WO2014029007A1

WO2014029007A1 - Protein kinase inhibitors

Info

Publication number: WO2014029007A1
Application number: PCT/CA2013/000727
Authority: WO
Inventors: Alain Laurent; Yannick Rose
Original assignee: Pharmascience, Inc.
Priority date: 2012-08-22
Filing date: 2013-08-21
Publication date: 2014-02-27
Also published as: CA2787472A1

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, having the general formula (1): A-Y-E-W-Z (1.)

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

4187891 vl 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 JM. Cell Signal. 2010,22: 1175-84). The Tec family includes Tec, Bruton's tyrosine kinase (Btk), inducible T-cell kinase (Itk), resting lymphocyte kinase (Rlk/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 antiinflammatory 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

4187891 vl 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 a\. 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 Src protein kinase families, more particularly Btk.

One aspect of the present invention is directed to a compound of the following formula :

A-Y-E-w-z (1) wherein

A is:

4187891 vl X¹ is selected from NH or O;

X² is selected from N or CH;

n is an integer from 0 to 2;

Y is selected from :

E is oxygen,

W is selected from:

Xi and X₂ are independently selected from hydrogen and halogen;

m and m' are integers from 0 to 2;

R and Z are independently selected from:

1) alkyl,

2) aralkyl,

3) heteroaralkyl,

4) -OR³,

5) -OC(0)R⁴,

6) -OC(0)NR⁵R⁶,

7) -CH₂0-R\ 8) -NR⁵R⁶,

9) -NR²C(0)R⁴,

10) -NR²S(0)_nR⁴,

11) -NR²C(0)NR⁵R⁶; wherein the alkyl, aralkyi and heteraralkyi 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, aralkyi 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 Z is selected from -OR³ and R³ is selected from substituted or unsubstituted aralkyi, or substituted or unsubstituted heteroaralkyl.

Preferred embodiment includes compounds of Formula 1 where A is selected from the group consisting of:

4187891 vl Preferred embodiment includes compounds of Formula 1 where Z is selected from the group consisting of:

More preferred embodiment includes compounds of Formula 1 where Z is selected from the group consisting of:

More preferred embodiment includes compounds of Formula 1 whe

selected from the group consisting of:

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

4187891 vl 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

4187891 vl 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, compositions, 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 β-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

4187891 vl 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.

4187891 vl 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, l,3-butadien-2-yl), 2,4-pentadienyl, and l,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 -(CH₂)i-io- 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:

4187891 vl O

^ N

R⁹ wherein R⁹, R¹⁰ 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:

wherein R⁹, R¹⁰ and R¹⁰ each independently represent a hydrogen, an alkyl, an alkenyl, -(CH₂)_m-R⁸, or R⁹ and R¹⁰ 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 R¹⁰ can be a carbonyl, e.g., R⁹, R¹⁰, and the nitrogen together do not form an imide. In even more preferred embodiments, R⁹ and R¹⁰ (and optionally R^10') 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 pK_a > 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)_n-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

4187891 vl 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 "carbocyclyl" 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-l-yl, cycloheptyl.

The term "carbonyl" is art- recognized and includes such moieties as can be represented by the general formula :

A-*- wherein X is a bond or represents an oxygen or a sulfur, and R^u represents a hydrogen, an alkyl, an alkenyl, -(CH₂)_m-R⁸ or a pharmaceutically acceptable salt. Where X is an oxygen and R¹¹ is not hydrogen, the formula represents an "ester". Where X is an oxygen, and R¹¹ 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

4187891 vl 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 "heterocyclyl" 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 "heterocyclyl" 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 "polycyclyl" or "polycyclic" refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or

4187891 vl 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

4187891 vl 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:

scheme 1

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 3:

4187891 vl

Scheme 2

Step 1: Intermediate 2-b

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 MgS0₄, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 2-b as a yellow oil.

Step 2: Intermediate 2-c

To a solution of intermediate 2-b (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.

4187891 vl Water and ethyl acetate were added to the residue, the organic layer was separated, washed with brine, dried over MgS0₄, filtered and concentrated under reduced pressure to provide intermediate 2-c as yellow oil.

