WO2014198844A1

WO2014198844A1 - Protein kinase inhibitors

Info

Publication number: WO2014198844A1
Application number: PCT/EP2014/062273
Authority: WO
Inventors: Wenche Stensen; Florian SCHEVENELS; Istvan E. Marko; John Sigurd Mjøen SVENDSEN
Original assignee: Lytix Biopharma As
Priority date: 2013-06-12
Filing date: 2014-06-12
Publication date: 2014-12-18
Also published as: GB201310464D0

Abstract

The present invention provides a compound incorporating a group of formula (I) wherein: 1 of X, Y and Z is nitrogen and the other 2 are carbon; V is sulpur or carbon; R₃ is oxygen or fluorine; R₄ is an optionally present C_1-3 alkyl group optionally substituted by fluorine; R₅ is an optionally present cyclic group with 5-7 heavy atoms in the ring which may be carbocyclic or heterocyclic and aromatic or aliphatic and is optionally substituted, e.g. by a halogen, C_1-2 alkyl or fluoroalkyl, OH, OR₆, cyano, COOR₆, CONHR₆, sulfonamide or NHR₆, in which R₆ is a C_1-2 alkyl or fluoroalkyl; at least one of R₄ and R₅ is present and R₄ and R₅ may both be present; m and n are each 1 or 2 depending on the identity of V and R₃; when n=2 each R₃ may be the same or different but are preferably the same, when m=2, each R₄ and each R₅ may be the same or different but are preferably the same; and W represents hydrogen, carbon, nitrogen, oxygen or sulphur; or incorporating a salt, hydrate or solvate of a group of formula (I); as well as therapeutic uses of these compounds, in particular as inhibitors of protein kinase activity and in the treatment of inflammation, inflammatory conditions and cancer.

Description

PROTEIN KINASE INHIBITORS

The present invention relates to novel chemical compounds, more particularly to novel pyrrolopyridines with activity as protein kinase inhibitors and thus of therapeutic use.

Protein kinases regulate a variety of cellular processes by adding phosphate groups to substrate proteins. The majority of kinases phosphorylate threonine, serine or tyrosine residues. Kinases play an important role in signal transduction and co-ordination of complex functions such as the cell cycle. There are over 500 human protein kinases, most of which have some sequence similarity. The general structure comprises an N- and C-terminal lobe joined at a hinge region close to an ATP binding site; phosphate groups are transferred from bound ATP to a protein substrate.

Kinases are of great therapeutic interest and many are targets for cancer treatment, treatment of inflammation and other conditions. Indeed, more than 400 human diseases have been connected to protein kinases. Kinase targets in cancer therapy include bcr-Abl, EGFR (epidermal growth factor receptor), Raf, VEGFR (vascular endothelial growth factor receptor), PDGFR (platelet-derived growth factor receptor), KIT and Aurora. It is estimated that the market for kinase inhibitors in cancer treatment will grow from $769 million in 2006 to more than $7.5 billion in 2016. Chemical discovery work on kinases has produced ATP-competitive compounds, allosteric regulators, irreversible binders and highly specific inhibitors. Although an inhibitor with high specificity for a single protein kinase may seem the most desirable, many clinical-stage kinase inhibitors are rather non-selective. In the cancer field, for example, clinicians may prefer multi-targeted drugs in order to maximise the chance for clinical antitumour activity and they will manage attendant toxicities. Kinase inhibition is a growing area and new cancer treatments are still highly sought after, thus a need for new protein kinase inhibitors/(PKIs) of narrow or broad specificity exists.

A range of PKIs are already available or in trial, usually with specific kinase targets, e.g. Imatinib against Bcr-Abl or Gefitinib against EGFR, although Dasatinib has multiple targets. Most existing PKIs are small molecules but monoclonal antibodies such as Beracizumab (against VEGF) and Trastuzumab (against Erb2) also exist in growing numbers.

US 2008/0139606 describes a series of substituted pyrrolopyridines which inhibit kinases, in particular FAK, KDR, Tie2, Aurora A, Aurora B and CDK2. These molecules are described as being of particular utility in the treatment of cancer. These compounds incorporate two cyclic moieties linked to each other, optionally via a molecular linker as a single substituent on the pyrrole group. Independently, the present inventors have surprisingly found that a phenol group as pyrrole substituent results in PKIs of broad specificity. In addition, without wishing to be bound by theory, it is thought that by incorporating a hydrogen bond acceptor (i.e. an atom with an electron (lone pair, such as oxygen or fluorine) on the indole nitrogen, flexibility of substituting groups at this position is possible which allows for further optimisation in terms of efficacy, specificity etc..

Thus, in one aspect, the present invention provides a compound

incorporating a group of formula (I)

(I) wherein:

1 of X, Y and Z is nitrogen and the other 2 are carbon, preferably Y or X is nitrogen, more preferably Y is nitrogen;

the hydroxyl of the phenol moiety can be located in the ortho, meta or para position, preferably in the meta or para position, more preferably in the para position; V is sulpur or carbon;

R₃ is oxygen or fluorine (preferably oxygen);

R₄ is an optionally present linking moiety between V and (where present) R₅, thus R₄ is an optionally present Ci_-3 alkyl group (including cyclopropyl) optionally substituted by fluorine;

R₅ is an optionally present cyclic group with 5-7 heavy atoms in the ring which may be carbocyclic or heterocyclic and aromatic or aliphatic and is optionally substituted, e.g. by a halogen (e.g. F or CI), Ci_-2 alkyl or fluoroalkyl, OH, OR₆, cyano, COOR₆, CONHR₆, sulfonamide or NHR₆, in which R₆ is a Ci_-2 alkyl or fluoroalkyl;

at least one of R₄ and R₅ is present and R₄ and R₅ may both be present; m and n are each 1 or 2 depending on the identity of V and R₃; when n=2 each R₃ may be the same or different but are preferably the same, when m=2, each R₄ and each R₅ may be the same or different but are preferably the same; and

W represents hydrogen, carbon, nitrogen, oxygen or sulphur;

or a salt, hydrate or solvate of a group of formula (I).

Other than when W is hydrogen (forming an aldehyde at this position), the compound of the invention incorporating a group of formula (I) will not consist of a compound of formula (I) as the atom represented by W will be attached to another atom or atoms, thus forming a carboxylic acid derivative, suitable carboxylic acid derivatives are discussed below in the context of the definition of R' in compounds of formula (II).

The present invention thus also provides a compound of formula (II)

(II) wherein:

the hydroxyl of the phenol moiety can be located in the ortho, meta or para position, preferably in the meta or para position, more preferably in the para position;

R , together with the carbonyl group to which it is attached, is a carboxylic acid derivative, preferably a carboxylic acid derivative selected from an aldehyde, a ketone, an amide, an ester, a thioester, an acyl phosphate, and an acyl

hydroxamate, more preferably the carboxylic acid derivative is an amide or ester, with esters being most preferred; and

V is sulpur or carbon;

R₃ is oxygen or fluorine (preferably oxygen);

at least one of R₄ and R₅ is present and R₄ and R₅ may both be present; m and n are each 1 or 2 depending on the identity of X and R₃; when n=2 each R₃ may be the same or different but are preferably the same, when m=2, each R₄ and each R₅ may be the same or different but are preferably the same;

or a salt, hydrate or solvate of a compound of formula (II).

All potential stereoisomers of compounds incorporating a group of formula (I) or of formula (II) are encompassed within the invention and products may be racemic mixtures or enriched partially or essentially completely for one

stereoisomer or enantiomer.

R' (in formula (II)), or W and the atoms attached thereto (in formula (I)), has a molecular weight up to 500 Da, preferably up to 300 Da. This group may comprise 0-4, preferably 0-3 cyclic groups more preferably 1 -3, most preferably 1 or 2 cyclic groups (typically of 5 to 6 but possibly with 7 atoms in the ring). Where two or more cyclic groups are present two or more of the cyclic groups may be fused but they may conveniently be linked rather than fused, the cyclic groups may be linked directly or there may be a linking group. Suitable linking groups typically comprise 1 -10 non-hydrogen atoms, preferably up to 7 non-hydrogen atoms, suitable linking groups include -CH₂-, -NR-, -0-, -S-,

-CHRCHR-, -CH=CH-, -C=0-0-, -C=0-NR-, -0-CHR-, -NR-CHR-, -NR-C=0-NR-, -NR-C=0-0- and -CH₂CH₂CH₂-; wherein R is H or Ci_-2 alkyl. Linking groups are typically 1 to 4 atoms in length.

The cyclic groups are preferably, but may not be, aromatic, including heteroaromatic; if the group comprises two or more aromatic groups, then preferably no more than one of these groups is carbocyclic. Suitable cyclic groups include cyclohexyl, phenyl, pyridine, pyrazole, imidazole, thiazole, oxazole, furan, piperazine, pyrroline, pyrrolidine, diazolone, morpholine and piperidine. Two of the rings may be fused (e.g. phenyl and pyridine to form quinoline).

The cyclic groups may be unsubstituted or substituted, substituents typically comprise no more than 8, preferably no more than 6, more preferably no more than 4 non-hydrogen atoms and include: -OH, -OR, -NRR, -C=0-OR, -C=0-NRR, -S0₂- NRR, -NR-SO2R, -SO2R, -NR-C=OR, a halogen (e.g. F or CI), d_-3 alkyl or fluoroalkyl; wherein each R (which may be the same or different) is H or Ci_-3 alkyl. Typically there will be up to 3, more usually 1 or 2 substituting groups.

In certain preferred embodiments R' (in formula (II)), or W and the atoms attached thereto (in formula (I)), comprises no more than 18 non-hydrogen (heavy) atoms, preferably no more than 16 non-hydrogen atoms, more preferably no more than 12 non-hydrogen atoms, e.g. no more than 10 non-hydrogen atoms.

R' (in formula (II)), or W and the atoms attached thereto (in formula (I)), is preferably selected from hydrogen, -NH₂ ,- OR-i, -Ri or -NR₁R₂ in which R₂ is preferably hydrogen but may be a further R-_\ group and Ri may be a C-_\.-_\-_\

(preferably Ci_-6) alkyl, alkenyl, alkynyl or alkylamine, which may be linear or branched; Ri may comprise or consist of cyclic groups as described above which may be fused or linked, again as described above.

By way of example R' (in formula (II)), or W and the atoms attached thereto (in formula (I)), may be hydrogen, -OCH₃, -OCH₂CH₃, -OCH₂CH₂CH₃ , - OCH₂CH₂CH₂CH₃ , -NHCH₃, -NHCH₂CH₃, -NHCH₂CH₂CH₃, -NHCH₂CH₂OH or -HCH₂CH₂N(CH₂CH₃)₂; hydrogen, -OCH₃, -OCH₂CH₃, or -NHCH₂CH₂OH are particularly preferred. ln other embodiments, Ri moieties comprising or consisting of cyclic groups are especially preferred and suitable groups are discussed above. A preferred set of substituents are represented by the formula R' = -NHR-i and some representative examples are provided below:

Preferred R₅ groups are phenyl, pyrrole, pyridine, pyrimidine, thiazole, oxazole, piperidine and morpholine; preferred -V(R₃)_n (R₄R5)_m moieties are phenylsulfonyl, cyclohexylsulfonyl, pyrrolylsulfonyl, pyridylsulfonyl,

pyrimidinesulfonyl, thiazolylsulfonyl and oxazolylsulfonyl.

The meta or para position of the hydroxyl group is preferred in combination with all options at other positions allowing variation in substituents.

Likewise, Y being nitrogen and X and Z being carbon is a preferred configuration in combination with all options at other positions allowing variation in substituents.

Likewise, R' (in formula (II)), or W and the atoms attached thereto (in formula (I)), being selected from hydrogen, -OCH₃, -OCH₂CH₃ or

-NHCH2CH2OH is preferred in combination with all options at other positions allowing variation in substituents.

Compound 1227, described in the Examples, is particularly preferred. As shown in the Examples, kinase inhibitory activity is very significantly influenced by the presence of hydroxyl in the phenyl moiety, as compared, for example, to an unsubstituted benzene ring or methoxy or amino substituent. The Examples also demonstrate the surprisingly broad specificity of the compounds of the present invention.

As mentioned above, it seems that the presence of a hydrogen bond acceptor at the indole nitrogen permits binding with the target protein kinase in a way which allow for a large substituent at this position. -V(R₃)_n(R₄R5)_m may therefore incorporate up to 25 or more non-hydrogen atoms, typically up to 18 non-hydrogen atoms, preferably up to 12 non-hydrogen atoms. This specific interaction with, and orientation within, the protein kinase appears to explain why a benzyl substituent at the indole nitrogen (compounds 1286 and 1395) is so much less effective than a phenylsulfonyl group (compound 1227).

In a further aspect is provided the compounds of the present invention for use in therapy, particularly for use as a protein kinase inhibitor and thus for the treatment of diseases or other pathological conditions associated with aberrant protein kinase activity or which would benefit from a reduction in protein kinase activity. More specifically, the compounds of the invention may be of use in the treatment of inflammation and inflammatory conditions and most especially in the treatment of cancer.

Methods of treating diseases or other pathological conditions associated with aberrant protein kinase activity or which would benefit from a reduction in protein kinase activity, which methods comprise administration to a human or animal patient of an effective amount of a compound of the invention, constitute further aspects of the present invention.

Methods of treating cancer, inflammatory conditions or inflammation, which methods comprise administration to a human or animal patient of an effective amount of a compound of the invention, constitute further aspects of the present invention.

The patient will typically have been identified as in need of treatment.

Treatments may be prophylactic but generally will not be.

In a further aspect, the present invention provides a method of inhibiting a protein kinase and/or a reaction catalysed by a protein kinase, the method comprising contacting said kinase with a compound of the present invention. Such methods may be in vivo or ex vivo, e.g. in vitro.

In a further aspect, the present invention provides the use of a compound of the invention in the manufacture of a medicament for the treatment of cancer, inflammatory conditions or inflammation.

In a further aspect, the present invention provides the use of a compound of the invention in the manufacture of a medicament for the treatment of a disease or other pathological condition associated with aberrant protein kinase activity or which would benefit from a reduction in protein kinase activity.

Animals which may be treated include domestic animals, in particular cats and dogs and livestock animals such as pigs, cows, sheep or goats as well as horses, also elephants. Treatment of humans is nevertheless preferred. Methods for the synthesis of compounds of the invention are described in the Examples hereto, non-exemplified compounds can be prepared by methods which are analogous to the schemes and protocols described herein.

Methods of synthesising compounds of the invention, in particular methods described in the Examples, constitute a further aspect of the present invention. More particularly, in a further aspect, the present invention provides a method of preparing a compound of formula (II) comprising reaction step II below, optionally and where appropriate, preceded by reaction step I.

