CA2779184A1 - Protein kinase inhibitors - Google Patents

Abstract

The present invention relates to a novel family of inhibitors of protein kinases.
In particular, the present invention relates to inhibitors of the members of the Tec and Src protein kinase families, more particularly Btk.

Description

PROTEIN KINASE INHIBITORS
FIELD OF INVENTION
The present invention relates to a novel family of inhibitors of protein kinases. In particular, the present invention relates to inhibitors of the members of the Tec and Src protein kinase families, more particularly Btk.
BACKGROUND OF THE INVENTION
Protein kinases are a large group of intracellular and transmembrane signaling proteins in eukaryotic cells. These enzymes are responsible for transfer of the terminal (gamma) phosphate from ATP to specific amino acid residues of target proteins. Phosphorylation of specific tyrosine, serine or threonine amino acid residues in target proteins can modulate their activity leading to profound changes in cellular signaling and metabolism. Protein kinases can be found in the cell membrane, cytosol and organelles such as the nucleus and are responsible for mediating multiple cellular functions including metabolism, cellular growth and division, cellular signaling, modulation of immune responses, and apoptosis. The receptor tyrosine kinases are a large family of cell surface receptors with protein tyrosine kinase activity that respond to extracellular cues and activate intracellular signaling cascades (Plowman et al. (1994) DN&P, 7(6):334-339).
Aberrant activation or excessive expression of various protein kinases are implicated in the mechanism of multiple diseases and disorders characterized by benign and malignant proliferation, excess angiogenesis, as well as diseases resulting from inappropriate activation of the immune system. Thus, inhibitors of select kinases or kinase families are expected to be useful in the treatment of cancer, autoimmune diseases, and inflammatory conditions including, but not limited to: solid tumors, hematological malignancies, arthritis, graft versus host disease, lupus erythematosus, psoriasis, colitis, illeitis, multiple sclerosis, uveitis, coronary artery vasculopathy, systemic sclerosis, atherosclerosis, asthma, transplant rejection, allergy, dermatomyositis, pemphigus and the like.

Examples of kinases that can be targeted to modulate disease include receptor tyrosine kinases such as members of the platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR) families and intracellular proteins such as members of the Syk, SRC, and Tec families of kinases.
Tec kinases are non-receptor tyrosine kinases predominantly, but not exclusively, expressed in cells of hematopoietic origin (Bradshaw 3M. Cell Signal. 2010,22:1175-84). The Tec family includes Tec, Bruton's tyrosine kinase (Btk), inducible T-cell kinase (Itk), resting lymphocyte kinase (RIk/Txk), and bone marrow-expressed kinase (Bmx/Etk). Btk is a Tec family kinase which is important in B-cell receptor signaling. Btk is activated by Src-family kinases and phosphorylates PLC gamma leading to effects on B-cell function and survival. Additionally, Btk is important in signal transduction in response to immune complex recognition by macrophage, mast cells and neutrophils. Btk inhibition is also important in survival of lymphoma cells (Herman, SEM. Blood 2011, 117:6287-6289) suggesting that inhibition of Btk may be useful in the treatment of lymphomas. As such, inhibitors of Btk and related kinases are of great interest as anti-inflammatory as well as anti-cancer agents.
cSRC is the prototypical member of the SRC family of tyrosine kinases which includes Lyn, Fyn, Lck, Hck, Fgr, Blk, Syk, Yrk, and Yes. cSRC is critically involved in signaling pathways involved in cancer and is often over-expressed in human malignancies (Kim LC, Song L, Haura EB. Nat Rev Clin Oncol. 2009 6(10):587-9). The role of cSRC in cell adhesion, migration and bone remodeling strongly implicate this kinase in the development and progression of bone metastases. cSRC is also involved in signaling downstream of growth factor receptor tyrosine kinases and regulates cell cycle progression suggesting that cSRC inhibition would impact cancer cell proliferation.
Additionally, inhibition of SRC family members may be useful in treatments designed to modulate immune function. SRC family members, including Lck, regulate T-cell receptor signal transduction which leads to gene regulation events resulting in cytokine release, survival and proliferation. Thus, inhibitors of Lck have been keenly sought as immunosuppressive agents with potential application in graft rejection and T-cell mediated autoimmune disease (Martin et al. Expert Opin Ther Pat. 2010, 20:1573-93).
Inhibition of kinases using small molecule inhibitors has successfully led to several approved therapeutic agents used in the treatment of human conditions. Herein, we disclose a novel family of kinase inhibitors. Further, we demonstrate that modifications in compound substitution can influence kinase selectivity and therefore the biological function of that agent.
SUMMARY OF THE INVENTION
The present invention relates to a novel family of kinase inhibitors.
Compounds of this class have been found to have inhibitory activity against members of the Tec and Scr protein kinase families, more particularly Btk.
One aspect of the present invention is directed to a compound of Formula 1:
NH2 y_z_w N N, Formula 1 wherein n is an integer from 0 to 2;
X is selected from the group consisting of:
1) hydrogen, 2) alkyl, 3) heteroalkyl, 4) carbocyclyl, 5) heterocyclyl;
wherein the alkyl, heteroalkyl, carbocyclyl and heterocyclyl may be further substituted by the groups consisting of:
1) hydroxy, 2) alkoxy, 3) alkyl, 4) -0C(0)R4, 5) -0C(0)NR5R6, 6) -C(0)R4, 7) -C(0)NR5R6, 8) -NR5R6, 9) -NR2C(0)R4, 10) -NR2S(0)nR4, 11) -NR2C(0)NR5R6;
Y is selected from:
1 o (X2)n Z is selected from:
X1 and X2 are independently selected from hydrogen, halogen or cyano;

W is independently selected from:
1) alkyl, 2) aralkyl, 3) heteroaralkyl, 4) -0R3, 5) -0C(0)R4, 6) -0C(0)NR5R6, 7) -CH2O-R4, 8) -NR5R6, 9) -NR2C(0)R4, 10) -NR2S(0)R4, 11) -NR2C(0)NR5R6;
wherein the alkyl, aralkyl and heteraralkyl may be further substituted;
R2 is selected from hydrogen or alkyl;
R3 is selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, aralkyl or heteroaralkyl;
R4 is selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, carbocyclyl, heterocyclyl, aryl or heteroaryl;
R5 and R6 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl or R5 and R6 can be fused to form a 3 to 8 membered heterocyclyl ring system.