Step 3: Intermediate 2-d

To a solution of intermediate 2-c (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 μΙ, 5.03 mmol) drop wise at room temperature; the reaction was stirred at reflux for 2 hours then cooled to room temperature. A saturated aqueous solution of ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgS0₄, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 2-d as a colorless oil.

Step 4: Intermediate 2-e

To a solution of intermediate 2-d (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. A saturated aqueous solution of ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgS0₄, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 2-e as a white solid.

Step 5: Intermediate 2-g

To a solution of 4-iodoaniline (1 g, 4.57 mmol), 4-chloro-6,7- dimethoxyquinoline (1.12 g, 5.02 mmol) in iPrOH (22.8 ml) was added 4N

4187891 vl HCI in 1,4-dioxane (0.57 ml, 2.28 mmol) and the reaction was heated in a sealed tube at 120 °C for 1 hour. Volatiles were removed under reduced pressure. MeOH and ethyl acetate were added to the residue; a precipitate formed and was collected by filtration to provide intermediate 2-g as white solid.

Step 6: Compound 3

To a solution of intermediate 2-e (178 mg, 0.79 mmol), intermediate 2-g (200 mg, 0.45 mmol) in 1,4-dioxane, was added cesium carbonate (294 mg, 0.90 mmol), N-N-dimethylaminoglycine (14 mg, 0.13 mmol) and copper (I) iodide (17 mg, 0.09 mmol). The reaction was degassed with argon for 10 minutes, stired at reflux for 36 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 MgS0₄, filtered and concentrated under reduced pressure. Purification by reverse phase chromatography eluting with 1% HCI/methanol gradient provided compound 3-HCI as beige solid. MS (m/z) M+H= 504.2

Synthesis of Compound 4:

4187891 vl

°^'° Compound 4

2-g

Scheme 3

Step 1: Intermediate 3-b

To a solution of Resorcinol (11.83 g, 107 mmol) in DMF (50 ml) cooled to 0°C was added imidazole (15.36 g, 226 mmol) and tert- butylchlorodimethylsilane (17.0 g, 113 mmol). The reaction was then stirred at room temperature overnight. A saturated aqueous solution of 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 MgS0₄, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 3-b as colorless oil.

Step 2: Intermediate 3-c

To a solution of intermediate 3-b (1.94 g, 8.68 mmol) and thiazol-5- ylmethanol (1.0 g, 8.68 mmol) in THF (20 ml) were sequentially added triphenylphosphine (3.42 g, 13.0 mmol) and DIAD (2.52 ml, 13.0 mmol) at room temperature and the reaction was then stirred at room temperature

4187891 vl overnight. Volatiles were removed under reduced pressure. Purification by silica gel chromatography provided intermediate 3-c as yellow oil.

Step 3: Intermediate 3-d

To a solution of intermediate 3-c (1.60 g, 4.98 mmol) in THF (20 ml) was added a 1.0 M solution of TBAF in THF (5.47 ml, 5.47 mmol) and the reaction was stirred at room temperature for 1 hour. A saturated aqueous solution of ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgS0₄, filtered and concentrated under reduced pressure. Diethyl ether was added to the residue; a precipitate formed and was collected by filtration to provide intermediate 3-d as white solid.

Step 4: Compound 4

To a solution of intermediate 3-d (100 mg, 0.48 mmol), intermediate 2-g (175 mg, 0.43 mmol) in 1,4-dioxane, was added cesium carbonate (314 mg, 0.96 mmol), N-N-dimethylaminoglycine (50 mg, 0.48 mmol) and copper (I) iodide (33 mg, 0.17 mmol). The reaction was degassed with argon for 10 minutes, stirred at reflux for 24 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 MgS0₄, filtered and concentrated under reduced pressure. Purification by reverse phase chromatography eluting with 1% HCI/methanol gradient provided compound 4-HCI as beige solid. MS (m/z) M + H= 486.1

Synthesis of Compound 7:

4187891 vl

2-g Compound 7

Scheme 4

Step 1: Intermediate 4-a

To a solution of 2-(bromomethyl)benzonitrile (2.01 g, 10.25 mmol) and intermediate 3-b (2.30 g, 10.25 mmol) in acetone (103 ml) was added cesium carbonate (6.68 g, 20.50 mmol) and the reaction was stirred at reflux for 2 hours and then cooled to room temperature. A saturated aqueous solution of ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgS0₄, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 4-a as yellow oil.