Intermediate compounds of the invention as represented by formulae (ΙΙΓ), (III) and (IV) represent further aspects of the present invention. X, Y, Z, R', V, R₃, R₄ , R_5, n and m in compounds of formulae (ΙΙΓ), (III) and (IV) are as defined herein for compounds of formula (II).

(Ill) (ii) Formulations comprising one or more compounds of the invention in admixture with a suitable diluent, carrier or excipient constitute a further aspect of the present invention. Such formulations may be for pharmaceutical use. Suitable diluents, excipients and carriers are known to the skilled man.

The compositions according to the invention may be presented, for example, in a form suitable for oral, nasal, parenteral, intravenal, topical or rectal administration.

As used herein, the term "pharmaceutical" includes veterinary applications of the products of the invention.

The active compounds defined herein may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, inhalers, solutions, emulsions, liposomes, powders, capsules or sustained release forms.

Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms. Tablets may be produced, for example, by mixing the active ingredient or ingredients with known excipients, such as for example with diluents, such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as magnesium stearate or talcum, and/or agents for obtaining sustained release, such as carboxypolymethylene,

carboxymethyl cellulose, cellulose acetate phthalate, or polyvinylacetate.

The tablets may if desired consist of several layers. Coated tablets may be produced by coating cores, obtained in a similar manner to the tablets, with agents commonly used for tablet coatings, for example, polyvinyl pyrrolidone or shellac, gum arabic, talcum, titanium dioxide or sugar. In order to obtain sustained release or to avoid incompatibilities, the core may consist of several layers too. The tablet coat may also consist of several layers in order to obtain sustained release, in which case the excipients mentioned above for tablets may be used.

Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as

p-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are then filled into injection vials or ampoules. Nasal sprays administration may be formulated similarly in aqueous solution and packed into spray containers either with an aerosol propellant or provided with means for manual compression.

Capsules containing one or several active ingredients may be produced, for example, by mixing the active ingredients with inert carriers, such as lactose or sorbitol, and filling the mixture into gelatin capsules.

Suitable suppositories may, for example, be produced by mixing the active ingredient or active ingredient combinations with the conventional carriers envisaged for this purpose, such as natural fats or polyethyleneglycol or derivatives thereof.

The pharmaceutical compositions may additionally comprise further active ingredients, including, for example, other anti-inflammatory or chemotherapeutic agents. Likewise the medical uses and methods of treatment may additionally comprise further active ingredients, including, for example, other anti-inflammatory or chemotherapeutic agents. When the disease being treated is cancer, the therapeutic regimen may also include radiation therapy.

In employing such compositions systemically (intra-muscular, intravenous, intraperitoneal), the active molecule is generally present in an amount to achieve a serum level of the active molecule of at least about . A preferred serum level is about 1 - 10 micromolar Such serum levels may be achieved by incorporating the bioactive molecule in a composition to be administered systemically at a dose of from 50 mg - 250 mg.

It is appreciated that appropriate dosages will vary from patient to patient dependent on age, sex, previous treatments, severity of symptoms presented etc.

The above description describes numerous features of the present invention and in most cases preferred embodiments of each feature are described. It will be appreciated that each preferred embodiment of a given feature may provide a molecule, use, method etc. of the invention which is preferred, both when combined with the other features of the invention in their most general form and when combined with preferred embodiments of other features. The effect of selecting multiple preferred embodiments may be additive or synergistic. Thus all such combinations are contemplated unless the technical context obviously makes them mutually exclusive or contradictory. In general each feature and preferred embodiments of it are independent of the other features and hence combinations of preferred embodiments may be presented to describe sub-sets of the most general definitions without providing the skilled reader with any new concepts or information as such.

The invention will now be further described with reference to the following non-limiting Examples. Not all of the compounds synthesised and tested are within the scope of the present invention, some are provided for comparative purposes and highlight the efficacy of the molecules of the invention.

Examples

A. Synthesis Examples Reaction schemes are given throughout the Examples and three or four digit arabic numbers are given for each end product which is then tested. Roman numerals are used in the schemes and Examples for the intermediate compounds.

Example 1 . Preparation of 3-phenyl-1 H-pyrrolo[2,3-fe]pyridine-2-carbaldehyde (801 )

Step 1 . Preparation of /V-(pyridin-2-yl)pivalamide (I)

To a solution of 1.88 g (20.0 mmol, 1 eq.) of 2-aminopyridine and 3.6 mL (26.0 mmol, 1.3 eq.) of triethylamine in 30 mL of DCM, cooled to 0°C, were added 2.7 mL (22.0 mmol, 1.1 eq.) of trimethylacetyl chloride in 4 mL of DCM. After 2 hours, the mixture was stirred one hour at room temperature. Then, 40 mL of water were added. The aqueous layer was extracted with 20 mL of dichloromethane. The organic phase was washed with 20 mL of aqueous NaHC0₃, dried, filtered and concentrated. Purification over silica gel provided 2.40 g (13.5 mmol, 67%) of the title compound as a white solid. R_f=0.49 (PE/EtOAc 2:1 ). ¹H NMR (300 MHz): δ = 8.24 (m, 2 H), 8.00 (broad, 1 H), 7.67 (t, J = 7.9 Hz, 1 H), 7.01 (m, 1 H), 1 .31 (s, 9 H) ppm. ¹³C NMR (75 MHz): δ = 177.2, 151.7, 147.8, 138.4, 1 19.8, 1 14.0, 39.9, 27.6 ppm.

Step 2. yV-(3-(hydroxy(phenyl)methyl)pyridin-2-yl)pivalamide (II)

To a solution of 2.67 g (15.0 mmol, 1 eq.) of pivalamide I and 6.5 mL (43.1 mmol, 3 eq.) of TMEDA in 1 15 mL of diethylether, cooled to -78°C, were added 17.3 mL (43.1 mmol, 3 eq., 2.5 M in hexane) of BuLi. After 15 minutes, the solution was brought back to -24°C and stirred 2 hours. Then, the mixture was cooled down to - 78°C before addition of 3.2 mL (31 .6 mmol, 2.2 eq) of benzaldehyde in 50 mL of THF. The reaction was brought back to 0°C and stirred 2 hours before being stirred at room temperature over night. Then, 100 mL of water were added. The aqueous layer was extracted with 3 x 60 mL of dichloromethane. The organic phase was dried, filtered and concentrated. Purification over silica gel provided 1.92 g (6.8 mmol, 47%) of II as a white solid. R_f=0.20 (PE/EtOAc 1 :2). ¹H NMR (300 MHz): δ = 8.62 (s, 1 H), 8.30 (dd, J = 4.7 Hz, J = 1 .6 Hz, 1 H), 7.54 (dd, J = 7.7 Hz, J = 1 .5 Hz, 1 H), 7.29 (m, 5 H), 7.08 (dd, J = 7.6 Hz, J = 4.9 Hz, 1 H), 5.85 (s, 1 H), 4.93 (s, 1 H), 1.21 (s, 9 H) ppm. ¹³C NMR (75 MHz): δ = 178.6, 149.0, 147.7, 141.8, 138.9, 132.6, 128.4, 127.6, 126.4, 121 .5, 72.2, 39.7, 27.5 ppm.

Step 3. (2-aminopyridin-3-yl)(phenyl)methanone (III)

A solution of 1 .90 g (6.7 mmol, 1 eq.) of alcohol II and 2.90 g (33.4 mmol, 5 eq.) of activated Mn0₂ in 67 mL of DCM was refluxed 40 hours. The mixture was filtered on celite and the solvent was evaporated. Then, 130 mL of 3 M aqueous HCI were added and refluxed 24 hours. The reaction was cooled down to 0°C and 60 mL of aqueous ammonia were added. The aqueous layer was extracted with 3 x 50 mL of DCM. The organic phase was dried, filtered and concentrated. Purification over silica gel provided 1 .06 g (5.4 mmol, 80%) of III as a white solid. R_f=0.42 (PE/EtOAc 1 :2). ¹H NMR (300 MHz): δ = 8.24 (dd, J = 4.7 Hz, J = 1 .7 Hz, 1 H), 7.76 (dd, J = 7.8 Hz, J = 1.6 Hz, 1 H), 7.60 (m, 2 H), 7.55 (m, 1 H), 7.46 (t, J = 7.2 Hz, 2 H), 6.91 (broad, 2 H), 6.59 (dd, J = 7.8 Hz, J = 4.8 Hz, 1 H) ppm. ¹³C NMR (75 MHz): δ = 197.8, 159.9, 154.0, 143.0, 139.2, 131.6, 129.1 , 128.4, 1 12.9, 1 12.1 ppm.

Step 4. 3-phenyl-1 H-pyrrolo[2,3-fe]pyridine-2-carbaldehyde (801 )

To a solution of 2.0 mL (14.1 mmol, 4 eq.) of diisopropylamine in 12 mL of THF, cooled to 0°C, were added 5.7 mL (14.1 mmol, 4 eq., 2.5 M in hexane) of BuLi. The mixture was cooled to -78°C and 0.44 mL (5.3 mmol, 1 .5 eq.) of dichloroethylene were added. The system was brought back to 0°C and 700 mg (3.5 mmol, 1 eq.) of aminophenone III in 10 mL of THF were added. After one hour, 25 mL of aqueous NH₄CI were added. The aqueous layer was extracted with 3 x 25 mL of

dichloromethane. The organic phase was dried, filtered and concentrated. The crude mixture was dissolved in 70 mL of dichloroethane and 70 mL of 0.1 M aqueous sulphuric acid were added. After 16 hours under reflux, the mixture was cooled down to room temperature and the pH was brought to 1 1 with Na₂C0₃. The aqueous layer was extracted with 2 x 70 mL of dichloromethane. The organic layer was dried, filtered and concentrated. Purification over silica gel provided 120 mg (0.5 mmol, 15%) of compound 801 as a brown solid. R_f=0.53 (PE/EtOAc 2:1 ). ¹H NMR (300 MHz): 5 = 13.06 (broad, 1 H), 10.04 (s, 1 H), 8.92 (dd, J = 4.7 Hz, J = 1.5 Hz, 1 H), 8.22 (dd, J = 8.1 Hz, J = 1 .1 Hz, 1 H), 7.62 (m, 2 H), 7.56 (m, 2 H), 7.48 (m, 1 H), 7.26 (dd, J = 8.1 Hz, J = 4.8 Hz, 1 H) ppm. ¹³C NMR (75 MHz): δ = 183.2, 149.3, 149.0, 132.0, 131 .7, 130.6, 129.2, 128.4, 127.3, 1 19.8, 1 17.6 ppm. MS (CI): m/z = 222.1 . I.R. (neat): v 2800, 1661 . H.R.M.S. (CI): Calcd for Ci₄HnN₂0;

223.0871. Found: 223.0865. mp 197 °C.

Example 2. Preparation of 3-phenyl-1 H-pyrrolo[2,3-c]pyridine-2-carbaldehyde (799) Step 1. W-(pyridin-3-yl)pivalamide (IV)

To a solution of 1 .88 g (20.0 mmol, 1 eq.) of 3-aminopyridine and 3.6 mL (26.0 mmol, 1.3 eq.) of triethylamine in 30 mL of DCM, cooled to 0°C, were added 2.7 mL (22.0 mmol, 1.1 eq.) of trimethylacetyl chloride in 4 mL of DCM. After 2 hours, the mixture was stirred one hour at room temperature. Then, 40 mL of water were added. The aqueous layer was extracted with 20 mL of dichloromethane. The organic phase was washed with 20 mL of aqueous NaHC0₃, dried, filtered and concentrated. Recrystallisation in PE/EtOAc 10:1 provided 3.14 g (17.6 mmol, 88%) of IV as a white solid. R_f=0.13 (PE/EtOAc 2:1 ). ¹H NMR (300 MHz): δ = 8.55 (d, J = 2.4 Hz, 1 H), 8.29 (dd, J = 4.8 Hz, J = 0.9 Hz, 1 H), 8.1 1 (d, J = 8.1 Hz, 1 H), 7.76 (broad, 1 H), 7.21 (dd, J = 8.3 Hz, J = 4.7 Hz, 1 H), 1 .29 (s, 9 H) ppm. ¹³C NMR (75 MHz): 5 = 177.5, 145.2, 141.7, 135.1 , 127.7, 123.7, 39.8, 27.6 ppm.

Step 2. yV-(4-(hydroxy(phenyl)methyl)pyridin-3-yl)pivalamide (V)

To a solution of 2.67 g (15.0 mmol, 1 eq.) of pivalamide IV and 6.75 mL (45.0 mmol, 3 eq.) of TMEDA in 120 mL of diethylether, cooled to -78°C, were added 18.0 mL (45.0 mmol, 3 eq., 2.5 M in hexane) of BuLi. After 15 minutes, the solution was brought back to -24°C and stirred 2 hours. Then, the mixture was cooled down to - 78°C before addition of 3.35 mL (33.0 mmol, 2.2 eq) of benzaldehyde in 60 mL of THF. The reaction was brought back to 0°C and stirred 2 hours before being stirred at room temperature over night. Then, 120 mL of water were added. The aqueous layer was extracted with 3 x 80 mL of dichloromethane. The organic phase was dried, filtered and concentrated. Recrystallisation in DCM provided 1.60 g (5.6 mmol, 37%) of V as a white solid. R_f=0.16 (PE/EtOAc 1 :2). ¹H NMR (300 MHz): δ = 9.33 (s, 1 H), 8.80 (s, 1 H), 8.20 (d, J = 4.9 Hz, 1 H), 7.34 (m, 5 H), 7.04 (d, J = 4.9 Hz, 1 H), 5.89 (s, 1 H), 4.54 (s, 1 H), 1.13 (s, 9 H) ppm. ¹³C NMR (75 MHz): δ = 177.1 , 144.9, 144.7, 140.0, 139.9, 133.9, 128.9, 128.5, 126.5, 123.1 , 74.9, 39.7, 27.5 ppm.

Step 3. (3-aminopyridin-4-yl)(phenyl)methanone (VI)

A solution of 1 .50 g (5.3 mmol, 1 eq.) of alcohol V and 2.30 g (26.4 mmol, 5 eq.) of activated Mn0₂ in 50 mL of DCM was refluxed 40 hours. The mixture was filtered on celite and the solvent was evaporated. Then, 100 mL of 3 M aqueous HCI were added and refluxed 16 hours. The reaction was cooled down to 0°C and 50 mL of aqueous ammonia were added. The aqueous layer was extracted with 3 x 50 mL of DCM. The organic phase was dried, filtered and concentrated. Purification over silica gel provided 1 .00 g (5.0 mmol, 96%) of VI as a yellow solid. R_f=0.28

(PE/EtOAc 1 :2). ¹H NMR (300 MHz): δ = 8.28 (s, 1 H), 7.93 (d, J = 5.2 Hz, 1 H), 7.68 (d, J = 7.2 Hz, 2 H), 7.58 (t, J = 7.4 Hz, 1 H), 7.48 (t, J = 7.4 Hz, 2 H), 7.22 (d, J = 5.2 Hz, 1 H), 5.87 (broad, 2 H) ppm. ¹³C NMR (75 MHz): δ = 198.3, 144.5, 141 .4, 138.3, 136.8, 132.3, 129.5, 128.5, 125.1 , 122.0 ppm. Step 4. 3-phenyl-1 H-pyrrolo[2,3-c]pyridine-2-carbaldehyde (799)

To a solution of 2.55 mL (18.2 mmol, 4 eq.) of diisopropylamine in 15 mL of THF, cooled to 0°C, were added 7.3 mL (18.2 mmol, 4 eq., 2.5 M in hexane) of BuLi. The mixture was cooled to -78°C and 0.55 mL (6.8 mmol, 1 .5 eq.) of dichloroethylene were added. The system was brought back to 0°C and 900 mg (4.5 mmol, 1 eq.) of aminophenone VI in 7 mL of THF were added. After one hour, 30 mL of aqueous NH₄CI were added. The aqueous layer was extracted with 3 x 30 mL of

dichloromethane. The organic phase was dried, filtered and concentrated. The crude mixture was dissolved in 80 mL of dichloroethane and 80 mL of 0.1 M aqueous sulphuric acid were added. After 16 hours under reflux, the mixture was cooled down to room temperature and the pH was brought to 1 1 with Na₂C0₃. The aqueous layer was extracted with 2 x 120 mL of dichloromethane. The organic layer was dried, filtered and concentrated. The crude mixture was recrystallised in DCM providing 495 mg (2.2 mmol, 49%) of 799 as a brown solid. R_f=0.19 (PE/EtOAc 1 :2). ¹ H NMR (300 MHz): δ = 10.03 (s, 1 H), 9.57 (broad, 1 H), 9.04 (s, 1 H), 8.41 (d, J = 5.6 Hz, 1 H), 7.73 (d, J = 5.5 Hz, 1 H), 7.55 (m, 5 H) ppm. ¹³C NMR (75 MHz): 5 = 183.6, 140.2, 137.0, 133.6, 133.3, 131 .4, 131 .0, 130.4, 129.3, 128.7, 127.7, 1 16.0 ppm. MS (ESI): m/z = 222.0. I.R. (neat): v 2718, 1672. H.R.M.S. (El): Calcd for Ci₄H₁₀N₂O; 222.0788. Found: 222.0793. mp >220 °C.

Example 3. Preparation of 3-phenyl-1 H-pyrrolo[3,2-c]pyridine-2-carbaldehyde (800) Step 1 . W-(pyridin-4-yl)pivalamide (VII)

To a solution of 1 .88 g (20.0 mmol, 1 eq.) of 4-aminopyridine and 3.6 mL (26.0 mmol, 1 .3 eq.) of triethylamine in 30 mL of DCM, cooled to 0°C, were added 2.7 mL (22.0 mmol, 1 .1 eq.) of trimethylacetyl chloride in 4 mL of DCM. After 2 hours, the mixture was stirred one hour at room temperature. Then, 40 mL of water were added. The aqueous layer was extracted with 20 mL of dichloromethane. The organic phase was washed with 20 mL of aqueous NaHC0₃, dried, filtered and concentrated. Recrystallisation in PE/EtOAc 8:1 provided 2.50 g (14.0 mmol, 70%) of VII as a white solid. R_f=0.07 (PE/EtOAc 2:1 ). ¹H NMR (300 MHz): δ = 8.46 (d, J = 5.5 Hz, 2 H), 7.73 (broad, 1 H), 7.49 (dd, J = 4.9 Hz, J = 1 .4 Hz, 2 H), 1 .30 (s, 9 H) ppm. ¹³C NMR (75 MHz): δ = 177.5, 150.7, 145.3, 1 13.9, 40.1 , 27.5 ppm.

Step 2 and step 3. (4-aminopyridin-3-yl)(phenyl)methanone (IX) To a solution of 2.33 g (13.1 mmol, 1 eq.) of pivalamide VII and 5.9 mL (39.2 mmol, 3 eq.) of TMEDA in 105 mL of diethylether, cooled to -78°C, were added 15.7 mL (39.2 mmol, 3 eq., 2.5 M in hexane) of BuLi. After 15 minutes, the solution was brought back to -24°C and stirred 2 hours. Then, the mixture was cooled down to - 78°C before addition of 2.9 mL (28.8 mmol, 2.2 eq) of benzaldehyde in 50 mL of THF. The reaction was brought back to 0°C and stirred 2 hours before being stirred at room temperature over night. Then, 100 mL of water were added. The aqueous layer was extracted with 3 x 60 mL of dichloromethane. The organic phase was dried, filtered and concentrated. The crude mixture was purified over silica gel (PE/EtOAc 2:1 ) and the mixture (2 products, including VIII) was dissolved in 50 mL of DCM. Then, 2.30 g (26.4 mmol) of activated Mn0₂ were added and the reaction was refluxed 40 hours. The mixture was filtered on celite and the solvent was evaporated. The crude mixture was purified over silica gel (PE/EtOAc 1 :1 ). Then, 100 mL of 3 M aqueous HCI were added and refluxed 16 hours. The reaction was cooled down to 0°C and 50 mL of aqueous ammonia were added. The aqueous layer was extracted with 3 x 50 mL of DCM. The organic phase was dried, filtered and concentrated to yield 730 mg (3.7 mmol, 28%) of IX as a white solid. R_f=0.12 (PE/EtOAc 1 :2). ¹H NMR (300 MHz): δ = 8.58 (s, 1 H), 8.22 (d, J = 5.9 Hz, 1 H), 7.67 (m, 2 H), 7.57 (m, 1 H), 7.49 (m, 2 H), 6.60 (d, J = 5.9 Hz, 1 H), 6.56 (broad, 2 H) ppm. ¹³C NMR (75 MHz): δ = 198.5, 156.0, 155.6, 152.0, 139.0, 131.9, 129.3, 128.5, 1 15.0, 1 1 1 .3 ppm.

Step 4. 3-phenyl-1 H-pyrrolo[3,2-c]pyridine-2-carbaldehyde (800)

To a solution of 1 .8 mL (12.5 mmol, 4 eq.) of diisopropylamine in 10 mL of THF, cooled to 0°C, were added 5.0 mL (12.5 mmol, 4 eq., 2.5 M in hexane) of BuLi. The mixture was cooled to -78°C and 0.38 mL (4.7 mmol, 1 .5 eq.) of dichloroethylene were added. The system was brought back to 0°C and 620 mg (3.1 mmol, 1 eq.) of aminophenone IX in 10 mL of THF were added. After one hour, 20 mL of aqueous NH₄CI were added. The aqueous layer was extracted with 3 x 20 mL of

dichloromethane. The organic phase was dried, filtered and concentrated. The crude mixture was dissolved in 60 mL of dichloroethane and 60 mL of 0.1 M aqueous sulphuric acid were added. After 16 hours under reflux, the mixture was cooled down to room temperature and the pH was brought to 1 1 with Na₂C0₃. The aqueous layer was extracted with 2 x 80 mL of dichloromethane. The organic layer was dried, filtered and concentrated. The crude mixture was recrystallised in

DCM/PE 10: 1 providing 370 mg (1 .7 mmol, 53%) of 800 as a brown solid. R_f=0.15 (PE/EtOAc 1 :2). ¹ H N MR (300 MHz): δ = 10.49 (broad, 1 H), 9.94 (s, 1 H), 9.18 (s, 1 H), 8.52 (d, J = 5.8 Hz, 1 H), 7.65 (d, J = 6.9 Hz, 2 H), 7.53 (m, 3 H), 7.43 (d, J = 5.6 Hz, 1 H) ppm. ¹³C NMR (75 MHz): δ = 182.9, 146.5, 145.1 , 140.6, 132.2, 130.7, 130.5, 129.4, 129.2, 128.7, 123.9, 107.4 ppm. MS (ESI): m/z = 222.0. I .R. (neat): v 2824, 2685, 1651 . H.R.M.S. (El): Calcd for Ci₄H₁₀N₂O; 222.0788. Found: 222.0789. mp 199 °C.

Example 4. Preparation of 3-phenyl-1 H-pyrrolo[3,2-fe]pyridine-2-carbaldehyde (805)

Step 1 . yV-(2-bromopyridin-3-yl)pivalamide (X) To a solution of 1 .13 g (6.5 mmol, 1 eq.) of 3-amino-2-bromopyridine and 1 .2 mL (8.5 mmol, 1 .3 eq.) of triethylamine in 13 mL of DCM, cooled to 0°C, were added 0.9 mL (7.2 mmol, 1 .1 eq.) of trimethylacetyl chloride in 2 mL of DCM. After 3 hours at room temperature, 15 mL of water were added. The aqueous layer was extracted with 2 x 10 mL of dichloromethane. The organic phase was dried, filtered and concentrated. Purification over silica gel provided 1 .59 g (6.1 mmol, 93%) of X as a colourless oil. R_f=0.68 (PE/EtOAc 2: 1 ). ¹ H NMR (300 MHz): δ = 8.69 (dd, J = 8.1 Hz, J = 1 .6 Hz, 1 H), 8.07 (dd, J = 4.6 Hz, J = 1 .6 Hz, 1 H), 8.02 (broad, 1 H), 7.26 (dd, J = 8.1 Hz, J = 4.7 Hz, 1 H), 1 .35 (s, 9 H) ppm. ¹³C NMR (75 MHz): δ = Ml A, 144.4, 133.8, 133.5, 128.6, 123.7, 40.3, 27.6 ppm. Step 2 (and step 3 on the scheme). (3-aminopyridin-2-yl)(phenyl)methanone (XI)

To a solution of 1 .55 g (6.0 mmol, 1 eq.) of pivalamide X in 24 mL of THF, cooled to -78°C, were added 6.0 mL (15.1 mmol, 2,5 eq., 2.5 M in hexane) of BuLi. After one hour, 1 .50 g (9.0 mmol, 1 .5 eq.) of /V-methoxy-/V-methylbenzamide in 6 mL of THF were added dropwise. The reaction was stirred 18 hours at room temperature and 30 mL of water were added. The aqueous layer was extracted with 3 x 15 mL of dichloromethane. The organic phase was dried, filtered and concentrated. Then, 60 mL of 3 M aqueous HCI and 30 mL of methanol were added and refluxed 20 hours. The reaction was cooled down to 0°C and 30 mL of aqueous ammonia were added. The aqueous layer was extracted with 3 x 100 mL of DCM. The organic phase was dried, filtered and concentrated to yield 330 mg (1.6 mmol, 27%) of XI as a yellow solid. R_f=0.39 (PE/EtOAc 2:1 ). ¹H NMR (300 MHz): δ = 8.06 (dd, J = 4.1 Hz, J = 1 .4 Hz, 1 H), 7.93 (m, 2 H), 7.52 (m, 1 H), 7.43 (m, 2 H), 7.21 (dd, J = 8.4 Hz, J = 4.1 Hz, 1 H), 7.09 (dd, J = 8.4 Hz, J = 1 .3 Hz, 1 H), 6.1 1 (broad, 2 H) ppm. ¹³C NMR (75 MHz): 5 = 197.2, 147.5, 139.1 , 137.9, 134.8, 131 .7, 130.7, 127.9, 127.8, 125.0 ppm.

Step 4. 3-phenyl-1 H-pyrrolo[3,2-fe]pyridine-2-carbaldehyde (805)

To a solution of 0.77 mL (5.5 mmol, 4 eq.) of diisopropylamine in 5 mL of THF, cooled to 0°C, were added 2.2 mL (5.5 mmol, 4 eq., 2.5 M in hexane) of BuLi. The mixture was cooled to -78°C and 0.17 mL (2.0 mmol, 1 .5 eq.) of dichloroethylene were added. The system was brought back to 0°C and 270 mg (1.4 mmol, 1 eq.) of aminophenone XI in 3 mL of THF were added. After one hour, 10 mL of aqueous NH₄CI were added. The aqueous layer was extracted with 3 x 10 mL of

dichloromethane. The organic phase was dried, filtered and concentrated. The crude mixture was dissolved in 30 mL of dichloroethane and 30 mL of 0.1 M aqueous sulphuric acid were added. After 16 hours under reflux, the mixture was cooled down to room temperature and the pH was brought to 1 1 with Na₂C0₃. The aqueous layer was extracted with 2 x 30 mL of dichloromethane. The organic layer was dried, filtered and concentrated. Purification over silica gel provided 30 mg (0.14 mmol, 10%) of 805 as an orange oil. R_f=0.32 (PE/EtOAc 2:1 ). ¹H NMR (300 MHz): 5 = 10.05 (s, 1 H), 9.92 (broad, 1 H), 8.66 (dd, J = 7.4 Hz, J = 1.4 Hz, 1 H), 7.80 (m, 3 H), 7.52 (m, 2 H), 7.43 (m, 1 H), 7.33 (dd, J = 8.5 Hz, J = 4.4 Hz, 1 H) ppm. ^1JC NMR (75 MHz): δ = 183.7, 146.2, 143.4, 133.2, 131 .2, 131 .0, 130.6, 128.9, 128.6, 128.4, 121.9, 120.4 ppm. MS (CI): m/z = 222.1 . I.R. (neat): v 3285, 1643. H.R.M.S. (CI): Calcd for Ci₄HnN₂0; 223.0871 . Found: 223.0866.

Example 5. Preparation of 3-(4-hydroxyphenyl)-1 H-pyrrolo[2,3-c]pyridine-2- carbaldehyde (942)

Step 1 and 2. (3-aminopyridin-4-yl)(4-methoxyphenyl)methanone (XIII)

To a solution of 2.67 g (15.0 mmol, 1 eq.) of pivalamide IV and 6.8 mL (45.0 mmol, 3 eq.) of TMEDA in 120 mL of dry diethylether, cooled to -78°C, were added 18.0 mL (45.0 mmol, 3 eq., 2.5 M in hexane) of BuLi. After 15 minutes, the solution was brought back to -24°C and stirred 2 hours. Then, the mixture was cooled down to - 78°C before addition of 4.0 mL (33.0 mmol, 2.2 eq) of p-anisaldehyde in 60 mL of THF. The reaction was brought back to 0°C and stirred 2 hours before being stirred at room temperature over night. Then, 120 mL of water were added. The aqueous layer was extracted with 2 x 80 mL of dichloromethane. The organic phase was dried, filtered and concentrated. The crude mixture was purified over silicagel. The obtained white solid (estimation 9 mmol of XII) was dissolved in 90 mL of DCM. Then, 7.74 g (89.0 mmol, 10 eq.) of activated Mn0₂ were added and the reaction was refluxed 20 hours. The reaction was brought back to room temperature and 7.74 g (89.0 mmol, 10 eq.) of activated Mn0₂ were added again. After 20 hours to reflux, the mixture was filtered on celite and the solvent was evaporated. The crude mixture was dissolved in 180 mL of 3 M aqueous HCI were added and refluxed 16 hours. The reaction was cooled down to 0°C and 50 mL of aqueous ammonia were added. The aqueous layer was extracted with 3 x 90 mL of DCM. The organic phase was dried, filtered and concentrated. Purification over silica gel afforded 1.65 g (7.2 mmol, 48%) of XIII as a yellow solid. The yield varied from 38% to 48%. R_f=0.15 (PE/EtOAc 1 :2). ¹H NMR (300 MHz): δ = 8.26 (s, 1 H), 7.94 (d, J = 5.1 Hz, 1 H), 7.72 (m, 2 H), 7.22 (m, 1 H), 6.96 (d, J = 5.1 Hz, 2 H), 5.58 (s, 2 H), 3.88 (s, 3 H) ppm. ¹³C NMR (75 MHz): δ = 196.5, 163.4, 144.0, 141.1 , 137.1 , 132.2, 130.6, 124.7, 123.4, 1 13.8, 55.6 ppm. Step 3. 3-(4-methoxyphenyl)-1 H-pyrrolo[2,3-c]pyridine-2-carbaldehyde (936)

To a solution of 1 .7 mL (12.3 mmol, 3.5 eq.) of diisopropylamine in 12 mL of THF, cooled to 0°C, were added 4.9 mL (12.3 mmol, 3.5 eq., 2.5 M in hexane) of BuLi. The mixture was cooled to -78°C and 0.42 mL (5.3 mmol, 1 .5 eq.) of

dichloroethylene were added. After 5 minutes, 0.03 mL (0.35 mmol, 0.1 eq.) of aniline were added. The cooling bath was removed and 620 mg (3.1 mmol, 1 eq.) of aminophenone XIII in 7 mL of THF were immediately added. Then, the reaction was brought back to 0°C and stirred one hour. After this time, 15 mL of water were added. The aqueous layer was neutralised with diluted sulphuric acid. The aqueous layer was extracted with 2 x 20 mL of EtOAc and with 2 x 20 mL of DCM. The organic phase was dried, filtered (the solid was washed with acetone) and concentrated. The crude mixture was dissolved in 50 mL of dichloroethane and 50 mL of 0.1 M aqueous sulphuric acid were added. After 18 hours under reflux, the mixture was cooled down to room temperature. The solid was filtered. The filtrate was decanted and the organic layer was extracted with 2 x 30 mL of 3 M aqueous HCI. The combined aqueous layers were neutralised with K₂C0₃. Once again, the solid was filtered. The combined solids were recrystallised in 30 mL of

DCM/acetone 1 :2 providing 600 mg (2.4 mmol, 68%) of 936 as an ochre solid. The yield varied from 50% to 68%. R_f=0.30 (PE/EtOAc 1 :2). NMR data were taken in deuterated DMSO. ¹H NMR (300 MHz): δ = 12.45 (broad, 1 H), 9.93 (s, 1 H), 8.92 (s, 1 H), 8.24 (d, J = 5.6 Hz, 1 H), 7.68 (d, J = 5.6 Hz, 1 H), 7.62 (d, J = 8.6 Hz, 2 H), 7.12 (d, J = 8.6 Hz, 2 H), 3.84 (s, 3 H) ppm. ¹³C NMR (75 MHz): δ = 183.7, 159.2, 138.9, 137.3, 133.7, 133.4, 131 .5, 129.8, 125.5, 123.4, 1 15.3, 1 14.6, 55.3 ppm. MS (ESI): m/z = 252.1 . I.R. (neat): v 2999, 1651 . H.R.M.S. (ESI): Calcd for Ci₅H₁₃N₂0₂; 253.0977. Found: 253.0976. mp >220 °C.

Step 4. 3-(4-hydroxyphenyl)-1 H-pyrrolo[2,3-c]pyridine-2-carbaldehyde (942)

To a suspension of 320 mg (1.3 mmol, 1 eq.) of methyl ether 936 in 25 mL of DCM cooled to -78°C were added dropwise 0.62 mL (6.5 mmol, 5 eq.) of BBr₃ in 7 mL of DCM. After one hour, the reaction was brought back to 0°C and then stirred at room temperature overnight. Then, the reaction was poured on 30 g of ice. The two layers were decanted and the organic layer was extracted with 20 mL of 3 M aqueous HCI. The combined aqueous phases were neutralised (pH 6-7) with Na₂C0₃. The mixture was filtered and the obtained gel triturated with diethylether. Then, the solid was triturated in 10 mL of hot acetone. The solid was collected and dried under vacuum providing 151 mg (0.65 mmol, 50%) of 942 as brown solid. The yield varied from 36% to 50%. R_f=0.21 (EtOAc). NMR data were taken in deuterated DMSO. ¹H NMR (300 MHz): δ = 12.39 (s, 1 H), 9.92 (s, 1 H), 9.78 (s, 1 H), 8.90 (s, 1 H), 8.23 (d, J = 5.4 Hz, 1 H), 7.67 (d, J = 5.2 Hz, 1 H), 7.49 (d, J = 8.1 Hz, 2 H), 6.95 (d, J = 8.1 Hz, 2 H) ppm. ¹³C NMR (75 MHz): δ = 183.7, 157.6, 138.8, 137.3, 133.7, 133.3, 131.5, 129.8, 126.2, 121 .7, 1 16.0, 1 15.4 ppm. MS (ESI): m/z = 238.1 . I.R. (neat): v 3263, 1645. H.R.M.S. (ESI): Calcd for C HnNzOz; 239.0821. Found: 239.0825. mp > 220 °C.

Example 6. 3-(4-hydroxyphenyl)-1 H-pyrrolo[3,2-c]pyridine-2-carbaldehyde (940) Step 1 . yV-(3-(hydroxy(4-methoxyphenyl)methyl)pyridin-4-yl)pivalamide (XIV)

To a solution of 2.67 g (15.0 mmol, 1 eq.) of pivalamide VII and 6.8 mL (45.0 mmol, 3 eq.) of TMEDA in 120 mL of diethylether, cooled to -78°C, were added 18.0 mL (45.0 mmol, 3 eq., 2.5 M in hexane) of BuLi. After 15 minutes, the solution was brought back to -24°C and stirred 2 hours. Then, the mixture was cooled down to - 78°C before addition of 4.0 mL (33.0 mmol, 2.2 eq) of p-anisaldehyde in 60 mL of THF. The reaction was brought back to 0°C and stirred 2 hours before being stirred at room temperature over night. Then, 120 mL of water were added. The aqueous layer was extracted with 3 x 80 mL of dichloromethane. The organic phase was dried, filtered and concentrated. Precipitation in 30 mL of PE/DCM 1 :1 afforded 3.26 g (10.4 mmol, 69%) of XIV as a white powder. R_f=0.16 (PE/EtOAc 1 :2). ¹H NMR (300 MHz): δ = 9.57 (s, 1 H), 8.36 (d, J = 5.7 Hz, 1 H), 8.23 (d, J = 5.7 Hz, 1 H), 7.87 (s, 1 H), 7.18 (d, J = 8.5 Hz, 2 H), 6.89 (broad, 1 H), 6.83 (d, J = 8.3 Hz, 2 H), 5.78 (s, 1 H), 3.77 (s, 3 H), 1 .08 (s, 9 H) ppm. ¹³C NMR (75 MHz): δ = 177.9, 159.4, 149.8, 148.4, 145.6, 133.0, 127.5, 126.4, 1 15.3, 1 14.0, 73.2, 55.5, 40.2, 27.2 ppm.

Step 2. (4-aminopyridin-3-yl)(4-methoxyphenyl)methanone (XV) To a solution of 3.15 g (10.0 mmol, 1 eq.) of XIV in 100 mL of DCM, 8.71 g (100.0 mmol, 10 eq.) of activated Mn0₂ were added and the reaction was refluxed 40 hours. The mixture was filtered on celite and the solvent was evaporated. The crude mixture was dissolved in 200 mL of 3 M aqueous HCI were added and refluxed 16 hours. The reaction was cooled down to 0°C and 50 mL of aqueous ammonia were added. The aqueous layer was extracted with 3 x 100 mL of DCM. The organic phase was dried, filtered and concentrated yielding 1 .45 g (6.3 mmol, 63%) of XV as a white powder. R_f=0.09 (PE/EtOAc 1 :2). ¹H NMR (300 MHz): δ = 8.57 (s, 1 H), 8.18 (d, J = 5.9 Hz, 1 H), 7.69 (m, 2 H), 6.96 (m, 2 H), 6.58 (d, J = 5.9 Hz, 1 H), 6.49 (broad, 2 H), 3.87 (s, 3 H) ppm. ¹³C NMR (75 MHz): δ = 196.8, 162.9, 155.4, 155.3, 151 .7, 131 .9, 131 .4, 1 15.4, 1 13.8, 1 1 1.2, 55.6 ppm.

Step 3. 3-(4-methoxyphenyl)-1 H-pyrrolo[3,2-c]pyridine-2-carbaldehyde (937)

To a solution of 3.4 mL (23.7 mmol, 4 eq.) of diisopropylamine in 15 mL of THF, cooled to 0°C, were added 9.5 mL (23.7 mmol, 4 eq., 2.5 M in hexane) of BuLi. The mixture was cooled to -78°C and 0.71 mL (8.9 mmol, 1 .5 eq.) of dichloroethylene were added. The system was brought back to 0°C and 1 .35 g (5.9 mmol, 1 eq.) of aminophenone XV in 15 mL of THF were added. After one hour, 40 mL of aqueous NH₄CI were added. The aqueous layer was extracted with 3 x 40 mL of

dichloromethane. The organic phase was dried, filtered and concentrated. The crude mixture was dissolved in 120 mL of dichloroethane and 120 mL of 0.1 M aqueous sulphuric acid were added. After 16 hours under reflux, the mixture was cooled down to room temperature and the pH was brought to 1 1 with Na₂C0₃. The aqueous layer was extracted with 2 x 150 mL of dichloromethane. The organic layer was dried, filtered and concentrated. The crude mixture was recrystallised in

DCM/PE 2:1 providing 970 mg (3.8 mmol, 65%) of 937 as an ochre solid. R_f=0.20 (PE/EtOAc 1 :2). NMR data were taken in deuterated DMSO. ¹H NMR (300 MHz): δ = 12.39 (broad, 1 H), 9.83 (s, 1 H), 9.00 (s, 1 H), 8.37 (d, J = 5.7 Hz, 1 H), 7.67 (d, J = 8.5 Hz, 2 H), 7.43 (d, J = 5.6 Hz, 1 H), 7.12 (d, J = 8.5 Hz, 2 H), 3.84 (s, 3 H) ppm. ¹³C NMR (75 MHz): δ = 182.7, 159.5, 145.6, 144.3, 140.5, 132.1 , 131.7,

127.2, 123.1 , 123.1 , 1 14.7, 107.8, 55.3 ppm. MS (ESI): m/z = 252.1 . I.R. (neat): v 3327, 1666. H.R.M.S. (ESI): Calcd for Ci₅H₁₃N₂0₂; 253.0977. Found: 253.0984. mp 182 °C. Step 4. 3-(4-hydroxyphenyl)-1 H-pyrrolo[3,2-c]pyridine-2-carbaldehyde (940)

To a suspension of 770 mg (3.0 mmol, 1 eq.) of methyl ether 937 in 60 mL of DCM cooled to -78°C were added dropwise 1 .5 mL (15.3 mmol, 5 eq.) of BBr₃. After one hour, the reaction was brought back to 0°C and then stirred at room temperature overnight. Then, the reaction was cooled to 0°C and 5 mL of water were added dropwise, followed by 50 mL of 3 M aqueous HCI. The organic layer was extracted with 50 mL of 3 M aqueous HCI. The aqueous layers were neutralised with Na₂C0₃. The precipitate was collected and washed with 50 mL of acetone. The filtrate was evaporated and the brown solid was precipitated in 5 mL of acetone. The solid was removed by filtration and the filtrate was evaporated providing 370 mg (1.6 mmol, 51 %) of 940 as an ochre solid. R_f=0.12 (PE/EtOAc 1 :2). NMR data were taken in deuterated DMSO. ¹H NMR (300 MHz): δ = 12.45 (broad, 1 H), 9.84 (s, 2 H), 9.04 (s, 1 H), 8.37 (d, J = 5.9 Hz, 1 H), 7.56 (d, J = 8.4 Hz, 2 H), 7.47 (d, J = 5.9 Hz, 1 H), 6.97 (d, J = 8.4 Hz, 2 H) ppm. ¹³C NMR (75 MHz): δ = 182.7, 157.9, 145.1 , 143.2, 140.7, 132.2, 131 .8, 128.0, 123.1 , 121.2, 1 16.1 , 108.8 ppm. MS (ESI): m/z = 238.1 . I.R. (neat): v 3265, 1643. H.R.M.S. (ESI): Calcd for Ci₄HnN₂0₂; 239.0821. Found: 239.0826. mp 220 °C.

Step 1 Step 2 Step 3

Product

R=Me XIX 1285

XVIII

R=Bn XX 1286

Example 7. Preparation of 3-(4-hydroxyphenyl)-1 -(phenylsulfonyl)-1 H- pyrrolo[2,3-c]pyridine-2-carbaldehyde (1285) Step 1. 3-(4-(ieri-butyldimethylsilyloxy)phenyl)-1 H-pyrrolo[2,3-c]pyridine-2- carbaldehyde (XVIII)

To a solution of 750 mg (3.1 mmol, 1 eq.) of hydroxypyrrolopyridine 942 in 15 mL of DMF were added 860 mg (12.6 mmol, 4 eq.) of imidazole and 1.14 g (7.6 mmol, 2.4 eq.) of TBSCI. After 20 hours, 100 mL of water were added. The aqueous layer was extracted with 3 x 75 mL of DCM. The organic phase was dried, filtered and concentrated. The DMF was then removed under high vacuum. Purification over silica gel afforded 600 mg (1 .7 mmol, 54%) of XVIII as an ochre solid. R_f=0.38 (EtOAc). ¹H NMR (300 MHz): δ = 10.28 (s, 1 H), 10.03 (s, 1 H), 9.09 (s, 1 H), 8.42 (d, J = 5.6 Hz, 1 H), 7.73 (d, J = 5.5 Hz, 1 H), 7.47 (m, 2 H), 7.02 (m, 2 H), 1 .03 (s, 9 H), 0.28 (s, 6 H) ppm. ¹³C NMR (75 MHz): δ = 183.8, 156.4, 139.7, 137.0, 133.6, 131 .6, 131 .4, 127.8, 123.9, 120.9, 1 16.2, 25.8, 18.4, -4.2 ppm. MS (ESI): m/z = 352.2. I.R. (neat): v 2951 , 1674. H.R.M.S. (ESI): Calcd for C₂oH₂₅N₂0₂Si; 353.1680. Found: 353.1680. mp 210 °C. Step 2. 3-(4-((ferf-butyldimethylsilyl)oxy)phenyl)-1 -methyl-1 H-pyrrolo[2,3- c]pyridine-2-carbaldehyde (XIX)

To a solution of 1 .3 mg (3.2 μηιοΙ, 0.016 eq.) of Aliquat 336 in 0.6 mL of DCM, were added 0.3 mL of water, 40 mg (1 .0 mmol, 5 eq.) of NaOH and 71 mg (0.20 mmol, 1 eq.) of pyrrolopyridine XVIII. Then, 0.02 mL (0.22 mmol, 1 .1 eq.) of dimethylsulfate were added dropwise. The mixture was stirred one hour before addition of 5 mL of DCM and 5 mL of water. The organic layer was decanted, dried, filtered and concentrated. Purification over silica gel afforded 26 mg (0.07 mmol, 36%) of XIX as an orange oil. R_f=0.56 (EtOAc). ¹H NMR (300 MHz): δ = 10.02 (s, 1 H), 8.97 (s, 1 H), 8.35 (d, J = 5.5 Hz, 1 H), 7.62 (dd, J = 5.6 Hz, J = 0.6 Hz, 1 H), 7.36 (m, 2 H), 6.98 (m, 2 H), 4.22 (s, 3 H), 1 .02 (s, 9 H), 0.27 (s, 6 H) ppm. ¹³C NMR (75 MHz): δ = 185.2, 156.2, 139.6, 135.5, 135.4, 132.4, 131 .9, 130.1 , 129.8, 123.9, 120.7, 1 15.6, 32.4, 25.8, 18.4, -4.2 ppm. MS (ESI): m/z = 366.2. I.R. (neat): v 2949, 2926, 2856, 1666. H.R.M.S. (ESI): Calcd for C₂iH₂₇N₂0₂Si; 367.1836. Found: 367.1835.

Step 3. 3-(4-hydroxyphenyl)-1 -methyl-1 H-pyrrolo[2,3-c]pyridine-2- carbaldehyde (1285) To a solution of 55 mg (0.15 mmol, 1 eq.) of pyrrolopyridine XIX in 0.75 mL of THF were added 0.22 mL (0.23 mmol, 1.5 eq., 1 M in THF). After 15 minutes, 5 mL of water and 5 mL of DCM were poured in the reaction. The aqueous layer was extracted with 3 x 5 mL of DCM. The combined organic layers were dried, filtered and concentrated. Recrystallization in 3 mL of PE/DCM 2:1 afforded 24 mg (0.09 mmol, 63%) of 1285 as an ochre-orange solid. NMR data were taken in deuterated DMSO. R_f=0.38 (EtOAc). ¹H NMR (300 MHz): δ = 9.93 (s, 1 H), 9.78 (s, 1 H), 9.13 (s, 1 H), 8.27 (d, J = 5.3 Hz, 1 H), 7.61 (d, J = 5.4 Hz, 1 H), 7.39 (d, J = 8.4 Hz, 2 H), 6.94 (d, J = 8.4 Hz, 2 H), 4.15 (s, 3 H) ppm. ¹³C NMR (75 MHz): δ = 184.7, 157.6, 139.1 , 136.1 , 134.9, 131 .7, 128.8, 128.4, 121.2, 1 15.9, 1 14.9, 32.1 ppm. MS (ESI): m/z = 252.1 . I.R. (neat): v 1668. H.R.M.S. (ESI): Calcd for Ci₅H₁₃N₂0₂; 253.0971. Found: 252.0971. mp >220 °C.

Example 8. Preparation of 1 -benzyl-3-(4-hydroxyphenyl)-1 H-pyrrolo[2,3- c]pyridine-2-carbaldehyde (1286) Step 1. XVIII is already described in example 7.

Step 2. 1 -benzyl-3-(4-((ferf-butyldimethylsilyl)oxy)phenyl)-1 H-pyrrolo[2,3- c]pyridine-2-carbaldehyde (XX)

To a solution of 1 .3 mg (3.2 μηιοΙ, 0.016 eq.) of Aliquat 336 in 0.6 mL of DCM, were added 0.3 mL of water, 40 mg (1.0 mmol, 5 eq.) of NaOH and 71 mg (0.20 mmol, 1 eq.) of pyrrolopyridine XVIII. Then, 0.04 mL (0.30 mmol, 1 .5 eq.) of dimethylsulfate were added dropwise. The mixture was stirred three hours before addition of 5 mL of DCM and 5 mL of water. The organic layer was decanted, dried, filtered and concentrated. Purification over silica gel afforded 26 mg (0.07 mmol, 36%) of XX as an orange oil. R_f=0.15 (EtOAc/MeOH 10:1 ). ¹H NMR (300 MHz): δ = 10.40 (s, 1 H), 8.70 (s, 1 H), 7.77 (d, J = 6.8 Hz, 1 H), 7.40 (m, 6 H), 7.24 (m, 2 H), 6.93 (m, 2 H), 5.47 (s, 2 H), 1.01 (s, 9 H), 0.25 (s, 6 H) ppm. ¹³C NMR (75 MHz): δ = 190.6, 155.1 , 154.8, 142.7, 135.8, 135.2, 134.1 , 131.1 , 129.7, 129.6, 128.0, 126.8, 125.2, 122.6, 120.2, 1 17.4, 63.2, 25.9, 18.4, -4.2 ppm. MS (ESI): m/z = 442.2. I.R. (neat): v 2926, 1674. H.R.M.S. (ESI): Calcd for C27H31 N2O2S1; 443.2149. Found: 443.2149. mp 177 °C.

Step 3. 1 -benzyl-3-(4-hydroxyphenyl)-1 H-pyrrolo[2,3-c]pyridine-2- carbaldehyde (1286)

To a solution of 34 mg (0.077 mmol, 1 eq.) of pyrrolopyridine XX in 0.40 mL of THF were added 0.12 mL (0.12 mmol, 1.5 eq., 1 M in THF). After 15 minutes, 5 mL of water and 5 mL of DCM were poured in the reaction. The aqueous layer was extracted with 3 x 5 mL of DCM. The combined organic layers were dried, filtered and concentrated. Recrystallization in 3 mL of DCM afforded 14 mg (0.043 mmol, 55%) of 1286 as an orange solid. NMR data were taken in deuterated DMSO.

R_f=0.08 (EtOAc/MeOH 10:1 ). ¹H NMR (300 MHz): δ = 10.22 (s, 1 H), 9.45 (broad, 1 H), 9.31 (s, 1 H), 7.88 (d, J = 6.7 Hz, 1 H), 7.75 (d, J = 6.8 Hz, 1 H), 7.30 to 7.48 (m, 7 H), 6.84 (d, J = 8.5 Hz, 2 H), 5.68 (s, 2 H) ppm. ¹³C NMR (75 MHz): δ = 190.4, 156.0, 153.2, 141 .6, 137.0, 136.3, 133.7, 130.7, 129.0, 128.6, 128.0, 126.7, 124.5, 120.4, 1 16.6, 1 15.1 , 61 .1 ppm. MS (ESI): m/z = 328.1 . I.R. (neat): v 3298, 1672, 1610. H.R.M.S. (ESI): Calcd for C₂iH₁₇N₂0₂; 329.1285. Found: 329.1287. mp >220 °C.

Example 9. Preparation of yV-(2-(diethylamino)ethyl)-3-(4-hydroxyphenyl)-1 H- pyrrolo[2,3-c]pyridine-2-carboxamide (1356)

Step 1. methyl 3-(4-((ferf-butyldimethylsilyl)oxy)phenyl)-1 H-pyrrolo[2,3- c]pyridine-2-carboxylate (XXI)

To a solution of 21 mg (0.06 mmol, 1 eq.) of XVIII in 1 .5 mL of methanol were added 20 mg (0.30 mmol, 5 eq.) of KCN. Then, 100 mg (1 .2 mmol, 20 eq.) of Mn0₂ were carefully added (ignition) followed by 0.01 mL (0.18 mmol, 3 eq.) of acetic acid. After 4 hours, the solid was filtered and washed with 20 mL of DCM. The filtrate was washed with 15 mL of water. The organic phase was dried, filtered and concentrated to yield 22 mg (0.06 mmol, 96%) of XXI as a yellow solid. R_f=0.32 (EtOAc). ¹H NMR (300 MHz): δ = 10.13 (broad, 1 H), 9.00 (s, 1 H), 8.34 (d, J = 5.4 Hz, 1 H), 7.59 (d, J = 5.5 Hz, 1 H), 7.41 (m, 2 H), 6.95 (m, 2 H), 3.86 (s, 3 H), 1.03 (s, 9 H), 0.27 (s, 6 H) ppm. ¹³C NMR (75 MHz): δ = 162.2, 155.5, 139.1 , 135.8, 132.7, 132.6, 131.6, 125.7, 125.2, 123.1 , 1 19.8, 1 16.0, 52.3, 25.8, 18.4, -4.2 ppm. MS (CI): m/z = 383.2. I.R. (neat): v 2957, 2928, 2897, 171 1. H.R.M.S. (CI): Calcd for C₂iH₂₇N₂0₃Si; 383.1786. Found: 383.1785. mp 206 °C. Step 2. yV-(2-(diethylamino)ethyl)-3-(4-hydroxyphenyl)-1 H-pyrrolo[2,3- c]pyridine-2-carboxamide (1356)

To a solution of 92 mg (0.24 mmol, 1 eq.) of ester XXI in 0.25 mL of DCM were added 0.70 mL (4.8 mmol, 20 eq.) of diethylethylenediamine. The mixture was heated to 40°C in order to evaporate the DCM. Then, the mixture was sealed for 34 hours. The reaction was cooled to room temperature. The solvent was

concentrated. The amine was removed under vacuum at room temperature. The orange oil was dissolved in 3 mL of THF. Then, 0.36 mL (0.36 mmol, 1.5 eq., 1 M in THF) of TBAF were added. After 30 minutes, the solvent was evaporated. The red oil was dissolved in 5 mL of DCM before addition of 5 mL of water. The mixture was stirred for 2 minutes. Then, 10 mL of diethylether and 20 mL of petroleum ether were added. The mixture was cooled to 0°C for 30 minutes. The solid was collected, washed with petroleum ether and dried under vacuum to give 58 mg (0.17 mmol, 69%) of 1356 as an ochre solid. NMR data were taken in deuterated

DMSO. R_f=0.02 (EtOAc). ¹H NMR (300 MHz): δ = 12.12 (broad, 1 H), 9.59 (broad, 1 H), 8.81 (s, 1 H), 8.13 (d, J = 4.2 Hz, 1 H), 7.38 (d, J = 5.0 Hz, 1 H), 7.29 (d, J = 8.5 Hz, 3 H), 6.90 (d, J = 8.5 Hz, 2 H), 3.29 (q, J = 5.8 Hz, 2 H), 2.43 (m, 6 H), 0.86 (t, J = 7.0 Hz, 6 H) ppm. ¹³C NMR (75 MHz): δ = 160.9, 157.0, 138.4, 135.7, 132.1 , 131 .2, 130.9, 130.6, 122.6, 1 15.8, 1 14.2, 50.5, 45.9, 36.7, 1 1 .1 ppm. MS (ESI): m/z = 353.2. I.R. (neat): v 2970, 1632. H.R.M.S. (ESI): Calcd for CzoH^Oz; 353.1972. Found: 353.1973. mp >210 °C.

Example 10. Preparation of 1 -benzyl-W-(2-(diethylamino)ethyl)-3-(4- hydroxyphenyl)-1 H-pyrrolo[2,3-c]pyridine-2-carboxamide (1395)

Step 1. methyl 1 -benzyl-3-(4-((ieri-butyldimethylsilyl)oxy)phenyl)-1 H- pyrrolo[2,3-c]pyridine-2-carboxylate (XXII)

To a solution of 195 mg (0.44 mmol, 1 eq.) of XX in 1 1 mL of methanol were added 140 mg (2.2 mmol, 5 eq.) of KCN. Then, 765 mg (8.8 mmol, 20 eq.) of Mn0₂ were carefully added (ignition) followed by 0.07 mL (1 .3 mmol, 3 eq.) of acetic acid. After 3 hours, the solid was filtered and washed with 40 mL of DCM. The filtrate was washed with 30 mL of water. The organic phase was dried, filtered and

concentrated to yield 200 mg (0.42 mmol, 96%) of XXII as a brown solid. R_f=0.23 (EtOAc/MeOH 1 :1 ). ¹H NMR (300 MHz): δ = 8.57 (broad, 1 H), 7.60 (s, 1 H), 7.36 (s, 7 H), 7.20 (s, 1 H), 6.89 (d, J = 6.4 Hz, 2 H), 5.41 (broad, 2 H), 3.90 (s, 3 H), 1 .00 (s, 9 H), 0.23 (s, 6 H) ppm. ¹³C NMR (75 MHz): δ = 165.7, 154.3, 149.6, 141.5, 135.3, 134.4, 134.3, 131 .1 , 129.5, 129.4, 127.8, 125.2, 121.3, 1 19.6, 1 16.2, 62.9, 51.9, 25.8, 18.3, -4.3 ppm. MS (ESI): m/z = 472.2. I.R. (neat): v 2953, 2928, 2899, 2878, 2854, 1713. H.R.M.S. (ESI): Calcd for C₂₈H₃₃N₂0₃Si; 473.2255. Found:

473.2255. mp 156 °C. Step 2. 1 -benzyl-yV-(2-(diethylamino)ethyl)-3-(4-hydroxyphenyl)-1 H-pyrrolo[2,3- c]pyridine-2-carboxamide (1395)

To a solution of 194 mg (0.41 mmol, 1 eq.) of ester XXII in 0.41 mL of DCM were added 1 .15 mL (8.2 mmol, 20 eq.) of diethylethylenediamine. The mixture was heated to 40°C in order to evaporate the DCM. Then, the mixture was sealed for 34 hours. The reaction was cooled to room temperature. The solvent was

concentrated. The amine was removed under vacuum at room temperature. The orange oil was dissolved in 6 mL of THF. Then, 0.62 mL (0.62 mmol, 1.5 eq., 1 M in THF) of TBAF were added. After 30 minutes, the solvent was evaporated. The red oil was dissolved in 5 mL of DCM before addition of 5 mL of water. The mixture was stirred for 2 minutes. Then, 20 mL of diethylether and 10 mL of petroleum ether were added. The solid was collected, washed with ether and dried under vacuum to give 120 mg (0.27 mmol, 66%) of 1395 as a yellow solid. NMR data were taken in deuterated DMSO at 90°C. R_f=0.03 (EtOAc/MeOH 10:1 ). ¹H NMR (300 MHz): δ = 8.93 (broad, 1 H), 8.18 (broad, 1 H), 7.68 (s, 1 H), 7.62 (s, 1 H), 7.37 (m, 8 H), 6.80 (s, 2 H), 5.62 (s, 2 H), 3.40 (s, 2 H), 2.55 (m, 6 H), 1 .01 (s, 6 H) ppm. ¹³C NMR (75 MHz): 5 = 165.0, 154.9, 152.9, 135.9, 133.1 , 130.3, 128.4, 127.9, 127.3, 126.2, 125.8, 1 17.5, 1 14.2, 60.5, 51.7, 46.3, 36.8, 1 1.5 ppm. MS (ESI): m/z = 442.2. I.R. (neat): v 2968, 1610. H.R.M.S. (ESI): Calcd for C27H31 N4O2; 443.2442. Found: 443.2442. mp 198 °C.

Example 1 1 . Preparation of 3-(4-hydroxyphenyl)-1 -(phenylsulfonyl)-1 H- pyrrolo[2,3-c]pyridine-2-carbaldehyde (1227)

Step 1 and step 2. 3-(4-methoxyphenyl)-1 -(phenylsulfonyl)-1 H-pyrrolo[2,3- c]pyridine-2-carbaldehyde (XXIV)

To a solution of 570 mg (2.5 mmol, 1 eq.) of aminophenone XIII in 10 mL of pyridine were added 0.42 mL (3.3 mmol, 1 .3 eq.) of benzenesulfonyl chloride. The reaction was refluxed 2 hours before addition of 30 mL of water. The solide was filtered, washed with water and dried under vacuum providing 885 mg of crude sulfonamide (80% purity, estimation 2.0 mmol of XXIII). This solid was suspended in 20 mL of THF before addition of 0.23 mL (2.8 mmol, 1 .4 eq.) of dichloroethylene. Then, 860 mg (7.6 mmol, 3.8 eq.) of tBuOK were added and the mixture was stirred two hours. After this time, 10 mL of water were poured in the reaction and the pH was brought back to 7 with diluted sulphuric acid. The aqueous layer was extracted with 3 x 20 mL of DCM and with 30 mL of EtOAc. The organic phase was dried, filtered and concentrated. The dark brown oil was dissolved in 40 mL of dichloroethane before addition of 40 mL of 1 M aqueous sulphuric acid. After 66 hours under reflux, the reaction was neutralised with Na₂C0₃ at room temperature and 40 mL of water were added. The aqueous layer was extracted with 2 x 60 mL of DCM. The organic layer was dried, filtered and concentrated. Purification over silica gel afforded 340 mg (0.87 mmol, 34%) of XXIV as an orange solid. R_f=0.68 (EtOAc). ¹H NMR (300 MHz): δ = 10.23 (s, 1 H), 9.65 (s, 1 H), 8.50 (d, J = 5.4 Hz, 1 H), 7.99 (m, 2 H), 7.59 (m, 1 H), 7.48 (m, 3 H), 7.41 (m, 2 H), 6.99 (m, 2 H), 3.83 (s, 3 H) ppm. ¹³C NMR (75 MHz): δ = 182.4, 160.5, 143.5, 138.5, 137.6, 134.6, 134.5, 134.0, 133.6, 133.3, 131 .8, 129.3, 127.3, 121 .0, 1 16.0, 1 14.1 , 55.3 ppm. MS (ESI): m/z = 392.1 . I.R. (neat): v 2932, 1682. H.R.M.S. (ESI): Calcd for C₂iH₁₇N₂0₄S; 393.0903. Found: 393.0903. mp degradation over 100 °C.

Step 3. 3-(4-hydroxyphenyl)-1 -(phenylsulfonyl)-l H-pyrrolo[2,3-c]pyridine-2- carbaldehyde (1227)

To a suspension of 157 mg (0.40 mmol, 1 eq.) of methyl ether XXIV in 6 mL of DCM cooled to -78°C were added dropwise 0.23 mL (2.4 mmol, 6 eq.) of BBr₃ in 2 mL of DCM. After one hour, the reaction was brought back to 0°C and then stirred at room temperature overnight. Then, the reaction was poured on 10 g of ice. The aqueous layer was extracted with 2 x 30 mL of DCM. The aqueous layer was then neutralised with diluted Na₂C0₃ and further extracted with 2 x 30 mL of DCM. The combined organic phases were dried, filtered and concentrated. The crude mixture was purified over silica gel. After evaporation, the orange solid was dissolved in 15 mL of DCM. The solid was triturated before filtration. The filtrate was evaporated, providing 55 mg (0.14 mmol, 36%) of 1227 as an ochre solid. R_f=0.64 (EtOAc).

NMR data were taken in deuterated DMSO. ¹H NMR (300 MHz): δ = 10.19 (s, 1 H), 9.92 (s, 1 H), 9.48 (d, J = 0.7 Hz, 1 H), 8.53 (d, J = 5.3 Hz, 1 H), 8.05 (m, 2 H), 7.75 (m, 1 H), 7.64 (m, 3 H), 7.41 (m, 2 H), 6.90 (m, 2 H) ppm. ¹³C NMR (75 MHz): δ = 183.1 , 158.6, 143.8, 137.7, 136.6, 135.1 , 134.3, 133.3, 132.6, 131 .9, 129.9, 127.1 , 1 19.0, 1 16.3, 1 15.5 ppm. MS (ESI): m/z = 378.1 . I.R. (neat): v 3057, 2934, 1688. H.R.M.S. (ESI): Calcd for C₂oH₁₅N₂0₄S; 379.0747. Found: 379.0748. mp 146 °C.

ASSAY METHODOLOGIES. 1.MKK1 assay

This is a two-step assay where inactive MAPK (0.06 mg/ml) is activated by MKK1 (diluted in 25 mM Tris, 0.1 mM EGTA, 0.1 % b-mercaptoethanol, 0.01 % Brij35, 1 mg/ml BSA) in 25.5 μΙ containing 25 mM Tris, 0.1 mM EGTA, 0.01 % Brij35, 10 mM magnesium acetate and 0.005 mM ATP. After incubating at room temperature for 30 min, 5 μΙ from the first reaction is pipetted into 20 μΙ of the second reaction mix containing (final concentration) 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM

Na3V04, 0.66 mg/ml myelin basic protein (MBP), 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (500 -1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

2. MAPK2/ERK2 assay.

MAPK/ERK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3V04, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5 μΙ in 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP](500 -1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

3. JNK1a1/SAPK1 c assay.

JNK1 a1/SAPK1 c (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % b-mercaptoethanol) is assayed against ATF2 (activating transcription factor in a final volume of 25.5 μΙ in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- Mercaptoethanol, ATF2 (3 μΜ), 10 mM magnesium acetate and 0.02 mM [33P-g- ATP] (500 -1000 cpm/pmole) and incubated for 30 min at room temperature.

Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 4. SAPK 2a/p38 assay.

SAPK 2a/p38 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3V04, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5 μΙ containing 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 5. SAPK 2b/p38 2 assay.

SAPK 2b/p38U2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3V04, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 6. SAPK 3/p38g assay.

SAPK 3/p38g (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3V04, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 25mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.005mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 7. SAPK 4/p38S assay.

SAPK 4/p38d (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3V04, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5 μΙ containing 25 mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 8. MAPKAP-K1 a assay. MAPKAP-K1 a (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01 % Brij35, 5% glycerol, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against KKLNRTLSVA in a final volume of 25.5 μΙ containing 50 mM Na-b- glycerophosphate pH 7.5, 0.5 mM EDTA, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 40 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

9. MAPKAP-K2 assay.

MAPKAP-K2 (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01 % Brij35, 5% glycerol, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against

KKLNRTLSVA in a final volume of 25.51 containing 50 mM Na-b-glycerophosphate pH 7.5, 0.5 mM EDTA, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

10. MSK1 assay.

MSK1 (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01 % Brij35, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against a modified Crosstide peptide GRPRTSSFAEGKK in a final volume of 25.5 μΙ containing 8 mM MOPS pH7.0, 0.2 mM EDTA, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM

[33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

11. PRAK assay. PRAK (5-20mU diluted in 50 mM Na-b-glycerophosphate pH 7.5, 0.1 mM EGTA, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against KKLRRTLSVA in a final volume of 25.5 μΙ containing 50 mM Na-b-glycerophosphate pH 7.5, 0.1 mM EGTA, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

32. PKA assay.

PKA (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01 % Brij35, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against Kemptide (LRRASLG) in a final volume of 25.5 μΙ containing 8 mM MOPS pH 7.5, 0.2 mM EDTA, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

13. PKCa assay.

PKCa (5-20 mU diluted in 20 mM Hepes pH 7.4, 0.03% Triton X-100) is assayed against Histone H1 in the presence of PtdSerine and DAG (0.1 mg/ml. and 10 μg/ml) and 0.1 mM CaCI2. The assay is carried out in a final volume of 25.5 μΙ containing 20 mM Hepes pH 7.4, 0.03% Triton X-100, 0.1 mg/ml Histone H1 , 10 mM magnesium acetate and 0.02 mM[33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. PtdSer/DAG preparation:- PtdSer stock = 10 mg/ml in MeOH/Chloroform (1 :2). Dry down required amount. Resuspend in appropriate volume of 10 mM Hepes pH 7.4. Vortex and briefly sonicate. (2 x 10-15 seconds at 10-15 seconds apart). DAG stock = 10 mg/ml in MeOH/chloroform (1 :2). Dry down required amount. Add sonicated PtdSer solution. Vortex and sonicate. 14. PDK1 assay.

PDK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 1 mg/ml BSA) is assayed against PDKtide (KTFCGTPEYLAPEVRREPRILSEEEQ- EMFRDFDYIADWC) in a final volume of 25.5 μΙ containing 50 mM Tris pH 7.5, 0.05% b-mercaptoethanol, 100 μΜ substrate peptide, 10mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

15. APH-PKBa-S473D assay.

APH-PKBa-S473D (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against a modified Crosstide peptide GRPRTSSFAEGKK in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

16. SGK assay. SGK (5-20mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01 % Brij35, 5% glycerol, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against a modified Crosstide peptide GRPRTSSFAEGKK in a final volume of 25.5 μΙ containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

17. S6K1/ P70 S6K assay. S6K1/P70 S6K (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01 % Brij35, 5% glycerol, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against substrate peptide (KKRNRTLTV) in a final volume of 25.5 μΙ containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 0.1 mM substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 18. GSK3b assay.

GSK3b (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01 % Brij35, 5% glycerol, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against Phospho-GS2 peptide (YRRAAVPPSPSLSRHSSPHQS(P04)EDEEE) in a final volume of 25.5 μΙ containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 20 μΜ Phospho GS2 peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

19. ROCK-II (ROKa) assay.

ROCK-II (ROKa) (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against Long S6 substrate peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) in a final volume of 25.5 μΙ containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 30 μΜ Long S6 substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

20. AMPK assay.

AMPK (5-20 mU diluted in 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35) is assayed against SAMS substrate peptide (HMRSAMSGLHLVKRR) in a final volume of 25.5 μΙ containing 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35, 0.4 mM SAMS peptide, 0.196 mM AMP, 10 mM magnesium acetate and 0.05 mM

[33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

21. CHK1 assay. CHK1 (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.1 % b- mercaptoethanol, 0.01 % Brij-35, 5% glycerol, 1 mg/ml BSA) is assayed against CHKtide substrate peptide (KKKVSRSGLYRSPSMPENLNRPR) in a final volume of 25.5 μΙ containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 μΜ CHKtide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP](50-1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M

(3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

22. CK2 assay.

CK2 (5-20 mU diluted in 20 mM Hepes pH7.5, 0.15 M NaCI, 0.1 mM EGTA, 0.1 % Triton X-100, 5 mM DTT, 50% glycerol) is assayed against CKII peptide

(RRRDDDSDDD) in a final volume of 25.5 μΙ containing 20 mM Hepes pH 7.5, 0.15 M NaCI, 0.1 mM EDTA, 5 mM DTT, 0.1 % Triton-X 100, CKII peptide (0.165 mM), 10 mM magnesium acetate and 0.005 mM [33P-g-ATP](500 -1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 33. PBK assay.

PBK (5-20 mU diluted in 50 mM Na-b-glycerophosphate pH 7.0, 0.1 % b- mercaptoethanol) is assayed against phosphorylase b peptide (KRKQISVRGL) in a final volume of 25.5 μΙ containing 50 mM Tris pH 8.6, 50 mM Na-b- glycerophosphate, 0.04 mM CaCI2, phosphorylase b peptide (0.196 mM), 10 mM magnesium acetate, 0.02 mM [33P-g-ATP] (500 -1000 cpm/pmole) then incubated for 15 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

31. LCK assay.

LCK (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01 % Brij35, 5% glycerol, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against Cdc2 peptide (KVEKIGEGTYGWYK) in a final volume of 25.5 μΙ containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3Vo4, Cdc2 peptide (0.25 mM), 10 mM magnesium acetate and 0.05mM [33P-g-ATP](500-1000 cpm/pmole) and incubated for 15 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

25. CSK assay.

CSK (5-20 mU diluted in 20 mM MOPS pH7.5, 1 mM EDTA, 0.01 % Brij35, 5% glycerol, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against Cdc2 peptide (KVEKIGEGTYGWYK) in a final volume of 25.5 μΙ containing 8 mM MOPS pH7.0, 0.2 mM EDTA, Cdc2 peptide (0.25 mM), 10 mM magnesium acetate and 0.02 mM [33P-g-ATP](500 -1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

26. CDK2/cyclin A assay. CDK2/cyclin A (5-20 mU diluted in 50 mM Hepes pH 7.5, 1 mM DTT, 0.02% Brij35, 100 mM NaCI) is assayed against Histone H 1 in a final volume of 25.5 μΙ containing 50 mM Hepes pH7.5, 1 mM DTT, 0.02% Brij35, 100 mM NaCI, Histone H1 (1 mg/ml), 10 mM magnesium acetate and 0.02 mM [33P-g-ATP](500-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

27. DYRK 1 A assay.

DYRK 1A (5-20 mU of diluted in 50 mM Tris pH7.5, 0.1 mM EGTA) is assayed against Woodtide (KKISGRLSPIMTEQ) in a final volume of 25.5μΙ containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 350 μΜ substrate peptide, 10 mM Magnesium acetate and 0.05 mM [33P-g-ATP](50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%)

orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

28. CK1 assay.

CK1 (5-20 mU diluted in 20 mM Hepes pH7.5, 0.15 M NaCI, 0.1 mM EGTA, 0.1 % Triton X-100, 5 mM DTT, 50% glycerol) is assayed against CKI peptide

(RRKDLHDDEEDEAMSITA) in a final volume of 25.5 μΙ containing 20 mM Hepes pH 7.5, 0.15 M NaCI, 0.1 mM EDTA, 5 mM DTT, 0.1 % Triton-X 100, CKI peptide (0.5 mM), 10 mM magnesium acetate and 0.02 mM [33P-g-ATP](500 -1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 29. NEK6 assay.

NEK6 (5-20 mU diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %,b- Mercaptoethanol) is assayed against NEK6 peptide (FLAKSFGSPNRAYKK) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.01 % Brij, 0.1 %, b-Mercaptoethanol, NEK6 peptide (0.3 mM), 10 mM magnesium acetate and 0.05 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 30. NEK2a assay.

5-20mU of NEK2a (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %,b-Mercaptoethanol) is assayed against NEK2a peptide (RFRRSRRMI) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.01 % Brij,

0.1 %, b-Mercaptoethanol, 300μΜ NEK2a peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

36. MAPKAP-K1 b/RSK2 assay.

MAPKAP-K1 b (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01 % Brij35, 5% glycerol, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against substrate peptide (KKLNRTLSVA) in a final volume of 25.51 containing 50 mM Na- b-glycerophosphate (pH 7.5), 0.5 mM EDTA, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

38. IKKb assay. 5-20mU of IKKb (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %, b-Mercaptoethanol) is assayed against substrate peptide

(LDDRHDSGLDSMKDEEY) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 %, b-Mercaptoethanol, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

40. smMLCK assay 5-20mU of smMLCK (diluted in 50mM Hepes (pH 7.5), 0.1 mM EGTA, 1 mg/mlBSA, 0.1 %,b-Mercaptoethanol) is assayed against substrate peptide

(KKRPQRATSNVFA) in a final volume of 25.5μΙ containing 50mM Hepes (pH 7.5), 0.1 mM EGTA, 5mM CaCI2, 10μΜ Calmodulin, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid. 42. PRK2 assay

5-20mU of PRK2 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %,b-Mercaptoethanol) is assayed against Long S6 peptide

(KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 %, b-Mercaptoethanol, 30μΜ Long S6 peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP]( 500- 1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

44. MNK2 alpha assay

5-20mU of MNK2 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %, b-Mercaptoethanol) is assayed against substrate peptide (elF4E) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 %, b- Mercaptoethanol, 0.5mg/ml substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

47. CAMK-1 assay 5-20mU of CAMK-1 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %,b-Mercaptoethanol) is assayed against substrate peptide (YLRRRLSDSNF) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.5mM CaCI2, 0.3μΜ calmodulin, 0.1 %, b-Mercaptoethanol, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid. 50. PIM2 assay

5-20mU of PIM2 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %, b-Mercaptoethanol) is assayed against substrate peptide (RSRHSSYPAGT) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.5mM CaCI2, 0.3μΜ calmodulin, 0.1 %, b-Mercaptoethanol, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

52. NEK7 assay

NEK7 (5-20 mU diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %,b- Mercaptoethanol) is assayed against substrate peptide (FLAKSFGSPNRAYKK) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.01 % Brij,

0.1 %, b-Mercaptoethanol, substrate peptide (0.3 mM), 10 mM magnesium acetate and 0.02 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

53. JNK3 alpha 1 assay

JNK3 alpha 1 (5-20 mU diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % b-mercaptoethanol) is assayed against ATF2 (activating transcription factor in a final volume of 25.5 μΙ in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- Mercaptoethanol, ATF2 (3 μΜ), 10 mM magnesium acetate and 0.05 mM [33P-g- ATP] (500 -1000 cpm/pmole) and incubated for 30 min at room temperature.

Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

54. MAPKAP-K3 assay 5-20mU of MAPKAP-K3 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %,b-Mercaptoethanol) is assayed against substrate peptide

(KKLNRTLSVA) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 %, b-Mercaptoethanol, 30μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

55. ERK8 assay

5-20mU of ERK8 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %, b-Mercaptoethanol) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 %, b-Mercaptoethanol, 0.33mg/ml MBP, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP]( 500-1000

cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

56. MNK1 assay

5-20mU of MNK1 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %, b-Mercaptoethanol) is assayed against substrate peptide (elF4E) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 %, b- Mercaptoethanol, 0.5mg/ml substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid. 57. SRPK1 assay

5-20mU of SRPK1 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %,b-Mercaptoethanol) is assayed against substrate peptide

(RSRSRSRSRSRSRSR) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 %, b-Mercaptoethanol, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

58. ΔΡΗ-PKBbeta (S474D) assay

APH-PKBbeta-S474D (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against a modified Crosstide peptide (GRPRTSSFAEGKK) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

46. Aurora B assay

Aurora B (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide

(LRRLSLGLRRLSLGLRRLSLGLRRLSLG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

49. CHK2 assay

CHK2 (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.1 % b- mercaptoethanol, 0.01 % Brij-35, 5% glycerol, 1 mg/ml BSA) is assayed against CHKtide substrate peptide (KKKVSRSGLYRSPSMPENLNRPR) in a final volume of 25.5 μΙ containing 8 mM MOPS pH 7.0, 0.2 mM EDTA, 200 μΜ CHKtide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP](50-1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 51. Src assay

Src (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide (KVEKIGEGTYGWYK) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50- 1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 59. EF2K assay

EF2K (5-20mU diluted in 50 mM Hepes pH 6.6, 0.1 % b-mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide (RKKFGESKTKTKEFL) in a final volume of 25.5 μΙ containing 50mM Hepes pH 6.6, 0.2mM CaCI2, 0.3μΜ

Calmodulin, 0.05% b-mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 60. MARK3 assay

MARK3 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against CHKtide substrate

(KKKVSRSGLYRSPSMPENLNRPR) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

61. MST2 assay

MST2 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 100μΜ Vanadate) is assayed against MBP in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 0.33mg/ml MBP, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

62. PKD1 assay

PKD1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed substrate peptide (KKLNRTLSVA) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50- 1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

63. PLK1 assay

PLK1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA, 100μΜ Vanadate) is assayed against a substrate peptide (ISDELMDATFADQEAKKK) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 10μΜ Vanadate, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

39. DYRK2 assay DYRK2 (5-20 mU of diluted in 50 mM Tris pH7.5, 0.1 mM EGTA) is assayed against Woodtide (KKISGRLSPIMTEQ) in a final volume of 25.5μΙ containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 350 μΜ substrate peptide, 10 mM Magnesium acetate and 0.05 mM [33P-g-ATP](50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%)

37. JNK2 assay JNK2 1 (5-20 mU diluted in 50 mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % b-mercaptoethanol) is assayed against ATF2 (activating transcription factor in a final volume of 25.5 μΙ in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- Mercaptoethanol, ATF2 (3 μΜ), 10 mM magnesium acetate and 0.02 mM [33P-g- ATP] (500 -1000 cpm/pmole) and incubated for 30 min at room temperature.

79. DYRK3 assay

DYRK3 (5-20 mU of diluted in 50 mM Tris pH7.5, 0.1 mM EGTA) is assayed against Woodtide (KKISGRLSPIMTEQ) in a final volume of 25.5μΙ containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 350 μΜ substrate peptide, 10 mM Magnesium acetate and 0.005 mM [33P-g-ATP](50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

64. HIPK2 assay

5-20mU of HIPK2 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %, b-Mercaptoethanol) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 %, b-Mercaptoethanol, 0.33mg/ml MBP, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP]( 500-1000

65. HIPK3 assay

5-20mU of HIPK3 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %, b-Mercaptoethanol) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 %, b-Mercaptoethanol, 0.33mg/ml MBP, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

66. PAK4 assay

PAK4 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide

(RRRLSFAEPG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b- mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room

temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

67. PAK5 (PAK7) assay PAK5 (PAK7)(5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide

(RRRLSFAEPG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b- mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room

68. PAK6 assay

PAK6 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide

69. CAMKKa assay

5-20mU of CAMKKa (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %,b-Mercaptoethanol) is assayed against substrate peptide

(AKPKGNKDYHLQTCCGSLAYRRR) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.5mM CaCI2, 0.3μΜ calmodulin, 0.1 %, b- Mercaptoethanol, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

104. CAMKKb assay 5-20mU of CAMKKb (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %,b-Mercaptoethanol) is assayed against substrate peptide

(DGEFLRTSCGSPNYAARRR) in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.5mM CaCI2, 0.3μΜ calmodulin, 0.1 %, b-Mercaptoethanol, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

71. PIM1 assay

PIM1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide

(RSRHSSYPAGT) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

72. PIM3 assay

PIM3 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide

73. PLK1 assay PLK1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA, 100μΜ Vanadate) is assayed against a substrate peptide (ISDELMDATFADQEAKKK) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 10μΜ Vanadate, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 74. BRSK2 assay

BRSK2 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide

(KKLNRTLSFAEPG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

75. MELK assay

MELK (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide

(KKLNRTLSFAEPG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5,

0.05% b-mercaptoethanol, 200 μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

76. PKC zeta assay

PKC zeta (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA, 100μΜ Vanadate) is assayed against a substrate peptide (ERMRPRKRQGSVRRRV) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 10μΜ Vanadate, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

77. Aurora C assay Aurora C (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % b- mercaptoethanol, 1 mg/ml BSA) is assayed against a substrate peptide

(LRRLSLGLRRLSLGLRRLSLGLRRLSLG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.05% b-mercaptoethanol, 300 μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

78. ERK1 assay

5-20mU of ERK1 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA, 0.1 %,b-Mercaptoethanol) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.1 %, b-Mercaptoethanol, 0.33mg/ml MBP, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP]( 500-1000

80. FGF-R1 assay

FGF-R1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 5mM MnCI₂, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000

cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

81. IRR assay

5-20mU of IRR (diluted in 50mM Hepes (pH 7.5), 0.1 mM EGTA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Hepes (pH 7.5), 0.1 mM EGTA, 0.33mg/ml MBP, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

82. EPH-A2 assay EPH-A2 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

83. MST4 assay 5-20mU of MST4 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.33mg/ml MBP, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

84. SYK assay

SYK (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

85. YES1 assay

YES1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

88. IGF-1 R assay

IGF-1 R (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (KKKSPGEYVNIEFG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

89. VEG-FR assay

VEG-FR (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (KKKSPGEYVNIEFG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 90. BTK assay

BTK (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (KVEKIGEGTYGVVYK) in a final volume of

25.5 μΙ containing 50mM Tris pH 7.5, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

91. IR-HIS assay

IR-HIS (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (KKSRGDYMTMQIG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

94. EPH-B3 assay

EPH-B3 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.005 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

34. TBK1 (DU 12569) assay

TBK1 (DU12569) (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (KKKKERLLDDRHDSGLDSMKDEE) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 300μΜ substrate peptide, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

35. IKKepsilon (DU14231 ) assay

5-20mU of IKKepsilon (DU14231 )(diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.33mg/ml MBP, 10 mM magnesium acetate and 0.05 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

109. GCK assay

GCK (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris pH 7.5,0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%)

110. IRAK4 assay

IRAK4 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%)

orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 111. NUAK1 assay

NUAK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against ALNRTSSDSALHRRR in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.3mM

ALNRTSSDSALHRRR, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50- 1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

112. MLK1 assay

MLK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%)

113. Ml NK1 assay

MINK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.05mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%)

114. MLK3assay

MLK3 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%)

115. LKB1 assay

LKB1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against

LSNLYHQGKFLQTFCGSPLYRRR in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.2mM LSNLYHQGKFLQTFCGSPLYRRR, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

116. HER4 assay HER4 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against Poly Glut Tyr in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml Poly Glut Tyr, 10 mM magnesium acetate and 0.005mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

117. TTK assay TTK (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against RSRSRSRSRSRSRSR in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.3mM

RSRSRSRSRSRSRSR, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50- 1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

120. RIPK2 assay

RIPK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%)

121. Aurora A assay

Aurora A (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against

LRRLSLGLRRLSLGLRRLSLGLRRLSLG in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.3mM

LRRLSLGLRRLSLGLRRLSLGLRRLSLG, 10 mM magnesium acetate and

0.005mM [33P-Y-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

122. PAK2 assay

PAK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against RRRLSFAEPG in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.3mM RRRLSFAEPG, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

123. BRSK1 assay BRSK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against KKLNRTLSFAEPG in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.3mM

KKLNRTLSFAEPG, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

65. HIPK3 assay

HIPK3 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%)

126. HIPK1 assay

HIPK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%)

52. JNK3a1 assay

JNK3 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % β- mercaptoethanol) is assayed against ATF2 (activating transcription factor in a final volume of 25.5 μΙ in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β-Mercaptoethanol, ATF2 (3 μΜ), 10 mM magnesium acetate and 0.02 mM [33Ρ-γ-ΑΤΡ] (500 -1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 54. MAPKAP-K3 assay

MAPKAP-K3 (5-20 mU diluted in 20 mM MOPS pH 7.5, 1 mM EDTA, 0.01 % Brij35, 5% glycerol, 0.1 % β-mercaptoethanol, 1 mg/ml BSA) is assayed against

KKLNRTLSVA in a final volume of 25.51 containing 50 mM Na-3-glycerophosphate pH 7.5, 0.5 mM EDTA, 30 μΜ substrate peptide, 10 mM magnesium acetate and 0.02 mM [33P-Y-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

129. MARK2 assay

MARK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against

KKKVSRSGLYRSPSMPENLNRPR in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.3mM KKKVSRSGLYRSPSMPENLNRPR, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

130. MARK4 assay

MARK4 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against

KKKVSRSGLYRSPSMPENLNRPR in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.3mM KKKVSRSGLYRSPSMPENLNRPR, 10 mM magnesium acetate and 0.05mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

131. EPH-B4 assay

EPH-B4 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 10mM MnCI, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 10mM MnCI, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.05 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 132. JAK2 assay

JAK2 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% β- mercaptoethanol, 1 mg/ml BSA) is assayed against PDKtide

(KTFCGTPEYLAPEVRREPRILSEEEQ-EMFRDFDYIADWC) in a final volume of 25.5 μΙ containing 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.05% β-mercaptoethanol, 100 μΜ substrate peptide, 10mM magnesium acetate and 0.005 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 133. EPH-A4 assay

EPH-A4 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 10mM MnCI, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.05 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

134. TAK1 assay

TAK1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (RLGRDKYKTLRQIRQ) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β-Mercaptoethanol, 300μΜ substrate peptide, 10 mM magnesium acetate, 0.5mM MnCI and 0.005 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

135. TrkA assay TrkA (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 10mM MnCI, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.02 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

136. MEKK1 assay

MEKK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.33 mg/ml MBP, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%)

137. MARK1 assay

MARK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA) is assayed against

142. CLK2 assay

CLK2 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (RNRYRDVSPFDHSR) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.3mM peptide, 10mM DTT, 10 mM magnesium acetate and 0.005 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 143. DAPK1 assay

DAPK1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide KKLNRTLSFAEPG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.3mM peptide, 10mM DTT, 10 mM magnesium acetate and 0.005 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

144. EPH-B2 assay

EPH-B2 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 1 mg/ml substrate peptide, 10 mM magnesium acetate and 0.02 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 169. TSSK1 assay

TSSK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA, 10mM DTT) is assayed against

KKKVSRSGLYRSPSMPENLNRPR in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.3mM KKKVSRSGLYRSPSMPENLNRPR, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 170. TESK1 assay

TESK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA, 10MM DTT) is assayed against Cofilin 2 in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.2mg/ml Cofilin 2, 10 mM magnesium acetate and 0.05mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

171. TTBK1 assay

TTBK1 (5-20 mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 % β- mercaptoethanol, 1 mg/ml BSA, 10mM DTT) is assayed against

RRKDLHDDEEDEAMSITA in a final volume of 25.5μΙ containing 50mM Tris pH 7.5, 0.1 mM EGTA, 0.3mM RRKDLHDDEEDEAMSITA, 10 mM magnesium acetate and 0.005mM [33P-Y-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

172. MST3 assay

5-20mU of MST3 (diluted in 50mM Tris (pH 7.5), 0.1 mM EGTA) is assayed against MBP in a final volume of 25.5μΙ containing 50mM Tris (pH 7.5), 0.1 mM EGTA, 0.33mg/ml MBP, 10 mM magnesium acetate and 0.02 mM [33P-g-ATP]( 500-1000 cpm/pmole) and incubated for 30 mins at room temperature. Assays are stopped by addition of 5μΙ of 0.5M (3%) orthophosphoric acid. Assays are harvested onto P81 Unifilter plates using a wash buffer of 50mM orthophosphoric acid.

173. CSNK1 G2 assay.

CSNK1 G2 (5-20 mU diluted in 20 mM Hepes pH7.5, 0.15 M NaCI, 0.1 mM EGTA, 0.1 % Triton X-100, 5 mM DTT, 50% glycerol) is assayed against

RRKDLHDDEEDEAMSITA in a final volume of 25.5 μΙ containing 20 mM Hepes pH 7.5, 0.15 M NaCI, 0.1 mM EDTA, 5 mM DTT, 0.1 % Triton-X 100,

RRKDLHDDEEDEAMSITA (0.5 mM), 10 mM magnesium acetate and 0.02 mM

[33P-g-ATP](500 -1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

174. QIK assay

QIK (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA,1 mg/ml Poly Glut Tyr), 10 mM magnesium acetate and 0.05 mM [33P-y-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

175. QSK assay

QSK (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against a substrate peptide (Poly Glut Tyr) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 1 mg/ml Poly Glut Tyr, 10 mM magnesium acetate and 0.05 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

177. WNK1 assay

WNK1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against OSR1 in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 10mM MnCI, 1 mg/ml OSR1 , 10 mM magnesium acetate and 0.02 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 178. CDK9/Cyclin T1 assay

CDK9/Cyclin T1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against a substrate peptide (YSPTSPSYSPTSPSYSPTSPKKK) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 1 mg/ml BSA, 0.3mM YSPTSPSYSPTSPSYSPTSPKKK, 10 mM magnesium acetate and 0.05 mM [33P- γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 179. DDR2 assay

DDR2 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against a substrate peptide (KKSRGDYMTMQIG) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 0.3mM substrate peptide 10 mM magnesium acetate and 0.005 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 180. OSR1 assay

OSR1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against a substrate peptide (RRHKKKDTHTNTYYLRTFGHNTRR) in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 0.3mM substrate peptide, 0.12mg/ml GST- M025, 10 mM magnesium acetate and 0.005 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 181. ULK1 assay

ULK1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against MBP in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 0.33mg/ml substrate, 10 mM magnesium acetate and 0.02 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

182. ULK2 assay

ULK1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against MBP in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 0.33mg/ml substrate, 10 mM magnesium acetate and 0.005 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

183. TTBK2 assay

TTBK2 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against substrate peptide RRKDLHDDEEDEAMSITA in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 0.3mM substrate, 10 mM magnesium acetate and 0.005 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

184. MAP4K3 assay

MAP4K3 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against MBP in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 0.33 mg/ml substrate, 10 mM magnesium acetate and 0.02 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

185. MAP4K5 assay

MAP4K5 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against MBP in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 0.33 mg/ml substrate, 10 mM magnesium acetate and 0.02mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

187. PDGFRA assay

PDGFRA (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against substrate peptide KKKKEEIYFFFG in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 5mM MnCI₂ 0.3mM substrate, 10 mM magnesium acetate and 0.005 mM [33Ρ-γ- ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

189. TGFBR1 assay

TGFBR1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against substrate peptide RRKVLTQMGSPSIRCSS^*VS in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 1 mM DTT, 5mM MnCI₂, 0.3mM substrate, 10 mM magnesium acetate and 0.05 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

190. ERK5 assay

ERK5 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against MBP in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 0.33mg/ml substrate, 10 mM magnesium acetate and 0.05mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid. 194. PINK1 assay

PINK1 (5-20mU diluted in 50 mM Tris pH 7.5, 0.1 mM EGTA, 1 mg/ml BSA, 0.1 % Mercaptoethanol) is assayed against GST PARK2 TV3 in a final volume of 25.5 μΙ containing 50mM Tris pH 7.5, 0.1 mM EDTA, 10mM DTT, 0.3mg/ml substrate, 10 mM magnesium acetate and 0.005 mM [33Ρ-γ-ΑΤΡ] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 μΙ of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.

Results Data showing residual kinase activity at 100 micromolar inhibitor concentration is given in the table which follows. The values are on an absolute scale such that 100 is no change in the presence of putatitic inhibitor, any value over 100 shows kinase activation. Any value less that 25 is considered to be highly significant inhibition.

DATA SHOWS RESIDUAL KINASE ACTIVITY AT 100 MICROMOLAR INHIBITOR CONCENTRATION

PKBa 50 52 97 75 86 49 50 14 25 17 8 53 74

PKBb 17 31 57 41 53 23 25 11 8 36 2 76 66

SGK1 45 51 81 82 72 44 38 19 33 31 23 84 24

S6K1 44 71 84 62 84 74 17 15 15 15 29 58 20

PKA 22 81 96 100 67 79 66 7 26 11 80 39 83

ROCK 2 13 64 86 77 74 36 14 11 17 11 93 29 58

PRK2 17 53 78 81 71 54 39 9 35 15 87 12 49

PKCa 27 82 77 82 81 87 73 7 12 5 90 31 82

PKCy 56 79 76 77 82 86 68 23 15 28 116 54 66

PKCz 46 72 101 80 92 79 54 6 39 33 91 92 34

PKD1 35 44 76 29 68 43 17 11 31 31 51 74 79

STK33 31 60 67 79 81 67 42 25 35 25 101 46 82

MSK1 28 26 73 74 83 41 9 7 14 8 33 57 45

MNK1 27 23 69 25 72 44 26 20 21 11 81 4 76

MNK2 30 25 71 58 75 66 22 16 27 19 94 21 90

MAPKAP-K2 70 77 90 74 85 69 79 55 99 94 93 68 21

MAPKAP-K3 36 44 86 53 76 52 44 49 30 54 33 87 62

PRAK 82 89 107 80 101 72 56 36 52 72 78 81 73

CAMKKb 19 36 83 46 71 47 47 17 20 20 69 45 74

CAMK1 8 23 39 56 83 50 17 7 8 19 32 60 38

YES1 8 39 99 60 55 24 48 5 4 3 99 67 94

ABL 11 56 99 87 40 37 61 5 11 27 67 82 103

BTK 17 24 36 41 11 34 28 45 8 3 14 32 69

JAK2 48 70 79 82 89 70 73 18 28 22 104 23 86

SYK 29 57 87 66 81 44 66 17 34 11 26 93 84

ZAP70 84 90 98 93 93 94 83 43 123 56 188 125 130

TIE2 52 101 108 101 79 98 94 34 9 10 57 60 60

BRK 2 13 51 35 4 17 4 2 4 24 39 33 107

EPH-A2 9 38 81 57 20 55 47 24 15 13 62 50 93

EPH-A4 9 47 69 51 37 42 44 13 21 17 66 61 79

EPH-B1 6 63 83 86 39 76 73 5 47 22 81 79 158

EPH-B2 2 41 112 65 56 46 38 2 5 4 66 81 120

EPH-B3 5 37 59 58 12 55 46 6 6 6 30 9 124

EPH-B4 14 72 109 90 88 74 69 10 6 4 67 81 90

FGF-R1 6 26 75 70 61 36 37 0 5 6 26 64 89

HER4 3 33 83 62 10 40 39 2 1 2 52 47 118

IGF-1R 3 9 55 57 24 15 13 4 3 2 16 70 5

IR 1 15 75 57 31 21 26 1 4 3 50 114 64

IRR 11 39 55 43 60 38 35 11 10 5 56 30 74

TrkA 5 26 64 40 23 26 33 6 9 5 11 62 53

Claims

1 . A compound incorporating a group of formula (I)

(I) wherein:

1 of X, Y and Z is nitrogen and the other 2 are carbon;

V is sulpur or carbon;

R₃ is oxygen or fluorine;

R₄ is an optionally present Ci_-3 alkyl group optionally substituted by fluorine;

R₅ is an optionally present cyclic group with 5-7 heavy atoms in the ring which may be carbocyclic or heterocyclic and aromatic or aliphatic and is optionally substituted;

W represents hydrogen, carbon, nitrogen, oxygen or sulphur;

or incorporating a salt, hydrate or solvate of a group of formula (I).

2. A compound as claimed in claim 1 of formula (II)

(II) wherein:

X, Y, Z, m, n, V, R₃, R₄ and R₅ are as defined in claim 1 ; and

R\ together with the carbonyl group to which it is attached, is a carboxylic acid derivative, preferably a carboxylic acid derivative selected from an aldehyde, a ketone, an amide, an ester, a thioester, an acyl phosphate and an acyl

hydroxamate;

or a salt, hydrate or solvate of a compound of formula (II).

3. A compound as claimed in claim 1 or claim 2 wherein Y or X is nitrogen, preferably Y is nitrogen.

4. A compound as claimed in any preceding claim wherein the hydroxyl of the phenol moiety is in the meta or para position, preferably the para position.

5. A compound as claimed in any preceding claim wherein R₃ is oxygen.

6. A compound as claimed in any preceding claim wherein R₅ is a cyclic group substituted by a halogen, Ci_-2 alkyl or fluoroalkyl, OH, OR₆, cyano, COOR₆, CONHR₆, sulfonamide or NHR₆, in which R₆ is a Ci_-2 alkyl or fluoroalkyl.

7. A compound as claimed in any preceding claim wherein the cyclic group of R₅ is selected from the group consisting of phenyl, pyrrole, pyridine, pyrimidine, thiazole, oxazole, piperidine and morpholine.

8. A compound as claimed in any preceding claim wherein -V(R₃)_n (R4R₅)_m is selected from the group consisting of phenylsulfonyl, cyclohexylsulfonyl, pyrrolylsulfonyl, pyridylsulfonyl, pyrimidinesulfonyl, thiazolylsulfonyl and oxazolylsulfonyl.

9. A compound as claimed in any one of claims 2 to 8 wherein R' is selected from hydrogen, -NH₂ ,- OR-i , -Ri or -NR₁ R₂ in which R₂ is preferably hydrogen but may be a further R-i group and Ri is a Ci-n alkyl, alkenyl, alkynyl or alkylamine, which may be linear or branched, or R-i may comprise or consist of 1 -3 cyclic groups which may be fused or linked.

10. A compound as claimed in claim 9 wherein R' comprises no more than 18 non-hydrogen atoms.

1 1 . A compound as claimed in claim 9 or claim 10 wherein Ri incorporates 1 -3 cyclic groups, preferably 1 or 2 cyclic groups.

12. A compound as claimed in claim 1 1 wherein one or more of the cyclic groups is selected from the group consisting of cyclohexyl, phenyl, pyridine, pyrazole, imidazole, thiazole, oxazole, furan, piperazine, pyrroline, pyrrolidine, diazolone, morpholine and piperidine.

13. A compound as claimed in claim 1 1 or 12 wherein the cyclic group is substituted by one or more groups selected from -OH, -OR, -NRR, -C=0-OR, -C=0-NRR, -SO2-NRR, -NR-SO2R, -SO2R, -NR-C=OR, a halogen, and d_-3 alkyl or fluoroalkyi, wherein each R which may be the same or different, is H or Ci_-3 alkyl.

14. A compound as claimed in any one of claims 1 1 to 13 wherein two cyclic groups are linked by a linking group of 1 -10 non-hydrogen atoms.

15. A compound as claimed in claim 14 wherein the linking group is selected from -CH2-, -NR-, -0-, -S-, -CHRCHR-, -CH=CH-, -C=0-0-, -C=0-NR-, -0-CHR-, -NR-CHR-, -NR-C=0-NR-, -NR-C=0-0- and -CH₂CH₂CH₂-; wherein R is H or Ci_-2 alkyl.

16. A compound as claimed in claim 2 wherein R' is hydrogen, -OCH₃, - OCH₂CH₃, -OCH₂CH₂CH₃ , -OCH₂CH₂CH₂CH₃ , -NHCH₃, -NHCH₂CH₃, - NHCH₂CH₂CH₃, -NHCH₂CH₂OH or -HCH₂CH₂N(CH₂CH₃)₂.

17. A compound as claimed in any preceding claim for use in therapy.

18. A compound for use as claimed in claim 17 for the treatment of a disease or other pathological condition associated with aberrant protein kinase activity or which would benefit from a reduction in protein kinase activity.

19. A compound for use as claimed in claim 17 or 18 for the treatment of inflammation, inflammatory conditions or cancer.

20. A method of inhibiting a protein kinase and/or a reaction catalysed by a protein kinase, the method comprising contacting said kinase with a compound as defined in any one of claims 1 to 16.

21 . A pharmaceutical formulation comprising a compound as defined in any one of claims 1 to 16 in admixture with a suitable diluent, carrier or excipient.