Preferred embodiment includes compounds of Formula 1 where W is selected from -0R3 and R3 is selected from substituted or unsubstituted aralkyl, or substituted or unsubstituted heteroaralkyl.
Preferred embodiment includes compounds of Formula 1 where W is selected from the group consisting of:
N, ¨0 = ¨0 II 00 ¨0 ¨0 Ho ¨0 /
lit ¨0 N
rN rN
--0/ / \
s-or Preferred embodiment includes compounds of Formula 1 where X is selected from the group consisting of:

JVVV JVL/11 'NW
JVV,/
.Aftn/
VVIIV
NH
H, Me, acetyl, cis.? 0 0 , 0 ONV
JVW

Ny0< N rN) ( , ./VVV
N) , ,or More preferred embodiment includes compounds of Formula 1 where W is selected from the group consisting of:
or More preferred embodiment includes compounds of Formula 1 where Z is selected from the group consisting of:
j Another aspect of the present invention provides a pharmaceutical composition comprising an effective amount of a compound of Formula 1 and a pharmaceutically acceptable carrier, diluent or excipient.
In another aspect of the present invention, there is provided a use of the compound of Formula 1 as an inhibitor of protein kinase, more particularly, as an inhibitor of Btk.

Another aspect of the present invention provides a method of modulating kinase function, the method comprising contacting a cell with a compound of the present invention in an amount sufficient to modulate the enzymatic activity of a given kinase or kinases, such as Btk, thereby modulating the kinase function.
Another aspect of the present invention provides a method of modulating the target kinase function, the method comprising a) contacting a cell with a compound of the present invention in an amount sufficient to modulate the target kinase function, thereby b) modulating the target kinase activity and signaling.
Another aspect of the present invention provides a probe, the probe comprising a compound of Formula 1 labeled with a detectable label or an affinity tag. In other words, the probe comprises a residue of a compound of Formula 1 covalently conjugated to a detectable label. Such detectable labels include, but are not limited to, a fluorescent moiety, a chemiluminescent moiety, a paramagnetic contrast agent, a metal chelate, a radioactive isotope-containing moiety, or biotin.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention relates to novel kinase inhibitors. These compounds are found to have activity as inhibitors of protein kinases: including members of the tyrosine kinases Aurora, SRC (more specifically Lck) and Tec (more specifically Btk) kinase families.
Compounds of the present invention may be formulated into a pharmaceutical composition which comprises an effective amount of a compound of Formula 1 with a pharmaceutically acceptable diluent or carrier.
For example, the pharmaceutical compositions may be in a conventional pharmaceutical form suitable for oral administration (e.g., tablets, capsules, granules, powders and syrups), parenteral administration (e.g., injections (intravenous, intramuscular, or subcutaneous)), drop infusion preparations, inhalation, eye lotion, topical administration (e.g., ointment), or suppositories. Regardless of the route of administration selected the compounds may be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.
The phrase "pharmaceutically acceptable" is employed herein to refer to those ligands, materials, cornpositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material.

Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation, including the active ingredient, and not injurious or harmful to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch, potato starch, and substituted or unsubstituted (3-cyclodextrin; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc;
(8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
The term "pharmaceutically acceptable salt" refers to the relatively non-toxic, inorganic and organic acid addition salts of the compound(s). These salts can be prepared in situ during the final isolation and purification of the compound(s), or by separately reacting a purified compound(s) in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, laurylsulphonate salts, and amino acid salts, and the like (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:
1-19).
In other cases, the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases.
The term "pharmaceutically acceptable salts" in these instances refers to the relatively non-toxic inorganic and organic base addition salts of a compound(s). These salts can likewise be prepared in situ during the final isolation and purification of the compound(s), or by separately reacting the purified compound(s) in its free acid form with a suitable base, such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary, or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts, and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like (see, for example, Berge et al., supra).
As used herein, the term "affinity tag" means a ligand or group, linked either to a compound of the present invention or to a protein kinase domain, that allows the conjugate to be extracted from a solution.
The term "alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups, including haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl, etc. Representative alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, cyclopropylmethyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. The terms "alkenyl" and "alkynyl" refer to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
Representative alkenyl groups include vinyl, propen-2-yl, crotyl, isopenten-2-yl, 1,3-butadien-2-y1), 2,4-pentadienyl, and 1,4-pentadien-3-yl.
Representative alkynyl groups include ethynyl, 1- and 3-propynyl, and 3-butynyl. In certain preferred embodiments, alkyl substituents are lower alkyl groups, e.g., having from 1 to 6 carbon atoms. Similarly, alkenyl and alkynyl preferably refer to lower alkenyl and alkynyl groups, e.g., having from 2 to 6 carbon atoms. As used herein, "alkylene" refers to an alkyl group with two open valencies (rather than a single valency), such as -(0-12)1-10- and substituted variants thereof.
The term "alkoxy" refers to an alkyl group having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxy.
The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group, thereby forming an ether.
The terms "amide" and "amido" are art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:

wherein R9, R1 are as defined above. Preferred embodiments of the amide will not include imides, which may be unstable.
The terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, e.g., a moiety that can be represented by the general formulae:

