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(11) Patent Number: K.E 5 84
(45) Date of grant: 01/07/2013   
(51) Int.Cl.8: A 61K 31/4985, A 61P 19/02, C 07D 487/04

(2l)Application Number KE/P/2009/ 000875

(22) Filing Date: 11/09/2007

(30)    Priority data:  60/825,168  11/09/2006 us

(86)  PCT data

PCT/US07/078103 11/09/2007 wo 2008/033798 20/03/2008

(73) Owner:SANOFI A VENTIS of 174 Avenue de France, 75013Paris, France

(72) Inventors:GILLESPY, Timothy A., 55 Corporate Drive, Bridgewater, New Jersey 08807, US; EYNOTT, Paul, 55 Corporate Drive, Bridgewater, New Jersey 08807, US; ALLEN Elizabeth M., 55 Corporate Drive, Bridgewater, New Jersey 08807, US; YU, Kin T., 55 Corporate Drive, Bridgewater, New Jersey 08807, US and ZILBERSTEIN,Asher, 55 Corporate Drive, Bridgewater, New Jersey 08807, us .

(74) Agent/address for correspondence:

Kaplan & Stratton Advocates, P.O. Box 40111-00100, Nairobi
(54)    Title: KINASE INHIBITOR.

(57)    Abstract: The invention is directed to a compound of formula (I) and the prodrugs, and pharmaceutically acceptable salts and solvates of such compounds and their prodrugs. Such a compound has valuable pharmaceutical properties, in particular the ability to inhibit protein kinases.






This invention is directed to• substituted azaindoles;, their preparation, pharmaceutical compositions containing these compounds, and their pharmaceutical use in the treatment of disease states capable ofbeing modulated by the inhibition of the protein kinases.



"Protein kinases participate. in the signalling events which control the activation, growth and

30    differentiation of cells in response to extracellular mediators and to changes in the environment. In general, these kinases fall into several groups; those which preferenti'ally phosphorylate serine and/or threonine residues and .those which preferentially phosphorylate
tyrosine residues [S.K.Hanks and T.Hunter, FASEB. J., 19..95, 9, pages 576-596]. The serine/threonine kinases include for example, protein kinase C isotonns [A.C.Newton, J. Bioi.

35    Chern., 1995, 270, pages 28495-28498] and a group of cyclin-depen~ent kinases such as cdc2 [J .Pines, Trends in Biochemical Sciences, 1995, I 8, pages 1QS-197]. The tyrosine kinases 'include membrane-spanning growth factor receptors such a:s the epidermal growth factor receptor [S.Iwashita and M.Kobayashi, Cellular Signalling, 1.992, 4, pages 123-132], and


cytosolic non-receptor kinases such as p56tck, p59tYn, ZAP-70 and csk kinases [C. Chan et. al., Ann. Rev. ImmuMI., 1994, 12, pages 555-592].

Inappropriately high protein"kinase activity has been implicated in many diseases resulting

5    from abnormal cellular function. This might arise either directly or indirectly, for example by failure of the proper control mechanisms for the kinase, related for example to mutation, over-expression or inappropriate activation of the enzyme; or by over- or underproduction of

cytokines or growth factors also participating in the tr-ansduction of signals upstream or downstream of the kinase. tn an of these instances, selective inhibition of the action of the

10    kinase might be expected to have a beneficial effect.

Syk is a 72-kDa cytoplasmic protein tyrosine kinase that is expressed in a variety of hematopoietic cells and is an essential element in several cascades that couple antigen receptors to cellular responses. Thus, Syk plays a pivotal role in signalling ofthe• high affinity

15 IgE receptor, FceRl, in mast cells and in receptor antigen signalling in T and B lymphocytes. The si!,'llal. transduction pathways pre~ent in mast, T and B cells have common features. The ligand binding domain of the receptor lacks intrinsic tyrosine. kinase activity. However,-they interact with transducing subunits that contain immunoreceptor tyrosine based activation motifs (!TAMs) [M.Reth, Nature, 1989, 338, pages 383-384]. These motifs are present in both

20    • the p andy subunits of the FceRl, in the s-subunit the ofT cell receptor (TCR) and in the lgGa and IgG p subunits of the B cell receptor (BCR). [N.S.van Oers and A. Weiss, Seminars in Immunology, 1995,7, pages 227-236] Upon binding of antigen and multimcrization, th~ IT AM residues are phosphorylated by protein tyrosine kinases of the Src family. Syk belongs to a unique class of tyrosine kinases that have two tandem Src homology 2 (SH2) domains and

25    a C terminal catalytic domain. These SH2 domains bind with high affinity to !TAMs and this SH2 -mediated association of Syk with an activated receptor stimulates Syk kinase activity and localises Syk to the plasma membrane.

In Syk deficient mice, mast cell degranulation is inhibited, suggesting that this is an important

30    target for the development of mast cell stabilising agents [P.S.Costello, Oncogene, 1996, 13, pages 2595-2605]. Similar studies have. demonstrated a critical role for Syk in BCR and TCR signalling [A.M.Cheng, Nature, 1995,378, pages 303-306, {1995)and D.H.Chu ct al.,.


Immunological Reviews, 1998, 165, pages 167-180]. Syk also appears• to be involved in eosinophil survival in response to lL-S.and GM-CSF [S.Yousefi et al., J. Exp. Med., 1996, 183, pages 1407-1414]. Despite the key role ofSyk in mast cell, BCR and T cell signalling, little is known about the mechanisin bywhich Syk transmits downstream effectors. Two

5    adaptor proteins, BLNK (B cell Linker.protein, SLP-65) and SLP-76 have been shown .to be substrates of Syk in B cells and mast cells respectively and have been postu'lated to interface Syk with downstream effectors [M.Ishiai et al., Immunity, 1999, I 0, pages 117-125 and
L.R.Hendricks-Taylor et at., J.Biol. Chern, 1997,272, pages 1363-1367].  In addition Syk

appears to play ~n impqrtant role in the CD40 signalling pathway, which plays an important

10    role in B cell proliferation [M.Faris et al., J .Exp. Med., 1994, 179, pages 1923-1931].

Syk is further involved in the activation of platelets stimulated via the low.:.affinity lgG receptor (Fe gamma-RIIA) or stimulated by collagen [F.Yanaga et al., Biochem. J., 1995, 311, (Pt. 2) pages 471-478].


Focal adhesion kinase (F AK) is a non-receptor tyrosine kinase involved in integrin-mediated signal transduction pathway~. FAK co localizes with integrins in focal contact sites and FAK activation and its tyrosine phosphorylation have been shown in many cell types to be dependent on integrins binding to their extracellular ligands. Results from several studies

20    support the hypothesis that FAK inhibitors could be useful in cancer treatment. For example, FAK-deficient cells migrate poorly in response to chemotactic signals and ovcrexpression of C-tem1inal domain ofFAK blocks cell spreading as well as chemotactic migration ( al, J. Cell Science,1999, 112, 2677-2691; Richardson A. and Parsons T., Cell,.1997, 97, 221-231) ; in addition, tumor cells treated with FAK antisense oligonucleotides lost their attachment and

25    underwent apoptosis (Xu et al, Cell Growth Differ. 1996, 4, 413-418). FAK has been reported to be overexpressed in prostate, breast, thyroid, colon and lung cancers. The level of expression ofFAK. is directly correlated with tumors demonstrating the most aggressive phenotype.

