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(11) Patent Number: K.E 610   

(45) Date of grant: 25/09/2013   
               
(73) Owner: A 61K 31/437, A 61P 27/02, C 07D 471/04    FOVEA PHARMACEUTICALS SA of , Institut de la Vision, 17 Rue Moreau, F-75012 Paris,France       

(21)Application Number: KE/P/ 2011/001389   

(22) Filing Date: 09/02/2010   

(72) Inventors:    LERICHE, Caroline, 7, rue Antoine Chantin, F-   

(30) Priority data: 75014 Paris, France; AUCLAIR, Eric, 119 Rue 09360013.8  13/02/2009 EP    Paul Fort, F-91310 Montlhery, France; LEROUX,    Jacques, 18 Rue Garibaldi, F-93310 LePre Saint   
   
(86)  PCT data    Gervais, France and MIDDLEMISS, David, 61,    PCT/EP10/051556    09/02/2010    Thorley Hill, Bishop's Stortford Hertfordshire    wo 2010/092041    19/08/2010    CM23  3NF, Great Britain   

(74) Agent/address for correspondence:    Kaplan & Stratton Advocates, P.O. Box 40111-    00100, Nairobi   

(54)    Title: [1, 2, 4] TRIAZOLO [1, 5 -A] PYRIDINES AS KINASE INHIBITORS

(57) Abstract:The invention is directed to certain novel compounds, methods for producing them and methods for treating or ameliorating a kinase-mediated disorder. More particularly, this invention is directed to substituted triazolopyridine compounds useful as selective kinase inhibitors, methods for producing such compounds and methods for treating or ameliorating a kinase-mediated disorder. In particular, the methods relate to treating or ameliorating a kinase-mediated disorder including cardiovascular diseases, diabetes, diabetes-associated disorders, inflammatory diseases, immunological disorders, cancer and diseases of the eye such as retinopathies or macular degeneration or other vitreoretinal diseases, and the like.
 
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[1, 2, 4] TRIAZOLO [1, 5 -A] PYRIDINES AS KINASE INHIBITORS
10

15 The invention is directed to certain novel compounds, methods for producing them and methods for treating or ameliorating a disorder involving tyrosine kinase

.dysregulation such as disorder associated with increased vascular permeability or angiogenesis. More particularly, this invention is directed to substituted triazolopyridine compounds useful as selective kinase inhibitors, methods for producing such compounds
20    and methods for treating or ameliorating a kinase~mediated disorder. In particular, the methods relate to treating or ameliorating a disorder involving tyrosine kinase dysregulation including cardiovascular diseases, diabetes, diabetes-associated disorders, inflammatory diseases, immunological disorders, cancer and diseases of the eye such as retinopathies or macular degeneration or other vitreoretinal diseases, and the like.

25    Passage of fluid and cells out of blood vessels is a significant contributing factor to

inflammation,  tissue  injury,  oedema  and  death  in  a  variety  of circumstances.  These

include  ischemic  injury,  toxic  shock,  burns,  trauma,  allergic  and  immune  reactions.

Vascular permeability is regulated in part by cell~cell adhesions between endothelial cells.

The endothelial eel! monolayer lining the vasculature forms a barrier that maintains the

30    integrity of the blood fluid compartment, but permits passage of soluble factors and leukocytes in a regulated manner. Dysregulation of this process results in vascular leakage into surrounding tissues, which accompanies the inflammation associated with

pathological oedematous conditions. Vascular permeability is a finely-tuned function that

can  positively contribute to protective immune responses and wound healing;  however,

35    in a number of pathological situations, massive and/or chronic leakage of fluid as well as migration of immune cells into tissues can have serious, and sometimes, life-threatening consequences.

Abnormal retinal vascular permeability leading to oedema in the area of the macula is the leading cause of vision loss in diseases such as diabetic retinopathy1

40 exudative macular degeneration, retinal vascular occlusions, and inflammatory and neoplastic conditions. Although a variety of disease processes may lead to increased •vascular permeability through different mechanisms, the cytokine VEGF is known to play

wo 2010/092041 PCT/EP2010/051556 2

a major role as inducer of vascular leakage. VEGF was first described as a potent vascular permeability factor (VPF) secreted by tumour cells that stimulated a rapid and reversible increase in microvascular permeability (Senger et al., 1983, Science., 25, 219,

983~5). Increased  vascular permeability  in  ischemic retinopathies and  possibly  also  in

"5 exudative macular degeneration and•uveitis, for example, correlated with VEGF levels (Fine et al., 2001, Am. J. Ophtha•lmol., 132, 794-796 ; Boyd et al., 2002, Arch Ophthalmol. , 1201 1644-1650) and VEGF antagonists have been successfully used to reduce retinal/macular oedema in neovascular eye diseases such as age-related macular degeneration leading to stabilization or even improvement of visual acuity in a subset of

10    affected patients. The way by which VEGF. induces vascular permeability has recently been unravelled (Gavard and Gutkind, 2006, Nat Cell Bioi., 8, 1223~1234) and it has been shown that VEGF-induced vascular leakage is mediated by cytoplasmic protein kinase members of the Src proto oncogene family.

Protein  kinases  play a central  role in the regulation and maintenance of a wide

15    variety of cellular processes and cellular functions. For example, kinase activity acts as a molecular switch regulating cell proliferation, activation, and/or differentiation. It is now widely accepted that many diseases result from abnormal cellular responses triggered by overactive protein kinase-mediated pathways.

Src kinases form a family of membrane-attached non receptor-dependent tyrosine

20    kinases encompassing eight members in mammals: Src, Fyn, Yes, Fgr, Lyn, Hck, Lck, and Blk (Bolen eta!., 1997, Annu. Rev. Immunol, 15, 371) which have important roles in

receptor signalling and cellular communication (Thomas and Brugge, 1997, Annu Rev Cell Dev Bioi., 13, 513-609). While most Src kinases are broadly expressed (i.e. Src, Fyn, Yes), certain members of the family such as Hck, Blk or Lck exhibit a restricted

25    expression. Src kinases play a pivotal role as membrane-.attached molecular switches that link a variety of extracellular cues to intracellular signalling pathways. This is the basis for the involvement of Src kinases in cell proliferation and differentiation as well as cell adhesion and migration (Thomas SM and JS Brugge, 1997, supra).

It has been well-documented  that Src protein  levels and  Src kinase activity are

30    significantly elevated in human cancers including breast cancers, colon cancers, pancreatic cancers, certain B-cell leukemias and lymphomas, gastrointestinal cancer, non-small cell lung cancers, bladder cancer, prostate and ovarian cancers, melanoma and

    sarcoma (Summy and Gallick, 2003, Cancer Metastasis Rev,  22, 337-58). Thus, it has               
    been anticipated that blocking signalling through the inhibition of the kinase activity of               
35    Src will  be  an  effective  means    of modulating  aberrant  pathways that  drive  oncologic               
    transformation of cells  (Abram et al., 2000,  Exp. Cell  Res., 254,  1; Russi et al.r-.:.2:0'0b,~--::"':"~~.:.       
                    . -:.            •-...;:   
    JPET, 318, 161-172; Jallal et al.,    2007,  Cancer Research,  67, 1580-1588)///                - •:.:,~•\\   

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Similarly, it is well documented that Src-family kinases are also important for signalling downstream of immune cell receptors. Fyn, . like Lck, is involved in TCR signalling in T cells (Appleby et al., 1992, Cell, 70, 751). Hck and Fgr are involved in Fey receptor signalling leading to neutrophil activation (Vicentini et al., 2002, J. Immunol.,

5    1681 6446). Lyn and Src also participate •in•Fey receptor signaling leading to release of histamine and other allergic mediators (Turner and Kinet, 1999, Nature, 402, 824). These findings suggest that Src family kinase inhibitors may be useful in treating allergic diseases and asthma.


In accordance with the effect of VEGF on vascular perm_eability, several  reports

10    support a role of Src kinase in the development of oedema. For instance, Src but not Fyn deficiency or blockade of Src reduced brain oedema by about 55% following permanent

cerebral ischemia in mice (Paul et al. 1 2001, Nat Med. 1 7(2):222-7). Recently1 PP1, a Src tyrosine kinase inhibitor was found to decrease oedema, to decrease breakdown of the brain-blood barrier (BBB), to reduce expression of VEGF (Jadhav et al., 2007, J

15    Neurosurg., 106, 680-686). Similarly, Scheppke eta!. (2008, J Clin Invest., 118, 2337-2346) have shown that Src kinases are critical mediators of VEGF- and ischemia-induced retinal vascular leakage.

Furthermore,  Src  tyrosine  kinases  fully  mediate  VEGF  receptor  signalling  in

vascular endothelial cells. Thus,  activation  of Src kinases  resulting  from  stimulation. of

20    VEGF receptor or other growth factor located on endothelial cells or progenitors triggers angiogenesis, a response which can be deleterious in retinal and corneal diseases and which markedly contributes to tumor development and metastasis migration.

Several classes of compounds have been disclosed that modulate or, more specifically, inhibit kinase activity as potential treatments of kinase-mediated disorders,

25    particularly cancer.

For example, W02001038315 describes aminoquinazolines as inhibitors of cyclin-dependent kinases.

W02008068507 describes pyridinylquinazolines as Raf serine/threonine kinase inhibitors for treating cancer.

30 W02008079988 describes quinazolines as PDK1 kinase inhibitors for treating proliferative diseases such as cancer.

W02006118256 describes quinazoline derivatives as p38MAPK inhibitors for inhalation and for treating various inflammatory diseases and cancer.

WOZ006039718 describes aryl nitrogen-containing bicyclic compounds for use in

35    treating protein kinase-mediated disease, including inflammation/ cancer and related conditions.
 
r\
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        4   
           
    W02005037285    describes   2,6-disubstituted   bicyclic   heterocycles   as   Raf   
    serine/threonine kinase inhibitors for treating disorders such as cancer.   
    W02004065378    describes  2-aminopyridines  as  cdk4  inhibitors  for  treating  cell   
    proliferative disorders such as cancer, atherosclerosis and restenosis.   

5 Interestingly, W02006024034 describes h-eterocyclic compounds derived from benzotriazine, triazines, triazoles and oxadiazoles, such as benzotriazine compounds (W02005096784) or pyrimidine compounds (W02006101977) which are capable of inhibiting kinases, such as members of the Src kinase family. Nevertheless, these drugs while they are•claimed as potentially useful as for treatment of various ophthalmological

10    diseases (e.g. age-related macular degeneration, diabetic retinopathy, diabetic macular oedema, cancer, and glaucoma) are lipophilic and.water insoluble (see W02006133411). According to the inventors of W02006133411; these specific properties are particularly

advantageous,  particularly  for  ophthalmic  uses,  since  these  drugs  being  insoluble  in

water (water solubility of less t~an about 0.1 mg/ml at a pH range of 4-8) possess high

15    efficiency of loading and negligible leakage due to high partitioning of the drug into the liposome used for delivering them compared to the water.

All the patents and publications mentioned above and throughout are incorporated in their entirety by reference herein.

The eye is a tightly protected organ. In this respect, treating diseases of the back-

20    of-the-eye is probably the most difficult and challenging task of drug discovery as evidenced by the paucity of therapeutic options. One of the• most convenient and safest

form of drug delivery to the eye is eye drops, since it is non invasive, does not require medical assistance and requires small volumes of drug solution. However, in order to be suitable for topical instillation, molecules have to be potent enough towards their

25    molecular target, to present physico-chemica! properties allowing crossing of cell membranes, and to be sufficiently soluble in aqueous medium to be applied as solution onto the cornea. In addition, it is crucial that such drug molecules are as colourless as

possible to prevent staining of ocular tissue which ultimately may interfer with vision. Furthermore, patients enrolled in clinical trials must not be aware of the nature of their

30    treatment, which is obviously biaised when the preparation of the active ingredient is highly coloured. Additionally, due to the multiple cross reactivity between kfnases, it is highly desirable that said drug molecules inhibit the targeted kinases with a high degree of selectivity.

    Another feature  of the  present invention  is to  provide  novel  compound$,-Whicli'.,.,....,   .
35    have increased water solubility compared to competitors.    . ->~

/
 

,./
 
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5

Another feature of the•present invention is to provide compounds that are highly potent, particularly towards src and lyn kinase inhibitors.

Another feature of the present invention is to provide compounds which are useful for treating a disorder, including an ophthalmic disorder, involving tyrosine kinase

5    dysregulation such , as disorder associated with increased vascular permeability or angiogenesis.


Another feature of the present invention is to provide compounds which are colourless or almost colourless, especially in solution.

Additional features and  ~dvantages of the present invention will  be set forth  in

10    part in the description that follows; and in part will be apparent from the description, or may be learned by practice of the present invention. The objectives and other advantages of the present invention will be realized and attained by means of the elements and combinations particularly pointed out in the description and appended claims.


15 According to one embodiment, the invention concerns compounds having the structure (I) as well as a pharmaceutically acceptable salt, hydrate or solvate thereof :

25
wherein :

A1  and A2  is N or C, with the proviso that one of A1  or A2  is N and one of Al  or A2 is

carbon,
30
R1 and R2 are hydrogen, Cl-C4 alkyl, aryl, heteroaryl, -CN, -halogen, -CF3 ,  -0R4,

R3 is hydrogen, C1-C4 alkyl, cycloalkyl,  heterocycloalkyl, aryl, heteroaryl, -CN,  -CF3 ,   -

OR4,-0COR4 -COR4, -NR4R5, -NR4COR5, -NR4COOR5, -(Cl-C4 alkyi)OR4, -(Cl-C4 alkyi)COR4, -(C1-C4 alkyl) NR4R51 -(Cl-C4 alkyl)NR4COR5, -(Cl-C4 alkyi)NR4COOR5,

35    X is a bond, or (CH2)aW(CH 2)b, (CH 2)aW(CH 2 )bY(CH2)c or-[(CH 2)aW(CH 2)b]m-(Z)e-[( CH 2)cY(CH 2)d]n wherein :
 

a1   b, c and d are independently 0, 1, 2 or 3,
 

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(~'    6
e is 0 1   1 or 21  and

n and m are independently 0 or 11  and


and

Y is -C0-1-0-,-S02-~-CH2-,-CHOH-or-NR6-, NR7CONR8 or NR?S02NR8 and

Z is selected from the group consisting o(cycloalkyl, heterocycloalkyl 1 aryl and heteroaryl, and when e is 2 1 then each Z moiety is selected independently from one another

R4, RS and R6 are independently hydrogen1  C1-C4 alkyl and where R4 and RS

10    together can form a 5-7 membered ring 1

R7 and R8 are independently hydrogen, C1-C4 alkyl and where R7 and R8 together can form a 5-7 membered ring.

As used herein throughout the entire application 1 the terms "a" and "an" are used in the sense that they mean "at least one"1 "at least a first/{, "one or more" or "a

15    plurality" qf the referenced compounds or steps1 unless the context dictates otherwise. More specifically, "at least one" and "one or more" means a number which is one or greater than one, with a special preference for one1 two or three.

The term "and/or" wherever used herein includes the meaning of "and" 1 "or" and "all or any other combination of the elements connected by said term".

20 The term "about" or "approximately" as used herein means within 20%, preferably within 10%1 and more preferably within 5% of a given value or range.

As used herein, the term "comprising", "containing" when used to define products, compositions and methods, is intended to mean that the products, compositions and methods include the referenced compounds or steps, b1,1t not excluding others.

25 As used herein, the term "halogen" as a group or part of a group is generic for fluoro, chloro, bromo or iodo.

The term "cycloalkyl" means a saturated monocyclic carbocycle containing from 3 to 7 carbon atoms, more preferably from to 5 carbon atoms. Examples of monocyclic cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl and the like.

30 The term "heterocycloalkyl" means a saturated mono- or bicyclic heterocycle. having from 3 to 14 ring members, preferably from 5 to 10 ring members and more preferably from 5 to 6 ring members, which contains one or more heteroatom ring members selected from nitrogen, oxygen and sulphurand which is optionally substituted
 
(.'


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with R9 and/or R10 moities. Exam.ples of heterocycloalkyl are pyrrolidine, piperidine, piperazine, morpholine and the like.

The term "aryl" includes mono- and. bicyclic aromatic carbocycles, optionally substituted with R9 and/or R10 moities. Examples. of a.ryl include phenyl, 1-naphthyl, 2-

5    naphthyL

The term "heteroaryl" nieans an aromatic mono- or bicyclic heterocycle having from 5 to 10 ring members, preferably from 5 to 6 ring members, which contains one or more heteroatom ring members selected from nitrog~n, o.xygen and sulphur and which is optionally substituted with R9 and/or RlO moities. Examples of heteroaryl are pyridine,

10    indole, benzofuran, oxazole, triazole, pyrimidine and the like.

R9/R10 are independently selected from hydrogen, C1-C4 alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -CN, -halogen, -CF3, =0, -OR4, -NR4R5, -NR4COR5, - NR4COOR5, -(C1-C4 alkyi)OR4, -(C1-C4 alkyl) NR4R5, -(C1-C4 alkyi)NR4CORS, -(C1-C4 alkyi)NR4COORS, -COOH, COOR4 with R4 and R5 as defined above.

15 The compounds of the invention may contain one or more chiral centres, because of the presence of asymmetric carbon atoms, and they may therefore exist as a number of diastereoisomers with R or S stereochemistry at each chiral centre. The invention includes all such diastereoisomers and mixtures thereof.

Prodrug  forms  of  the  compounds  of  Formula  I  are  also  part  of  the  present

20    invention. A prodrug may be a pharmacologically inactive derivative of a biologically active substance (the "parent drug" or "parent molecule") that requires transformation within the body in order to release the active drug, and that has improved delivery properties over the parent drug molecule. The transformation in vivo may be, for

example,  as  the  result  of  some  metabolic  process,  such  as  chemical  or  enzymatic

25    hydrolysis of a carboxylic, phosphoric or sulphate ester, or reduction or oxidation of a susceptible functionality.

The term "compound" herein is in general referring to compounds of formula I, or pharmaceutically acceptable salt, hydrate, solvate, crystal form, individual diastereomers and prodrugs thereof.

30 For use in accordance with the in.vention, the following structural characteristics are currently preferred, in any compatible combination, in the compounds (I):

Rl is preferably an aryl, more preferably a phenyl.

Rl is preferably substituted with R9 and R10 wherein R9/R10 is C1-C4 alkyl (preferably CH 3 ), halogen (preferably -CI), or -OH.

35    Rl is preferably a phenyl and is substituted with R9 and R10 in positions 2, 5 or 6.

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    8

R2 is preferably hydrogen or

    •R2 is preferably C1-C4 alkyl (preferably CH 3 ).   
    X is preferably (CH 2)aW(CH 2)b with a is 0, b is• 2, W is -0-.   
    Alternatively X is preferably (CH 2)aW(CH2 )bY(CH2 )c with a is 0, b is 1 and c is 0,
5    w is -o- arid Y is -co-.   
    Alternatively X is preferably -[( CHz)aW(CH 2 )b]m-Z-[( CH2 )cY(CH 2 )d]n with    m is
    0,  n is 1, c is 0, d is 0 or 2, Y  is -co- or is absent and  Z is imidazoline-2-one    or a
    piperazine.   
    R3 is preferably a heterocycloalkyl, preferably a pyrrolidine.   
10    R3 is preferably substituted with R9 wherein R9 is preferably -COOH, -N[CH 3 h or
    -COOR4 wherein R4 is preferably C1-C4 alkyl.   
    Alternatively R3 is preferably an h~teroaryl, preferably a pyridine.   
    Compounds of the invention include those •of the Examples herein,  in  particular
    the following, and their salt, hydrate, solvate :   
15    4-Chloro-3-{2- [ 4-(2-pyrrolidin-1-yl-ethoxy)-phenylamino ]-[1,2,4]triazolo[ 1, 5-
    a]pyridin-6-yl}-phenol, hydrochloride   
    2-{ 4- [ 6-(2-Chloro-5-hydroxy-phenyl)-[ 1,2,4]triazolo[ 1,5-a] pyridin- 2-ylami no]-
    phenoxy}-1-( ( R)-3-dimethylamino-pyrrolidin-1-yl)-ethanone, hydrochloride   
    1-{4-[6-(2-Chloro-5-hydroxy-phenyl)-[1,2,4 ]triazolo[1,5-a] pyridin-2-ylamino ]-
20    phenyl}- 3-(2-pyrrolidin-1-yl-ethyl)-imidazolidin- 2-one   
    (S)-1-(2-{ 4- [6-(2-Chloro-5-hydroxy-phenyl)-[1,2,4]triazolo[ 1,5-a] pyridi n-2-   
    ylamino]-phenoxy}-acetyl)-pyrrolidine-2-carboxylic acid   
    4-Chloro- 3-{ 8-methyl-2-[4-(2-pyrrolidin-1-yl-ethoxy)-phenylamino ]-   
    [1,2,4]triazolo[1,5-a]pyridin-6-yl}-phenol, hydrochloride   
25    4-Chloro- 3-{2-[ 4-(2-pyrrolidin-1-yl-ethoxy)-phenylamino ]- [1,2,4 ]triazolo[l,S-
    a]pyridin-7-yl}-phenol, hydrochloride   
    4-Chloro-3-{ 2- [ 4-( 2-morphol in-4-yl-ethoxy )-phenyla mino ]- [ 1, 2,4]triazolo[ 1,5-
    a] pyridin-6-yl}-phenol   
    4-Chloro- 3-{ 2- [ 3- ( 2 -morpho! in-4-yl-ethoxy )-phenyla mino ]- [ 1,2,4]triazolo[ 1,5-
30    a]pyridin-6-yl}-phenol   
    4-Chloro-3-[2- ( 3-pyrazol-1-yl-phenylamino )-[ 1,2,4]triazolo [ 1,5-a] pyrid i n-6-yl]-

phenol

4-Chloro-3-{2-[4-(2-dimethylamino-ethoxy)-phenylamino]-[1,2,4]triazolo[1,5-a]pyridin-6-yl}-phenol
 

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    9   
    4-Chloro-3-{2-[3-(2-dimethylamino-ethoxy)-phenylamino]-[1,2,4]triazolo[1,5-
    a]pyridin-6-yl}-phenol   
    4-Chloro-3-{2-[3-(2-pyrrolidi n-1-yl-ethoxy)-pheriylamino ]- [1 1 2A ]triazolo[1 1 5-
    a]pyridin-6-yl}-phenol   
5    4-Chloro-3-[2-( 4-hydroxymethyl-phenylamino )-[1,2A ]triazolo[1,5-a] pyridin-6-
    yl]-phenol  •   
    4-Ch Ioro- 3- [2- (3-hyd roxymethyl- phenyla mino )- [ 1,2,4 ]triazolo[ 1 (5-a] pyri di n-6-
    yl]-phenol   
    4- [ 6-(2 -Chloro- 5-hyd roxy- phenyl)- [ 1,2,4 ]triazolo [ 1 ,5-a] pyridin- 2-ylamino]-
10    benzenesulfonamide   
    4-Chloro- 3-{ 2- [ 4- ( 2 -hyd roxy-ethoxy)- phenylamino ]- [ 1,2,4]triazolo[1, 5-a] pyridin-
    6-yl}-phenol   
    1-{4- [ 6-( 2-Chloro- 5-hyd roxy-phenyl)- [ 1,2,4 ]triazolo [ 1, 5-a] pyridin- 2-ylami no]-
    phenyl}-!midazolidin-2-one   
15    3-[6-(2-Chloro-5-hydroxy-phenyl)- [1,2A ]triazolo[1,5-a] pyridin-2-ylamino ]-
    benzenesu lfona m ide   
    2-{ 4- [ 6- (2 -ChI oro- 5-hyd roxy-phenyl)- [ 1 ,2A ]triazolo[1, 5-a] pyridi n-2 -ylam i no]-
    phenoxy}-acetamide   
    2 -{ 3- [ 6- ( 2 -ChI oro- 5-hydroxy-phenyl)- [ 1 ,2,4 ]triazolo[ 1,5-a] pyridi n- 2 -yla m i no]-
20    phenoxy}-acetamide   
    4-Chloro- 3-[2-( 4-trifluoromethoxy-phenylamino )- [1,2,4 ]triazolo[1,5-a]pyridin-6-
    yl]-phenol   
    4-Chloro-3-[2-(4-phenoxy-phenylamino )- [ 1,2A]triazolo[ 1,5-a] pyridin-6-yl]-
    phenol   
25    4-Chloro-3-[2 -( 4-methanesulfonyl-phenylamino )- [1,2,4 ]triazolo[1,5-a ]pyridi n-6-
    yl]-phenol   
    3- [2- ( 4- Benzyloxy-phenylami n•o)- [ 1,2,4]triazolo [ 11 5-a] pyridi n-6-yl] -4-ch Ioro-
    phenol   
    1-{4-[6-(2-Chloro-5- hyd roxy-phenyl)-[1,2,4 ]triazolo[1 1 5-a] pyridin-2-ylam ino ]-
30    phenyl}-3-(2-pyrrolidin-1-yl-ethyl)-imidazolidin-2-one   
    5- [ 6-(2 -Chloro- 5-hyd roxy- phenyl)-[ 1,2,4]triazolo[ 1,5-a] pyridi n- 2-ylami no]-
    benzofuran-2-carboxylic acid   
    1-{4- [6-(2-Chloro-5-hydroxy-phenyl)-[ 1,2,4]triazolo [1,5-a] pyridin- 2-ylamino ]-
    phenyl}-3-methyl-imidazolidin-2-one   
3 5    1-{4- [ 6-(2 -Chloro- 5-hyd roxy- phenyl)- [ 11 2,4] triazolo[ 1, 5-a] pyridi n-2-ylam i no]-
    phenyl}-3-(2-methoxy-ethyl)-imidazolidin-2-one   

1- [ ( 4-{[ 6-(2-chloro-5-hydroxyphenyl)-[1,2,4 ]triazolo[ 11 5-a] pyridin- 2-yl]amino}benzene)sulfonyl]-3-ethylurea
 
(


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1-(4-{ 4-[6-(2-Chloro-5-hydroxy-phenyl)- [1,2,4 ]triazolo[1 ,5-a]pyridin-2-ylamino] -phenyl}-piperazin-1-yl)- 2-methoxy-ethanone

( 4-{ 4- [ 6-(2-Ch Ioro- 5-hyd roxy-phenyl)- [ 1 ,2,4 ]triazolo[ 1,5-a] pyridin- 2 -ylami no]-phenyl}-piperazin-1-yl)-pyridin-4-yl-methanone

5 1-{4- [ 6-(2-Chloro- 5-hydroxy-phenyl)-[1,2,4 ]triazolo[1, 5-a] pyridin-2-ylamino ]-phenyl}- 3- ( 2-pyrrolidi n-1-yl-ethyl)-u rea

4-Ch loro-3- { 2- [ 4-( 2- pi perazin-1-yl-ethoxy)-phenyla mi no]- [ 1,2,4]triazolo [ 1, 5-a]pyridi n-6-yl}-phenol

10 According to one embodiment, the compound of the Invention is a salt of compound of formula I.


According to one preferred embodiment, the compounds of the Invention have a •watersolubility over 01 1 mg/ml at a pH range of 4-8, preferably pH range of 5-7, such as over about 0,5 mg/ml at a pH range of 5-7, for example over about 1 mg/ml at a pH

15    rangeofS-7.

According to one embodiment, the compounds of the Invention have a limited colour, preferably they are uncoloured or pale yellow.

Preferred  compounds  of the  present  invention  act  primarily  on  src and/or  lyn

kinase.

20 According to one embodiment, the compounds of the Invention are src and/or lyn kinase inhibitors.


According to one embodiment, the compounds of the Invention have an ICSO towards Src of less than about 15 nM, advantageously less than about 10 nM, more preferably less than about 1 nM, advantageously less than about 0,9 nM, more preferably

25    less than about 0,5 nM.

According to one embodiment, the compounds of the Invention have an ICSO towards Lyn of less than about 15 nM, advantageously less than about 11 nM, more preferably less than about 4 nM, advantageously less than about 3 nM, more preferably less than about 1 nM.


30 According to one embodiment, there are provided compositions including one or more compound of the Invention and a pharmaceutically acceptable carrier or aqueous medium.

As used herein, the term "pharmaceutically acceptable" refers to carriers that do not produce an adverse, allergic or other unwanted reaction when administered. to an

35    animal, or human, as appropriate. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, anti!Jacterial and antifungal
 

wo 2010/092041 PCT/EP2010/051556 11

agents, isotonic and absorption delaying agents and the like. The use of such carriers for pharmaceutical active substances is well known in th•e art. Examples of suitable pharmaceutical carriers are described in "Remington'sPharmaceutical Sciences" by E. W. Martin. In a preferred embodiment, the compounds of the Invention are formulated in

5    accordance with routine procedures as a pharmaceutical composition adapted for administration to the eye. Supple.mentary active ingredients, such as anti-inflammatory agent, chemotherapeutic. agent, anti-cancer agent, immunomodulatory agent, gene-based therapeutic vaccine, immunotherapy product, therapeutic antibody and/or protein kinase inhibitors can also be incorporated into the compositions.

10 According to one embodiment, the compounds of the present invention will be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. The preparation of an aqueous composition that contains a compound or compounds of the Invention will be within the skill of those in the art, in light of the present disclosure.

15    Typically, such compositions can be prepared as injectables, either as liquid solutions or

.suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a• liquid prior to injection can.also be prepared; and the preparations can also be emulsified.

According to another embodiment, the compounds of the present invention will be

20    formulated for topical administration of the compounds of the Invention, especially for the treatment of ophthalmic disorders. The preparation of a composition that contains a compound or compounds of the Invention will be within the skill of those in the art, in

light of the present disclosure. Typically, such compositions for topical administration can

be  prepared  as  ointment,  gel  or  eye  drops.  The  topical  ophthalmic composition  may

25    further be an in situ gel formulation. Such a formulation comprises a gelling agent in a concentration effective to promote gelling upon contact with the eye or with lacrimal fluid in the exterior of the eye. Suitable gelling agents include, but are not limited to,

thermosetting  polymers such as tetra-substituted ethylene diamine block copolymers of

ethylene    oxide   and   propylene   oxide   (e.g.,   poloxamine);   polycarbophil;   and

30    polysaccharides such as gellan, carrageenan (e.g., kappa-carrageenan and iota-carrageenan), chitosan and alginate gums. The phrase "in situ gellable" as used herein

embraces not only liquids of low viscosity that form gels upon contact with the eye or with lacrimal fluid in the exterior of the eye, but also more viscous liquids such as semi-fluid and thixotropic gels that exhibit substantially increased viscosity or gel stiffness

35    upon administration to the eye.

According to another embodiment, the compounds of the present invention will be formulated for oral administration of the compounds of the Invention. The preparation
 
(--,_


wo 2010/092041 PCT/EP2010/051556 12

of a composition that contains a compound or compounds of the Invention will be within the skill of those in the art, in light of the present disclosure. Typically, such compositions for oral administration can be prepared as liquid solutions or suspensions, tablets, time release capsules and other solids for oral administration.

5 According. to another ~mbodiment, the compounds of the present invention will be formulated for intratumoral •administration of the compounds of the Invention. The preparation of a composition that contains a comppund or compounds of the Invention will be within the skill of those in the art, in light of the present disclosure. Typically, such compositions for intratumoral ~dministration can be prepared as disclosed above for the

10    other routes of administration.

According to another embodiment, the compounds of the present invention will be formulated for inhaled administration of the compounds of the Invention. The preparation of a composition that contains a compound or compounds of the Invention will be within the skill of those in the art, in light of the present disclosure. Typically, such compositions

15    for inhalation can be prepared as disclosed above for the other routes of administration.

According to another embodiment, the compounds of the present invention will be combined with ophthalmologically acceptable preservatives, viscosity enhancers, penetration enhancers, buffers1 sodium chloride, and Water to form an aqueous, sterile ophthalmic suspension or solution. Ophthalmic solution formulations may be prepared by

20    dissolving a compound in a physiologically acceptable isotonic aqueous buffer. Further, the ophthalmic solution may include an ophthalmologically acceptable surfactant to assist in dissolving the compound. Furthermore, the ophthalmic solution may contain an agent

to    increase   viscosity,   such   as   hydroxymethylcellulose,   hydroxyethylcellulose,

hydroxypropylmethylcellulose1    methylcellulose,  polyvinylpyrrolidone,  or  the  like,  to

25    improve the retention of the formulation in the conjunctival sac. Gelling agents can also be used, including/ but not limited to, gellan and xanthan gum. In order to prepare sterile ophthalmic ointment formulations, the active ingredient is combined with a

preservative  in  an  appropriate  vehicle,  such  as,  mineral  oil 1    liquid  lanolin,  or  white

petrolatum. The compounds are preferably formulated as topical ophthalmic suspensions

30    or solutions, with a pH of about 5 to 81 and more preferably from about 6.5 to about 7.5. The compounds will normally be contained in these formulations in an amount 0.001% to 5% by weight, but preferably in an amount of 0.025% to 2% by weight. Thus, for topical presentation 1 to 2 drops of these formulations would be delivered to the surface of the eye 1 to 4 times per day according to the discretion of a skilled clinician.
 

35 In another embodiment, there are provided methods of treating a disorder involving tyrosine kinase dysregulation such as disorder associated with increased vascular permeability or angiogenesis/ including the administration of a therapeutically
 

wo 2010/092041    PCT/EP2010/0515513

effective amount of one or more compound of the Invention to a subject in need of such treatment.


As used herein, the term "treatment" or "treating" encompasses prophylaxis and/or therapy. Accordingly the compositions and methods of the present invention are

5    not limited to therapeutic applications and can be used in prophylaxis ones. Therefore "treating" or "treatment" of a state, disorder or condition includes: (i) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition

developing in a subject that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the

10    state, disorder or condition, (ii) inhibiting the state, disorder or condition, i.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof, or (iii) relieving the disease, i.e. causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

As  used  herein,  the  terms  "patient"  "subject  in  need  thereof"  are  meant  any

15    animal; preferably, the a•nimal is a vertebrate; more particularly a member of the mammalian species and includes, but (s not limited to, domestic animals (e.g. cows, hogs, sheep, horses, •dogs, and cats), primates including humans. The terms "patient"

"subject in• need thereof" are in no way limited to a special disease status, it encompasses both patients who have already developed a disease of interest and

20    patients who are not sick.

As used herein, the terms "therapeutically effective amount" are meant any amount of compound or composition that will elicit the biological response of a tissue, animal1 or human, cell, organ ...

According to one embodiment, the said disorder involving tyrosine kinase 25 dysregulation is a disorder associated with increased vascular permeability.

According to another embodiment1 the said disorder involving tyrosine kinase dysregulation is a disorder associated with angiogenesis.

In preferred embodiment, the disorder involving tyrosine kinase dysregulation is a disorder associated with a src and/or lyn kinase dysregulation.

30 According to one embodiment, the said disorder involving tyrosine kinase dysregulation is selected in the group consisting of myocardial infarction1 stroke, congestive heart failure, an ischemia or reperfusion injury, trauma, cancer, oedema, arthritis or other arthropathy, retinopathy or vitreoretinal disease, diabetic retinopathy, macular oedema, including diabetic macular oedema, macular degeneration, glaucoma,

35    autoimmune disease, vascular leakage syndrome1 inflammatory disease, oedema, transplant rejection, burn, or acute or adult respiratory distress syndrome (ARDS).
 
(


wo 2010/092041 PCT/EP2010/051556 14

In another embodiment, there are provided methods of treating an ophthalmic disorder associated with increased va•scular permeability 1 including the administration of a therapeutically effective amount of one or more compound of the Invention to a subject in need ofsu~h treatment.

5 In another embodiment, there are provided me~hods of treating a subject having or at risk of having cancer including administering to the subject a therapeutically effective amount of one or more compound of the Invention thereby treating the subject.

In another embodiment, there are provided methods of treating a subject having or at risk of having oedema and/or angiogenesis including administering to the subject a

10    therapeutically effective amount of one or more compound of the Invention 1 thereby treating the subject.

In another embodiment, there are provided methods of treating a subject having or at risk of having macular degeneration including administering to the subject a therapeutically effective amount of one or more compound of the Invention, thereby

15    treating the subject.

In another embodiment, there are provided methods .of treating a subJect ~aving or •at risk of having diabetic retinopathy including administering to the subject a therapeutically effective amount of one or more compound of the Invention1 thereby treating the subject.

20 In another embodiment, there are provided methods of treating a subject having or at risk of having macular oedema/ including diabetic macular oedema, including administering to the subject a therapeutically effective amount of one or more compound of the Invention1 thereby treating the subject.

In another embodiment, there are provided methods of treating a subject having

25    or at risk of having glaucoma including administering to the subject a therapeutically effective amount of one or more compound of the Invention, thereby treating the subject.

In another embodiment, there are provided methods of treating a subject having

or at risk of having retinopathy including administering to the subject a therapeutically

30    effective amount of one or more compound of the Invention, thereby treating the subject.


In another embodiment1 there are provided methods of treating a subject having or at risk of having vitreoretinal disease including administering to the subject a therapeutically effective amount of one or more compound of the Invention1 thereby

35    treating the subject.
 

wo 2010/092041 PCT!EP2010/051556 15

In another embodiment, there are provided methods of treating a •subject having

•    or at risk of having inflammatory disease, including administering to the subject a therapeutically effective amount of one or more compound of the Invention, thereby treating the subject. .

5 In yet another embodiment, there are provided methods of treating a disorder, including an ophthalmic disorder and cancer, associated with compromised vascular permeability including the administration of a therapeutically effective amount of one or more compound of the Invention in combination with an anti-inflammatory agent~ chemotherapeutic agent1 antitumoral agent1 immunomodulatory agent1 gene-based

10    therapeutic vaccine, immunotherapy product, therapeutic antibody and/or a kinase inhibitor, to a subject in need of such treatment.

Administration of the compounds of the Invention 1 especially for ophthalmic applications1 is preferably by topical administration. However, the invention is not limited to topicai delivery in that it also includes for example intraocular and periocular injection,

15    systemic delivery (e.g. oral or other parenteral route such as for example subcutaneous, intramuscular, intravenous administrations) or intratumoi-al delivery.

In yet another embodiment1 there are provided methods of delivering a compound of the Invention to the back of the eye, the method including preparing a composition including a pharmaceutically effective amount of at least one compound of the Invention

20    and delivering said composition to the eye of a subject in need of such delivery.

In yet another embodiment, there are provided methods of delivering a compound of the Invention intratumoraly, the method including preparing a composition including a pharmaceutically effective amount of at least one compound of the Invention and delivering said composition to the tumor of a subject in need of such delivery.

To prepare a composition of the Invention, and more specifically an ophthalmic composition or antitumoral composition, a therapeutically effective amount of one or more compound of the Invention is placed in a vehicle as is known in the art. For example, topical ophthalmic formulations containing steroids are disclosed in US
5,041,434, whilst sustained release ophthalmic formulations of an ophthalmic drug and a

30    high molecular weight polymer to form a highly viscous gel have been described in US 4,271 1 143 and US 4,407,792. Further GB 2007091 describes an ophthalmic composition in the form of a gel comprising an aqueous solution of a carboxyvinyl polymer, a water-

soluble basic substance and an ophthalmic drug. Alternatively, US 4,615,697, discloses a controlled release composition and method of use based on a bioadhesive and a treating

35    agent, such as an anti- inflammatory agent.
 

wo 2010/092041 PCT/EP2010/051556 16

The amount of the compounds of the Invention to be administered and its concentration in the compositions used in the method of the Invention depend upon the selected dissolving agent, delivery system or device, clinical condition of the patient, side effects arid stability of the compound within the composition. Thus, the physician

5    employs the appropriate preparation containing• the appropriate concentration of the compounds of the Invention and selects t.he amount of formulation administered, depending upon clinical experience with a given patient or with similar types of patients.

In•another embodiment, there are •provided processes for making one or more compound• of the Invention or its pharmaceutically acceptable salt, hydrate, solvate,
10    crystal form .salt and individual diastereomers thereof.

There are multiple synthetic routes for the preparation of the compounds of the invention, but all rely on chemistry known to the synthetic organic chemist. Thus, compounds represented by Formula I can be synthesized according to •procedures described in the literature and are well-known to•one skilled in the art. Typical literature

15    sources are "Advanced organic chemistry", 4th Edition (Wiley), J March, "Comprehensive Organic Transformation", 2nd Edition (WileyL R. C. Larock, "Handbook of Heterocyclic Chemistry", 2nd Edition (Pergamon), A. R. Katritzky), review. articles such as found in

"Synthesis", "Ace. Chern. Res."1  "Chern. Rev", or primary literature sources identified by

standard  literature  searches  online  or  from  secondary  sources  such  as  "Chemical

20    Abstracts" or "Beilstein". Compounds of the invention can be synthesized by methods analogous to those exemplified in the Examples herein for certain representative compounds. Using the procedures described in the Examples section, and well known procedures, one skilled in the art can prepare the compounds disclosed herein.

In another embodiment, there are provided kit including packaging material and a

25    composition contained within the packaging material, wherein the packaging material includes a label which indicates that the composition can be used for treatment of disorders associated with compromised vascular permeability and wherein the composition includes one or more compound of the Invention.

In another embodiment, there are provided kit including packaging material and a

30    composition contained within the packaging material, wherein the packaging material includes a label which indicates that the composition can be used for treatment of disorders associated with compromised vascular permeability and selected from

myocardial infarction, stroke, congestive heart failure, an ischemia or reperfusion injury,

cancer,  arthritis  or  other  arthropathy/  retinopathy  or  vitreoretinal  disease,  macular

35    degeneration, autoimmune disease, vascular leakage syndrome, inflammatory disease, edema, transplant rejecti.on, burn, or acute or adult respiratory distress syndrome (ARDS) and wherein the composition includes one or more compound of the Invention.
 

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17

In one preferred embodiment, there are provided kit including packaging material and •a composition contained within the packaging material, wherein the packaging material includes a label which indicates that the composition can be used for treatment of ophthalmic disorders associated with compromised vascular permeability and wherein

5    the composition includes one or more compound of the Invention or its pharmaceutically acceptable salt, hydrate, solvate, crystal form salt and individual diastereomers thereof .

Those  skilled  in  the  art  will  appreciate. that  the  invention  described  h~rein is

susceptible to variations and  modifications other than those specifically described. The

invention includes all such variation and modifications. The invention also includes all of

10    the steps, features, formulations •and compounds referred to or indicated in the specification, individually or collectively and any and all combinations or any two or more of the steps or features.

Each  document,  reference,  patent  application  or  patent  cited  in  this  text  is

expressly incorporated herein in their entirety by reference, which means that it should

15    be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

The present invention is not to be. limited in scope by the specific embodiments

described herein, which are intended for the purpose of exemplification only. Functionally

20    equivalent products, formulations and methods are clearly within the scope of the invention as described herein.

The invention described herein may include one or more range of values (eg size,

concentration etc). A range of values will be understood to include all values within the

range, including the values defining the range, and values adjacent to the range which

25    lead to the same or substantially the same outcome as the values immediately adjacent to that value which defines the boundary to the range.

The following examples are given to illustrate the preparation of compounds that are the subject of this invention but should not be construed as implying any limitations to the claims. The proton magnetic resonance spectrum of each compound of the

30    Examples was consistent with the assigned structure.

EXAMPLES

1 -  SYNTHESIS OF COMPOUNDS OF GENERAL FORMULA (I)

1.1. General method

Step A -  Coupling of 7-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine or

35    6-Bro~o-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine  to    1    eq    of    optionally
 

wo 2010/092041    PCTIEP2010/051556
18

substituted 81,82-phenyl boronic acid in a polar solvent at -100 to 300°C, most preferably 50-150°C


5 Step 8 - Coupling of 3 or 4;..substituted bromo-phenyl to 1 eq of optionally substituted 81~82-7 or 6-phenyl--[1,2,4]triazolo[1,5-a]pyridin-2-ylamine in a polar solvent at -100°C to 300°C, most preferably 50-150°C
s,-(')~vI            D-s3 B,        v::::,..       
B    ?'    A-N        Br               
2                    1           
    •    I'QI        -    A- N        B   
                    1 Q  I           
    R2    A,.  / --..    NH2        A2.N/-- ..N        3   
        - N        R2    H           
                               
10                    Cpn           


The compounds of the formula I and also the starting materials for their preparation, are prepared by methods as described in the examples or by methods known per se, as described in the literature (for example in standard works, such as

15    Houben-Weyl, Methoden der Organischen Chemie [Methods of Organic Chemistry], Georg Thieme Verlag, Stuttgart; Organic Reactions, John Wiley & Sons, Inc., New York), to be precise under reaction conditions which are known and suitable for the said reactions. Use can also be made here of variants which are known per se, but are not mentioned here in greater detail.


20 The starting materials for the claimed process may, if desired, also be formed in situ by not isolating them from the reaction mixture, but instead immediately converting them further into the compounds of the formula I. On the other hand, it is possible_ to carry out the reaction stepwise.


Preferably,  the  reaction  of the compounds  is carried  out  in  the  presence  of a

25    suitable solvent, which is preferably inert under the respective reaction conditions. Examples of suitable solvents are hydrocarbons, such as hexane, petroleum ether,
benzene,  toluene  or xylene;  chlorinated  hydrocarbons,  such  as trichlorethylene,  1~3..:~...,.•~":-~~~
dichloroethane,  tetrachloromethane,  chloroform  or dichloromethane;  alcohols, .~yc'!f'as .    •  . -~~

methanol, ethanol, isopropanol[  n-propanol,  n-butanol or tert-butanol;  ether,?';such    as    ..        \   

wo 2010/092041    PCT/EP2010/051556
19
 
diethyl ether, diisopropyl ether, tetrahydrofuran (THF) or dioxane~ glycol ethers, such as
.    .

ethylene  glycol  monomethyl  or  monoethyl  ether  or  ethylene  glycol  dimethyl  ether

(diglyme); ketones, such as aceton~ or butanone; amides, such as acetamide, dimethylacetamide, dimethylformamide (DMF) or N-methyl pyrr.olidinone (NMP); nitriles,

5    such as acetonitrile; sulfoxides, such as dimethyl sUlfoxide (DMSO); nitro compounds,• such as nitromethane or nitrobenzene; esters, such as ethyl acetate, or mixtures. of the said solvents or mixtures with water. Polar solvents are in general preferre.d. Examples

for suitable polar solvents are chlorinated hydrocarbons, alcohols, glycol ethers, nitriles, amides and sulfoxides or mixtures thereof. More preferred are amides, especially
10    dimethylformamide (DMF).

As stated above, the reaction temperature is between about -100°C and 300°C, depending on the reaction step and the conditions used.

Reaction times are generally in the range between some minutes and several days, depending on the reactivity of the respective compounds and the respective

15    reaction conditions. Suitable reaction times are readily determinable by methods known in the art, for example reaction monitoring. Based on the reaction temperatures given above, suitable reaction times generally lie in the range between 10 min and 48 hrs.

Every reaction step described herein can optionally. be followed  by one or more

working up procedures and/or isolating procedures. Suitable such procedures are known

20    in the art, for example from standard works, such as Houben-Weyl, Methoden der organischen Chemie [Methods of Organic Chemistry], Georg-Thieme-Verlag, Stuttgart). Examples for such procedures include, but are not limited to evaporating a solvent,

distilling,  crystallization,  fractionised  crystallization,  extraction  procedures,  washing

procedures,    digesting    procedures,    filtration    procedures,    chromatography,

25    chromatography by HPLC and drying procedures, especially drying procedures in vacuo and/or elevated temperature.

List of Abbreviations and Acronyms:

AcOH acetic acid, anh anhydrous, atm atmosphere(s), BOC tert-butoxycarbonyl CDI 1,1'-carbonyldiimidazole, cone concentrated, d day(s), dec decomposition, DMAC

30    NN-dimethylacetamide, DMPU 1,3-dimethyl-3,4,5,6-tetrahydro-2(IH)-pyrimidinone, DM•F NN-dimethylformamide, DMSO dimethylsulfoxide, DPPA diphenylphosphoryl azide, EDCI

1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, EtOAc ethyl acetate, EtOH ethanol (100%), Et2 0 diethyl ether, Et3N triethylamine, h hour(s), MeOH methanol, pet. ether petroleum ether (boiling range 30-60°C), temp. temperature, THF tetrahydrofuran, TFA

35    trifluoroAcOH, Tf trifluoromethanesulfonyl.
 

r    ',
(
 
wo 2010/092041 PCT/EP2010/051556 20

The compounds of general formula I of the present invention can be prepared according to the procequres of the following Steps A and B above disclosed and the examples. In all preparative methods, all starting material is known or may easily be prepared from known starting materials.

1.2. Intermediates

In all preparative methods, all starting materials are known or may be prepared from known starting materials by the following general methods:

                0       
            /' - )l    N~.   
Br'Cl    o       
I        N        -    ~s   
    ,.-:;;    NH           
            2           
Br'Cl-........    °    NH20H, HCI   
CH31    I    N           
~        ,.-:;;    N~NJlO~ DIPEA   
H
 



10 The compounds can be prepared by the general method, following procedures depicted in W02007/095588 (Novartis).


Synthesis    of    intermediate    1:    6-{2-Chloro-5-methoxy-phenyl)-

[ 1,2,4 ]triazolo[l,S-a ]pyridin-2-ylamine

15    To a solution of 2-Chloro-5-methoxy-phenylboronic acid (3.38g, 22.5 mmol, l.Seq), 6-Bromo-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine (3.2g, 15 mmol, leq) and Na2C03

(6.36g, 60 mmol, 4eq) in a mixture of 40ml DMF/10ml EtOH/10ml H20, was added 1. 733g (1.5 mmol, 0.1 eq) of tetrakis(triphenylphospine) palladium. The reaction was refluxed for 2 hours under argon. It was then cooled off to room temperature and the

20    product was precipitated by water, filtered, rinsed with water, ether and pentane to give a pale yellow powder (3.21 g, 13 mmol, 90% yield).

wo 2010/092041 PCTIEP2010/051556 21

Intermediate    2:    7-(2-Chloro-5-methoxy-phenyl)-[1,2,4]triazolo[1,5-

a]pyridin-2-ylamine  has  been  synthesized  according  to  the. method  disclosed  for

Intermediate 1 starting from 7-Bromo-[1,2A]triazolo[1,5-a]pyridin-2-ylamine.



5    Synthesis  of  intermediate  3:  3-{2-Amino-[1,2,4]triazolo[11 5-a]pyridin-

6-yl)-4-chloro-phenol

To a suspension of 5.560g (20.24 mmol1 1eq) of 6-(2-Chloro-5-methoxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-ylamine in 90 ml of •dichloromethane cooled to ooc was added carefully 60 ml of a 1M solution of 1M BBr3. The solution is stirred for

10    2hrs. The pH is then adjusted to pHS by adding a sturated solution of NaHC03. The precipitated product is filtered and washed with ether and dried to give 4.856g (19 mmol1 92%) of a white powder.

Intermediate    4:    3-(2-Amino-[1,2,4]triazolo[1,5-a]pyridin-7-yl)-4-

chloro-phenol has qeen synthesi~ed according to the method disclos~d for Intermediate 15 3 startin•g from 7-(2-Chloro-5-methoxy-phenyl)-[1,2,4]triazolo[1,5-a] pyridin-2-ylamine.



INTERMEDIATES        81    82        R2    Al        A2    LC/MS   
                                       
                                               
Intermediate 1        2-CI    5-        H    N        c    M+1= 274.9   
            OCH3                       
                                           
                                               
intermediate 2        2-CI    5-        H    c        N    M+1=274.9   
            OCH3                       
                                           
                                           
Intermediate 3        2-CI    5-0H    H    N        c    M+1=260.9   
                                           
Intermediate 4        2-CI    5-0H    H    c        N    M+1=260.9   
                                               



20    1.3. Compounds of the Invention


Synthesis of compound of the Invention N°5

To 54mg (0.06 mmol, 0.03eq) of Pd2(dba)3, 18 mg (0.04 mmol, 0.02eq) of 5-(Di-tert-butyl-phosphanyl)-1',3'r5'-triphenyi-1'H-[1,4']bipyrazolyland 265 mg (4.73

25    mmol, 2.15 eq) of KOH, was added 3 ml tertamylacohol and 4001JI of water and the suspension is stirred for 10 minutes. 573 mg (2.20 mmol, 1eq) of 6-(2-Chloro-5-methoxy-phenyl)-[1,2,4]triazolo[115-a]pyridin-2-ylamine and 713 mg (2.64 mmol,
 

wo 2010/092041    PCT!EP2010/051556
    22   
.I    1.2eq)  of  1-[2-(4-Bromo-phenoxy)-ethyl]-pyrrolidine    are  then •added, followed  by

another 3ml of tertamyl alcohol and 400j..il of water and the mixture is stirred at 80°C under a•rgon for 3 hours. The compound is extracted by 3 times Ethyl acetate, washed with brine. The organic layers are then dried over Na2S04 , filtered and evaporated. The

5    compound is then dissolved in methanol and HCI 1M •in ether is added. The precipitated compound is then filtered, washed with ethyl acetate and 'etherto give 549 mg {1.13 mmol, 51%) of a white powder.

1-[2-(4-Bromo-phenoxy)-ethyl]-pyrrolidine  could  be  purchased  from  Sigma-

Aldrich.  Other derivatives  could  be  synthetically  obtained  using  classical  methods  of

10    organic synthesis.

Some compounds could also be purified by prep HPLC. We have used an Agilent

1200 series semi-prep with UV detector monitoring at 254 nm. Co.mpounds were purified

on  a  ZORBAX,  SB-C18 .column  (21,2mmx100mm,  51Jm).  The  gradient  was  typically

performed using a H20/Acetonitrile gradient (from a range starting from 5 to 50% water

15    to 95% acetonitrile) at a flow rate of SOml/mn during 15 min.

The  following  compounds  of the  Invention  were  made  in  a similar way  as  described

above:

20


Examples    Name    MS

NMR (200MHz, DMSOd6)

compound    4-Chloro-3-{2-[4-    M+1= 450.1
5    (2-pyrrolidin-1-yl- NMR: 10.89 (bb, 1H); 9.58 (bb, lH); 8.84 (t, 1H);
7.62    (m, 4H); 7.38 (d, 1H); 6.84-7.01 (m, 4H);
    ethoxy)-    4.31 (t, 2H); 3.55 (m, 4H); 3.10 (m, 2H); 2.17-       
    phenylamino]-    1.74 (m, 4H)           
                           
    [1,2,4 ]triazolo[1,5-                       
    a]pyridin-6-yl}-                       
    phenol,                       
    hydrochloride                       
                           
compound    2-{ 4-[6-(2-    Chloro-    M+1= 507.1           
6    5- hydroxy-phenyl)-                       
    [ 1,2,4]triazolo[ 1,5-                ~~-~   
    a]pyridin-2-                   
    ylamino]-phenoxy}-            ~C:"•,0~. ~\}I> • . ;  ~ )J ,,.>J   

    wo 2010/092041    PCT/EP2010/051556   
                23   
                   
            1-((R)-3-       
            dimethylamino-       
            pyrrolidin-1-yl)-       
            ettianone,       
            hydrochloride       
                   
compound        1-{4-[6-(2-Chloro-    M+1= 518.1   
7        5-hyd roxy- phenyl)-    NMR: 9.95 (bb, 1H, OH); 9.51 (s, 1H, NH); 8.82   
            (t, 1H); 7.64 (d, 2H); 7.58 (d, 2H); 7.46 (d, 2H);   
                   
            [1,2A ]triazolo[1,5-    7.38 (d, 1H);  6.87 {m, 2H);  3.75 (m, 2H); 3.50   
            a]pyridin-2-    (m, 2H); 3.28. (m, 4H);  2.52 (m, 4H); 1.67 (m,   
                4H)   
                   
            ylamino]-phenyl}-       
            3-(2-pyrrolidin-1-yl-       
            ethyl)-imidazolidin_:       
            2-one       
                   
compound        (S)-1-(2-{4-[6-(2-    M+1= 508.0   
8        Chloro- 5-hydroxy-    NMR: 9.95 (bb 1  1H, OH); 9.44 (s, 1H, NH); 8.82   
            (s 1H); 7.58 (m, 4H); 7.38 (d, 1H);  6.87 (m,   
            phenyl)-       
                4H);  4.70 (s, 2H); 4.27 (m, lH); 3.59 (m, 2H);   
            [ 1,2,4 ]triazolo[1,5-    2.17-1.90 (m, 4H)   
                   
            a]pyridin-2-       
            ylamino ]-phenoxy}-       
            acetyl)-pyrrolidine-       
            2-carboxylic acid       
                   
~--------~----~----~--~-------------------------------------
compound    4-Chloro-3-{8-    M+1= 464.1

g             methyl-2-[4-( 2-        NMR: 10.86 (bb, 1H); 9.58 (bb, lH); 8.64 (s, lH); 7.62 (d, 2H); 7.42 (s, 1H); 7.36 (d,  1H); pyrrolidin-1-yl-       6.83-7.00 (m, 4H);  4.30 (t, 2H); 3.55 (m, 4H);

ethoxy)-    3.10 (m, 2H); 2.17-1.77 (m, 4H)

phenylamino]-[1,2,4 ]triazolo[1,5-a]pyridin-6-yl}-phenol, hydrochloride

compound    4-Chloro-3-{2-[4-    M+1= 450.1   
10    (2-pyrrolidin-1-yl-    NMR: 10.92 (bb, 1H, OH); 9.64 (bb 1   1H, NH);   
        8.81 (d, lH); 7.64 (d, 2H); 7.55 (s,  lH); 7.38 (d,   
    ethoxy)-       
        1H); 6.86-7.10 (m, 5H);  4.32 (t, 2H); 3.55 (m,   
    phenylamino]-    4H); 3.10 (m, 2H); 1.96 (m, 4H)   
           
    [ 1,2,4]triazolo[1,5-       
    a]pyridin-7-yl}-       
    phenol,       
    hydrochloride       
 

            wo 2010/092041    PCT/EP2010/051556                       
                                    24                           
                                                               
                                                               
                compound        4-Chloro-3-{2-[4-    M + 1 = 466.1                           
    11        (2-morphol i n-4-yl-    NMR: 9.95 (s, 1H); 9.45 (s, 1H); 8.84 (s,                       
                1H);7.63(m, 4H); 7.40 (d, 1H); 6.90 (m, 4H);                       
                                ethoxy)-.                           
                                    4.05 (t, 2H); 3.58 (m, 4H);  2.68 (t, 2H); 2.504                       
                                phenyl~mino]­    (m, 4H)                           
                                                               
                                [1,2,4 ]triazolo[1,5-                               
                                a]pyridin-6-yl}-                               
                                phenol                               
                                                               
                compound        • 4-Chloro-3-{2-[3-    M +: 1 = 466.1                           
    12        (2-morpholin-4-yl-.    NMR: 9.95 (s, 1H); 9.67 (s, 1H); 8.89 (s,                       
                1H);7.63(s, 2H}; 7.41 (m, 2H); 7.23 (m, 2H);                       
                                ethoxy)-                           
                                    6.87 (m, 2H); 6.50 (d, 1H); 4.07 (t, 2H); 3.59 (t,                       
                                phenyl•amino]-    4H);  2.70 (t, 2H); 2.50 (m, 4H)                       
                                                               
                                [1,2,4 ]triazolo[1,5-                               
                                a]pyridin-6-yl}-                               
                                phenol                               
                                                               
                compo1,1nd        4-Chloro-3- [2-(3-    M + 1 = 403.1                           
    13        pyrazol-1-yl-    NMR: 9.95 (m, 2H); 8.91 (s, 1H);8.41 (s,1H);                       
                8.23(s, 1H); 7.69 (m, 4H); 7.37 (m, 3H); 6.89                       
                                phenyl amino)-                           
                                    (m,  2H); 6.55 (s,  1H)                       
                                [ 1,2,4]triazolo[1 ,5-                               
                                a] pyridi n-6-yiJ-                               
                                phenol                               
                                                               
                compound        4-Chloro-3-{2-[ 4--    M + 1 = 424.1                           
    14        (2-dimethylamino-    NMR: 9.96 (s, 1H); 9.43 (s, 1H); 8.82 (s,                       
                1H);7.60(s, 4H); 7.38 (d, 1H); 6.88 (m, 4H); 3.99               
                                ethoxy)-                   
                                    (t, 2H); 2.59 (t, 2H);  2.21 (s, 6H)                       
                                phenyl amino]-                               
                                [ 1 ,2,4 ]triazolo[ 1,5-                               
                                a] pyrid i n-6-yl }-                               
                                phenol                               
                                                           
                compound        4-Ch !oro-3-{ 2- [ 3-    M + 1 = 424.1                           
    15        (2-dimethylamino-    NMR: 9.65 (s, 1H); 8.83 (s, 1H);7.60(s, 2H); 7.41        ....-:~~.!..--~;   
                (m, 1H); 7.23 (m, 3H); 6.84 (m, 2H); 6.46 (d,                   
                                ethoxy)-                       
                                    1H); 4.02 (t, 2H); 2.62 (t, 2H);    2.19 (s, 6H)    /'        .   
                                phenylamino]-                        .   
                                                           
                                                           
                                [1,2,4]triazolo[1,5-                            ':IJS~cu"\.   
                                a]pyridin-6-yl}-                            ~~ ~ .t~;j~   
                                                            •~)  t   
                                                            ~;:3.-o$.   
                                phenol            ••~~   
    l ------- 1        ----- , ----- : - : --- = --- l ------------- = ---------- j  ••.. -::--::'; ~-. '~. m   
               
                compound        4-Chloro-3-{2-[3-    M + 1 = 450.1    •,.;;.....,~:-:.~.2.~   
    16        ( 2-pyrrolidin- 1-yl-    NMR: 10.01 (s, 1H); 9.68 (s, 1H);8.90(s, 2H);    "-"'   
                                    7.64 (m, 1H); 7.43 (m, 2H); 7.23 (m, 2H); 6.89                       
                                ethoxy)-    (m, 2H); 6.49 (d, 1H); 4.07 (t, 2H); 2.81 (t, 2H);                       
                                    2.51 (m  4H)• 1.71 (m, 4H)                       
 

        wo 2010/092041    25        PCT!EP2010/051556                           
                                                                       
                                                                           
                    phenylamino]-                                                       
                    [ 1,2,4 ]triazolo[1,5-                                                       
                    a]pyridin-6-yl}-                                                       
                    phenol                                                       
                                                                           
        compound        4-Chloro-3-[2-(4-                M + 1 = 367.0                                   
    17            hydroxymethyl-        NMR: 8.47 (s, 1H); 7.50 (s, 1H); 6.51(d, 2H);                           
                        6.38 (, s,2H);6.06 (d, 2H); 5.85 (d, 1H); 5.31 (m,                           
                    phenylamino )-                                   
                                    2H); 3.25 (s, 2.H)                                   
                    [1,2,4 ]triazolo[1,5-                                                       
                    a] pyridin-6-yl]-                                                       
                                                                           
                    phenol                                                       
                                                                           
        compound        4-Chloro-3-[2.-( 3-                M + 1 = 367.0                                   
    18            hydroxymethyl-                                                       
                    phenylamino)-                                                       
                                                                           
                    [1,2,4 ]triazolo[1,5-                                                       
                                                                           
                    a]pyridin-6-yl]-                                                       
                                                                           
                                                                           
                    phenol                                                       
                                                ..........               
        compound        4-[6-(2-Chloro-5-                M + 1 = 416.0                           
        19            hydroxy-phenyl)-                                        .,""           
                                                                       
                    [1,2,4 ]triazolo[1,5-        I                    ~~~       
                                                       
                                                       
                    a]pyridin-2-                                ~•J.t:&~\    'y~~•        ~   
                                                                   
                                                                   
                    ylamino]-        ~        \.)':.> ~ ;vt.;:j~>/ "lry~h           
                                               
                    benzenesulfonamide        1[.    .    .:. .J'y\•    -~           
                                    if••~-,;__--        G~   
                                               
                                        ~••                   
        compound        4-Chloro-3-{2-[ 4-                M + 1- 397.1 .   -~J. . l(!;W.IU.                       
                                                           
        20            (2-hydroxy-        I                                   
                                                               
                    ethoxy)-                                           
                                                                           
                    phenylamino]-                                                       
                    [1,2,4 ]triazolo[1,5-        I                                   
                                                               
                    a]pyridin-6-yl}-        I                                   
                            I                                   
                            I                                   
                    phenol    I                                   
                                                           
                            I                                   
        compound        1-{4-[6-(2-Chloro-                M + 1 =  421.1                                       
21    5- hydroxy-phenyl)- NMR:  9.51 (s, 1H); 8.80 (s, 1H); 7.61(m, 4H);
7.46    (, d,2H); 7.34 (d, 1H); 6.83 (m, 3H); 4.00

[1,2,4 ]triazolo[1,5- (t, 1H); 3.82 (t, 2H); 3.69 (t, 1H) a]pyridin-2-

ylamino ]-phenyl}- I imidazolidin-2-one

compound    3-[6-(2-Chloro-5-    M + 1 =  416.0
22    hydroxy-phenyl)-   
    [1,2,4 ]triazolo[1,5-   
 
\r


    wo 2010/092041    PCT/EP2010/051556
            26
           
        a]pyridin-2-    I
        •  ylamino]-    '1
        benzenesulfonamide   
           
    compQund    2-{4-[6-(2-Chloro-    M + 1 = 410.1
23    5-hydroxy-phenyl)-   
        [1,2A]triazolo[1,5- •
        a]pyridin-2-   
        ylamino]-phenoxy}-   
        acetamide   
           
    compound    2-{3-[6-(2-Chloro-    M + 1 = 410.1

24    5-hydroxy-phenyl)-[ 1,2,4]triazolo[ 1,5-a]pyridin-2-

        ylamino]-phenoxy}-           
        acetamide           
                   
    compound    4-Chloro-3-[2-( 4-        M + 1 = 421.0   
25    trifluoromethoxy-    NMR; 10.00 (s, 2H); 8.90 (s, 1H); 7.81 (d, 2H);   
            7.65 (s,2H); 7.36 (m, 3H); 6.87 (m, 2H)   
                   
        phenylamino )-           
        [1,2,4 ]triazolo[1,5-           
        a] pyridin-6-yl]-           
        phenol           
                   
    compound    4-Chloro-3-[2-( 4-        M + 1 = 429.1   
26    phenoxy-    NMR: 9.94(s, 1H); 9.71 (s, 1H); 8.85 (s,1H); 7.74   
        (d,    2H); 7.61 (s,2H); 7.34 (m, 4H); 7.00 (m, 6H)   
                   
        phenylamino )-           
        [1,2,4 )triazolo[l,S-           
        a]pyridin-6-yl)-           
        phenol           
                   
    compound    4-Chloro-3-[2-( 4-        M + 1 = 415.0   
27    methanesulfonyl-           
        phenylamino )-           
        [1,2,4 ]triazolo[1,5-           
        a]pyridin-6-yl]-           
                   
        phenol           
                   
    compound    3-[2-(4-Benzyloxy-        M + 1 = 443.1   
28    phenylamino)-           
        [ 1,2,4]triazolo[ 1,5-           
        a]pyridin-6-yl]-4-           
        chloro-phenol           
                   
 

wo 2010/092041    PCT/EP2010/051556
        27
       
compound    1-{4-[6-(2-Chloro-    M + 1 = 518.3

29    5-hydroxy-phenyl)-[1,2,4 ]triazolo[1,5-

    a]pyridin-2-           
    ylamino ]-phenyl}-           
    3-(2-pyrrolidin-1-yl-           
    ethyl)-imidazolidin-           
    2-one           
               
               
compound    5- [6-(2-CI:lloro-5-    M + 1 = 421.0       
30    hydroxy:- phenyl)-           
    [ 1 ,2,4 ]triazolo [ 1,5-           
    a]pyridin-2-           
    ylamino]-           
    benzofu ran- 2-           
    carboxylic acid           
               
compound    1-{4-[6-(2-Chloro-    M + 1 = 435.1       

31    5-hydroxy-phenyl)-[ 1,2,4]triazolo [ 1,5-a]pyridin-2-

yla mi no]- phenyl}-3-methyl-

i    midazolidin- 2-one

            compound    1-{4-[6-(2-Chloro-        M + 1 = 479.1   
                           
32    5-hydroxy-phenyl)-                       
                [1,2,4 ]triazolo[1,5-                       
                a]pyridin-2-                I   
                                       
                ylamino]-phenyl}-                       
                3-(2-methoxy-                       
I    ethyl)-imidazolidin-                       
I    2-one                       
                               
            compound    4-[6-(2-Chloro-5-            M + 1 = 487.1       
33    hydroxy- phenyl)-                       
                [1,2,4 ]triazolo[ 1,5-                       
                a] pyridi n- 2-                       
                ylamino]-                       
                benzenesulfonyl-                       
                ethyl urea                       
                                       
            compound    1-(4-{4-[6-(2-            M + 1    = 493.2       
                        ..               
                                       
 

wo 2010/092041    PCT/EP2010/051556
28

34    Chloro-5-hydroxy-

phenyl)-

[1,2,4 ]triazolo[1,5-a]pyridin-2-ylamino]-phenyl}-piperazin-1-yl)-2-methoxy-ethanone

compound( 4-{ 4-[6-(2-Chloro-    M + 1 = 526.1

35    5-hydroxy-phenyl)'-[1 1 21 4 ]triazolo[1,5-a]pyridin-2-. ylamino]-phenyl}-piperazin-1-yl)-pyridin-4-yl-methanone

compound1-{4-[6-(2-Chloro-    M + 1 = 492.2

36    5-hydroxy-phenyl)-[1,2,4 ]triazolo[1,5-a]pyridin-2-ylamino]-phenyl}-

            3-(2-pyrrolidin-1-yl-       
            ethyl)-urea       
                   
        compound    4-Chloro-3-{2-[ 4-    M + 1 = 465.1   
    37    (2-piperazi n-1-yl-       
            ethoxy)-       
                   
            phenylamino]-       
            [1,2,4 ]triazolo[1,5-       
                   
            a]pyridin-6-yl}-       
                   
            phenol       
                   
                   




2 - SOLUBILITY ANALYSIS OF COMPOUNDS OF THE INVENTION

Solubility of Compounds was determined in aqueous medium using the following 5 procedure.

Two mg of Compound (2 mg) was added to 200 JJI buffer (acetic acid/KOH) solution at pH 5. ~olution was then stirred for 24 h at room temperature a_nd then centrifuged 10 min at 16,000 rpm. Corresponding supernatants were analyzed by HPLC
 
0

C)    wo 2010/092041    PCTJEP2010/051556   
    .    29   
    and UV detection.  Calculation  of a given  Compound  concentration  was  performed  by   
    reporting area under the experimental slope onto a calibration slope obtained separately   
    using DMSO-solubilised Compound at different concentrations.   
    The tested compounds are :       
5    The  reference  compound  in  the    following  is  as  disClosed  in  W02005096784   
    (compound CL) •       

3 -  Measurement of inhibition constants of the compounds of the

Invention.
 

wo 2010/092041    PCT/EP2010/051556
31

The screening and profiling experiments described here were performed using Caliper Life Sciences'proprietary LabChip™ technology. Caliper LC3000 and EZ Reader II instruments are widely used throughout the drug discovery process for assay development, primary screening, selectivity screening, generation c:>f Structure-Activity

5    Rerationships (SARs) and Mechanism of Action (MOA) studies. The LabChip ™ technology is particularly well suited for enzymatic 'targets'such as kinases, proteases, phosphatases, histone deacetylases (HDAC), phosphodiesterases (PDE), and• acyl-transferases. The key benefit of the technology is the separation and direct measurement of substrates and products, which allows for higher signal-to-noise ratios and fewer false

10    positive/negative results. This direct measurement also allows for the identification and elimination of enzymatic activities that are not associated with the kinase reaction of interest.

General:

The  off-chip  incubation  mobility-shift  kinase  assay  uses a microfluidic chip to

15    measure the conversion of a fluorescent peptide substrate to a phosphorylated product. The reaction mixture, from a microtiter plate well, is introduced through a capillary sipper onto the chip, where the nonphosphorylated substrate and phosphorylated product are
separated by electrophoresis and detected via laser-induced fluorescence. The signature

of  the  fluorescence  signal  over  time  reveals  the  extent •of  the  reaction.  The

20    phosphorylated product migrates through the chip faster than the non-phosphorylated substrate, and signals from the two forms of the peptide appear as distinct peaks. Caliper'sdata analysis software (HTSWA) determines peak heights, from which the ratio

of product to the peak sum P/(P+S) and percent (% ) conversion is calculated. This value is used to compare compound wells to control wells present on the plate, and

25    thereby determine the % inhibition values for the compound. The formula used to calculate % inhibition is as follows, where C10o% is the average % conversion of the 100% activity wells and Co% is the average % conversion of the 0% activity wells:

(1-(%conversionofsample- Co%)/(ClOo%-Co%))*100

Specific:

30    LC3000 Src and Lyn Assays

Compounds were dissolved in 100% DMSO and diluted to 25X the final desired screening concentration. Serial dilutions were performed to obtain the concentrations specified for particular studies. One !JL of each concentration was transferred, in duplicate, to a 384-well Greiner microtiter plate. Generally, 12 !JL of enzyme buffer

35    containing purified kinase (various suppliers), 100 mM HEPES, pH 7.5, 1 mM DTT (Ca!biochem, 2333153), 10 mM• MgCI2 •(Sigma, M-1028) or 10 •mM MnCb (Sigma, M-
 

wo 2010/092041    PCTJEP2010/051556
32

1787) (assay specific), and 0.002% Brij-35 (Sigma, B4184) was added to each well. Compound and •enzyme were allowed to pre-incubate for 15 minutes. 12 !JL of peptide/ATP buffer containing 100 mM HEPES, pH 7.5, 1.5 !JM fluorescein-labeled peptide (specific to kinase of interest), ATP (at KM apparent, Sigma1 A9187), and 0.002% Brij-35 •

5    was then added to each well to initiate the •reaction. Generally, reactions were incubated for 1 - 1.5 hours at room temperature to obtain adequate (15-40%) conversion of peptide to phosphorylated product in the linear range of the reaction. Reactions were terminated with the addition of 45. !JL of Stop Buffer (containing 20 mM EDTA). Plates were then read on the LabChip 3000 using a 12-sipper LabChip. % conversion values
10    and %  inhibition values were obtained as described and IC50  curves of compounds were

generated using Graphpad Prism Version 4 or 5.01. A nonlinear curve fit using the sigmoidal dose response - variable slope fit was used to graph IC50 curves and determine IC50 values and hillslopes.

It has been shown that the compounds of the Invention have ICSO against Src

15    and Lyn kinases of < 100 nM.

4 -  Cell-based assays of compounds of the Invention.

20

4.1- CeiiTiter-Gio (ATP) Viability /Proliferation Assay


MDA-MB-231  is a human breast cancer cell  line which  is highly dependent on

Src  kinase  pathway  for  viability  and  proliferation.  Thus,  Compounds  of  the  present

25    invention were evaluated for their capacity to reduce viability/proliferation of MDA-MB-231 cells, using two different methods that both address cell metabolic activity. In addition, some Compounds of the present invention were tested for their inhibitory against VEGF-induced proliferation of human vascular endothelial cells (HUVECs).

30    Assay Characteristics:

MDA-MB-231 cells are maintained as adherent cultures of no greater than 80% confluent in 185 cm2 vented culture flask in the medium specified for the cell line supplemented with 10% fetal bovine serum (FBS) at 370 C in 5% C02 • For proliferation assays the adherent cells are collected from culture flask with typsin-EDTA and

35    resuspended in respective medium containing 0.1%- 5% FBS for assay.

The cellular content of ATP (CeiiTiter-Gio reagent from Promega) is measured by luminescent emission based on the following principle:•
 

wo 2010/092041    PCT/EP2010/051556
    33

In the presence of ATP (provided by the cell) luciferin is converted to oxylociferin and light is emitted. The ATP content within the cell is proportional to the amount of oxyluciferin and• luminescence produced.

5    Incubation Conditions:

0.1 ml of cells in suspension at 1,0.00 cells per 0.1 ml is plated on white flat bottom 96 well plates. Cells are allowed to adhere to plates for 2-4 hours before the addition of test compounds.
0.05 ml of test compounds suspended in medium are added to wells to give final

10    volumes of 0.15 mi. Cultures are incubated with the test compounds for 3-4 days before the cultures are assayed for cell viability. If incubation periods are longer than 4 days the .

• final culture volume should. be increased to 0.2 mi.

At the termination of treatments 0.05 ml of the culture medium are removed from each well with a multichannel pipetter, pi petting from the surface of the well.

15 In low light 0.1 ml of the CeiiTiter-Glo reagent is added to each well and the contents of each wen are gently mixed by pipetting up and down (Cover plates with foil until each plate is read on t~e Envision plate reader.)



20    Reading:

The luminescence is read on an Envision 2103 Multi-label Reader (PerkinEimer)


Calculation of Data:

Cell proliferation is expressed as percent of control wells (untreated).


It has been shown that the compounds of the Invention inhibit cell proliferation

with an IC50<500nM.


4.2- WST-1 (mitochondrial metabolism) Viability/Proliferation Assay

30

Assay Characteristics:

The assay measures mitochondrial metabolic activity of cultured cells is based on the rate of conversion of WST-1 substrate to a product with an optical density measured at 440 nm.

35
 

MDA-MB-231 are maintained as adherent cultures of no greater than 80% confluent in 185 cm2 vented culture flask in the medium specified for the. cell line supplemented with 10% fetal bovine serum (FBS) at 370 C in 5% C02 • For proliferation
 

wo 2010/092041    PCT/EP2010/051556
    34

assays the adherent cells are collec.ted from culture flask with typsin-EDTA and resuspended in_respective medium containing 0.1%- 5% FBS for assay.

WST-1  assay  (WST-1  reagent  from  Roche)   is  based  on  the  mitochondrial

5    metabolism of the substrate (4-[3-(4-Iodophenyl)-2-(4-nitrophenyi)-2H-5-tetrazolio]-1,3-benzene disulfonate) to fcirmazan and measurement of its absorbance at 440 nm.

Incubation Conditions:

Aliquots of 0.1 ml of cells are plated into wells. Cells are plated at a density of

1.0    500-1,000 cells per 0.1 ml on clear flat bottom 96-well plates. Cells are allowed to ~dhere to plates for 2-4 hours before the addition of test compounds.

0.5    ml of test compounds suspended in medium are added to wells to give final

.volumes of 0.15 mi. Cultures are incubated with the test compounds for 3-4 days before the ~ultures are assayed for cell viability. If incubation periods are longer than 4 days the
15    final culture volume showed be increased to.0.2 mi.

At the termination of treatments 0.015 ml of the WST-1 solution is added to each well. Plates are returned to the C0 2 incubator and incubated at 37 oc for 1-3 hours. After incubation,the plates are removed from the incubator and placed on a micro-titer plate shaker and gently shaken for 2 minutes.

20

Readings:

The optical density at 440 nm of each well is determined using a Spectra-max plus 384 plate reader.

25    Calculation of Data:

Cell proliferation is expressed as percent of control wells (untreated).


It has been shown that the compounds of the Invention inhibit proliferation with an IC50<500nM.


30    5 . In vivo data

Inhibition of vascular leakage in a rat model of blood-retinal barrier breakdown


We investigated the efficacy of topical administration of compound 5 of the invention in reducing the retinal leakage in a VEGF-induced blood-retinal barrier

35    breakdown in the rat. Rats were treated by a single intravitreal injection of 51-JI (100ng) recombinant rat VEGF164 (RD systems) into each eye.
 
,(


wo 2010/092041    PCT!EP2010/051556
35

During twenty-seven hours following VEGF injection, 0.58% test compound 5 of the invention (5.8mgfml buffer pH 5) and control without compound of the invention were administered six times by topical administration (10!-11) in eyes of sixteen rats.

• Twenty-seven hours after the VEGF challenge, Evans blue dye ( 45mg/kg) was • 5 injected intravenously and the dye w.as allowed tp circulate during two hours.

Theri, each rat was infused with O.OSM citrate buffer pH 3.5 (37 °C) for 2 minutes to allow clearance of the dye. Immediately after said perfusion, both eyes were enucleated and Evans blue dye was extracted by incubating each retina in formamide (Qaum et al Invest. Ophthalmol. Vis. Sci.2001, Vol 42, No 10). Afterward, the
10    absorbance was measured with a spectrophotometer at 620nm.

Breakdown of the blood-retinal ba~rier was proportional to the concentration of Evans blue in the retina normalized by Evans blue concentrations in the plasma.

Results

We  found  that  compound  of  invention  reduced  vascular  leakage  by  71%

15    compared to control providing evidence that the compounds of the invention are useful to reduce vascular permeability and more particularly vascular permeability associated with vitreojretinal diseases such as diabetic retinopathy, retinal vein occlusion and wet age-related macular degeneration.
 
36


Proposed amended claims for the entry of the national phases


CLAIMS



5 1 . Compounds having the structure (I) as well as pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof :

(I)

15

wherein :

A1  and A2 is N or C, with the proviso that one of A1  or A2 is N and one of A1 or A2 is

carbon,

20

Rl and R2 are hydrogen; Cl-C4 alkyl; aryl, which may be optionally substituted with R9 and/or RlO, whereby R9/R10 are independently selected from hydrogen, Cl-C4 alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -CN, halogen, -CF3, =0, -OR4, -NR4RS, - NR4CORS, -NR4COORS, -(Cl-C4 alkyi)OR4, -(Cl-C4 alkyl) NR4RS, -(Cl-C4

25    alkyi)NR4CORS, -(Cl-C4 alkyi)NR4COORS, -COOH, -COOR4; heteroaryl; -CN; -halogen;

-CF3; -OR4;

R3 is hydrogen, Cl-C4 alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -CN, -CF3, - OR4,-0COR4 -COR4, -NR4RS, -NR4CORS, -NR4COORS, -(Cl-C4 alkyi)OR4, -(Cl-C4
alkyi)COR4, -(Cl-C4 alkyl) NR4RS,  -(Cl-C4 alkyi)NR4CORS, -(Cl-C4 alkyi)NR4COORS,
---••----... -    •••--    --•••-    --•-••----•--    •---    --•••-
30    X is a bond, or (CH2)aW(CH2)b, (CH2)aW(CH2)bY(CH2)c or-[(CH2)aW(CH2)b]m-(Z)e-[( CH2)cY(CH 2)d]n wherein :

a, b, c and dare---Independently 0,.-1, 2 or 3, e is 0, 1 or 2, and

n and m are independently 0 or 1, and

w is -co-,-o-,-so2-,-  CH2-,-CHOH-, -NR6-,  NR7CONR8 or NR7S02NR8, ,
 
37


Y is -C0-,-0-,-S02-,-CH2-,-CHOH-or-NR6-, NR7CONR8 or N'R7S0NR8 and 2

Z is selected from the group consisting of cycloalkyl, heterocycloalkyl, aryl and heteroaryl, and when e is 2, then each Z moiety is selected independently from one another

5 R4, R5 and R6 are independently hydrogen, C1-C4 alkyl and where R4 and R5 together can form a 5-7 membered ring ,

R7 and RS are independently hydrogen, C1-C4 alkyr and where R7 and RS together can form a 5-7 membered ring.

2.  Compound  of claim  1,  wherein  R1  is an  aryl,  more  preferably a phenyl or

10    pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.

3.    Compound of claim 1 or 2 wherein R1 is substituted with R9 and R10 and wherein R9/R10 is C1-C4 alkyl (preferably CH3), halogeri (preferably -CI), or-OH or pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.

4.  Compound of claim 3, wherein R1 is a phenyl and is substituted with R9 and

15    R10 in positions 2, 5 or 6 or pharmaceutically acceptable salt, hydrate, solvate

or prodrug thereof.
 

5.    Compound of any of previous claims wherein R2 is•selected in the group of hydrogen and Cl-C4 alkyl or pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.

6.    Compound of any of previous claims wherein X is (CH2)aW(CH 2)b with a is 0, b

is 2, W is -0- or pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.

7. Compound of any of previous claims wherein X is (CH2)aW(CH 2 )bY(CH2)c with a is 0, b is 1 and c is 0, W is -0- and Y is -co- or pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.

8.    Compound of any of--.pr..e.vious claims -wherein X is -[(.. -Crh)aW(CH2)b]m-•Z.~H•­ CH2)cY(CH2)d]n with m is 0, n is 1, c is 0, d is 0 or 2, Y is -co- or is absent and z is imidazoline-2-one or a piperazine or pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.

9.    Compound of any of previous claims wherein R3 is a heterocycloalkyl, preferably a pyrrolidine or pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.
 
38


10.    Compound of any of previous claims wherein R3 is substituted with R9 wherein R9 is preferably -COOH , -N[CH3 h or -COOR4 wherein R4 is preferably C.1-C4 alkyl or pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.

    11.  Compound of any of previous claims wherein R3 is an heteroaryl, preferably a
5    pyridine  or  pharmaceutically  acceptable  salt,  hydrate,  solvate  or  prodrug
    thereof.
    12. Compound of any of claim 1 wherein it is selected in the group consisting of :
    4-Chloro-3-{2- [ 4-(2-pyrrolidin-1-yl-ethoxy)-phenylamino ]-[1,2,4 ]triazolo[ 1,5-
    a]pyridin-6-yl}-phenol, hydrochloride
10    2-{ 4-[ 6-( 2-Chloro-5-hydroxy-phenyl)-[ 1,2,4]triazolo[ 1,5-a] pyridin-2-ylamino ]-
    phenoxy}-1-( (R)-3-d imethylamino-pyrrolidin-1-yl)-ethanone, hydrochloride
    1-{4-[ 6- ( 2 -Chiaro- 5-hyd roxy-phenyl)- [ 1,2,4]triazolo[ 1,5-a] pyridin- 2-yla mi no]-
    \
    phenyl}-3-(2-pyrrolidin-1-yl-ethyl)-imidazolidin-2-one
    (S)-1-(2-{ 4- [ 6-( 2 -Chiaro-5-hydroxy- phenyl)- [ 1,2,4]triazol o[1,5-a] pyridin- 2-
15    ylamino]-phenoxy}-acetyl)-pyrrolidine-2-carboxylic acid
    4-Chloro- 3-{ 8-methyl-2- [ 4- (2-pyrrolidin-1-yl-ethoxy)-phenylami no]-
    [1,2,4]triazolo[1,5-a]pyridin-6-yl}-phenol, hydrochloride
    4-Chloro-3-{ 2- [ 4- ( 2- pyrrolidin-1-yl-ethoxy )- phenyla mine]-[ 1,2,4]triazolo[ 1,5-
    a]pyridin-7-yl}-phenol1 hydrochloride
20    4-Chloro- 3-{2- [ 4- ( 2-morphol in-4-yl-ethoxy)-phenyla m ino]- [ 112,4 ]triazolo[ 1,5-
    a]pyridin-6-yl}-phenol
    4-Ch Ioro-3-{ 2 -[3- ( 2-morpholin-4-yl-ethoxy)- phenylamino] -[ 112,4 ]triazolo [ 1,5-
    a]pyridin-6-yl}-phenol
    4-Chloro-3- [2- ( 3- pyrazol-1-yl-phenyla mi no)- [1 1 2,4 ]triazolo[ 115-a] pyrid i n-6-yl]-
25    phenol
    4-Chloro-3-{2-[4-(2-dimethylamino-ethoxy)-phenylamino]-[112,4]triazolo[1,5-
    a]pyridin-•6•••yl}-phenol
    4-Chloro-3-{2-[3-(2-dimethylamino-ethoxy)-phenylamino]-[1 12,4]triazolo[1 15-
    a]pyridin-6-yl}-phenol
30    4-Chloro-3-{2-[3-(2-pyrrolidin-1 ::Yt-ethoxy)-phe~YJ.?.mino ] -[ 112,4 ]triazolo[1,5-
    a] pyridin-6-yl}-phenol
    4-Chloro-3-[2-( 4-hydroxymethyl-phenylamino )- [1,2,4 ]triazolo[ 1,5-a] pyridin-6-
    yl]-phenol

4-[6-(2-Chloro-5-hydroxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-ylamino]-benzenesulfonamide

4-Chloro- 3-{2- [ 4-( 2 -hyd roxy-ethoxy)- phenyl ami no]- [ 1,2,4 ]triazolo [ 1,5-a] pyridin-6-yl}-phenol

5 1-{4-[6-(2-Chloro-5-hyd roxy-phenyl)-[1,2,4 ]triazolo[1,5-a]pyridin- 2-ylamino]-phenyl}-imidazolidin-2-one

3-[6-(2-Chloro-5-hydroxy-phenyl)-[1,2,4]triazolo[1,5-a]pyridin-2-ylamino]-benzenesulfonamide

2 -{4- [ 6- ( 2-Chloro- 5-hyd roxy- phenyl)- [ 1,2,4]triazolo [ 1, 5-a] pyridin- 2-ylami no]-

10    phenoxy}-acetamide

2-{3- [ 6- (2 -Chloro- 5-hyd roxy-phenyl)- [ 1 ,2,4 ]triazolo[ 1,5•-a]pyrid in-2-ylam ino]-phenoxy}-acetamide

4-Ch Ioro-~- [2-( 4-trifluoromethoxy- phenyl amino)- [1, 2,4 ]triazolo[ 1, 5-a] pyrid in-6-yl]-phenol
15    4-Chloro-3-[2-(4-phenoxy-phenylamino)-[1,2,4 ]triazolo[1,5-a]pyridin-6-yl]-

phenol

4-Chloro-3- [2- ( 4-metha nesu lfonyl-phenylamino )- [ 1,2,4]triazolo[ 1,5-a] pyridin-6-yl]-phenol

3-[2-(4-Benzyloxy-phenylamino)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]-4-chloro-

4-Chloro-3-{2-[4-(2-pi perazin-1-yl-ethoxy)-phenylamino ]-[1,2,4 ]triazolo[ 1,5-a]pyridin-6-yl}-phenol, or pharmaceutically acceptable salt, hydrate, solvate or produrug thereof.



5    13.   Compound of claim 1, selected from

4-Chloro-3-{2-[ 4-(2-pyrrolidin-1-yl-ethoxy)-phenylamino ]-[1,2,4 ]triazolo[ 1,5-a]pyridin-6-yl}-phenol, hydrochloride;

2 -{4- [ 6- ( 2-Chloro- 5-hyd roxy-phenyl)- [ 1,2,4]triazolo[ 1,5-a] pyridi n-2 -ylam i no]-phenoxy}-1-( (R)-3-dimethylamino-pyrrolidin-1-yl)-ethanone, hydrochloride;

10 4-Chloro-3-{2-[4-(2-pyrrolidin-1-yl-ethoxy)-phenylamino ]-[ 1,2,4]triazolo[ 1,5-a]pyridin-7-yl}-phenol, hydrochloride;

4-Chloro- 3-{ 2- [3- (2 -pyrrolidin-1-yl-ethoxy )-phenyla mino]- [ 1,2,4]triazolo[ 1,5-a]pyridin-6-yl}-phenol, and

4-Chloro-3-{2-[ 4-(2-piperazin-1-yl-ethoxy)-phenylamino ]-[-1,2,4 ]triazolo[1,5-15 a]pyridin-6-yl}-phenol, or prodrug thereof.

14.    Compound of claim 1, which is

4-Chloro- 3-{ 2- [ 4-(2-pyrrolidin-1-yl-ethoxy)-phenyla m ino ]- [ 1,2,4]triazolo[ 1,5-a]pyridin-6-yl}-phenol, hydrochloride or prodrug thereof.

20

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