or ¨N¨+
Rl Rlo R1o.
wherein R9, Rim and Rim' each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2)m-R8, or R9 and R' taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; R8 represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocyclyl or a polycyclyl; and m is zero or an integer from 1 to 8. In preferred embodiments, only one of R9 or 111 can be a carbonyl, e.g., R9, R10, and the nitrogen together do not form an imide. In even more preferred embodiments, R9 and R1 (and optionally R10') each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2)m-R8. In certain embodiments, the amino group is basic, meaning the protonated form has a pKa > 7.00.
The term "aralkyl", as used herein, refers to an alkyl group substituted with an aryl group, for example -(CH2)n-Ar.
The term "heteroaralkyl", as used herein, refers to an alkyl group substituted with a heteroaryl group, for example -(CH2)õ-Het.
The term "aryl" as used herein includes 5-, 6-, and 7-membered substituted or unsubstituted single-ring aromatic groups in which each atom of the ring is carbon. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, anthracene, and phenanthrene.
The terms "carbocycle" and "carbocyclyl", as used herein, refer to a non-aromatic substituted or unsubstituted ring in which each atom of the ring is carbon. The terms "carbocycle" and "carbocycly1" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is carbocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Representative carbocyclic groups include cyclopentyl, cyclohexyl, 1-cyclohexenyl, and 3-cyclohexen-1-yl, cycloheptyl.
The term "carbonyl" is art-recognized and includes such moieties as can be represented by the general formula:

x,R"
wherein X is a bond or represents an oxygen or a sulfur, and RH represents a hydrogen, an alkyl, an alkenyl, -(CH2)m-R8 or a pharmaceutically acceptable salt. Where X is an oxygen and RH is not hydrogen, the formula represents an "ester". Where X is an oxygen, and Ril is a hydrogen, the formula represents a "carboxylic acid".
The terms "heteroaryl" includes substituted or unsubstituted aromatic 5- to 7-membered ring structures, more preferably 5- to 6-membered rings, whose ring structures include one to four heteroatoms. The term "heteroaryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, isoxazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, and sulfur.
The terms "heterocycly1" or "heterocyclic group" refer to substituted or unsubstituted non-aromatic 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. The term terms "heterocycly1" or "heterocyclic group" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heterocyclic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.
Heterocyclyl groups include, for example, tetrahydrofuran, tetrahydropyran, piperidine, piperazine, pyrrolidine, morpholine, lactones, and lactams.
The term "hydrocarbon", as used herein, refers to a group that is bonded through a carbon atom that does not have a =0 or =S substituent, and typically has at least one carbon-hydrogen bond and a primarily carbon backbone, but may optionally include heteroatoms. Thus, groups like methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to be hydrocarbyl for the purposes of this application, but substituents such as acetyl (which has a =0 substituent on the linking carbon) and ethoxy (which is linked through oxygen, not carbon) are not. Hydrocarbyl groups include, but are not limited to aryl, heteroaryl, carbocycle, heterocycle, alkyl, alkenyl, alkynyl, and combinations thereof.
The terms "polycycly1" or "polycyclic" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Each of the rings of the polycycle can be substituted or unsubstituted.
As used herein, the term "probe" means a compound of the invention which is labeled with either a detectable label or an affinity tag, and which is capable of binding, either covalently or non-covalently, to a protein kinase domain. When, for example, the probe is non-covalently bound, it may be displaced by a test compound. When, for example, the probe is bound covalently, it may be used to form cross-linked adducts, which may be quantified and inhibited by a test compound.
The term "substituted" refers to moieties having substituents replacing a hydrogen on one or more carbons of the backbone. It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. Substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.

Compounds of the invention also include all isotopes of atoms present in the intermediates and/or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include deuterium and tritium.
General Synthetic Methods General Synthetic Method A:
W W W

Base, ligand, IW ir W catalyst 0 4:S: NaOH
) oxalyl chloride 0 ---b- 0 OH a Bo2c w Ho2c ir cloc le Br .' 1-i 1-iii 1-iv 1-v 1-ii 40w 0=w 0 =
1-v malononitrile io TMS-diazomethane 0 hydrazine . W
r- r NC
i \
CN meo -,.. CN ,N

CN CN H
1-vi 1-ii 1-viii 0 = 0 =
formamidine ________ 3. NH2 = W __________________ fa W
Ph3P, DIAD
1-viii . NH2 1,1 N N N 1-x R, 1-ix Scheme 1 Ulmann condensation of phenol 1-i with ester 1-ii provided intermediate 1-iii.

Saponification of intermediate 1-iii yielded intermediate 1-iv. Conversion of intermediate 1-iv to its acid chloride, using for example oxalyl chloride and DMF, provided intermediate 1-v. Condensation of intermediate 1-v with malononitrile yielded intermediate 1-vi. Methylation of intermediate 1-vi with TMS-diazomethane provided intermediate 1-vii. Condensation of 1-vii with hydrazine yielded intermediate 1-viii. Condensation of intermediate 1-viii with formamidine yielded intermediate 1-ix. Intermediate 1-ix was treated with alcohol R1OH, under Mitsunobu conditions, to provide the desired compounds or intermediates of general formula 1-x.
General Synthetic Method B:
malononitrile TMS-diazomethane hydrazine CN
,c) CN
NC
\

CN

2-i 2-ii 2-ili 2-iv X=I, Br X 0*
X
2-iv formamidine Ph3P, DIAD, NH2 Base, ligand, = NH2 catalyst NH2 N \ \
RICH L ,N 1 N IN
I
N". \N N, N N
10 w , R1 N N
2-vii 2 OH-v 2-vi 2-viii Scheme 2 Benzoyl chlorides of formula 2-i were condensed with malononitrile to provide intermediate 2-ii. Methylation of intermediate 2-ii with TMS-diazomethane provided intermediate 2-iii. Condensation of intermediate 2-iii with hydrazine provided intermediate 2-iv. Further condensation of intermediate 2-iv with formamidine provided intermediate 2-v. Intermediate 2-v was treated with alcohol WON, under Mitsunobu conditions, to provide intermediate 2-vi. Ullmann condensation of intermediate 2-vi with phenolic intermediates 2-vii provided the desired compounds or intermediates of general formula 2-viii.