Step 2: Intermediate 4-b

To a solution of intermediate 4-a (3.40 g, 10.01 mmol) in THF (40 ml) was added a 1.0 M solution of TBAF in THF (10.01 ml, 10.01 mmol) and the reaction was stirred at room temperature for 30 minutes. A saturated aqueous solution of ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgS0₄, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 4-b as yellow oil.

4187891 vl Step 3: Compound 7

To a solution of intermediate 4-b (102 mg, 0.45 mmol), intermediate 2-g (200 mg, 0.45 mmol) in 1,4-dioxane, was added cesium carbonate (294 mg, 0.90 mmol), N-N-dimethylaminoglycine (14 mg, 0.13 mmol) and copper (I) iodide (17 mg, 0.09 mmol). The reaction was degassed with argon for 10 minutes, stirred at reflux for 24 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 MgS0₄, filtered and concentrated under reduced pressure. Purification by reverse phase chromatography eluting with 1% HCI/methanol gradient provided compound 7-HCI as yellow solid. MS (m/z) M + H= 504.1

Synthesis of Compound 8:

Scheme 5

Step 1: Intermediate 5-b

Ethyl chloroacetate (50.0 g, 0.41 mol) and ethyl formate (30.2 g, 0.41 mol) were taken in anhydrous toluene (500 mL) and cooled to 0°C. Sodium

4187891 vl ethoxide (35.1 g, 0.49 mol) was added portion wise. The reaction mixture was stirred at 0 °C for 5 hours and then at room temperature overnight. The reaction mixture was quenched with water (250 ml_) and washed twice with diethyl ether. The aqueous layer was cooled to 0 °C and acidified to pH 4-5 using 1 N HCI. The aqueous layer was extracted twice with diethyl ether; the combined organic layers were dried over MgS0₄ filtered and concentrated under reduced pressure to provide intermediate 5-b as beige oil.

Step 2: Intermediate 5-c

To a solution of ethyl 2-chloro-3-oxopropanoate 5-b (34.7 g, 230 mmol) in toluene (250 ml) was added thioacetamide (26.0 g, 346.0 mmol). The reaction was stirred at 90°C overnight and then cooled to room temperature, diluted with water (300 mL) and then neutralized to PH=7 with a saturated aqueous solution of NaHC0₃. Ethyl acetate was added, the organic layer was separated, washed with brine, dried over MgS0₄, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 5-c as beige oil.

Step 3: Intermediate 5-d

To a solution of intermediate 5-c (22.2 g, 130.0 mmol) in THF (430 ml) cooled to 0°C was added a 1.0 M solution LiAIH₄ in THF (91.0 ml, 91.0 mmol) and the solution was slowly warmed to room temperature and stirred for 2 hours. Water (3.5 ml) was slowly added, followed by 3.5 ml 15% NaOH (3.5 ml) and water (10.5 ml) and the mixture was stirred for 1 hour. The reaction was filtered over celite and volatiles were removed under reduced pressure to provide intermediate 5-d as yellow oil.

Step 4: Intermediate 5-f

4187891 vl To a solution of l-fluoro-3,5-dimethoxybenzene (12.5 g, 80 mmol) in dichloromethane (80 ml) cooled to 0°C was added dropwise over a period of 30 minutes a 1.0 M solution of BBr₃ in dichloromethane (200 ml, 200 mmol). The reaction was stirred for 1 hour at 0°C and then slowly warmed to room temperature and stirred for 18 hours. The reaction was cooled to 0°C and quenched by slow addition of MeOH and water. After stirring at room temperature for 1 hour the mixture was filtered and volatiles were removed under reduced pressure. Ethyl acetate was added to the residue; a precipitate formed and was collected by filtration to provide intermediate 5-f as orange solid.

Step 5: Intermediate 5-g

To a solution of intermediate 5-f (10.25 g, 80.0 mmol) in DMF (50 ml) cooled to 0°C was added imidazole (5.99 g, 88.0 mmol) and tert- butylchlorodimethylsilane (13.27 g, 88.0 mmol). The reaction was then stirred at room temperature overnight. A saturated aqueous solution of 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 MgS0₄, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 5-g as yellow oil.