30    Angiogenesis or the formation of new blood vessels by sprouting from the preexisting vasculature is of central importance for embryonic development and organogenesis. Abnormal enhanced neovascularization is observed in rheumatoid arthritis, diabetic retinopathy and

during tumor development (Folkman, Nat. Med., 1995, 1, 27•31.). Angiogenesis is a complex multistage process which includes activation, migration, proliferation and survival of endothelial cells. Extensive studies in the field of tumor• angiogenesis in. the past two decades have identified a number of therapeutic targets including kinases, proteases and integrins

5    resulting in 'the discovery•ofmany new anti-angiogenic agents, including KDR inhibitors some of which are currently under clinical evaluation (Jekunen, et al Cancer Treatment Rev. 1997 , 23, 263-286.). Angiogenesis irihibitors may be used in frontline, adjuvant and even preventive settings for the emergence or regrowth of malignancies.

10    Several proteins involved-in chromosome segregation and spindle assembly have been identified in yeast and drosophila. Disruption of these proteins results-in chromosome missegregation and monopolar or disrupted spindles. Among these kinases are the Ipll and aurora kinases from S.ccrevisiae and drosophila respectively, which are required for
centrosome separation and chromosome segregation.  One human homologue of yeast Ipll

15    was recently cloned and •characterized-by different laboratories. This kinase termed Aurora2, STKl 5 or BT AK belongs to the serine/threonine kinase family. Bischoff et al showed that Aurora2 is oncogenic and is-amplified in human colorectal cancers (EMBO J_, 1998, 17, 3052-3065). It has also been exemplified in cancers involving epithelial tumors such as breast


We have now found a novel substituted azaindole, which has valuable pharmaceutical properties; in particular, the ability to inhibit protein kinases, more particularly, the ability to selectively inhibit Syk kinase. This 117,:aindole compound is related to those disclosed in United States Patent 6~ 770,643 but i"s not specifically disclosed in that patent.



The present invention relates to a compound offonnula I:
a phannaceutically- acceptable salt or prodrug thereof, or a solvate of such a compound, its salt or its prodrug.

The invention is aiso directed to a pharrp.!lceutical composition comprising a compound of

10    fonnula I, and method for using the compound of formula I for or treating or preventing a physiological condition related to Syk in a patient.

The invention is also directed to a process for preparing.a compound that is an intermediate useful in preparing a compound offormula I.


In another aspect, the present invention is directed to pharmaceutical compositions com•prising compounds of general formula (I):

the corresponding N-oxide, and the prodrug; and a pharmaceutically acceptable salt and solvate (e.g. hydrate) of such a compound; and theN-oxide and the pro drug; together with one or more .pharmaceutically acceptable carrier or excipient.



FIGURE 1: Reaction scheme for making a compound of Formula l.


FIGURE 2: Mean ankle joint diameter ofrats injected with collagen type II from bovine nasal in Freund's incomplete adjuvant and treated with A003397769, the compound ofFonnula I

(3 .0, 1 o, or 30 mglkg b.i.d.) from day 6 through day 21. Mean ankle joint diameters of female LEW rats sensitized by intra~dennal injection of collagen in Freund's incomplete adjuvant

5    (400ug/400ul/rat) on Days 0 and 7.  Animals dosed from Day 6-21.

FIGURE 3: Analysis of bone erosion (ratio ofbone surface to bone volume) in the calcaneus of CIA rats treated with A003397769 (3.0, 10 or '30 mglkg).

10    FIGURE 4: Mean ankle joint diameter of rats injected with collagen type U from bovine nasal in Freund's incomplete adjuvant and treated with A0033977(i9 (3.0 mgfkg) alone or in combination with Methotrexate (0.1 or 0.2 mglkg) froin day 6 through day 20 or 21 and compared to vehicle dosed animals. Effects of SYK Inhibitor, A003397769 (3.0 mglkg b.i.d.), as Monotherapy or in Combination with Methotrexate on Rat CIA.


FIGURE 5: Mean ankle joint diameter of rats injected with collagen type 11 from bov.ine nasal in Freund's incomplete adjuvant and treated with A003397769 (10 mglkg) alone or in combination with Methotrexate (0.1 or 0.2 mglkg) from day 6 through day 20 or 21 and compared to vehicle dosed animals. Effects ofSYK Inhibitor, A003397769 (10 mglkg b.i.d.),

20    as Monotherapy or in Combination with Methotrexate on Rat CIA.

FIGURE 6:  Analysis ofbone erosion (ratio of bone surface to bone volume) in the calcaneus

of CIA rats treated with A003397769 (3.0 or 10 mglkg) alqne orin combination with Methotrexate (0.1 or 0.2 mglkg). •


FIGURE 7: Mean ankle joint diameter of rats injected with collagen type II from bovine nasal in Freund's incomplete adjuvant and treated therapeutically with A003397769A (10 and 30 mglkg b.i.d) from day 1.2 through day 21. Mean Ankle Joint Diameters ofFemale Lewis Rats Sensitized by Intra-dermal Injection of Collagen in Freund's Incomplete Adjuvant (400

30    ug/400ul/rat) on Days 0 and 7.  Average-of Both Paws.

FIGURE 8: Analysis of bone erosion (ratio ofbone surface tp bone volume) in the calcaneus of CIA rats treated therapeutically with A003397769A (10 or 30 mg/kg b.i.d.).

35    FIGURE 9: Effect ofthe compound ofFormula I on rat weight.

•FIGURE 10: Effects ofthe compound ofFonnula I on hemoglobin concentration.



Thus, in one aspect, the present invention i~ directed to phannaceudcal compositions comprising a compound of general fonnula (1):

which also may be known as: 2-[4-(7-Ethyl-5H-pyrrolo[2,3-b]pyrazin-6-yl)-phenyl]-propan-2-ol.


In the present specificat.ion, the term "compound ofthe invention", and equivalent expressions, are meant to embrace a compound of general formula (I) as hereinbefore
15• described, which expression includes th~ prodrugs, the pharmaceutically acceptable salts, and the solvates, e.g. hydrates,. where the context so permits. Similarly, reference to intennediates, whether or not they themselves are claimed, is meant to embrace their salts, and solvates, where the context so permits. For the sake of clarity, particular instances when the context so permits are sometimes indicated in the text, but these instances are•purely illustrative and it is

20    not intended to exclude other instances when the:context so permits. Abbreviatibns used herein:
ATP adenosine triphosphate

25    DTT dithiothreitol

PBS phosphate buffered saline

As used above, and throughout the description of the invention, the following terms, unless otherwise• indicated, shall be understood to have the following meanings:

"Patient" includes both human and other mammals.


"Prodrug" means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound of formula (I), including N-oxides there0f. For example an ester of a compound of formula (I) containing a hydroxy group may be convertible by hydrolysisin_ vivo to the parent molecule. Alternatively, an ester of a compound of formula (I) containing a
10    carboxy group may be convertible by hydrolysis in vivo to the parent molecule.

Suitable esters of compounds of formula (I) containing a hydroxy group are, for example acetates, citrates, lactates, tartrates, malonates, •oxalates, salicylates, propionates, succinates,
fumarates, maleates, methylene-bis-~-hydroxynaphthoates, gentisates, iscthionates,

15    di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesultonates, p-toluenesultonates, cyclohexylsulfamates and quinates.

An especially useful class of esters of compounds of formula (I) containing a hydroxy group,

may be formed from acid moieties selected from those described by Bundgaard et. al., J. Med.

20    Chern., 1989, 32 , page 2503-2507, and include substituted (aminomethyl)-benzoates, for example dialkylamino-methylbcnzoates in which the two alkyl groups may be joined together and/or interrupted by an oxygen atom or by an optionally substituted nitrogen atom, e.g. an alkylatcd nitrogen atom, more especially (morpholino-methyl)benzoates, e.g. 3- or 4-(morpholinomethyl)-benzoates, and. (4-alkylpiperazin-1-yl)benzoates, e.g. 3- or
25    4-( 4-alkylpiperazin-1-yl)benzoates.