Exemplification The following synthetic methods are intended to be representative of the chemistry used to prepare compounds of Formula 1 and are not intended to be limiting.
Synthesis of Compound 1:
,L0 )LOH 0 OBn OH K2CO3 Al OBn CuCI, Cs2CO3 ________________ = ______________________ =
Benzyl bromide IP 0 0 EtO2C
3-a Br 3-b OBn S I. OBn NaOH oxaly1 chloride 3-b ________ .. o __________ = o Ho2c CIOC
3-c 3-d DIPEA TMS-diazomethane 3-d ________ 0.
malononitrile (1101 0 0 \ CN \ CN
HO Me0 CN CN
3-e 3-f 0 . 0 *
hydrazine 0 formamidine 0 3-f _______ =
NC NH2 *
N .
/ \ N
= \
H2N N N'' I , H N [NI
3-g Compound 1 Scheme 3 Step 1: Intermediate 3-a Benzyl bromide (27.0 ml, 227 mmol) was added drop wise to a stirred suspension of resorcinol (25.0 g, 227 mmol) and potassium carbonate (31.4 g, 227 mmol) in acetone (150 ml) and the reaction was heated under reflux overnight. Volatiles were removed under reduced pressure. Water and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure.
Purification by silica gel chromatography provided intermediate 3-a as a beige oil.
Step 2: Intermediate 3-b To a solution of 3-a (15.0 g, 74.9 mmol) in 1,4-dioxane (200 ml) were sequentially added ethyl 4-bromobenzoate (20.59 g, 90 mmol), N,N-dimethylglycine (4.25 g, 41.2 mmol), copper(I) chloride(3.71 g, 37.5 mmol) and cesium carbonate (61.0 g, 187 mmol). The reaction mixture was stirred at reflux overnight and then cooled to room temperature. Water and ethyl acetate were added, the organic layer was separated, washed with saturated aqueous NaHCO3, brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 3-b as a colorless oil.
Step 3: Intermediate 3-c To a solution of 3-b (17.5 g, 50.2 mmol) in THF (200 ml) and Me0H (100 ml) was added 2N sodium hydroxide (100 ml, 200 mmol) and the reaction was stirred at room temperature overnight. Volatiles were removed under reduced pressure. 10% Aqueous HCI and ethyl acetate were added to the residue, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure to provide intermediate 3-c as beige solid.

Step 4: Intermediate 3-d To a suspension of 3-c (16.1 g, 50.3 mmol) in dichloromethane (100 ml) were added DMF (0.1 ml, 1.29 mmol) and oxalyl chloride (4.4 ml, 50.3 mmol). The solution was stirred at room temperature for 2 hours. Volatiles were removed under reduced pressure to provide intermediate 3-d as beige solid.
Step 5: Intermediate 3-e To a solution of intermediate 3-d (16.5 g, 48.9 mmol) in toluene (50 ml) and THF (7 ml), cooled to -10 C, were added malononitrile (3.19 ml, 50.2 mmol) and DIPEA (17.5 ml, 100 mmol) in toluene (50 mL), drop wise, over a period of 30 minutes. After the addition was completed, the reaction was stirred for 1 hour at 0 C and room temperature overnight. Volatiles were removed under reduced pressure. 1M aqueous HCI and ethyl acetate were added, the organic layer was separated, washed with 1M HCI and brine, dried over MgSO4, filtered and concentrated under reduced pressure to provide intermediate 3-e as beige solid.
Step 6: Intermediate 3-f To a solution of intermediate 3-e (18.1 g, 49.1 mmol) in acetonitrile (177 ml) and methanol (19.0 ml), cooled to 0 C, were added DIPEA (10.3 ml, 59.0 mmol) and a 2M solution of (diazomethyl)trimethylsilane in hexanes (27.0 ml, 54.0 mmol). After the addition was completed, the reaction was stirred at room temperature overnight. Acetic acid (0.56 ml, 9.83 mmol) was added, the reaction was then stirred for 30 minutes and volatiles were removed under reduced pressure. Saturated aqueous NaHCO3 and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 3-f as yellow solid.
Step 7: Intermediate 3-g To a suspension of intermediate 3-f (8.05 g, 21.1 mmol) in ethanol (10.5 ml) was added a solution of hydrazine nionohydrate (2.76 ml, 56.8 mmol). The reaction was stirred at 100 C for 1 hour and then cooled to room temperature. Water was added; a precipitate formed and was collected by filtration, washed with diethyl ether and dried in vacuo to provide intermediate 3-g as an off-white solid.
Step 8: Compound 1 Intermediate 3-g (8 g, 20.92 mmol) was added to a solution of formamidine (58.4 ml, 1464 mmol) and the reaction was stirred at 180 C for 2 hours and then cooled to room temperature. Water and ethyl acetate were added; the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure to provide compound 1 as beige solid. MS (m/z) M+H= 410.2 Synthesis of Compound 2:
o * o *
NH2 * Ph3P, DIAD NH2 =
N \ OH N \
N
, 1_ I ,N
N N
Compound 1 Compound 2 Scheme 4 To a solution of cyclopentanol (316 mg, 3.66 mmol) in THF was added triphenylphosphine (961 mg, 3.66 mmol) and DIAD (712 id, 3.66 mmol). The yellow solution was stirred 5 minutes, compound 1 (1.0 g, 2.44 mmol) was added and the reaction was then stirred at room temperature overnight.
Volatiles were removed under reduced pressure. Purification by silica gel chromatography provided compound 2 as an off-white solid. MS (m/z) M+H=478.2 Synthesis of Compound 3:
0= 0=

Ph3P, DIAD
NH2 qk \ NV' \
N
I N
N
N
Compound 1 0 Compound 3 Scheme 5 To a solution of (S)-tert-butyl 3-hydroxypiperidine-1-carboxylate (5.65 g, 28.1 mmol) in THF was added triphenylphosphine (7.37 g, 28.1 mmol) and DIAD (5.46 ml, 28.1 mmol). The yellow solution was stirred 5 minutes, compound 1 (10 g, 24.42 mmol) was added and the reaction was then stirred at room temperature overnight. Volatiles were removed under reduced pressure. Purification by silica gel chromatography provided compound 3 as white foam. MS (m/z) M+H= 593.1 Synthesis of Compound 4:

0* 0*

NH2 HCI*
NH2 *
elkN "- N
N N
N N
a NH

Compound 4 Compound 3 Scheme 6 To a solution of compound 3 (1.88 g, 3.17 mmol) in dichloromethane was added 4N HCI in 1,4-dioxane (19.82 ml, 79.0 mmol) and the reaction was stirred at room temperature for 2 hours. Volatiles were removed under reduced pressure. Purification by reverse phase chromatography eluting with a 1% aqueous HCl/methanol gradient provided compound 4.2HCI as white solid. MS (m/z) M+H= 493.1 Synthesis of compound 5 * *

N "N N
I' 1 N
N N

L bN
Compound 4 Compound 5 Scheme 7 To a solution of compound 4.2HCI (100 mg, 0.17 mmol) in dichloromethane (2 ml) cooled to 0 C were sequentially added TEA (99 pl, 0.70 mmol) and acryloyl chloride (17.61 mg, 0.19 mmol). The reaction was stirred at 0 C for 1 hour. A saturated aqueous solution of ammonium chloride was added, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided compound 5 as white solid. MS (m/z) M+H= 547.1 Synthesis of compound 6 0* 0 =

TEA

\NI 0 2HCI N \ N , N N N N
LNH
Compound 4 Compound 6 Scheme 8 To a solution of compound 42HCI (1.8 g, 3.18 mmol) in dichloromethane (32 ml) cooled to 0 C were sequentially added TEA (1.77 ml, 12.73 mmol) and acetyl chloride (249 pl, 3.50 mmol). The reaction was stirred at 0 C for 1 hour and room temperature overnight. Saturated aqueous ammonium chloride was added, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure.
Purification by reverse phase chromatography eluting with 1% aqueous HCl/methanol gradient provided compound 6=FICI as beige solid. MS (m/z) M+H= 535.1 Compounds 7 and 8 were prepared in a similar manner to compounds 6 by acylation of compound 4 with butanoyl chloride and iso-butanoyl chloride, respectively.
Synthesis of intermediate 9-d Br Br Br DIPEA
malononitrile 1110 TMS-diazomethane CN
CN

CN
9-a 9-b Br Br hydrazine formamidine 9-b __________________________________________ NH2 NC N
N
\
NH N

9-c 9-d Scheme 9 Step 1: Intermediate 9-a To a solution of 4-bromobenzoyl chloride (25 g, 114 mmol) in toluene (200 ml) and THF (30 ml), cooled to -10 C, were sequentially added malononitrile (7.60 ml, 120.0 mmol) and DIPEA (39.8 ml, 228 mmol) in toluene (50 mL) drop wise over a period of 1 hour. After the addition was completed, the reaction was stirred for 1 hour at 0 C and room temperature overnight.
Volatiles were removed under reduced pressure. 1M HCI and ethyl acetate were added to the residue, the organic layer was separated, washed twice with 1M HCI and brine, dried over MgSO4, filtered and concentrated under reduced pressure to provide intermediate 9-a as yellow solid.

Step 2: Intermediate 9-b To a solution of intermediate 9-a (26.4 g, 106 mmol) in acetonitrile (300 ml) and methanol (35.0 ml), cooled to 0 C, was added DIPEA (22.2 ml, 127 mmol) and (diazomethyl)trimethylsilane (58.3 ml, 117 mmol). After the addition was completed, the reaction was stirred at room temperature overnight. Acetic acid (1.21 ml, 21.2 mmol) was added, the reaction was stirred for 30 minutes and volatiles were removed under reduced pressure.
Saturated aqueous NaHCO3 and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 9-b as a yellow solid.
Step 3: Intermediate 9-c To a suspension of intermediate 9-b (4.49 g, 17.07 mmol) in ethanol (8.5 ml) was added a solution of hydrazine monohydrate (2.23 ml, 46.1 mmol) and the reaction was stirred at 100 C for 1 hour and then cooled to room temperature. Volatiles were removed under reduced pressure to provide intermediate 9-c as a yellow solid.
Step 4: Intermediate 9-d Intermediate 9-c (4.49 g, 17.07 mmol) was added to a solution of formamidine (40.8 ml, 1024 mmol) and the reaction was stirred at 180 C
for 3 hours and then cooled to room temperature. Ethanol was added; a precipitate formed and was collected by filtration, dried in vacuo to provide intermediate 9-d as beige solid.
Synthesis of intermediate 10-a Br Ph3P, DIAD
9-d NH2 OH
IµV \ N
' N N
10-a Scheme 10 To a solution of intermediate 9-d (1.0 g, 3.45 mmol) in THF was added triphenylphosphine (1.35 g, 5.17 mmol), cyclopentanol (0.47 ml, 5.17 mmol) and DIAD (1.0 ml, 5.17 mmol) and the reaction was then stirred at room temperature overnight. Volatiles were removed under reduced pressure.
Purification by silica gel chromatography provided intermediate 10-a as white solid. MS (m/z) M+H= 359.6 =

Synthesis of Compound 9 CI
40 OH OTBS Ph3P, DIAD, roo 0 40 imidazole TBSCI CI
OH OH HO OTBS
11-a 11-b ci TBAF
11-b OH
11-c 0*

CuCI, CS2CO3, 1O-a+ 11-c ____________ = OH NH2 Wilk \ CI
1 NjN
1St N
Compound 9 Scheme 11 Step 1: Intermediate 11-a To a solution of resorcinol (15 g, 136 mmol) in DMF (100 ml), cooled to 0 C, were added imidazole (19.48 g, 286 mmol) and tert-butylchlorodimethylsilane (21.56 g, 143 mmol). The reaction was then stirred at room temperature overnight. Saturated aqueous ammonium chloride and ethyl acetate were added; the organic layer was separated, washed 3 times with a saturated aqueous solution of ammonium chloride and brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 11-a as a colorless oil.