Step 6: Intermediate 5-h

To a solution of intermediate 5-g (1.0 g, 105.0 mmol) and intermediate 5-d (352 mg, 2.73 mmol) in THF (20 ml) were sequentially added triphenylphosphine (1.07 g, 4.1 mmol) and DIAD (796 μΙ, 4.1 mmol) at room temperature and the reaction was then stirred at room temperature for 1 hour. Volatiles were removed under reduced pressure. Purification by silica gel chromatography provided intermediate 5-h as yellow oil.

4187891 vl Step 7: Intermediate 5-i

To a solution of intermediate 5-h (750 mg, 1.57 mmol) in THF (20 ml) was added a 1.0 M solution of TBAF in THF (1.72 ml, 1.72 mmol) and the reaction was stirred at room temperature for 1 hour. A saturated aqueous solution of ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgS0₄, filtered and concentrated under reduced pressure. Diethyl ether was added to the residue; a precipitate formed and was collected by filtration to provide intermediate 5-i as white solid.

Step 8: Compound 8

To a solution of intermediate 5-i (133 mg, 0.56 mmol), intermediate 2-g (227 mg, 0.56 mmol) in 1,4-dioxane, was added cesium carbonate (544 mg, 1.67 mmol), N-N-dimethylaminoglycine (86 mg, 0.83 mmol) and copper (I) iodide (53 mg, 0.28 mmol). The reaction was degassed with argon for 10 minutes, stirred at reflux for 24 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 MgS0₄, filtered and concentrated under reduced pressure. Purification by reverse phase chromatography eluting with 1% HCI/methanol gradient provided compound 8-HCI as yellow solid. MS (m/z) M + H= 518.2

4187891 v1 Synthesis of Compound 9:

Scheme 6

Step 1: Intermediate 6-b

To a solution of intermediate 6-a (2.0 g, 12.89 mmol) in THF (65 ml) cooled to 0°C was added a 1.0 M solution LiAIH₄ in THF (12.89 ml, 12.89 mmol) and the solution was slowly warmed to room temperature and stirred for 2 hours. Water (0.5 ml) was slowly added, followed by 15% aqueous NaOH (0.5 ml) and water (0.5 ml) and the mixture was stirred for 1 hour. The reaction was filtered over celite and volatiles were removed under reduced pressure to provide intermediate 6-b as yellow oil.

Step 2: Intermediate 6-c

To a solution of intermediate 5-g (1.42 g, 5.89 mmol) and intermediate 6-b (1.0 g, 8.84 mmol) in THF (20 ml) were sequentially added

4187891 vl triphenylphosphine (2.31 g, 8.84 mmol) and DIAD (1.71 ml, 8.84 mmol) at room temperature and the reaction was then stirred at room temperature for 1 hour. Volatiles were removed under reduced pressure. Purification by silica gel chromatography provided intermediate 6-c as yellow oil.

Step 3: Intermediate 6-d

To a solution of intermediate 6-c (1.10 g, 3.26 mmol) in THF (32 ml) was added a 1.0 M solution of TBAF in THF (3.59 ml, 3.59 mmol) and the reaction was stirred at room temperature for 1 hour. A saturated aqueous solution of ammonium chloride and ethyl acetate were added, the organic layer was separated, washed with brine, dried over MgS0₄, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 6-d as yellow oil.

Step 8: Compound 9

To a solution of intermediate 6-d (181 mg, 0.81 mmol), intermediate 2-g (330 mg, 0.81 mmol) in 1,4-dioxane, was added cesium carbonate (794 mg, 2.43 mmol), N-N-dimethylaminoglycine (126 mg, 1.22 mmol) and copper (I) iodide (77 mg, 0.40 mmol). The reaction was degassed with argon for 10 minutes, stirred at reflux for 24 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 MgS0₄, filtered and concentrated under reduced pressure. Purification by reverse phase chromatography eluting with 1% HCI/methanol gradient provided compound 9-HCI as yellow solid. MS (m/z) M + H= 502.2

4187891 vl 27 Synthesis of Compound 10:

Scheme 7

Step 1: Intermediate 7-b

To a solution of intermediate 5-g (3.02 g, 12.46 mmol) and intermediate 7-a (1.53 g, 13.71 mmol) in THF (20 ml) were sequentially added triphenylphosphine (3.92 g, 14.95 mmol) and DIAD (2.91 ml, 14.95 mmol) at room temperature and the reaction was then stirred at room temperature overnight. Volatiles were removed under reduced pressure. Purification by silica gel chromatography provided intermediate 7-c as yellow oil.

Step 2: Intermediate 7-c

To a solution of intermediate 7-b (4.0 g, 11.89 mmol) in THF (60 ml) was added a 1.0 M solution of TBAF in THF (14.3 ml, 14.3 mmol) and the reaction was stirred at room temperature for 1 hour. A saturated aqueous solution of ammonium chloride and ethyl acetate were added, the organic layer was

4187891 vl separated, washed with brine, dried over MgS0₄, filtered and concentrated under reduced pressure. Purification by silica gel chromatography provided intermediate 7-c as white solid.

Step 3: Compound 10

To a solution of intermediate 7-c (200 mg, 0.90 mmol), intermediate 2-g (366 mg, 0.90 mmol) in 1,4-dioxane, was added cesium carbonate (880 mg, 2.70 mmol), N-N-dimethylaminoglycine (139 mg, 1.35 mmol) and copper (I) iodide (86 mg, 0.45 mmol). The reaction was degassed with argon for 10 minutes, stirred at reflux for 24 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 MgS0₄, filtered and concentrated under reduced pressure. Purification by reverse phase chromatography eluting with 1% HCI/methanol gradient provided compound 10-HCI as yellow solid. MS (m/z) M+H= 501.2

Compounds 1, 2, 5, 6, 11, 12, 13, 14, 15, 16 and 17 were prepared using similar methods to those described above.

Table 1 summarizes representative compound according to Formula 1.

Table 1: Example Compounds of Formula 1

4187891 vl

4187891 vl

4187891 vl

4187891 vl 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 ™ FP Fluorescein Green Assay supplied from Millipore. Kinase reaction were performed at room temperature for 60 minutes in presence of 250 μΜ substrate, 10 μΜ 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 IC₅₀ 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 (Kl) and the concentration of inhibitor which causes 50 per cent inhibition (I₅₀) of an enzymatic reaction". Biochem Pharmacol 22 (23) : 3099- 108). kj values are reported in Table 2 :

Table 2 : Inhibition of Btk

4187891 vl 6 A

7 A

8 A

9 A

10 A

11 B

12 B

13 A

14 A

15 C

16 C

17 C

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, lOmM HEPES, 50uM beta mercaptoethanol) and incubated at 37 °C, 5% C0₂ 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% C0₂ for lh.

4187891 vl Splenocytes were pre-treated in triplicate with 10,000 nM curves of Formula 1 compounds for lh, followed by stimulation of B cell proliferation with 2.5ug/ml anti-IgM F(ab')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 3:

Table 3: Inhibition of splenic cell proliferation

4187891 vl 13 b

14 b

15 c

16 c

17 c a - Less than 100 nM; b - less than 1000 nM, c - more than 1000 nM

l

Claims

1. A compound of Formula 1 :

A-Y-E-W-Z wherein

X¹ is selected from NH or 0;

X² is selected from N or CH ;

n is an integer from 0 to 2;

Y is selected from :

E is oxygen,

W is selected from :

Xi and X₂ are independently selected from hydrogen and halogen ; m and m' are integers from 0 to 2;

R and Z are independently selected from :

1) alkyl,

2) aralkyl,

3) heteroaralkyl,

4) -OR³,

5) -OC(0)R⁴,

6) -OC(0)NR⁵R⁶,

4187891 vl 7) -CH₂0-R\

8) -NR⁵R⁶,

9) -NR²C(0)R⁴,

10) -NR²S(0)_nR\

11) -NR²C(0)NR⁵R⁶;

wherein the alkyl, aralkyi and heteraralkyl may be further substituted; R² is selected from hydrogen or alkyl;

2. Compound according to claim 1 wherein Z is selected from -OR³ and R³ is selected from substituted or unsubstituted aralkyi, or substituted or unsubstituted heteroaralkyl.

3. Compounds according to claim 1 or 2, wherein A is selected from the group consisting of: