td>A</td><td> 11-19</td><td>A</td>

<td>II-20</td><td>A</td><td> 11-21</td><td>C</td><td>II-22</td><td>A</td>

<td>II-23</td><td>A</td><td>II-24</td><td>A</td><td>II-25</td><td>C</td>

<td>il-26</td><td>A</td><td>II-27</td><td>A</td><td>II-28</td><td>A</td>

<td>II-29</td><td>C</td><td>II-30</td><td>A</td><td> 11-31</td><td>C</td>

<td>II-39</td><td>A</td><td>II-40</td><td>A</td><td> 11-41</td><td>A</td>

<td>II-42</td><td>A</td><td>II-43</td><td>A</td><td>II-44</td><td>A</td>

<td>II-45</td><td>A</td><td>II-46</td><td>A</td><td>II-47</td><td>A</td>

<td>II-48</td><td>A</td><td>II-49</td><td>A</td><td>II-50</td><td>A</td>

<td> 11-51</td><td>A</td><td>II-52</td><td>A</td><td>II-53</td><td>A</td>

<td>II-54</td><td>A</td><td>II-55</td><td>A</td><td>II-56</td><td>A</td>

<td>II-57</td><td>A</td><td>II-58</td><td>A</td><td>II-59</td><td>A</td>

<td>II-60</td><td>A</td><td> 11-61</td><td>A</td><td>II-62</td><td>A</td>

<td>II-63</td><td>A</td><td>II-64</td><td>A</td><td>II-65</td><td>A</td>

<td>II-66</td><td>A</td><td>II-67</td><td>A</td><td>II-68</td><td>A</td>

<td>II-69</td><td>A</td><td>II-70</td><td>A</td><td> 11-71</td><td>A</td>

<td>II-72</td><td>A</td><td>II-73</td><td>A</td><td>II-74</td><td>A</td>

<td>II-75</td><td>A</td><td>II-76</td><td>A</td><td>II-77</td><td>A</td>

<td>II-78</td><td>A</td><td>II-79</td><td>A</td><td>II-80</td><td>A</td>

<td> 11-81</td><td>A</td><td>II-82</td><td>A</td><td>II-83</td><td>A</td>

<td>II-84</td><td>A</td><td>II-85</td><td>A</td><td>II-86</td><td>A</td>

<td>II-87</td><td>A</td><td>II-88</td><td>A</td><td>II-89</td><td>A</td>

<td>II-90</td><td>A</td><td> 11-91</td><td>A</td><td>II-92</td><td>A</td>

<td>II-93</td><td>A</td><td>II-94</td><td>A</td><td>II-95</td><td>A</td>

<td>II-96</td><td>A</td><td>II-97</td><td>A</td><td>II-98</td><td>1</td>

<td>II-99</td><td>A</td><td> 11-100</td><td>A</td><td> 11-101</td><td>_i</td>

<td> 11-102</td><td>A</td><td> 11-103</td><td>A</td><td> 11-104</td><td>A s</td>

<td> 11-105</td><td>A</td><td> 11-106</td><td>A</td><td> 11-107</td><td>A</td>

<td> 11-108</td><td>A</td><td> 11-109</td><td>A</td><td> 11-110</td><td>A</td>

<td> 11-111</td><td>A</td><td> 11-112</td><td>A</td><td> 11-113</td><td>A</td>

<td> 11-114</td><td>A</td><td> 11-115</td><td>A</td><td> 11-116</td><td>B</td>

<td> 11-117</td><td>B</td><td> 11-118</td><td>B</td><td> 11-119</td><td>B</td>

<td> 11-120</td><td>B</td><td> 11-121</td><td>B</td><td> 11-122</td><td>B</td>

<td> 11-123</td><td>B</td><td> 11-124</td><td>B</td><td> 11-125</td><td>B</td>

<td> 11-126</td><td>B. I</td><td> 11-127</td><td>B</td><td> 11-128</td><td>B</td>

<td> 11-129</td><td>B</td><td> 11-130</td><td>B</td><td> 11-131</td><td>B</td>

<td> 11-132</td><td>B</td><td> 11-133</td><td>B</td><td> 11-134</td><td>B</td>

<td> 11-135</td><td>B</td><td> 11-136</td><td>B</td><td> 11-137</td><td>B</td>

<td> 11-138</td><td>B</td><td> 11-139</td><td>B</td><td> 11-140</td><td>B</td>

<td> 11-141</td><td>B</td><td> 11-142</td><td>B</td><td> 11-143</td><td>B</td>

<td> 11-144</td><td>B</td><td> 11-145</td><td>B</td><td> 11-146</td><td>B</td>

<td> 11-147</td><td>B</td><td> 11-148</td><td>B</td><td> 11-149</td><td>B</td>

<td> 11-150</td><td>B</td><td> 11-151</td><td>B</td><td> 11-152</td><td>B</td>

<td> 11-153</td><td>B</td><td> 11-154</td><td>B</td><td> 11-155</td><td>B</td>

<td> 11-156</td><td>B</td><td> 11-157</td><td>B</td><td> 11-158</td><td>B</td>

<td> 11-159</td><td>B</td><td> 11-160</td><td>B</td><td> 11-161</td><td>C</td>

<td> 11-162</td><td>C</td><td> 11-163</td><td>C</td><td> 11-164</td><td>C</td>

<td> 11-165</td><td>C</td><td> 11-166</td><td>C</td><td> 11-167</td><td>C</td>

<td> 11-168</td><td>C</td><td> 11-169</td><td>C</td><td> 11-170</td><td>C</td>

<td> 11-171</td><td>C</td><td> 11-172</td><td>C</td><td>il-285</td><td>B</td>

<td>il-286</td><td>C</td><td>il-287</td><td>C</td><td>il-288</td><td>B</td>

<td>il-289</td><td>C</td><td>il-290</td><td>B</td><td> 11-291</td><td>C</td>

<td>II-292</td><td>C</td><td>II-293</td><td>C</td><td>II-294</td><td>C</td>

<td>II-295</td><td>C</td><td>il-296</td><td>C</td><td>il-297</td><td>C</td>

<td>il-298</td><td>C</td><td>il-299</td><td>C</td><td></td><td></td>

example

Cell proliferation assay for ERK inhibition: Compounds were assayed for ERK inhibition using cell proliferation data. In these studies, complete medium was prepared by adding 10% fetal calf serum and penicillin / streptomycin solution to RPMI 1640 medium (JRH Biosciences). Colon cancer cells (HT-29 cell line) are added to each of 84 wells in a 96-well plate at 10,000 cells / well / 150 μΙ. Incubate at 37 ° C for 2 hours to allow cells to adhere to the plate. Preparation of the test compound in complete medium by serial dilution to such concentrations; 20 μΜ, 6.7 μΜ, 2.2 μΜ, 0.74 μΜ, 0.25 μΜ, and 0.08 μΜ. Add 50 μΙ of the test compound solution to each well containing 72 cells. Only 200 μ | is added to the other 12 wells complete medium to form a control group for maximum proliferation measurements. Full media is added to the remaining 12 wells to provide a carrier control group to measure the background. The plates are incubated at 37 ° C for 3 days. Freshly made³H-thymidine solution (1 mCi / ml, New England Nuclear, Bostos, MA) was diluted to 20 pCi / ml in RPMI medium, then 20 μΙ of this solution was added to each well. The plates are incubated for another 8 hours at 37 ° C and then measured in a liquid scintillation counter.³Absorption of H-thymidine.

Compounds of the present invention that inhibit ERK in colorectal cancer cell proliferation assays with IC₅o. less than 10 μΜ, II-43, II-48 and II-45.

example

JAK Inhibition Assay: The inhibition of JAK by the compounds of the present invention can be assayed using the procedures described in GRBrovvn et al., Bioorg. Med. Chem. Lett. 2000, vol. 10, p. 575-579. The Maxisorb plates are first coated with Poly (Glu, Ala, Tyr) 6: 3: 1 at 4 ° C, then rinsed with 0.5% sodium chloride in phosphate buffer and 2 μΜ ATP, 5 mM MgCl 2 are added.₂ and a solution of the compound in DMSO. The reaction is run on JAK and the plates are incubated at 30 ° C for 60 minutes. The plates were then washed with PBST, 100 μΙ HRP-conjugated antibody was added, and the plate was incubated at 30 ° C for 90 minutes. The plate is rinsed again with PBST, 100 μΙ TMB solution is added, and the plates are then incubated at 30 ° C for 30 minutes. Sulfuric acid (100 μΙ in 1 M almost) was added to stop the reaction and the optical density of the plate was measured at 450 nm to determine the IC₅o size.

example

JNK Inhibition Assay: To evaluate the ability of compounds to inhibit JNK, they were assayed using a coupled enzyme spectrophotometric method. In this procedure, a constant concentration (10 nM) of activated JNK was incubated with various concentrations of the compound in DMSO in fresh 0.1 M HEPES buffer, pH 7.5 containing 10 mM MgCl₂, 2.5 mM phosphoenolpyruvate, 200 μΜ NADH, 150 pg / mL pyruvate kinase, 50 pg / mL lactate dehydrogenase, and 200 μΜ EGF receptor peptide (containing the sequence KRELVEPLTPSGEAPNOALLR). The resulting mixture was incubated at 30 ° C for 10 minutes, after which the reaction was started by addition of 10 μΜ ATP. A decrease in absorbance at 340 nm with time was observed and the resulting data were processed using a competitive inhibition model to determine K i.

Table I shows the activity of the compounds of this invention in JNK inhibition assays.

The numbering of the compounds corresponds to that used in Table 1. The activity of the compounds is designated A if K, is less than 1 micromole; activity is denoted B if K is between 1 and 5 micromoles and activity is denoted B if K is greater than 5 micromoles.

table. Inhibited activity against JNK by selected compounds

<td>No.</td><td>Activity</td><td>No.</td><td>Activity</td>

<td>II-39</td><td>B</td><td>II-48</td><td>A</td>

<td>II-40</td><td>A</td><td> 11-51</td><td>B</td>

<td>II-43</td><td>A</td><td>li-55</td><td>A</td>

<td>II-46</td><td>A</td><td> 11-104</td><td>B</td>

<td>II-47</td><td>B</td><td> 11-112</td><td>C</td>

example

Aurora Inhibition Assay: To evaluate the ability of compounds to inhibit Aurora, they were assayed using a standard bound enzyme method. According to this procedure, fresh 0.1 M HEPES buffer, pH 7.5 containing 10 mM

MgCl₂, 2.5 mM phosphoenolpyruvate, 300 μΜ NADH, 30 pg / mL pyruvate kinase, 10 pg / mL lactate dehydrogenase, 40 μΜ ATP, and 800 μΜ peptide (containing LRRASLG, American Peptide, Sunnyvale, CA) 30 μΜ compound solution in DMSO was added. The resulting mixture was incubated at 30 ° C for 10 minutes, after which the reaction was started by the addition of 10 μΙ of 70 nM Aurora solution in 1 mM DTT. Reaction rate is obtained from absorbance at 340 nm over 5 minutes readings using a Bio Rad Ultramark plate reader (Hercules, CA). The IC50 value was determined from the rate-dependent concentration of inhibitor.

Table 4 shows the activity of the compounds of the present invention in Aurora2 inhibition assays. The numbering of the compounds corresponds to that used in Table 1. The activity of the compounds is denoted by A if IC₅₀ size less than 5 micromoles; activity is denoted B if the IC50 is between 5 and 10 micromoles and activity denoted C if JC₅₀ size greater than 10 micromoles.

table. Inhibitory activity against Aurora2 of selected compounds

<td>No.</td><td>Activity</td><td>No.</td><td>Activity</td><td>No.</td><td>Activity</td>

<td>II-48</td><td>A</td><td>II-89</td><td>A</td><td> 11-211</td><td>B</td>

<td> 11-51</td><td>B</td><td>II-93</td><td>A</td><td> 11-212</td><td>B</td>

<td>II-54</td><td>B</td><td>II-98</td><td>B</td><td> 11-213</td><td>B</td>

<td>II-57</td><td>A</td><td>II-99</td><td>A</td><td> 11-214</td><td>B</td>

<td> 11-61</td><td>A</td><td> 11-101</td><td>A</td><td> 11-215</td><td>B</td>

<td>II-64</td><td>A</td><td> 11-103</td><td>B</td><td> 11-216</td><td>B</td>

<td>II-66</td><td>B</td><td> 11-106</td><td>B</td><td> 11-218</td><td>B</td>

<td>II-70</td><td>B</td><td> 11-108</td><td>B</td><td>II-228</td><td>A</td>

<td>II-72</td><td>B</td><td> 11-112</td><td>A</td><td>II-252</td><td>B</td>

<td>II-76</td><td>A</td><td> 11-113</td><td>A</td><td>II-254</td><td>A</td>

<td>II-77</td><td>A</td><td> 11-114</td><td>A</td><td>II-255</td><td>B</td>

<td>II-80</td><td>C</td><td> 11-115</td><td>A</td><td>II-258</td><td>C</td>

<td> 11-81</td><td>A</td><td> 11-141</td><td>A</td><td>II-259</td><td>B</td>

<td>II-82</td><td>A</td><td> 11-142</td><td>A</td><td>II-260</td><td>C</td>

<td>II-85</td><td>B</td><td> 11-181</td><td>B</td><td>II-262</td><td>B</td>

<td>II-88</td><td>B</td><td> 11-188</td><td>C</td><td>II-266</td><td>B</td>

example

GSK-3 Inhibition Assay: To evaluate the ability of compounds to inhibit glycogen synthase kinase 3 (GSK-3), they were assayed using the standard coupled enzyme method (Fox et al., (1998) Protein Scil, 2249). In this procedure, fresh 0.1 M HEPES buffer, pH 7.5, containing 10 mM MgCl 2 was used₂, 25 mM NaCl, 2.5 mM phosphoenolpyruvate, 300 μΜ NADH, 1 mM DTT, 30 µg / ml pyruvate kinase, 10 pg / ml lactate dehydrogenase, 300 μΜ peptide (containing HSSPHQp-SEDEEE, American Peptide, Sunnyvale, CA) and 60 nM 30 μΜ of the test compound in DMSO was added to GSK-3. The resulting mixture was incubated at 30 ° C for 5 minutes, after which the reaction was started by addition of 10 μΜ ATP. Reaction speeds are obtained from absorbance at 340 nm over 5 minute scans using a Molecular Devices plate reader (Sunnyvale, CA). IC₅and size was determined from the rate-dependent concentration of inhibitor.

Table 4 shows the activity of the compounds of the present invention in GSK-3 inhibition assays. The numbering of the compounds corresponds to that used in Table 1. The activity of the compounds is denoted by jei if IC₅₀ size less than 10 micromoles; activity is denoted B if IC₅and the size is between 10 and 20 micromoles and the activity is denoted by C if IC₅₀ size greater than 20 micromoles.

table. Inhibitory activity of selected compounds on GSK-3

<td>No.</td><td>Activity</td><td>No.</td><td>Activity</td><td>No.</td><td>Activity</td>