Step 2: Intermediate 11-b To a solution of (4-chlorophenyl) methanol (1.52 g, 10.70 mmol) in THF (20 mL) were sequentially added intermediate 11-a (2.88 g, 12.84 mmol), triphenylphosphine (3.37 g, 12.84 mmol) and DIAD (2.53 ml, 12.84 mmol) drop wise at room temperature and the reaction was then stirred for 1 hour.
Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 11-b as a colorless oil.
Step 3: Intermediate 11-c Tetrabutylammonium fluoride trihydrate (3.93 g, 12.47 mmol) was added to a solution of intermediate 11-b (2.9 g, 8.31 mmol) in THF (15 mL) and the reaction was stirred at room temperature overnight. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 11-c as a colorless oil.
Step 4: Compound 9 A solution of intermediate 10-a (200 mg, 0.56 mmol), intermediate 11-c (229 mg, 0.977 mmol), quinolin-8-ol (16.21 mg, 0.112 mmol), copper (I) chloride (11.05 mg, 0.11 mmol) and cesium carbonate (546 mg, 1.67 mmol), in dimethylacetamide (1 ml), was degassed with argon for 10 minutes, sealed and heated in a sealed tube at 140 C overnight. The solution was cooled to room temperature, water and ethyl acetate were added, the organic layer was separated, the aqueous layer was extracted twice with ethyl acetate, the combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure.
Purification by reverse phase chromatography eluting with a 1%

HCl/methanol gradient provided compound 9.1-1CI as yellow solid. MS (m/z) M+H= 512.2 Synthesis of intermediate 12-a Br NH2 4Ik Ph3P, DIAD
9-d ________________________________ N
N
Me0H N
12-a Scheme 12 To a solution of intermediate 9-d (500 mg, 1.72 mmol), in THF (8.6 mL), were sequentially added methanol (105 pl, 2.59 mmol), triphenylphosphine (678 mg, 2.59 mmol) and DIAD (503 pi, 2.59 mmol), drop wise, at room temperature. The solution was then stirred at room temperature overnight.
A precipitate formed and was collected by filtration and dried in vacuo to provide intermediate 12-a as a white solid.
Synthesis of compound 16 0*
1410i, 23, 12-a + cueCSC0NH2 44Ik OH __ OH N1 ,N
3-a Compound 16 Scheme 13 A solution of intermediate 12-a (235 mg, 0.77 mmol), intermediate 3-a (271 mg, 1.35 mmol), quinolin-8-ol (22.4 mg, 0.15 mmol), copper (I) chloride (15.3 mg, 0.15 mmol) and cesium carbonate (755 mg, 2.31 mmol) in dimethylacetamide (1 ml) was degassed with nitrogen for 10 minutes, heated in a sealed tube at 140 C overnight and then cooled to room temperature. Water and ethyl acetate were added, the organic layer was separated, the aqueous layer was extracted twice with ethyl acetate, the combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by reverse phase chromatography eluting with 1% HCl/methanol gradient provided compound 16=HCI as a beige solid. MS (m/z) M+H= 424.2 Synthesis of compound 17 CHO CHO
DIPEA, TBSCI NaBH4 OH
OH OTBS OTBS
14-a 14-b 14-b ________ Ph3P, DIAD, 40 0 410 TBAF 0 CN CN
OTBS OH
HO
CN 14-c 14-d 0* CN
CuCI, CS2CO3, 0 12-a + 14-d N
\ Compound 17 Scheme 14 Step 1: Intermediate 14-a To a solution of 3-hydroxybenzaldehyde (14.73 g, 121 mmol) in dichloromethane (100 mL) were sequentially added triethylamine (25.08 ml, 181 mmol), tert-butylchlorodimethylsilane (20.0 g, 133 mmol), portion wise, and the reaction was stirred at room temperature overnight. 10% Citric acid was added, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 14-a as a yellow oil.
Step 2: Intermediate 14-b To a solution of intermediate 14-a (16.0 g, 67.7 mmol) in methanol (100 ml) cooled to 0 C was added portion wise sodium borohydride (1.28 g, 33.8 mmol). After the addition was completed the reaction was stirred at room temperature for 2 hours. Volatiles were removed under reduced pressure.
Water and ethyl acetate were added to the residue, the organic layer was separated, washed with brine, dried over Mg504, filtered and concentrated under reduced pressure to provide intermediate 14-b as a yellow oil.
Step 3: Intermediate 14-c To a solution of intermediate 14-b (1.0 g, 2.09 mmol) in THF (42 mL) were sequentially added 2-hydroxybenzonitrile (600 mg, 5.03 mmol), triphenylphosphine (1.32 g, 5.03 mmol) and DIAD (991 pl, 5.03 mmol) drop wise at room temperature; the reaction was then stirred at reflux for 2 hours then cooled to room temperature. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure.
Purification by silica gel chromatography provided intermediate 14-c as a colorless oil.

Step 2: Intermediate 14-d To a solution of intermediate 14-c (1.22 g, 3.62 mmol) in THF (36.0 ml) was added tetrabutylammonium fluoride (946 mg, 3.62 mmol) and the reaction was stirred at room temperature for 1 hour. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 14-d as a white solid.
Step 2: Compound 17 A solution of intermediate 12-a (200 mg, 0.6 mmol), intermediate 14-cl (259 mg, 1.15 mmol), quinolin-8-ol (19.0 mg, 0.13 mmol), copper (I) chloride (13.0 mg, 0.13 mmol) and cesium carbonate (643 mg, 1.97 mmol) in dimethylacetamide (3.0 ml) was degassed with argon for 10 minutes, heated in a sealed tube at 140 C overnight. After cooling to room temperature, water and ethyl acetate were added, the organic layer was separated, the aqueous layer was extracted twice with ethyl acetate, the combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided compound 17 as a white solid. MS (m/z) M+H= 449.3 Synthesis of compound 18 rN
LIAIH4 Ph3P, DIAD, HO
14-b 15-a 15-b Oc TBAF
OH
15-c 0*
CuCI, CS2CO3 12-a +15-cOH NH2 sz,N
NH
, N
N N\ Compound 18 Scheme 15 Step 1: Intermediate 15-a To a solution of ethyl 2-methylthiazole-5-carboxylate (5.82 g, 34.0 mmol) in THF (170 ml), cooled to 0 C, was added a 1.0M solution of LiAIH4in THF (34 ml, 34.0 mmol) and the reaction was slowly warmed to room temperature and stirred overnight. Water (1.3 ml) was slowly added, followed by 15%
NaOH (1.3 mL). The solution was stirred for 2 hours at room temperature then filtered on celite. The filtrate was concentrated under reduced pressure to provide intermediate 15-a as a yellow oil.
Step 2: Intermediate 15-b To a solution of intermediate 15-a (7.75 g, 34.5 mmol) and intermediate 14-b (4.25 g, 32.9 mmol), in THF (33 mL), were sequentially added triphenylphosphine (10.35 g, 39.5 mmol) and DIAD (7.68 ml, 39.5 mmol), drop wise, at room temperature. The reaction was then stirred for 18 hours.
Volatiles were removed in vacuo. Purification by silica gel chromatography provided intermediate 15-b as a colorless oil.

Step 3: Intermediate 15-c To a solution of intermediate 15-b (5.5 g, 16.39 mmol), in THF (82.0 ml), was added a 1.0M solution of tetrabutylammonium fluoride in THF (16.4 ml, 16.4 mmol) and the reaction was stirred at room temperature for 30 minutes. Saturated aqueous ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 15-c as beige solid.
Step 4: Compound 18 A solution of intermediate 12-a (200 mg, 0.65 mmol), intermediate 15-c (146 mg, 0.65 mmol), quinolin-8-ol (19.0 mg, 0.13 mmol), copper (I) chloride (13.0 mg, 0.13 mmol) and cesium carbonate (643 mg, 1.97 mmol) in dimethylacetamide (6.5 ml) was degassed with argon for 10 minutes, ealed, heated in a sealed tube at 140 C for 2 hours and then cooled to room temperature. Water and ethyl acetate were added, the organic layer was separated, the aqueous layer was extracted twice with ethyl acetate, the combined organic extracts were washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. Purification by reverse phase chromatography eluting with 1% HCl/methanol gradient provided compound 15.1-1CI as beige solid. MS (m/z) M+H= 445.1 Table 1 summarizes representative compound of Formula 1.

Table 1: Example Compounds of Formula 1 Compound Structure MS (m/z) 0*

1 NH2 [M+H]=410.2 \N
I N' 0=

NH2 =

[M+Hr=478.2 ,N
N
0=

NH2 th \ [M+H]+=593.1 N L ,N

UN-.?

0=

410 [M+H]=493.1 \
I ,N
N N
L\NH
0=

NH2 fa [M+H]=547.1 \
I ,N
N N

[M+H]=535.1 N "N
I , N N

0=

7 N' , I ,N [M+H]=563.1 N N
0*

8 N [M+H]=563.1 L ,N
N
L\N
*

[M+Hr=512.2 CI
N \N
L I =
N

0*

NH2 =
O [M+H]=508.1 N ' , \
L I ,N --0 d 0*
n.
NH2 = 0=
0, [M+H]=508.2 NV \
I'N
N N),...., U
_ 0*
CN
NH2 0 glis illit 12 [M+Hr=503.2 NI "
I ,NI
N N
d 0*

NH2 = =
13 [M+Hr=478.2 N' , "
I ,N
N N
d 0*
14 NH2 fi SN
[M+Hr=485.2 N' \N
I , N

[M+H]=503.3 N' \
I'N
N N
0*

441k NH2 41k [M+H]=424.2 N \N
I , N N

CN

17 NH2 [M+H]=449.3 N' \N
I , N N

0=

18 NH2 * [M+H]=445.1 \N
' N N
4Ik [M+H]=431.4 \
L ,N
N
4Ik [M+1-1]=492.1 , ,N
N N
0 =

21 'IN[M+Hr=494.2 N

0 .
CN

22 NH2 ik [M+Hr=519.2 N' , \
1 ,N
N N
o 0 .
u3 23 NH2 O Ilk [M+H]=562.1 N' \N
I , N
U

0=
O_______\
24 NH2 Th 410 sN
[M+Hr=501.2 N' , \
I ,N
N N
a0 0* CN

N' \ [M+H]=560.2 I ,N
N
UN-{

0*

N \N [M+H]=603.1 I N' 0*
S,f`J

N' \N [M+H]=542.2 I , N

O CN

[M+Hr=560.2 N7 "N
' N
UN( NH2 410 [M+Hr=546.2 "N
= I
N
0 =
CN

NH2 [M+H]=449.4 \
= I ,N
N N

NH2 [M+H]+=492.1 N7 \
I ,N
N N

32 NH2 =
[M+H]=546.1 N
I ,N
N N
0=
o CF3 NH2*
N , [M+H]=603.1 L ,N
N

0=
34 NH2 sN
[M+H]=488.3 , I ,N
N N
N--35 NH2 4. NC 410 [M+H]=519.2 \N
I ' N
0 =

36 NH2 44, F3C
[M+Hr=562.2 "N
I ' N
0 =
37 NH2 40 [M-I-H]=499.1 "N
I' N

0 =

[M+H]=515.1 'N
N N)Th 0 =

39 [M+H]=514.2 ,N
N N\
N,.
0*

N' 40 \ ,N1 [M+H]=543.1 N N\

0*
0'\ _ N
41 N \ [M+Fir=522.1 IN
N N
\---6 N
0 =

42 N' \ [M+H]=521.2 L, I ,N
N N

0*
NH2 fa N
43 N' , \ [M+H]=500.2 L... 1 ,N
N Nym C--.) N
H

0*
0"---_ NH2 4*
N
44 N \ N [M+H]=530.1 I , N Nv --A
(N---\
=--0/
0=

45 NH2 lli . [M+H]=452.1 NV \N
I , N N\
/=0 F
0*
0---A___ 46 NH2 * N [M+H]=519.1 NV \N
I' N r).Th \-- ) 0*
NH2, 47 INV \
I ,N [M+Hr=542.1 N N)Th 0=
, NI/