Some of the compounds of the present invention are basic, and such compounds are useful in

the form of the free base or in the fonn of a pharmaceutically acceptable acid addition salt



Acid addition salts are a more convenient form for use; and in practice, usc of the salt form inherently amounts to use of the free base fonn. The acids which can be used to prepare the

acid addition salts include preferably those which produce, when combined with the• free base, pharmaceutically acceptable salts, that is, salts whose anions are non-toxic to the patient in pharmaceutical doses of the salts, so that the beneficial inhibitory effects inherent in the free base are not vitiated by side effects ascribable to the anions. Although pharmaceutically

5    acceptable salts of said basic compounds are preferred, all acid addition salts are useful as sources of the free base form even if the particular salt, per sc, is desired only as an intermediate product as, for example, when the salt is formed only for purposes of

purification, and identification, or when it is used as intermediate in preparing a phannaceutically acceptable salt by ion exchange procedures. Pharmaceutically acceptable

to salts within the scope of the invention include those derived from mineral acids and organic acids, and include hydrohalides, e.g. hydrochlorides and hydrobromides,. sulfates, phosphates~ nitrates, .sulfamates, acetates, citrates, lactates, tartrates, malonates, oxalates, sa1icylates, propionates, succinates, fumarates, maleates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methane-sulfonates, ethanesulfonates, benzenesulfonates,
15    p-tolucnesulfonates, cyclohexylsulfamates and quinates.

As wel1 as being useful in themselves as:active compounds, salts of compounds of the

in vcntion are useful for the purposes of purification of the compounds, for example by exploitation of the solubility differences between the salts and the parent compounds, side
20    products and/or starting materials by techniques well known to-those skilled in the art.

The compounds of the invention exhibit useful pharmacological activity and accordingly are incorporated into pharmaceutical compositions and used in the treatment of patients suffering• from certain medical disorders. The present invention t~us provides, according to a further

25    aspect, compounds of the invention and compositions containing compounds of the invention for use in therapy.

Compounds within the scope of the present invention inhibit or block kinase catalytic activity according to tests described in the literature and described in vitro procedures hereinafter, and

30    which tests results are believed to correlate to pharmacological ~ctivity in humans and other mammals. Thus, in a further embodiment, the present invention provides compounds of the invention and. compositions containing compounds ofthe invention for use in the treatment of

a patient suffering from, or subject to, conditiGns which can be ameliorated by the administration ofproteinkinase inhibitors (e.g. Syk, FAK, KDR or Aurora2). For.example, compounds of the present invention ar~ useful in thetreatm.ent of inflammatory diseases.• for example asthma: inflammatory dermatoses {e:g. psoriasis; dematitis herpetiformis, eczema,

5    necrotizing a.nd cutaneous vasculitis, bullous dis.ease); allergic rhinitis and allergic. conjunctivitis; joint inflammation~.including arthritis, rheumatoid arthriti~ and other arthritic conditions such as rheumatoid spondylitis, gouty arthritis, traumatic arthritis, rubella arthritis, psoriatic arthritis and osteoarthritis. The compounds. are also useful in the treatment of Chronic Ol;>structive Pulmonary Disease (COPD), acute syn•ovitis, autoimmune diabetes,

10    autoimmune encephalomyelitis,. collitis, atherosclerosis, peripheral vw;cular disease, cardiovascular disease, multiple sclerosis, r:estenosis,.myocarditis, B cell lymphomas,
systemic lupus erythematosus,

!,'Taft versus host. disease and other transplant associated rejection events, cancers al).d tumors

(such as colorectal, prostate, breast, thyroid, colon and lung cancers) and  inflamma~oty bowel

15    disease. Additionally, the compounds are useful as tumor anti-angiogenic agents. And furthermore! the compounds of the invention are useful as.agcnts to control tumot cells.

A special embodiment. of the therapeutic methods of the present invention is the treating of joint inflammation.


Another special embodiment of the therapeutic methods of the present invention is the treating of rheumatoid arthritis.

A special-embodiment of the therapeutic methods of the present invention is the treating of

25    cancers, tumors an(,i other proliferative disorders.

Another special embodim~nt of the therapeutic methods of the present invention is the treating of cancers. involving liquid tumors:

30    Another special embodiment of the therapeutic methods of the presen~ invention is the treating of mantle .cell lymphoma.

Yet another special embodiment of the therapeutic methods of the present invention is the treating of disorders by inhibition of angiogenesis.

According to a further feature of the• invention there is provided,a method fcir the treatment of

5    a hw11an or animal patient suffering from, or subject to, conditions which can be ameliorated by the administration of a protein kinase inhibitor (e.g. Syk, F AK, KDR or Au.rora2) for example conditions as hereinbefore described, which comprises the administration to the

patient of an effective amount of a compound of the invention or a composition containing a

compound of the invention.  "Effective amount" is meant to describe an amount of compound

10    of the present invention effective in inhibiting "the catalytic a.ctivity a protein kinase, such as Syk, FAK, KDR or Aurora2, and thus producing the desired therapeutic effect.

References herein to treatment sho:tlld be understood to include prophylactic therapy as well as treatment of established conditions.


The present invention also includes within its scope phannaceutical compositions comprising a compound of the invention in association with a pharmaceutically acceptable carrier or excipient.

20    A compound of the invention may be administered by any suitable means. In practice a compound ofthe present invention may generally be administered parenterally, topically, rectally, orally or by inhalation; especially•by the oral route or by inhalation.

Compositions according to the invention may be prepared• according to the customary

25    methods, using one or more pharmaceutically acceptable adjuvants or excipients. The adjuvants comprise, inter alia, diluehts, sterile aqueous media and the various 1.10n-toxic organic solven~s. The compositions may be presented in the form of tablets, piUs, granules, powders, aqueous solutions or suspensions, injectable solutions, elixirs or syrups, and can
contain one or more agents chosen from the group comprising sweeteners, flavourings,

30    colourings, or stabilisers in order to obtain pharmaceutically acceptable preparations. The choice of vehicle and the content of active substance in the vehicle are generally determined in accordance with the solubility and chemical properties of the active compound, the particular


mode of administration and the provisions to be observed in phannaceutical practice. For example, excipients such as lactose, sodium citrate, calcium carbonate, dicalcium phosphate and disintegrating agents such as, alginic acids and certain complex silicates combined with lubricants such as magnesium stearate, sodium lauryl sulfate and tulc may be used for

5    preparing tablets. To prepare a capsule, it is advantageous to use• lactose and high molecular weight polyethylene glycols. When aqueous suspensions are used they can contain emulsifying agents or agents which facilitate suspension. Diluents such as sucrose, ethanol, polyethylene glycol, propylene glycol, glycerol and chloroform or mixtures thereof may also
be used.


For parenteral administration, emulsions, SU$pcnsions or solutions of the products according to the invention in vegetable oil,. for example sesame oil, groundnut oil or olive oil, or aqueous-organic solutions such as water and propylehe glycol, injectable organic esters such as ethyl oleate, as well as sterile aqueous solutions of the phannac.eutically acceptable salts,

15    are used. The solutions of the salts of the products according to the invention are especially useful for administration by intramuscular. or subcutaneous i.njection. The aqueous solutions, also comprising solutions of the salts in pure distilled water, may be used for intravenous administration with the proviso that their pH is suitably adjusted, that they are judiciously

buffered and rendered isotonic with a sufficient quantity of gJucose or sodium chloride and

20    that they are sterilised by heating, irradiation or microfiltration.

For topical administration, gels (water or alcohol based), creams or ointments containing compounds ofthe invention.maybc used. Compounds ofthe invention may also be

incorporated in a gel or matrix base for application in a patch, which would allow a controlled

25    release of compound through the transdermal barrier.

For administration by inhalation compounds of the invention may be dissolved or suspended in a suitable carrier for use. in a nebuliser or a suspension or solution aerosol, or may be absorbed or adsorbed onto a suitable solid carrier for use in a dry powder inhaler.

Solid compositions for rectal administration include suppositories formulated in accordance

with known methods and containing at least one compound of the invention.
The percentage of active ingredient in the compositions of the invention may be v•aried_, it being necessary that it should constitute a proportion suc)1 that a suitable dosage shall be obtained. Obviously,. several unit dosage forms may be administered at about the same time.

5    The dose employed will be determined by the physician, and depends upon the desired therapeutic effect, the route of administration and the duration of the treatment, and the condition of the patient. ln the adult, the doses are generally from about 0.001 to about 50, preferably about 0.001 to about 5, mglkg body weight per day by inhalation, from about 0.01

to about 100, preferably 0.1 to 70, more especially 0.5 to 10, mglkg body weight per day by

10    oral administration, and from about 0.001 to about 10, preferably•0.01 to 1, mglkgbody weight per day by intravenous administration. ln each particular case, the doses will be determined in accordance with the factors distinctive to the subject to be treated, such as age, weight, general state ofhealth and other characteristics which can influence the efficacy of the medicinal product.


The compounds according to the. invention may be adl'ninistered as frequently as necessary in order to obtain the desired therapeutic effect. Some. patients may respond rapidly to a higher or lower dose and may find much weaker maintenance doses adequate. For other patients, it may be necessary to have long-term treatments .at the rate of 1 to 4 doses per day, in

20    accordance with the physiological requirements of each 'particular patient. Generally, the active product may be administered orally 1 to 4 times per day. Of course, for some patients, it will be necessary to prescribe not more than one or two doses per day.

Compounds of the invention may be prepared by the application or adaptation of known

25    methods, by which is meant methods used heretofore or described in the literature, for example those described by R.C.Larock in Comprehensive Organic Transformations, VCH publishers, 1989.

In the reactions described hereinafter it may be necessary to protect reactive functional groups,

30    for example hydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions. Conventional protecting


groups may be used in accordance with standard practice; for examples see T.W. Greene and P.G.M.Wuts in "Protective Groups in Organic Chemistry" John Wiley and Sons, 1991.

It will be appreciated that compounds of the present invention may contain asymmetric

s centres. These asymmetric centres may independently be in either the R or S configuration. It will be apparent to those skilled in the art that certain compounds of the invention may also exhibit geometrical isomerism. It is to be understood that the present invention includes individual geometrical isomers and stereoisomers and mixtures thereof, including racemic mixtures, of compounds of formula Q) hereinabove~ Such isomers can be separated from their

10    mixtures,. by the application or adaptation pfknown methods, for example chromatographic techniques and recrystallisation techniques, or they arc separately prepared fi:om the appropriate isomers of their intermediates.

According to a further feature of the invention, acid addition salts of the compounds of this

15    invention may be prepared by reaction of the free base with the appropriate acid, by the application or adaptation ofknown methods. For example, the acid addition salts of the compounds of this invention may be prepared either by•dissolving the free base in water or

aqueous alcohol solution or other suitable solvents containing the appropri'ate acid and isolating the salt by•evaporating the sblution, or by reacting the free base and acid in an
20    organic solvent, in which •case the salt separates directly or be obtained by concentration •of the solution.

The acid addition salts of the compounds of this invention can be regenerated from the salts by

the application or adaptation of known methods.  For exa~ple, parent compounds of the

25    invention can be regenerated from their acid addition salts by treatment with an alkali, e.g. aqueous sodium bicarbonate solution or aqueous ammonia solution.

Compounds of this invention can be regenerated from their base addition salts by the application or aqaptation ofknown methods ..For example, parent compounds of the invention

30    can be regenerated .from their base addition salts by treatment with an acid, e.g. hydrochloric acid.


Compounds ofthe present invention m~y be conveniently prepared, or formed during the process of the invention, as solvates (e.g. hydrates). Hydrates of compounds of the present invention may be conveniently prepared by recrystallisation from an aqueous/organic solvent mixture, using organic solvents such as dioxan, tetrahydrofuran or methanol.


According to a further feature of the invention, base. addition salts of the compounds of this invention may be prepared by reaction acid with the appropriate base, by the application or adaptation ofknown methods. For example, the base addition salt&-ofthe• compounds of this invention may be prepared either by dissolving the free acid in water or

1o aqueous alcohol solution or other suitable solvents containing the appropriate base and isolating the salt by evaporating the solution, or by reacting the free acid and base in an organic solvent, in which case the salt sep~ates directly or- can be obtained by concentration of the solution.

15    The starting materials and intermediates may be prepared by the application or adaptation of known methods, for example methods as described in the Example or their obvious chemical equivalents.

20    The present invention is further Exemplified but not limited by the following illustrative Example.

300 MHz '1 H nuclear magnetic resonance spectra (NMR) were.recorded on a Varian Mercury

instruments.  ln the nuclear magnetic resonance spectra (NMR) the cht."'llical shifts (o) are

25    expressed in ppm relative to tetramethylsilane. Abbreviations have the following significances: s =singlet; d =doublet; t =.triplet; m =multiplet; q =quartet; dd =doublet of doublets; ddd =doublet of double doublets.

High Pressure Liquid Chromatography - Mass Spectrometry (LCMS) experiments to

30    determine retention times (RT) and associated mass ions are perfonned using the following method. Mass Spectra (MS) are recorded using a Micromass LCT time of flight mass spectrometer. The method is positive electrospray ionization, scanning mass m/z from 100 to


1000.  Liquid chromatography is performed on an Agilent™   I 100 Series Binary Pump &

Degasser; stationary phase: Phenomenex Synergi™ ~~L Hydro-RP 20 X 4.0mm column, mobile phase: A= 0.1% formic acid (FA) in water, B = 0.1% FA .in acetonitrile. Injection volume of 5~-tL by CTC Analytical PAL System. Flow is I mL/minutc. Gradient is 5% B to

5    90% B in 3 minutes and 90% B to 100% B in 2 minutes. Auxiliary detectors are: Agilent 1100 Series UV detector, wavelength= 220 run and Sedere SEDE)(TM 75 Evaporative Light Scattering (ELS) detector temperature= 46°C, Nitrogen pressure= 4bar.

The thin layer chromatography (TLC} Rp were determined using Merck™  silica plates.



A total of 6.0 g of 2-[ 4-(7-ethyl-5H-pyrrolo[2,3-b ]pyrazin-6-yl)phenyl]propan-2-ol (compound 1) was prepared in two steps from n-propylpyrazine (compound 2) and 4-acetylbenzonitrile (compound 3).


The synthesis of compound 1 was carried out as follows. Coupling of n-propylpyrazine (compound 2) and 4-acetylbenzonitrile (compound 3) with sodium bis(trimethylsilyl)amide in tetrahydrofuran at 40 °C gave the intermediate compound 4 in 30% )~eld. Reaqtion of compound 4 with methylmagnesium chloride in tctrahydrofuran at 0 °C gave the desired

25    compound compound 1 in 74% yield after recrystallization from 2-propanol. The synthesis is shown in Figure I.


2-[4.-(7-Ethy1-5H-pyrrolo[2,J-b]pyrazin-6-yl)phenyl]ethanone (compound 4). A solution of n-propylpyrazine (compound 2, 912 mg, 7.46 mmol) in tetrahydrofuran (5 mL) was added drop-

35    wise, over a period of seven minutes, to a solution of sodium bis(trimethylsilyl)amide (2M


solution in THF; 13 mL, 26 mmol, 3.5 equiv) at 20 °C. A deep, purple-red solution was obtained and the temperature fell to 16.4 °C. A solution of 4-acetylbenzonitrile (compound 3,

1.08 g, 7.4 mmol) in tetrahydrofuran (5 rnL) was added, over. a period of 18 minutes, at 13.3

''C. The temperature fell to 12.6 °C and a brown solution ensued: The mixture was stirred at s room temperature for one hour, heated to about 35 °C Jor six hours, then left to stir at room

temperature for about 60 hours. The mixture was poured into aqueous saturated sodium bicarbonate (200 mL) and extracted with ethyl acetate (2 x 150 mL). The ethyl acetate was washed with water (1 00 mL) and concentrated on the Buchi at a bath temperature of 40 °C and from 80 to 10 torr to give a deep-yellow solid. The solid was triturated with diethyl ether (25

1o mL), filtered -and washed with ether (25 mL). The solid was air dried to give 600 mg (30.3%) of compound 4 as a yellow solid: 1H NMR (300 MHz, CDCh, figure 1) 8 8.5 ( 1H, d, J=2 Hz), 8.15 (2H, d, J=8 Hz), 8.1 (1 H, d, 1=3 Hz), 7.9 (2H, d, 1=8 Hz), 3.1 (2H, q, J=9 Hz), 2.7 (3H, s), 1.4 (3H, t, J=9 Hz).

1-[ 4-(7-Ethyl-SH-pyrrolo[2,3-b]pyrazin-6-yl)phenyl]propan-2-ol (compound 1).  To •a cooled

(~5 °C) solution ofmethylmagnesiurn chloride (3M in THF; 81.3 mL, 244 mmol, 10 equiv.) in tetrahydrofuran (116 mL), was added a solution of eompound 4 (6.5 g, 24.4 mmo1) in tetrahydrofuran (348 mL) drop-wise, over ninety minutes, keeping the temperature at about-0

20    °C by the rate of addition. A bright yellow solution was observed on addition. After one hour, TLC (ethyl acetate/n-heptane 111) showed no starting material present and a new spot moving at a lower Rr. The batch was quenched by the carefui•addition of a saturated, aqueous solution of sodium bicarbonate (about 660 rnL). A thick mass ensued and ethyl acetate (250 mL) and water (250 mL) were added to the mixture. The aqueous layer was removed andre-extracted
25    with ethyl acetate (2 x 250 mL).  The ethyl acetate fractions were combined, washed with

water (2 x 200 mL) and concentrated on the Buchi at a bath temperature of 40 °C and from 80 to 10 torr, to give 7.5 g (Hi9%) ofl as a light-beige solid: 1H NMR (.DMSO-d6, figure 2) o

12.0 (lH, s), 8.35 (lH, d, J=J.5 Hz), 8.2 (lH, d, J=3..5 Hz), 7.6 (4H, s), 5.1 (lH, s), 2,9 (2H, q, J=8 Hz), 1.5 (6H, s), 1.3 (3H, t, J=8- Hz).


This material was combined with material from a previous experiment. The combined material (8.5 g) was refluxed with 2-propanol 0 50 mL) to give a clear, light brown solution, which was filtered hot through a hot Buchner funnel, under vacuum. A precipitate fanned in


the filter flask and the mixture wa~ heated to boiling to give a clear solution. The material was allowed to cool to room temperature with stirring, to give a light, yellow solid, which was fil~cred, washed with cold 2-propanol and dried in the vacuum oven at 50°C to give 6.0 g
(71 %) of compound 1 as a light, yellow solid:  LCMS: RT= 2.55 minutes, MS: 282 (M+H); 1H

s    NMR (DMSO-d6, figure 3) 5 12.0 (lH, s), 8.35 (l.H, d, J===3 Hz), 8.2 (l.H, d, J=3 •Hz),. 7.6 {4H,. s), 5. I (1 H, s), 2.9 (2H, q, J=9 Hz), 1.5 (6H, s), 1.3 (3.H, t, J::::9 Hz).

The elemental analysis of the compound l is shown in TABLE 1.

PER ELEMENT:    C 72.57%, H 6.81%, N 14.93%,0 5.69%

EXPERIMENTAL 1:    C 72.46%, H 7.05%, N 15.07%

15    TABLE 1



Assay Name:    Spleen Tyrosine (Y) Kinase

Short Name:    Syk

Assay Format:    Substrate Phosphorylation.

25    Detection Fonnat:    Streptavidin FlashPlate

Modulation:    Inhibition

Streptavidin FlashPlate Plus microplates from PerkinElmer Life Sciences™   are designed for

30    in-plate radiometric assays. The interior of each well is permanently coated with a thin layer of polystyrene-based scintillant followed by covalent binding of Streptavidin. These plates. are suitable for a wide variety of assay applications which utilize biotinylated capture molecules. Poly (Olu,Tyr) 4:1 (POT) is a random copolymer that can act as a substrate for tyrosine-specific protein kinases. The assay measures the phosphorylation of POT-Biotin substrate by


Syk. The enzyme transfers the [y33 P]-phosphate fro•m [l3P]-ATP t~ the polymeric substrate. The assay was run in solution in a non-:birtding_ 384-well plate. After stopping the reaction using phosphoric acid, the r~action mixture was transferred to a 384-well Streptavidin Flashplate. The biotinylated s~bstrate was c~ptured onto the plate ancj. other reagents including:

5    hot ATP were washed away. Each well was then counted for radioactivity.

The enzyme reaction was run in a 384-well non-binding plate. Final reagent concentration/well was: 7.77 nM Syk, 15.5 nM POT-Biotin substrate, 0.1 J.!.Ci 33P-ATP, 50 mM Tris, pH 7.5, 10 mM MgC}z, 3 mM MnCh, I mM DTT, 0.1 mglml y-Globulins. The

10    reaction volume was: 22 Jll. Reaction time: 120 minutes. Temperature: Room temperature. The reaction was stopped by addition of20 Jll of9% Phosphoric acid and 30 ~Ll of reaction mixture• was transferred to a 384-well Streptavidin Flashplate. After 90minutes ofincubati•on

at room temperature, the plate was washed with 0.02% ofTween-20 in 50 mM Tris, pH 7.5. Radioactivity was counted on a.Top Count™ scintillation counter.

.    15

Enzyme dilution was prepared and kept on ice prior- to. use.

MnCh and DIT was added fresh into the assay buffer prior to use.

Materials for the assay are shown in Tnble 2.


            1M MgCl2            Sigma        M-1028    95.2    Enzyme
            I MnCh            Sigma        M-l787    125.8    Enzyme
            DTT            Sigma        D-5545    154.2    Antioxida
            y-Globulins            Sigma.        G-5009        Protein
            Staurosporine                    .S-4400    466.5    Reference
Phosphoric acid (85%)            Sigma        P-6560    98.0    Stop


        1 mCi fy33P]-ATP    PerkinElmer        NEG6xx        Substrate   
            10 x PBS, pH 7.4    Fisher        BP399-1        Wash   
            Tween20    Fisher        BP337-500    1227.54    Detergent   
    384-well polypropylene    Coming        3657        Compoun   
            plate                        d plate   
    Non-binding 384-well    Corning        3652        Reaction   
            plate                        plate   
    384-well Strcptavidin    PcrkinElmer        SMP-410        Capture   
            Flashplate                (A)        plate   
    Top Seal A sealing film    Packard        6005185        Plate   
            Elx405 Automated    Bio-Tek                Plate   
            Washer                        washer   
            Top Count    Packard                Counter   
            FX Station    Beckman                Liquid   
            Beckman 2000    Beckman                Liquid   


Flag-tagged Syk (0.184 mglml, .MW = 35,531.81 Da) was produced and purified by methods

5    known in the ati.


Biotin conjugated Poly (Glu,Tyr) 4:1•was purchased from Cispio intemationalTM (catalog#

61.GTOBLB, lot# 16).


Assay solutions used are shown in Table 3.

    Reagent    Chemicals    Solvent            Concentration       
    Assay Buffer    Tris, pH 7.5    H20            SOmM       
    MnCh and DTI add    MnCh                3mM       
        MgCh                IOmM       
        DTT                lmM       
        y-globuJins            •0.1%       
    Enzyme & Substrate    Syk    Assay buffer            7:77 nM       
    Solution    PGT-Biotin                       
    Keep in non-binding                    15.5 nM       
    plates    substrate                       
    ATP I jjP-ATP        ATP/ 33P-A TP    Assay buffer    0.1 J!Ci/well
    Stop Solution        Phosphoric acid    H20    9%
    Wash Buffer        Tween-20    1 x PBS, pH 7.4    0.02%


Compound dilution:

1.    Compounds were received as 10 ~tl!well of lOmM in 100% DMSO in a 96-well U-bottom polypropylene plc!te with Row H empty. Added 90 J!l/well of 100% DMSO to yield I 00 J!l of 1 mM compound, resealed and stored the plate at room temperature in
10    the dark.

2.    Prepared compound destination plate: In a 384-well round-bottom polypropylene plate (Coming storage plate), added 23 J!l/well of H20 to eolumns 3 & 13, 20 J!llwell of 30% DMSO to columns 4 through 12 and columns 14 through 22 (leaving Rows 0 and

P empty).

15    3. Prepared compound dilutions (I 0 dilutions, 300 J!M, 100 ~tM, 30 J!M, --- etc) on Biomek 20QOTM using Kinase Profiling program: Using 20 J.d tips with 8'hone (Row

H)    removed, transfered lO J!l/well of 1 mM compound from Column 1 in source plate to Column 3 in destination plate to• make the first 300 J!M dilution. After that, in
destination plate, mixed and transfered 10 ullwell of300 J!M dilution from column 3 to

20 4 to make the 100 J!M dilution. Mixed and transfered 10 ul/well of 100 ~lM dilution from column 4 to 5 to make the 30 J!M dilution, ---etc. Made duplicate dilutions for each compound, for example, transfered 10 J!IIwell of compound from A I in source plate to A3 and B3 in destination plate. Repeated mixing and transfered. Then. transfered 10 !!1/well of 1 mM compound from Column 2 in source plate to Column 13

25    in destination plate. to• make the 300 J!M dilution. After that. in destination plate, mixed and transfered 10 ul/well of 300 J!M dilution from column 13 to 14 to make the 100 J!M dilution --- etc. One full 96-well source plate with Row H empty can make up to six compound destinatien plates.

4.    Prepared standard compound parent plate: Added 5 J!Vwell of 10 mM Roscovitine

30    solution in 100% DMSO to HI and H2 in a 96:-well U-bottom polypropylene plate, then diluted to 1 mM solution with 45 J!Vwell of 100% DMSO.


5.    Prepar~e standard compound destination•plate: In a 384-wcll round-bottom

polypropylene plate (Coming™  storage plate), added 23 ~tl/well ofHzO to columns 3

& 13, 20 Ill/well of30% DMSO to colum.ns-4 through 12 and columns 14 through 22 (Rows 0 and P only).
5    6.  Prepared standard compound dilutions (10 dilutions, 300 J.!M, 100 u J.tM, 30 u J.!M, ---

etc) on Biomek 200QTM •using Profiling Standard program: Using single 20 Ill tip transfered 10 J.!llwell of 1 mM standard compound from Hl in parent plate to 01 in
•destination plate to make the first 300 !lM dilution. After that, in destination pl~te, mixed and transfered.l 0 ul/wcll of 300 J.!M dilution from 03 to 04 to make the l 00

1o 1-1M dilution. Mixed and transfered 10 ullwell of 100 1-1M dilution from 04 to 05 to make the 30 ~tM dilution,--- etc. Made duplicate dilutions for each standard

. compound, for example, transfered I 0 Jll/well of compound from H 1 in parent plate to 03 and P3 in destination plate. Repeated mixing and transfered. Then, tmnsfered 10 pi/well of 1 mM compound from H2 in parent plate to 013 in de.stination plate to

15    make the 300 J.!M dilution. After that, in destination plate, mixed and transfered 10 ullwell of300 !lM dilution from 013 to 014 to make the 100 !lM dilution--- etc.

7.    In standard compound destination plate, added 20 ~!/well of 30% DMSO (high control) to A through H and 20 ~!/well of 45% H3P04 (low control) to 1 through J in column 23.


Assay procedure:

.1.  To the assay plate (Coming™  Non-bi.nding 3.84-well plate), added 10 Enzyme &

Substrate Solution, 2 ~tl of test compound, incubate. at room temperature for 30 min (enzyme/ compound pre-incubation step).
25    2.  Started Reaction by adding lO ~I ATP/ 33P-ATP Solution.

3.    Incubated at room temperature for 120 min.

4.    Stopped the reaction by adding 20 fll Stop Buffer.

5.    Transfered 30 Jll of reaction mixture to a 384-well Streptavidin Flashplate;

8.    Incubated temperature. for 90 min.

30    9. Washed Streptavidin Flashplate 2 times with 100 Ill/well of Wash Buffer using Elx405 Automated Washer.•

10.    Sealed and read the. plate (40 sec/well) on Top CounfrM scintillation counter.

*Ran assay from step. 1 to step "5 using Biomek™  FX station.


Equation for curve fitting in ICso detcnnination:

10    Y=Bottom +(Top-Bottom)/(!+ 10"((Log1Cso-X)*Hil1Slope))

X is the logarithm of concentration.

Y is the response.
Y starts at Bottom and goes to Top with a sigmoid shape.

15    This is identical to the 11 four parameter logistic" equation.

The compound ofFonnula I resulted in an ICsoof 1.7 nanoMolar with this assay.






This method is to detennine the viability of a liquid tumor cell line following treatment with a test compound. Tumor cells are maintained in suspension at log phase. growth. On the day of use, cells are resuspended to a density of 0.05 to 0.1 million/ml and cells are incubated in 96 well plate with the test compounds for 96 hours. Viability of cells was measured by incubating

30    cells with Promega's MTS reagent. The viability of the cell is proportional to the change in absorbance at 490 nm. By comparing the absorbance between control and compound-treated cells, the impact of the test compounds on the cell viability is detennined as a percent ofth~ control cell viability.


A. Materials

I.  Cells:

Liquid tumor cell lines are obtained from either American Tissue Culture Collection or from DSMZ (Deutsche Sammlung von Mikroorganismen und Z:ellkulturen GmbH).

2.  Culture medium:

5    Complete RPMI: RPMI-1649 medium with 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and L-glutamine (Gibco!Invitrogen™, Cat. #•22400-089) + 10% heat-inactivated fetal bqvine serum (FBS) (Gibco/Invitrogen™, Cat. # 16140-071) + lx Penicillin/Streptomycin (Gibco/ Inv'itrogen, Cat.# 15070-063) + 50uglml Plasmocin
(Invivogen™,    Cat.# ant-mpt)

10    Phenol red free cRPMI: Phenol red free RPMI-1640 medium L-glutamine (Gibco/ Invitrogen™, Cat. # Gibco/ Invitrogen, 11835-030) + 10% heat-inactivated fetal bovi~e •serum (FBS) (Gibco/ Invitrogen, Cat..# 16140-0TI) + lx Penicillin/Streptomycin (Gibco/ lnvitrogenTM, Cat. # 15070-063)

15    3.  Other liquid reagents:

Promega™ MTS reagent (Cell Titer 96 Aqueous Cat. # G358B) Dimethyl sulfoxiqe (DMSO) (SigmaTM, Cat.# D2650)

4.  Consumable supplies:

20    Sterile 96-well polystyrene tissue culture-treated clear plates with lid (Falcon™,  Cat.# 3072)

5.  Equipment:

Platereader, 96-well (SpectraMAX GeminiEM™,  Molecular Devices, USA)

25    B. Method

Day 1: Preparation of test compounds

Dissolve and serial-dilute test compounds in DMSO at 1OOOx concentration.

Dilute 1:100 test compounds in pheno.l red free cRPMI in sterile 96-well polystyrene tissue

30    culture plates

Use a 12-channel pipetor to tmnsfer I Oul of diluted compound solution to the an empty 96 well plate to be used for cell incubation


Following addition of 1OOul of cell cultures, the• final concentration of test compounds would

be 1x.

Dayl : Preparation ofliquid tumor cells for compound treatment

Harvest cells in complete RPMI from log-phase cultures at densities of approximately 0.3-0. 7

5    million/mi. Count cells and adjust to 0.1 million/ml in phenol red free cRPMI (For fast growing cells with doubling time of equal or less than 24 hours, e.g. K562, use 0.05 million/ml instead. to •avoid over-growing).

Transfer triplicates of l 00 J.l.l of cells to the wells of 96-well clear plates for a total of I 0000 cells/ well. Note that each well already contains 10 f.ll of 1Ox concentrated test compound
10    prepared as above.

Cell incubation

Incubate cells for 72-96 hours at 37°C in a tissue culture incubator containing 5% C02•

C- Measurement : Determination of cell viability•

15    l. Thaw the Promega's MTS reagent and add 20ul to each well with a repeat pipetor.

2.    Mix the reagent in the well by rocking the plate and place the plate to a C02 tissue culture incubator at 370C.

3.    Prepare the "n"o cell" blank control by adding the MTS reagent to a row of wells containing 1OOul of phenol red free cRPMI alone.

20    4. Incubate at 370C until the absorbance at 490 n111 for the control cells is > 1.5..

5.    Mix the cell culture by rocking to ensure the color is uniform in the wells, making sure there are no air bubbles. If present, centrifuge the plate at l.OOOxg on a bench top centrifuge to eliminate air bubbles.

6.    Read the absorbance in the Molecular Devices 96-well plate reader.


D-Analysis of results:

1.    Copy and paste the text data onto an Excel spreadsheet

2.    Average the "no cell" blank data and subtract this value from the absorbance from each well containing cells ...

30    3. Average the blank-corrected triplicate well data and compute standard deviation for replicate variation.
4. Avcrage of control cell data:

5. Calculate the averaged compound-treated cell data as a percent of the averaged control cell data as follows:

(Compound-treated value/control value)* 100 % of control value Results:

s We have determined the anti-proliferation activity of the compound of Formula I. As shown in Table 4, the compound of Formula I is consistently active• in the two MCL cell lines of Jeko-1 and Granta-519. Furthermore, the compound of Fom1Ula l appears to be inhibitory against a relatively wide spectrum of hematological malignant cell lines (6 out of 15).

                                                                ICSO (uM)        % Inhibition at max. cone. ( 1OuM)   
        Disease                Cell line        A003397769        A003397769   
                                                                N=4        N--4   
        AML                HL-60                            3.7, 8.1, >10, >10            35       
        A1vtL                    KG-1    1.9            91       
        AML                    ML-2    >1.0            28       
        B-ALL                Nalm-6    5.5            84       
        B-CLL                JVM-2    3.5, 3.6, 7.7, >10            60       
        B-CLL                JVM-2    6.8  7.0  8.4, >10            59       
        B-NHL            DLCL-2            1.4,>10,>10,>10            0       
        B-NHL            DOHH-2    4.5            75       
        CML                Jurl-MKl    1.2, >10, >10, >10            28       
        CML                    K562                    3.9, >10, >10, >10            21       
        MCL                Jeko-1                3.1            97       
        MCL                Grant a-            3.4            67       
        MM                L-363    5.5            61       
        MM                RPMI8226        >10            36       
        T-ALL                Jurkat    6.8, >10, >1.0, >10            43       


Table 4: Effects of Syk compo.und on viability of liquid tumor cell lines (MTS assay)

Collagen-induced arthritis (CIA) is a well characterized model of human rheumatoid arthritis

20    (RA) that can be induced in genetically susceptible rodents following immunization with type II collagen (ell) in adjuvant. Both CIA and RA exhibit severe swelling I inflammation of the joints, synovial hyperplasia and cartilage and bone erosion. This chronic inflammatory


arthritis induced by immunization with ell consists ofboth aT cell component, as evidenced by attenuated CIA in T cell-deficient mice and a B cell component. B cell-deficient mice, xid mice or mice with a null mutation in CXCR5 fail to develop CIA.

5    Methods:

Immunization and Challenge: •Female Lewis rats were immunized. on day 0 and challenged on day 7 with ell from bovine nasal mixed with Freund's incomplete adjuvant at a final collagen concentration of 1.0 mglml. The animals were. injected at the base ofthe tail with

400 Jlg of ell.


Prophylactic Dosing Regimen: The compound ofFormula I (3.0, 10, and 30 mg!kg) was orally dosed (p.o.) twice daily (b.i.d.) starting on day 6 and continued through day 21.

Combination Dosing Regimen:  Rats were dosed with the compound ofFormula I (3.0 and 10

15    mgfkg, p.o., bi.d.) or Methotrexate (MTX, 0.1 and 0.2 mg!kg, p.o., q.d.) as monothcrapy or in combinations consisting of each dose of the compound of Formula I with each dose ofMTX starting on day 6 and continuing through day 21 ..

'Therapeutic Dosing Regimen:  The compound of Formula I (1 0 and 30 mglkg, p.o., b.i.d.)

20    was orally administered starting on day 12 and continued through day 21.

Joint Pathology: Ankle joint swelling was measured to the nearest 0.01 mm by using

electronic digital calipers.  Measurements were recorded 7 times throughout the study starting

25    on day 6 and• ending on day 21. Body weights were recorded on the same days. On da:y 21, hind paws were removed at the hairline just above the ankle and fixed in 10% neutral buffered ibnnalin.

MicroCT analysis:  Ankle joints were examined using a cone-beam ~tCT scanner.  A scout

30    view scan was obtained t1rst for selection of the examination volume ofthe specimens, followed by positioning, measurement and computational reconstruction. In ankle joints, the ratio of bone surface to bone volume, describing the complexity of bone surface in a certain volume, was analyL:ed.

Statistical Analysis: For joint swelling I inflammation, data were• analyzed using Everstat v.S software and a 2-way repeated• measure AN OVA with Dunnett's post test. MicroCT data were analyzed by Evcrstat using a 1-way ANOV A and Newman-Keuls multiple comparison test. Data are presented as the mean± SEM and p values of <0.05 are considered significant.



Prophylactic Dosing:
to    Digital Caliper Measurements of joint swelling I inflammation (Figure 2).

-The compound ofFormu'!a I {3.0 mg/kg) significantly reduced ankle swelling/ inflammation compared to vehicle treated rats on day 12 only.

- The compound or Formula I {1 0 mg/kg) significantly reduced ankle swelling I inflammation compared to vehicle treated rats on days 12 through 19.

IS -The compound of.Formula I (30 mg/kg) significantly reduced ankle swelling I inflammation compared to vehicle treated rats from day 12 through day 21.

MicroCT analysis (Fi!,>Ure 3).

-The compound ofFormula I (3, 10 or 30mglkg, b.i.d.) demonstrated significant reductions in

20    bone erosion when compared to the vehicle-treated rats (measured by the ratio of bone surface to bone volume).

Combination Dosing:

25    Digital Caliper Measurements of joint swelling I inflammation (Figure 4, 5).

-The compo:und ofFormula I (I 0 mglkg) significantly reduced ankle swelling I inflammation compared to vehicle-treated rats from days 15 through .21.

-Combination therapy of the compqund ofFon:nula I (10 mglkg) plus MTX (either 0.2 or 0.1 mglkg) demonstrated a significant reduction in ankle swelling I inflammation from day 15 -

-The compound ofFonnula I (3.0 mglkg) as monoth~rapy or as combination therapy with MTX (0.1 mg/kg) failed to significantly impact on disease severity as measured by joint swelling I inflammation.
-The compound ofFonnula l (3.0 mglkg) as combination therapy with MTX (0.2 mglkg)

5    demonstrated a significant reduction in joint swelling I inflammation above what was observed when either drug was dosed as monotherapy on measurement taken from days 18

through 21.

MicroCT analysis (Figure 6):

-Three-dimensional images demonstrated significant bone erosion I destruction in joints of

10    vehicle-treated rats.

-Dosing of rats with the compound ofFormula I (10 mglkg, po, bid) resulted in a significant decrease in bone erosion versus vehicle-treated rats. A significant decrease was not observed
in rats dosed with 3 mg/ kg the. compound ofFonnula I.

-Mono therapy with MTX (0.2 mg!kg, po, qd) resulted in a •significant decrease in bone tS erosion. This effect was not seen in rats dosed with 0.1 mglkg MTX.

-When treated with combinations of the compound ofFonnula land MTX, all groups showed a significant decrease in the ratio of bone surface to bone volume compared to vehicle-treated rats.
-Combination therapy of the compound ofFonnula I (1 Omg/kg, bid, po) with MTX (0.2

20    mglkg, qd) resulted in additive protection from bone erosion v~rsus that observed in rats receiving monotherapy with either the compound of Formula I or MTX.

Therapeutic Dosing

Digital Caliper Measurem.ents of joint swelling I inflammation (Figure 7).

25    - Even when administered in a therapeutic regimen in which dosing was delayed until arthritis was visually evident, the compound of Formula I (10 and 30•mg!kg, p.o., b.i.d.) significantly reduced ankle swelling I inflammatibn from day 15-21

MicroCT analysis (Figure 8):

-    Compared to vehicle-treated, CIA rats dosed therapeuticaJJy with the compound of Formula I 30 ( 1Omglkg or 30mg/kg) exhibited a significant decrease in bone •erosion as measured by the
ratio ofbonc surface to bone volume.

These studies confirm that inhibition of syk kinase by the compound of Fonnula I can significantly delayed both the onset and progression of rat CIA as measured by decreased in joint swelling /inflammation and bone erosion. Importantly,. significant inhibition of disease

s progression and severity were .. observed 'in rats in which dosing was delayed until visible signs of arthritis were evident. New clinical therapies for RA are normally given in combination with MTX. Our data in a rodent model of arthritis demonstrate additive effects when the compound of Formula I is dosed in combination with MTX indicating that combination therapy of the compound •of Formula 1 and MTX could produce synergistic clinical effects in

10    RA patients.


Female Lewis rats (5 week-old, 150-175 g) were anesthetized using xylazine ( 4 mglkg) and

15    ketamine (80 mglkg). Cellulose sponges (diameter 10 mm, Vivoxid Ltd.™, Turku, Finland) containing 50 Jll of either FGF-2 (fibroblast•growth factor 2) solution (containing 400 ng of 'FGF-2) or vehicle (physiological salt solution, bovine serum albumin 0.08 %) were then implanted subcutaneously on the back of the. animals. During the two following days further angiogenesis was induced by daily injection through the skin and into the sponges of 50 J!l of
20    FGF-2 solution or vehicle (basal conditions). One week after sponge implantation, animals were euthanized using an overdose of pentobarbital and the sponges were dissected out. The sponges were then minced and homogenized in lysis buffer (NaCllSO mM, EDTA 1 mM, Triton Xl 00 1%, sodium deoxycholate 0.5%, NaF 10 mM, Tris/HC130 mM pH 7.8 containing

a protease inhibitor cocktail (P8340, Sigma-Aldrich™,  St Louis, USA)) using Lysing Matrix

25    D tubes (MP Biomedicals™, Illkirch, France) in a Fastprep homogenizer (QbiogeneTM, Illkirch, France). Hemoglobin concentration, indicative of vascular volume, was determined using the Dn:ibkin assay (Pierce BiotechrtologyTM, Rockford, Illinois, USA). Compounds were
administered by oral gavage as a suspension in aqueous methylcellulose 0.6%, Tween 80 0.5% solution.


The effects of the compound of Formula I on rat weight are shown in Figure 9. The effects of the compound of Formula I on hemoglobin concentration (mg/ml) are shown in Figure 10 ..

Claim Amendments (Clean Version):

1.    A compound of formula (I):


or a corresponding N-oxide, or a prodrug; or a pharmaceutically acceptable salt or solvate of

10    such compound; and theN-oxide and the prodrug, ofthe salt and solvate.

2. A pharmaceutical composition, comprising a compound according to claim 1, together with at least one pharmaceutically acceptable carrier or excipient.

15    3.    The use of a compound according to claim 1 in the manufacture of a medicament for

the treatment of joint inflammation.

4. The use of a compound according to claim 1 in the manufacture of a medicament for the treatment of rheumatoid arthritis.


5.    The use of a compound according to claim 1, in combination with methotrexate in the manufacture of a medicament for the treatment ofjoint inflammation.

6.    The use of a compound according to claim I, in combination with methotrexate, in the

25    manufacture of a medicament for the treatment of rheumatoid arthritis.

7.    The use of a compound according to claim 1 in the manufacture of a medicament for the treatment of a tumor.


8.    The use of a compound according to claim 1 in the manufacture of a medicament for

the treatment of mantle cell lymphoma.

5    9. The use of a compound according to claim 1 in the manufacture of a medicament for inhibiting angiogenesis.

10.    The compound according to claim 1, wherein the salt is selected from: hydrochloride,

hydrobromide, sulphate, phosphate, nitrate, sulfamate, acetate, citrate, lactate, tartrate,

10    malonate, oxalate, salicylate, propionate, succinate, fumarate, maleate, methylene-bis-b-hydroxynaphthoate, gentisate, isethionate, di-p-toluoyltartrate, methane-sulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate.

11.    The compound according to claim 1, wherein the salt is hydrochloride.


12.    The compound according to claim 1, wherein the salt is acetate.

13.    The compound according to claim 1, wherein the salt is citrate.


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