<td>II-89</td><td>C</td><td> 11-115</td><td>C</td><td>II-263</td><td>A</td>

<td>II-93</td><td>C</td><td> 11-127</td><td>B</td><td> 11-271</td><td>A</td>

<td>li-94</td><td>C</td><td> 11-199</td><td>C</td><td>il-278</td><td>A</td>

<td>II-99</td><td>A</td><td> 11-214</td><td>C</td><td> -</td><td> -</td>

<td> 11-108</td><td>B</td><td>il-227</td><td>B</td><td> -</td><td> -</td>

example

DRC Inhibition Assay: To evaluate the ability of compounds to inhibit glycogen synthase kinase 3 (GSK-3), they were assayed using the standard coupled enzyme method (Fox et al, (1998) Protein Sci 7, 2249). Assays are performed in a mixture of 200 mM HEPES 7.5; 10 mM MgCl2₂, 25 mM NaCl, 1 mM DTT, and 1.5% DMSO. The final substrate concentration in the assay was 300 μΜ ATP (Sigma Chemicals) and 10 μΜ poly E4Y (Sigma). Assays were performed at 37 ° C and 30 nM DRC. The final concentrations of the bound enzyme system were 2.5 mM phosphoenolpyruvate, 200 μΜ NADH, 30 μρ / ιτιΙ pyruvate kinase and 10 μg / ml lactate dehydrogenase.

A fresh assay buffer was prepared containing all of the above reagents except ATP and the test compound. Add 177 μΙ of fresh solution to a 96-well plate, then add 3 μΙ of 2 mM DMSO with test compound (final compound concentration 30 μΜ). The plate is incubated at 37 ° C for 10 minutes and the reaction is initiated by the addition of 20 μΙ ATP (final concentration 300 μΜ). . Reaction rates were calculated from data read over 5 min at 37 ° C using a Molecular Devices plate reader (Sunnyvale, CA). Compounds showing greater than 50% inhibition against standard wells containing test mixture and DMSO without test compound are titrated IC₅while setting.

Selected compounds of the present invention that inhibit KDR 2µΜ at concentrations greater than 40% are: II-43, li-48, li-304, and II-305.

example

AKT Inhibition Assay: To evaluate the ability of compounds to inhibit AKT, they were assayed using the standard bound enzyme method (Fox et al., (1998) Protein Sci7, 2249). Assays are performed in a mixture of 100 mM HEPES 7.5; 10 mM MgCl2₂, 25 mM NaCl, 1 mM DTT, and 1.5% DMSO. The final substrate concentration in the assay was 170 μΜ ATP (Sigma Chemicals) and 200 μΜ peptide (RPRAATF, American Peptide, Sunnyvale, CA). Assays were performed at 30 ° C and 45 nM AKT. The final concentrations of the bound enzyme system were 2.5 mM phosphoenolpyruvate, 300 μΜ NADH, 30 μg / mL pyruvate kinase, and 10 μρ ^ Ι lactate dehydrogenase.

Fresh assay buffer was prepared containing all of the above reagents except AKT, DTT and test compound. 56 μΙ of fresh solution is added to a 384-well plate, followed by addition of 1 μΙ of 2 mM DMSO with test compound (final compound concentration 30 μΜ). The plate is incubated at 30 ° C for 10 minutes and the reaction is initiated by adding 10 μΙ enzyme (45 nM final concentration). . Reaction rates were calculated from data read over 5 min at 30 ° C using a BioRad Ultramark plate reader (Hercules, CA). Compounds showing greater than 50% inhibition against standard wells containing test mixture and DMSO without test compound are titrated IC₅while setting.

Selected compounds of the present invention that inhibit AKT are: II-89, II-94, and II-305.

Although we have described several embodiments of the present invention, it is clear that the main examples may be applied to other embodiments using the compounds and methods of the present invention. It is, therefore, to be understood that the scope of the present invention will be defined more by the appended definition than by the specific embodiments provided in the form of examples.