48 [M+Hr=498.2 \N
' N

49 N \ [M +H] =600.1 L ,N
N
aN

0*
()Ars 50 NH2 * [M+I-1]+=503.2 N' 'N
N
0Il =

51 [M+Hr=500.2 N' N
I ' N N
L\NH
Kinase Binding Btk Kinase Inhibition Assay Fluorescence polarization-based kinase assays were performed in 384 well-plate format using histidine tagged recombinant human full-length Bruton Agammaglobulinemia Tyrosine Kinase (Btk) and a modified protocol of the KinEASE TM FP Fluorescein Green Assay supplied from Millipore. Kinase reaction were performed at room temperature for 60 minutes in presence of 250 11M substrate, 10 M ATP and variable test article concentrations. The reaction was stopped with EDTA/kinease detection reagents and the polarization measured on a Tecan 500 instrument. From the dose-response curve obtained, the ICso was calculated using Graph Pad Prisms using a non linear fit curve. The Km for ATP on each enzyme was experimentally determined and the Ki values calculated using the Cheng-Prusoff equation (see: Cheng Y, Prusoff WH. (1973) Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction". Biochem Pharmacol 22 (23): 3099-108).
k, values are reported in Tables 2:
Table 2: Inhibition of Btk Compound ki (nM) Compound k, (nM) Compound k(nM) 1 a 16 a 31 a 2 a 17 a 32 a 3 a 18 a 33 a 4 a 19 a 34 a a 20 a 35 a 6 a 21 a 36 a 7 a 22 a 37 a 8 a 23 a 38 a 9 a 24 a 39 a a 25 a 40 a 11 a 26 a 41 a 12 a 27 a 42 a 13 a 28 a 43 a 14 a 29 a 44 a a 30 a 45 a a - Less than 100 nM; b - less than 1000 nM, c - more than 1000 nM

Splenic Cell Proliferation Assay Splenocytes were obtained from 6 week old male CD1 mice (Charles River Laboratories Inc.). Mouse spleens were manually disrupted in PBS and filtered using a 70um cell strainer followed by ammonium chloride red blood cell lysis. Cells were washed, resuspended in Splenocyte Medium (HyClone RPMI supplemented with 10% heat-inactivated FBS, 0.5X non-essential amino acids, 10mM HEPES, 50uM beta mercaptoethanol) and incubated at 37 C, 5% CO2 for 2h to remove adherent cells. Suspension cells were seeded in 96 well plates at 50,000 cells per well and incubated at 37 C, 5% CO2 for 1h.
Splenocytes were pre-treated in triplicate with 10,000 nM curves of Formula 1 compounds for 1h, followed by stimulation of B cell proliferation with 2.5ug/m1 anti-IgM F(a131)2 (Jackson ImmunoResearch) for 72h. Cell proliferation was measured by Cell Titer-Glo Luminescent Assay (Promega).
EC50 values (50% proliferation in the presence of compound as compared to vehicle treated controls) were calculated from dose response compound curves using GraphPad Prism Software.
EC50 values are reported in Table 2:
Table 2: Inhibition of splenic cell proliferation Compound ki (nM) Compound k, (nM) Compound k1(nM) 1 b 16 b 31 b 2 b 17 b 32 a 3 b 18 a 33 a 4 b 19 b 34 b a 20 a 35 a 6 a 21 a 36 a 7 b 22 a 37 a 8 a 23 a 38 a 9 b 24 a 39 b b 25 a 40 b 11 b 26 a 41 12 a 27 a 42 a 13 b 28 a 43 b 14 a 29 a 44 a 15 a 30 a 45 a - Less than 100 nM; b - less than 1000 nM, c ¨ more than 1000 nM

Claims (11)

1. Compound of Formula 1:
wherein X is selected from the group consisting of:
6) hydrogen, 7) alkyl, 8) heteroalkyl, 9) carbocyclyl, 10) heterocyclyl;
wherein the alkyl, heteroalkyl, carbocyclyl and heterocyclyl may be further substituted by the groups consisting of:
12) hydroxy, 13) alkoxy, 14) alkyl, 15) -OC(O)R4, 16) -OC(O)NR5R6, 17) -C(O)R4, 18) -C(O)NR5R6, 19) -NR5R6, 20) -NR2C(O)R4, 21) -NR2S(O),R4, 22) -NR2C(O)NR5R6;
Y is selected from:
Z is selected from:
X1 and X2 are independently selected from hydrogen, halogen or cyano;
n is an integer from 0 to 2;
W is independently selected from:
1) alkyl,

2) aralkyl,

3) heteroaralkyl,

4) -OR3,

5) -OC(O)R4,

6) -OC(O)NR5R6,

7) -CH2O-R4,

8) -NR5R6,

9) -NR2C(O)R4,

10) -NR2S(O)n R4,

11) -NR2C(O)NR5R6;
wherein the alkyl, aralkyl and heteroaralkyl may be further substituted;
R2 is selected from hydrogen or alkyl;
R3 is selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl, aralkyl or heteroaralkyl;
R4 is selected from substituted or unsubstituted alkyl, alkenyl, alkynyl, heteroalkyl, carbocyclyl, heterocyclyl, aryl or heteroaryl;
R5 and R6 are independently selected from hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, carbocyclyl, heterocyclyl, aryl, heteroaryl or R5 and R6 can be fused to form a 3 to 8 membered heterocyclyl ring system.
2. Compound according to claim 1 wherein W is selected from -OR3 and R3 is selected from substituted or unsubstituted aralkyl, or substituted or unsubstituted heteroaralkyl.
3. Compound according to claim 1 wherein X is selected from H, Me, 4. Compound according to claim 1 wherein W is selected from the group consisting of 5. Compound of the following structure: