Thieno-pyridine derivatives as MEK inhibitors

A series of thieno[2,3-b]pyridine derivatives, attached at the 2-position to a substituted anilino moiety, which are substituted in the 3-position by a carbonyl group linked to a pyrrolidin-1-yl ring which in turn forms part of a heteroatom-containing fused bicyclic ring system, being selective inhibitors of human MEK (MAPKK) enzymes, are accordingly of benefit in medicine, for example in the treatment of inflammatory, autoimmune, cardiovascular, proliferative (including oncological) and nociceptive conditions.

The present invention relates to a class of thieno-pyridine derivatives and to their use in therapy. More particularly, the invention is concerned with thieno[2,3-b]pyridine derivatives which are substituted in the 2-position by a substituted anilino moiety. These compounds are selective inhibitors of MEK (MAPKK) enzymes, and are accordingly of benefit as pharmaceutical agents, especially in the treatment of adverse inflammatory, autoimmune, cardiovascular, proliferative (including oncological) and nociceptive conditions.

MEK enzymes are implicated in a variety of physiological and pathological functions that are believed to be operative in a range of human diseases. These functions are summarised in paragraphs [0004] and [0005] of US 2005/0049276 A1.

The compounds of use in the present invention, being potent and selective MEK inhibitors, are therefore beneficial in the treatment and/or prevention of various human ailments. These include autoimmune and inflammatory disorders such as rheumatoid arthritis, osteoarthritis, multiple sclerosis, asthma, inflammatory bowel disease, psoriasis and transplant rejection; cardiovascular disorders including thrombosis, cardiac hypertrophy, hypertension, and irregular contractility of the heart (e.g. during heart failure); proliferative disorders such as restenosis, and oncological conditions including leukaemia, glioblastoma, lymphoma, melanoma, and human cancers of the liver, bone, skin, brain, pancreas, lung, breast, stomach, colon, rectum, prostate, ovary and cervix; and pain and nociceptive disorders, including chronic pain and neuropathic pain.

In addition, the compounds of use in the present invention may be beneficial as pharmacological standards for use in the development of new biological tests and in the search for new pharmacological agents. Thus, the compounds of use in this invention may be useful as radioligands in assays for detecting compounds capable of binding to human MEK enzymes.

MEK inhibitors based on a fused bicyclic aromatic ring system attached to a substituted anilino moiety are known from the art, such as from WO 2007/088345.

Nowhere in the prior art, however, is there the precise disclosure of a class of thieno[2,3-b]pyridine derivatives, attached at the 2-position to a substituted anilino moiety, which are substituted in the 3-position by a carbonyl group linked to a pyrrolidin-1-yl ring which in turn forms part of a heteroatom-containing fused bicyclic ring system. It has now been found that such compounds are particularly valuable as selective inhibitors of MEK enzymes.

The compounds of the present invention are potent and selective MEK inhibitors having a binding affinity (IC50) for the human MEK1 and/or MEK2 enzyme of 50 μM or less, generally of 20 μM or less, usually of 5 μM or less, typically of 1 μM or less, suitably of 500 nM or less, ideally of 100 nM or less, and preferably of 20 nM or less (the skilled person will appreciate that a lower IC50figure denotes a more active compound). The compounds of the invention may possess at least a 10-fold selective affinity, typically at least a 20-fold selective affinity, suitably at least a 50-fold selective affinity, and ideally at least a 100-fold selective affinity, for the human MEK1 and/or MEK2 enzyme relative to other human kinases.

The compounds of the present invention possess high potency, and interesting pharmacokinetic properties owing to their improved solubility and clearance.

The present invention provides a compound of formula (I), or a pharmaceutically acceptable salt, solvate or N-oxide thereof:

wherein

Q represents the residue of a three-, four-, five- or six-membered heterocyclic ring optionally containing one or two double bonds, wherein the heterocyclic ring comprises one, two or three heteroatoms independently selected from oxygen, sulphur, nitrogen and phosphorus;

R3and R4, when both are attached to the same carbon atom, represent, when taken together with the carbon atom to which they are both attached, C3-7cycloalkyl or C3-7heterocycloalkyl, either of which groups may be optionally substituted by one or more substituents independently selected from C1-6alkyl, hydroxy and amino; and

R5, where present, represents hydrogen or C1-6alkyl.

The present invention also provides a compound of formula (I) as depicted above, or a pharmaceutically acceptable salt, solvate or N-oxide thereof, wherein

Q represents the residue of a three-, four-, five- or six-membered heterocyclic ring optionally containing a double bond, wherein the heterocyclic ring comprises one, two or three heteroatoms independently selected from oxygen, sulphur, nitrogen and phosphorus;

R3and R4, when both are attached to the same carbon atom, represent, when taken together with the carbon atom to which they are both attached, C3-7cycloalkyl or C3-7heterocycloalkyl, either of which groups may be optionally substituted by one or more substituents independently selected from C1-6alkyl, hydroxy and amino; and

For use in medicine, the salts of the compounds of formula (I) will be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds of the invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts which may, for example, be formed by mixing a solution of the compound of the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid or phosphoric acid. Furthermore, where the compounds of the invention carry an acidic moiety, e.g. carboxy, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g. sodium or potassium salts; alkaline earth metal salts, e.g. calcium or magnesium salts; ammonium salts; and salts formed with suitable organic ligands, e.g. quaternary ammonium salts.

The present invention includes within its scope solvates of the compounds of formula (I) above. Such solvates may be formed with common organic solvents, e.g. hydrocarbon solvents such as benzene or toluene; chlorinated solvents such as chloroform or dichloromethane; alcoholic solvents such as methanol, ethanol or isopropanol; ethereal solvents such as diethyl ether or tetrahydrofuran; or ester solvents such as ethyl acetate. Alternatively, the solvates of the compounds of formula (I) may be formed with water, in which case they will be hydrates.

Suitable alkyl groups which may be present on the compounds of the invention include straight-chained and branched C1-6alkyl groups, for example C1-4alkyl groups. Typical examples include methyl and ethyl groups, and straight-chained or branched propyl, butyl and pentyl groups. Particular alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl and 2,2-dimethylpropyl. Derived expressions such as “C1-6alkoxy”, “C1-6alkylthio”, “C1-6alkylsulphonyl” and “C1-6alkylamino” are to be construed accordingly.

Suitable C2-6alkynyl groups include ethynyl and prop-2-yn-1-yl.

Suitable aryl groups include phenyl and naphthyl, preferably phenyl.

The term “halogen” as used herein is intended to include fluorine, chlorine, bromine and iodine atoms.

Where the compounds of formula (I) have one or more asymmetric centres, they may accordingly exist as enantiomers. Where the compounds of the invention possess two or more asymmetric centres, they may additionally exist as diastereomers. The invention is to be understood to extend to all such enantiomers and diastereomers, and to mixtures thereof in any proportion, including racemates. Formula (I) and the formulae depicted hereinafter are intended to represent all individual stereoisomers and all possible mixtures thereof, unless stated or shown otherwise. In addition, compounds of formula (I) may exist as tautomers, for example keto (CH2C═O)enol (CH═CHOH) tautomers or amide (NHC═O)hydroxyimine (N═COH) tautomers. Formula (I) and the formulae depicted hereinafter are intended to represent all individual tautomers and all possible mixtures thereof, unless stated or shown otherwise.

It is to be understood that each individual atom present in formula (I), or in the formulae depicted hereinafter, may in fact be present in the form of any of its naturally occurring isotopes, with the most abundant isotope(s) being preferred. Thus, by way of example, each individual hydrogen atom present in formula (I), or in the formulae depicted hereinafter, may be present as a1H,2H (deuterium or3H (tritium) atom, preferably1H. Similarly, by way of example, each individual carbon atom present in formula (I), or in the formulae depicted hereinafter, may be present as a12C,13C or14C atom, preferably12C.

Suitable values of R1include hydrogen, halogen and C1-6alkyl. In one embodiment, R1represents hydrogen. In a particular embodiment, R1represents halogen, especially fluoro or chloro. In another embodiment, R1represents C1-6alkyl, especially methyl.

In one embodiment, the heterocyclic ring of which Q is the residue is a three-membered heterocyclic ring. In another embodiment, the heterocyclic ring of which Q is the residue is a four-membered heterocyclic ring. In a further embodiment, the heterocyclic ring of which Q is the residue is a five-membered heterocyclic ring. In an additional embodiment, the heterocyclic ring of which Q is the residue is a six-membered heterocyclic ring.

The heterocyclic ring of which Q is the residue will typically be a four-, five- or six-membered heterocyclic ring. The heterocyclic ring of which Q is the residue will suitably be a five- or six-membered heterocyclic ring.

In one embodiment, the heterocyclic ring of which Q is the residue is fully saturated, i.e. there is no double bond contained within the ring. In another embodiment, the heterocyclic ring of which Q is the residue is unsaturated, i.e. the ring contains one or two double bonds. In one aspect of that embodiment, the heterocyclic ring of which Q is the residue contains one double bond within the ring. In another aspect of that embodiment, the heterocyclic ring of which Q is the residue contains two double bonds within the ring.

Ideally, Q represents the residue of a three-, four-, five- or six-membered heterocyclic ring optionally containing a double bond, wherein the heterocyclic ring comprises one, two or three heteroatoms independently selected from oxygen, sulphur, nitrogen and phosphorus.

Typically, Q represents the residue of a four-, five- or six-membered heterocyclic ring optionally containing a double bond, wherein the heterocyclic ring comprises one, two or three heteroatoms independently selected from oxygen, sulphur, nitrogen and phosphorus.

Suitably, Q represents the residue of a five- or six-membered heterocyclic ring optionally containing a double bond, wherein the heterocyclic ring comprises one, two or three heteroatoms independently selected from oxygen, sulphur, nitrogen and phosphorus.

Depending upon the nature of the heterocyclic ring of which Q is the residue, it may not be possible to accommodate all three substituents R3, R4and R5around this ring. Thus, for certain values of Q, it will be appreciated that the substituent designated R5will be absent; and likewise, for certain other values of Q, it will be appreciated that the substituents designated R4and R5will both be absent. Moreover, for certain particular values of Q, it will be appreciated that the substituents designated R3, R4and R5will all be absent.

Alternatively, R3and R4may together form an optionally substituted spiro linkage. Thus, R3and R4, when taken together with the carbon atom to which they are both attached, may represent C3-7cycloalkyl or C3-7heterocycloalkyl, either of which groups may be unsubstituted, or substituted by one or more, typically by one or two, substituents independently selected from C1-6alkyl, hydroxy and amino. In this context, R3and R4, when taken together with the carbon atom to which they are both attached, may suitably represent an optionally substituted cyclopentyl, cyclohexyl, pyrrolidine or piperidine ring. In particular, R3and R4, when taken together with the carbon atom to which they are both attached, may suitably represent an optionally substituted piperidine ring.

Particular sub-classes of compounds in accordance with the present invention are represented by the compounds of formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IJ), (IK), (IL), (IM), (IN) and (IP):

wherein

T represents oxygen, sulphur or NH;

X represents oxygen, sulphur or N—R4a;

R6arepresents hydrogen or C1-6alkyl; and

Specific sub-classes of compounds in accordance with the present invention are represented by the compounds of formula (IA), (IB), (IC), (ID), (IE), (IF), (IG) and (IH) as depicted above, wherein R1, R2, T, X, Y, R3a, R4aand R6aare as defined above.

Suitably, T represents oxygen or sulphur.

In one embodiment, T represents oxygen. In another embodiment, T represents sulphur. In a further embodiment, T represents NH.

In one embodiment, X represents oxygen. In another embodiment, X represents sulphur. In a further embodiment, X represents N—R4a.

Suitably, Y represents oxygen, sulphur or N—R5, in which R5is as defined above.

Typically, Y represents oxygen or N—R5.

In one embodiment, Y represents oxygen. In another embodiment, Y represents sulphur. In another embodiment, Y represents N—R5. In a further embodiment, Y represents CH2.

The ring fusion between the pyrrolidine ring and its neighbouring fused ring in the compounds of formula (IA) to (IG) as depicted above, and also in the compounds of formula (IJ), (IK), (IL), (IN) and (IP) as depicted above, is suitably in the cis configuration, giving rise to the following compounds:

The ring fusion between the pyrrolidine ring and its neighbouring fused ring in the compounds of formula (IH) as depicted above may be in the cis or trans configuration, giving rise to the following compounds:

wherein R1, R2, T and R4aare as defined above.

One sub-class of compounds according to the invention is represented by the compounds of formula (IIA), and pharmaceutically acceptable salts, solvates and N-oxides thereof:

wherein

In one specific embodiment, R11is fluoro. In another specific embodiment, R11is chloro.

Specific novel compounds in accordance with the present invention include each of the compounds whose preparation is described in the accompanying Examples, and pharmaceutically acceptable salts and solvates thereof.

The present invention also provides a pharmaceutical composition which comprises a compound of formula (I) as defined above, or a pharmaceutically acceptable salt, solvate or N-oxide thereof, in association with one or more pharmaceutically acceptable carriers.

Pharmaceutical compositions according to the invention may take a form suitable for oral, buccal, parenteral, nasal, topical, ophthalmic or rectal administration, or a form suitable for administration by inhalation or insufflation.

For oral administration, the pharmaceutical compositions may take the form of, for example, tablets, lozenges or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methyl cellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium glycollate); or wetting agents (e.g. sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents, emulsifying agents, non-aqueous vehicles or preservatives. The preparations may also contain buffer salts, flavouring agents, colouring agents or sweetening agents, as appropriate.

Preparations for oral administration may be suitably formulated to give controlled release of the active compound.

The compounds of formula (I) may be formulated for parenteral administration by injection, e.g. by bolus injection or infusion. Formulations for injection may be presented in unit dosage form, e.g. in glass ampoules or multi-dose containers, e.g. glass vials. The compositions for injection may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising, preserving and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.

In addition to the formulations described above, the compounds of formula (I) may also be formulated as a depot preparation. Such long-acting formulations may be administered by implantation or by intramuscular injection.

For nasal administration or administration by inhalation, the compounds according to the present invention may be conveniently delivered in the form of an aerosol spray presentation for pressurised packs or a nebuliser, with the use of a suitable propellant, e.g. dichlorodifluoromethane, fluorotrichloromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas or mixture of gases.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack or dispensing device may be accompanied by instructions for administration.

For topical administration the compounds according to the present invention may be conveniently formulated in a suitable ointment containing the active component suspended or dissolved in one or more pharmaceutically acceptable carriers. Particular carriers include, for example, mineral oil, liquid petroleum, propylene glycol, polyoxyethylene, polyoxypropylene, emulsifying wax and water. Alternatively, the compounds according to the present invention may be formulated in a suitable lotion containing the active component suspended or dissolved in one or more pharmaceutically acceptable carriers. Particular carriers include, for example, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, benzyl alcohol, 2-octyldodecanol and water.

For ophthalmic administration the compounds according to the present invention may be conveniently formulated as microionized suspensions in isotonic, pH-adjusted sterile saline, either with or without a preservative such as a bactericidal or fungicidal agent, for example phenylmercuric nitrate, benzylalkonium chloride or chlorhexidine acetate. Alternatively, for ophthalmic administration compounds may be formulated in an ointment such as petrolatum.

For rectal administration the compounds according to the present invention may be conveniently formulated as suppositories. These can be prepared by mixing the active component with a suitable non-irritating excipient which is solid at room temperature but liquid at rectal temperature and so will melt in the rectum to release the active component. Such materials include, for example, cocoa butter, beeswax and polyethylene glycols.

The quantity of a compound of the invention required for the prophylaxis or treatment of a particular condition will vary depending on the compound chosen and the condition of the patient to be treated. In general, however, daily dosages may range from around 10 ng/kg to 1000 mg/kg, typically from 100 ng/kg to 100 mg/kg, e.g. around 0.01 mg/kg to 40 mg/kg body weight, for oral or buccal administration, from around 10 ng/kg to 50 mg/kg body weight for parenteral administration, and from around 0.05 mg to around 1000 mg, e.g. from around 0.5 mg to around 1000 mg, for nasal administration or administration by inhalation or insufflation.

The compounds of formula (I) above may be prepared by a process which comprises reacting a compound of formula (III) with a compound of formula (IV):

The leaving group L1is typically a halogen atom, e.g. fluoro.

The reaction is conveniently effected in a suitable solvent, e.g. a dipolar aprotic solvent such as N,N-dimethylformamide, or a chlorinated solvent such as dichloromethane, typically under basic conditions, e.g. in the presence of an organic base such as N,N-diisopropylethylamine or diisopropylamine.

The intermediates of formula (III) wherein L1is fluoro may suitably be prepared by reacting a compound of formula (V):

The reaction is conveniently effected in a suitable solvent, e.g. dichloromethane.

The intermediates of formula (V) above may suitably be prepared by reacting a compound of formula (VI) with a compound of formula (VII):

wherein R1and R2are as defined above, and L2represents a suitable leaving group.

The leaving group L2is typically a halogen atom, e.g. chloro.

The reaction is conveniently effected, at an elevated temperature if necessary, in a suitable solvent, e.g. tetrahydrofuran, typically under basic conditions, e.g. in the presence of lithium diisopropylamide.

The intermediates of formula (VII) above may be prepared by the procedure described in WO 2007/088345.

The compounds of formula (IC), the compounds of formula (IE) wherein T and Y both represent oxygen, and the compounds of formula (IH) wherein T represents oxygen, all as depicted above, may be prepared by a process which comprises reacting a compound of formula (VIII), (IX) or (X) respectively:

The reaction is conveniently effected in a suitable solvent, e.g. N,N-dimethylformamide.

The compounds of formula (IH) as depicted above wherein T represents sulphur may be prepared by a process which comprises reacting a compound of formula (X) as defined above with 1,1′-thiocarbonyldiimidazole. The reaction is conveniently effected in a suitable solvent, e.g. N,N-dimethylformamide.

The intermediates of formula (VIII), (IX) and (X) above may suitably be prepared by reacting a compound of formula (III) as defined above with a compound of formula (XI), (XII) or (XIII) respectively:

wherein R4ais as defined above; under conditions analogous to those described above for the reaction between compounds (III) and (IV).

Alternatively, the intermediates of formula (X) above may be prepared by reacting a compound of formula (III) as defined above with a compound of formula (XIV):

wherein R4ais as defined above; under conditions analogous to those described above for the reaction between compounds (III) and (IV); followed by reduction of the methyl ester group, typically by using a standard reducing agent such as an alkali metal borohydride, e.g. lithium borohydride.

The compounds of formula (ID) as depicted above may be prepared by a process which comprises reacting a compound of formula (VIII) as defined above with a compound of formula (XV):

wherein R6ais as defined above.

The reaction is suitably effected under acidic conditions, typically in the presence of a catalytic quantity of p-toluenesulphonic acid, in which case the reaction is conveniently carried out at ambient or elevated temperature in an inert organic solvent, e.g. a hydrocarbon solvent such as toluene.

The compounds of formula (IF) as depicted above may be prepared by a process which comprises reacting a compound of formula (VIII) as defined above with a phosphoric acid dihalide ester, e.g. methyl dichlorophosphate.

The reaction is conveniently effected at an elevated temperature in a suitable solvent, e.g. an ethereal solvent such as tetrahydrofuran, typically under basic conditions, e.g. in the presence of an organic base such as N,N-diisopropylethylamine.

The compounds of formula (IL) as depicted above wherein X represents oxygen or N—R4amay be prepared by a process which comprises reacting a compound of formula (VIII) or (IX) respectively, as defined above, with a compound of formula (XVI), or a carbonyl-protected form thereof:

wherein R5and R3aare as defined above; under conditions analogous to those described above for the reaction between compounds (VIII) and (XV).

Suitable carbonyl-protected forms of the compounds of formula (XVI) include the dimethyl acetal or ketal derivatives thereof. A particular carbonyl-protected compound of formula (XVI) is 2,2-dimethoxypropane.

The compounds of formula (IG) as depicted above wherein X represents oxygen or sulphur and R3arepresents C1-6alkylamino or C3-7cycloalkylamino may be prepared by a process which comprises reacting a compound of formula (XVII):

In an alternative procedure, the compounds of formula (IE) as depicted above wherein T represents sulphur, Y represents NH and R4arepresents C1-6alkyl or C3-7cycloalkyl may also be prepared by a process which comprises reacting a compound of formula (XVII) as defined above, wherein Xarepresents sulphur, with DAST.

The reaction between compound (XVII) and DAST is conveniently effected in a suitable solvent, e.g. a chlorinated solvent such as dichloromethane.

The intermediates of formula (XVII) above may suitably be prepared by reacting a compound of formula (IX) as defined above wherein R4arepresents hydrogen with the appropriate isocyanate or isothiocyanate derivative of formula Ra—N═C═Xa, wherein Raand Xaare as defined above. The reaction may conveniently be effected in a suitable solvent, e.g. a cyclic ether such as tetrahydrofuran, or a chlorinated solvent such as dichloromethane.

Alternatively, the intermediates of formula (XVII) above may be prepared by reacting a compound of formula (III) as defined above with a compound of formula (XVIII):

wherein Xaand Raare as defined above; under conditions analogous to those described above for the reaction between compounds (III) and (IV).

The intermediates of formula (XVIII) above may be prepared by reacting a compound of formula (XII) as defined above wherein R4arepresents hydrogen, or a protected derivative thereof, e.g. a compound of formula (XII) having a tert-butoxycarbonyl protecting group attached at the 1-position of the pyrrolidine ring, with the appropriate isocyanate or isothiocyanate derivative of formula Ra—N═C═Xa, wherein Raand X1are as defined above, under conditions analogous to those described above for the reaction between compound (IX) wherein R4arepresents hydrogen and the compound of formula R1—N═C═Xa; followed, where necessary, by deprotection. Where the protecting group is a tert-butoxycarbonyl group attached at the 1-position of the pyrrolidine ring, deprotection may be effected by treatment with an acid, e.g. a mineral acid such as hydrochloric acid, or an organic acid such as trifluoroacetic acid.

Where they are not commercially available, the starting materials of formula (IV), (VI), (XI), (XII), (XIII), (XIV), (XV) and (XVI) may be prepared by methods analogous to those described in the accompanying Examples, or by standard methods well known from the art.

It will be understood that any compound of formula (I) initially obtained from any of the above processes may, where appropriate, subsequently be elaborated into a further compound of formula (I) by techniques known from the art. By way of example, a compound of formula (IA), (IB), (IE), (IH), (IJ), (IK), (IN) or (IP) wherein R4arepresents tert-butoxycarbonyl may be converted into the corresponding compound wherein R4arepresents hydrogen by treatment with an acid, typically an organic acid such as trifluoroacetic acid, or a mineral acid such as hydrochloric acid.

A compound of formula (IE) wherein R4arepresents hydrogen may be converted into the corresponding compound wherein R4arepresents morpholin-4-ylmethyl by treatment with morpholine and formaldehyde, typically at an elevated temperature.

The pyridine-N-oxide derivative of a compound of formula (I) may be converted into the corresponding compound of formula (I) by treatment with triphenylphosphine and phosphorus trichloride.

Where a mixture of products is obtained from any of the processes described above for the preparation of compounds according to the invention, the desired product can be separated therefrom at an appropriate stage by conventional methods such as preparative HPLC; or column chromatography utilising, for example, silica and/or alumina in conjunction with an appropriate solvent system.

Where the above-described processes for the preparation of the compounds according to the invention give rise to mixtures of stereoisomers, these isomers may be separated by conventional techniques. In particular, where it is desired to obtain a particular enantiomer of a compound of formula (I) this may be produced from a corresponding mixture of enantiomers using any suitable conventional procedure for resolving enantiomers. Thus, for example, diastereomeric derivatives, e.g. salts, may be produced by reaction of a mixture of enantiomers of formula (I), e.g. a racemate, and an appropriate chiral compound, e.g. a chiral base. The diastereomers may then be separated by any convenient means, for example by crystallisation, and the desired enantiomer recovered, e.g. by treatment with an acid in the instance where the diastereomer is a salt. In another resolution process a racemate of formula (I) may be separated using chiral HPLC. Moreover, if desired, a particular enantiomer may be obtained by using an appropriate chiral intermediate in one of the processes described above. Alternatively, a particular enantiomer may be obtained by performing an enantiomer-specific enzymatic biotransformation, e.g. an ester hydrolysis using an esterase, and then purifying only the enantiomerically pure hydrolysed acid from the unreacted ester antipode. Chromatography, recrystallisation and other conventional separation procedures may also be used with intermediates or final products where it is desired to obtain a particular geometric isomer of the invention.

The following Examples illustrate the preparation of compounds according to the invention.

The compounds in accordance with this invention potently inhibit the activity of human MEK enzyme.

In Vitro MEK Assay

MEK1 activity was measured in a cascade assay initiated by active Raf, via activation of MEK, Erk2 and subsequent phosphorylation of fluorescein-labelled Erk-tide substrate in an assay based on fluorescence polarisation (IMAP). The assay was carried out in 20 mM Tris+5 mM MgCl2+2 mM DL-dithiothreitol+0.01% Tween 20 pH 7.2, containing 1.5 nM unactive MEK, 100 nM unactive Erk and 200 nM Erk-tide (all concentrations are final concentrations). Compounds, or DMSO controls, were tested at a final concentration of 2% DMSO, and the assay initiated in the presence of 5 μM ATP by addition of 1.25 nM active Raf in assay buffer. After 20 min at r.t., stop solution was added followed by IMAP binding beads, the assay mixture was then incubated for 90 min at r.t. (with shaking) and then read on a Molecular Devices LJL HT reader.

When tested in the above assay, the compounds of the accompanying Examples were all found to inhibit human MEK enzyme with IC50values of 10 μM or better.

EXAMPLES

Abbreviations

r.t.: room temperature

RT: retention time

HPLC: high performance liquid chromatography

LCMS: liquid chromatography mass spectrometry

All NMR spectra were obtained either at 300 MHz or 400 MHz.

Compounds were named with the aid of ACD Labs Name (v. 7.0) supplied by Advanced Chemical Development, Toronto, Canada.

Standard LCMS Method

The LC-MS system used comprises a Waters Alliance 2795 HT quaternary HPLC, Waters 996 Photo Diode Array (PDA) detector and Waters ZQ 4000 single quadrupole mass spectrometer. The ZQ can acquire data simultaneously in positive and negative electrospray ionisation modes.

ZQ Mass Spectrometer

Data were acquired in a full scan from 100 to 1000 m/z.

Analytical reverse phase separation was carried out on a Gemini C18 from Phenomenex 50×4.6 mm with 5 μm silica.

The LC system comprises a Waters 2525 quaternary pump, a Waters 996 Photo Diode Array (PDA) detector, a Waters 2700 sample manager, a Column Fluidics Organiser and a Waters Fraction Collector operating in reverse phase at one of two pH systems.

Low pH System (Approximately pH 3.2)

The reverse phase separation was carried out on a Luna C18 from Phenomenex 100×21.2 mm with 5 μm silica.

The reverse phase separation was carried out on a Gemini C18 from Phenomenex 150×21.2 mm with 10 μm silica.

Typical gradient profiles are described below:

To a 500 mL, round-bottom, 3-necked flask equipped with dropping funnel and magnetic stirrer and set for reflux was prepared a solution of 2-chloro-3-(hydroxymethyl)-pyridine (25.0 g, 174 mmol) in DCM (250 mL) under positive nitrogen atmosphere. The solution was cooled to 10° C. and thionyl chloride (31.0 g) was added dropwise over 25 minutes (exothermic). The reaction was then heated to reflux for 90 minutes, at which point the reaction was deemed complete by HPLC. The reaction mixture was cooled below boiling point and the equipment set for distillation. A total of 110 mL of DCM was initially removed and replenished with fresh DCM (110 mL), followed by another 80 mL of DCM before cooling the solution to 5-10° C. The acidic mixture was treated with a saturated solution of sodium bicarbonate (3 volumes) to pH 10. The lower organic phase was separated and the aqueous phase extracted with DCM (2 volumes). The organic phases were gathered, dried on sodium sulfate, filtered and concentrated in vacuo to afford the title compound as a pale yellow oil in excellent purity and yield (24.8 g, 88%). δH(d6-DMSO, 300 MHz) 8.45 (1H, dd), 8.10 (1H, dd), 7.50 (1H, dd), 4.85 (2H, s). LCMS (ES)+RT 3.00 min, m/e 162.1.

In a 3 L reactor, set for reflux under positive nitrogen pressure and using a bleach scrubber, was prepared a solution of potassium cyanide (68.32 g, 1.04M) in EtOH (136 mL) and water (255 mL). The mixture was heated to reflux, at which point a solution of 2-chloro-3-(chloromethyl)pyridine (Intermediate 1; 170.0 g, 1.04M) in EtOH (170 mL) was added dropwise over 30 minutes. The whole mixture was maintained at reflux for a further 150 minutes. The mixture was then allowed to cool just below boiling point and the equipment set for distillation. A total of 8.5 volumes of EtOH were removed. On cooling, half a volume of water was added. At a temperature of 40° C., the solution was seeded and crystallised instantaneously. The thick beige slurry was allowed to cool to ambient temperature and then to 0° C. This mixture was filtered, rinsed with cold water (2 vols) and dried at 45° C. in a vacuum oven overnight. The title compound was afforded as a beige solid in excellent yield and purity (126.9 g, 80%). δH(d6-DMSO, 300 MHz) 8.45 (1H, dd), 8.00 (1H, dd), 7.50 (1H, dd), 4.15 (2H, s). LCMS (ES)+RT 2.15 min, m/e 153.01 & 155.01 (M+1 & M+3, Product).

To a 2 L reactor, set for reflux, was stirred a pre-prepared 15% w/w solution of sodium hydroxide (5 vols) to which was added (2-chloropyridin-3-yl)acetonitrile (Intermediate 2; 276.4 g, 1.81M). The beige suspension was heated to reflux for 30 minutes, at which point the reaction was deemed complete by HPLC. The brown solution was then cooled to 0-5° C. and acidified to pH 1 with conc. HCl while keeping the temperature below 10° C., using concentrated hydrochloric acid (1.8 vols). An off-white solid precipitated and was left to mature for another hour before filtration. Once dried, the material was recrystallised from propan-2-ol (4 vols) to afford the title compound as an off-white material in excellent yield and purity (280.3 g, 90%). δH(d6-DMSO, 300 MHz) 12.70 (1H, s), 8.35 (1H, dd), 7.85 (1H, dd), 7.40 (1H, dd), 4.25 (2H, s). LCMS (ES)+RT 1.75 min, m/e 171.99 (M+1, Product).

To a stirred solution of diisopropylamine (35.3 mL, 250 mmol) in anhydrous THF (200 mL) cooled to −15° C. was added n-butyllithium (100 mL, 2.5M in hexanes, 250 mmol) slowly such that an internal temperature of between −10 and 0° C. was maintained. The resultant mixture was stirred at room temperature for 15 minutes before being cooled to 0° C. The solution of lithium diisopropylamide was added via cannula to a rapidly stirred suspension of (2-chloropyridin-3-yl)acetic acid (Intermediate 3; 21.4 g, 125 mmol) in anhydrous THF (400 mL) at 0° C. The temperature of the reaction mixture was maintained at 0° C. over the course of the addition. Upon complete addition of the lithium diisopropylamide solution the resultant bright yellow suspension was stirred at 0° C. for 15 minutes. A solution of 2-fluoro-4-iodo-1-isothiocyanatobenzene (WO 2007/088345) (34.9 g, 125 mmol) in anhydrous THF (200 mL) was then added to the reaction mixture via cannula and the mixture heated to 65° C. for 18 hours. The reaction mixture was cooled and the volatiles removed in vacuo. The resultant brown gum was redissolved in THF (200 mL), cooled to 0° C. and 10% aqueous acetic acid (500 mL) added slowly. Acetonitrile (˜200 mL) was added slowly until a brown solid developed; the solid was isolated by filtration and washed with successive portions of diethyl ether and acetonitrile to give the title compound as a yellow crystalline solid (11.0 g, 21%). δH(DMSO-d6) 8.42 (1H, d, J 6.7 Hz), 8.22 (1H, m), 7.73 (1H, m), 7.61 (1H, m), 7.46 (1H, t, J 8.6 Hz), 7.35-7.31 (1H, m). Exchangeable protons were not observed. LCMS (pH 10) RT 1.82 minutes, ES+415 (M+H)+, ES−413 (M−H)−.

Intermediate 4 (7.80 g, 18.7 mmol) was suspended in dichloromethane (200 mL). DAST (2.80 mL, 20.8 mmol) was added and the mixture was stirred at room temperature for 3 hours. Ice cold water (3 mL) was added and stirring was continued for 5 minutes. Sodium sulfate (˜25 g) was then added to absorb the water and dry solvent. After filtration, the filtrate was passed through a 70 g pre-packed silica column and eluted with dichloromethane (1 L). All the eluent was collected and concentrated in vacuo to give the title compound as a pale yellow solid (4.80 g, 61%). δH(DMSO-d6) 10.03 (1H, br s), 8.33 (1H, dd, J 1.4, 4.7 Hz), 8.09 (1H, d, J 8.1 Hz), 7.91 (1H, dd, J 1.8, 9.7 Hz), 7.73 (1H, dd, J 1.0, 8.3 Hz), 7.49-7.42 (2H, m).

To a solution of tert-butyl 2,5-dihydro-1H-pyrrole-1-carboxylate (10 g, 0.059 mol) in DCM (200 mL) was added MCPBA (14.5 g, 0.065 mol) and the mixture stirred for 18 h. After this time the mixture was washed with water (50 mL) and sodium bicarbonate solution (50 mL), dried (Na2SO4), filtered and the volatiles evaporated in vacuo. The crude product was purified by chromatography (SiO2; 10% ethyl acetate in DCM), yielding the title compound as a colourless oil (6.2 g, 59%). δH(DMSO-d6) 3.74 (2H, m), 3.57 (2H, m), 3.25 (2H, m), 1.38 (9H, s).

To a solution of Intermediate 11 (5.1 g, 17.1 mmol) in MeOH (50 mL) was added TFA (3 drops) and the mixture stirred for 2 h. After this time the mixture was treated with platinum oxide (100 mg, 0.44 mmol) and stirred under a hydrogen atmosphere for 18 h. The mixture was then filtered through celite and the solvent evaporated in vacuo. The crude solid was recrystallised from DCM to afford the title compound as a white solid (2.42 g, 71%). δH(CDCl3) 3.99 (1H, m), 3.70 (2H, m), 3.34 (2H, m), 3.12 (1H, m), 1.48 (9H, s).

In an alternative procedure, the hydrochloride salt of the title compound was prepared as a colourless solid from Intermediate 95 by hydrogenation under the conditions described below for the preparation of Intermediate 80.

Intermediate 16 (571 mg, 2.75 mmol) was treated with 4M HCl in 1,4-dioxane (5 mL) and allowed to stir for 3 h at room temperature. After this time the solvent was removed in vacuo to afford the hydrochloride salt of the title compound as an off-white solid (383 mg, 97%). δH(DMSO-d6) 9.30 (2H, m), 5.32 (2H, m), 4.09 (2H, m), 3.21 (2H, m), 2.95 (2H, m).

To a solution of Intermediate 19 (2.5 g, 8.4 mmol) in MeOH (50 mL) was added TFA (3 drops) and the mixture stirred for 2 h. After this time the mixture was treated with platinum oxide (100 mg, 0.44 mmol) and stirred under a hydrogen atmosphere for 18 h. The mixture was then filtered through celite and the solvent evaporated in vacuo. The crude solid was recrystallised from DCM to afford the title compound as a white solid (1.5 g, 89%). δH(CDCl3) 3.99 (1H, m), 3.70 (2H, m), 3.34 (2H, m), 3.12 (1H, m), 1.48 (9H, s).

To a solution of Intermediate 21 (814 mg, 2.75 mmol) in DCM (20 mL) was added triethylamine (0.46 mL, 3.3 mmol). After cooling to 0° C. methanesulfonyl chloride (0.24 mL, 3.0 mmol) was added and, after stirring for 5 minutes at 0° C., the mixture was allowed to warm up to room temperature and stirred for a further 2.5 h. DBU was then added and the reaction stirred for 30 minutes before the mixture was evaporated in vacuo. The crude product was purified by column chromatography (3:1 hexane:ethyl acetate) to afford the title compound as a colourless oil (397 mg, 57%). δH(CDCl3) 5.27 (1H, dd, J 7.3, 5.7 Hz), 4.84 (1H, t, J 7.5 Hz), 3.96-4.07 (1H, m), 3.89 (1H, d, J 12.4 Hz), 3.52-3.40 (2H, m), 1.47 (9H, s).

Intermediate 22 (397 mg, 1.42 mmol) was dissolved in MeOH (6 mL) and water (3 mL) and treated with potassium carbonate (1.18 g, 8.5 mmol) and left to stir for 18 h at room temperature. After this time the volatiles were removed by evaporation in vacuo, and the mixture then extracted with 9:1 chloroform:2-propanol solution (5×10 mL). The organic layers were combined, dried (Na2SO4), filtered and evaporated in vacuo before being recrystalised from DCM/hexane to give the title compound as a white powder (205 mg, 72%). δH(DMSO-d6) 4.95 (1H, s), 3.85 (1H, s), 3.37-3.15 (4H, m), 2.86-2.78 (1H, m), 1.49 (2H, s), 1.39 (9H, s).

Intermediate 23 (100 mg, 0.5 mmol) in methanol (5 mL) was treated with 4M HCl in 1,4-dioxane (5 mL) and allowed to stir for 6 h at room temperature. After this time the solvent was removed in vacuo to afford the hydrochloride salt of the title compound as an off-white solid (87 mg, quant.), which was used in the next reaction without further purification.

To a solution of Intermediate 39 (3.34 g, 14.63 mmol) in DCM (80 mL) cooled to 0° C. was added DIPEA (3.45 mL, 19.75 mmol), followed by methanesulfonyl chloride (1.47 mL, 19.01 mmol). The mixture was allowed to warm up to room temperature and stirred for a further 6 h. After that time the mixture was washed with saturated brine (2×100 mL), dried (Na2SO4), and concentrated in vacuo. The crude product was purified by chromatography (SiO2; 10-20% EtOAc/hexane) to obtain the title compound as a pale yellow oil (3.54 g, 79%). δH(CDCl3) 4.77-4.82 (1H, m), 4.02-4.15 (1H, m), 3.28-3.63 (4H, m), 2.92 (3H, s), 1.30 (9H, s).

To a solution of Intermediate 40 (2.08 g, 6.79 mmol) in DMF (40 mL) was added sodium azide (1.32 g, 20.37 mmol), and the mixture stirred at 80° C. for 20 h. After this time the reaction mixture was diluted with Et2O (50 mL) and washed with saturated brine (4×50 mL), dried (Na2SO4), and concentrated in vacuo. The crude product was purified by chromatography (SiO2; 10-30% EtOAc in hexane) to obtain the title compound as a pale yellow oil (1.05 g, 61%). δH(CDCl3) 4.04-4.13 (2H, m), 3.58-3.68 (2H, m), 3.35-3.53 (2H, m), 1.46 (9H, s).

To a solution of Intermediate 41 (138 mg, 0.54 mmol) was added 20% palladium hydroxide on carbon (15.3 mg, 0.11 mmol) and stirred under a hydrogen atmosphere for 18 h. The mixture was then filtered through celite, and washed with MeOH and the solvent evaporated in vacuo to obtain the title compound as a colourless oil (93.4 mg, 85%). δH(CDCl3) 3.52-3.78 (2H, m), 3.38-3.52 (2H, m), 3.14-3.37 (2H, m), 1.64-1.99 (4H, m), 1.48 (9H, s).

To a solution of Intermediate 43 (170 mg, 0.75 mmol) in MeOH (4 mL) was added 4M HCl in 1,4-dioxane (4 mL) and the mixture was stirred for 2 h. After this time the solvent was removed in vacuo and azeotroped with toluene to afford the hydrochloride salt of the title compound as a brown oil (95 mg, quant.). δH(DMSO-d6) 4.88-4.93 (2H, m), 3.44-3.51 (2H, m), 3.27-3.55 (2H, m), 2.12 (3H, s).

To a solution of Intermediate 42 (104 mg, 0.52 mmol) in DMF (3 mL) was added CDI (84 mg, 0.52 mmol) and the mixture stirred for 56 h. After this time the solvent was removed in vacuo. The residue was diluted with EtOAc (15 mL), extracted with water (3×15 mL), and then the aqueous was extracted with 10% MeOH/DCM (3×80 mL). The organics were dried (Na2SO4) and concentrated in vacuo to afford the title compound as a yellow/gold foam (82 mg, 60%), which was used in the next reaction without further purification.

To a solution of Intermediate 45 (82 mg, 0.36 mmol) in MeOH (10 mL) was added 4M HCl in 1,4-dioxane (10 mL) and the mixture was stirred for 1.5 h. After this time the solvent was removed in vacuo and azeotroped with toluene to afford the hydrochloride salt of the title compound as a brown oil (46 mg, quant.), which was used in the next reaction without further purification.

To a solution of Intermediate 12 (300 mg, 1.49 mmol) in DCM (50 mL) was added trimethylsilyl isocyanate (189 mg, 1.64 mmol) and the mixture was stirred at room temperature for 18 hours. After this time hexane (5 mL) was added to the reaction mixture and a pale precipitate formed. This was filtered and taken on to the next step as crude product (255 mg, 75%).

To a solution of Intermediate 53 (250 mg, 1.02 mol) in DCM (20 mL), cooled in an acetone/cardice bath, was added DAST (177 mg, 1.09 mmol) and stirred for 30 minutes before being allowed to warm up to room temperature. After 2 hours at r.t. the reaction mixture was diluted with DCM (50 mL) and washed with NaHCO3solution (50 mL). After drying (Na2SO4) the organics were evaporated in vacuo to give a residue which was purified by chromatography (SiO2; 15% MeOH, DCM) to afford the title compound as a white powder (200 mg, 87%). δH(DMSO-d6) 5.10 (1H, m), 4.45 (1H, m), 3.67 (1H, m), 3.48-3.18 (5H, m). LCMS RT 1.26 minutes, (ES+) 228 (M+H).

Intermediate 54 (250 mg, 1.11 mmol) was treated with 4M HCl in 1,4-dioxane (5 mL) and allowed to stir for 3 hours at room temperature. After this time a precipitate had formed which was filtered off to afford the dihydrochloride salt of the title compound as an off-white solid (222 mg, quant.). δH(DMSO-d6) 10.14 (3H, m), 9.33 (2H, m), 5.67 (1H, m), 4.83 (1H, m), 3.51-3.20 (4H, m).

To a solution of oxalyl chloride (66.5 μL, 0.79 mmol) in DCM (7 mL), cooled to −78° C., was added dimethylsulfoxide (112 μL, 1.57 mmol) and the mixture was stirred for 10 minutes. After this time Intermediate 43 (177 mg, 0.79 mmol) in DCM (10 mL) was added and the mixture was stirred for a further 30 minutes. Triethylamine (50 mL) was then added and the mixture allowed to warm up to r.t. over 30 minutes. After this time the mixture was washed with saturated sodium bicarbonate solution (20 mL), the aqueous was extracted with DCM (3×50 mL), and the combined organics were dried (Na2SO4) and concentrated in vacuo. The crude product was purified by chromatography (SiO2; 95:4.5:0.5→90:9:1 DCM/MeOH/28% aqueous NH3) to obtain the title compound as a brown oil (109 mg, 62%). LCMS RT 1.46 minutes, (ES+) 224 (M+H)+.

To a solution of Intermediate 62 (86 mg, 0.39 mmol) in MeOH (4 mL) was added 4M HCl in 1,4-dioxane (4 mL) and the mixture was stirred for 3 h. After this time the solvent was removed in vacuo and azeotroped with toluene to afford the hydrochloride salt of the title compound (47 mg, quantitative), which was used in the next reaction without further purification.

To a solution of Intermediate 68 (103 mg, 0.39 mmol) in MeOH (5 mL) was added 4M HCl in 1,4-dioxane (5 mL) and the mixture was stirred for 2.5 h. After this time the solvent was removed in vacuo and azeotroped with toluene to afford the hydrochloride salt of the title compound (69 mg, quantitative). δH(CD3OD) 5.09 (2H, dd, J 2.4, 3.6 Hz), 3.75-3.67 (2H, m), 3.63-3.53 (2H, m), 1.38 (9H, s).

Anhydrous pyridine (8.5 mL, 105 mmol) and dry DCM (100 mL) were added to a 3-necked round-bottomed flask under a nitrogen atmosphere. The reaction flask was then cooled to about −20° C. with a dry ice/ethylene glycol bath. Trifluoromethanesulfonic anhydride (17 mL, 100 mmol) was added, whereupon a white/pink precipitate formed immediately. 2-Bromoethanol (7.1 mL, 100 mmol) was added after 5 minutes. The precipitate disappeared and then after a few minutes a new white precipitate formed. The reaction mixture was stirred for 20 minutes during which time it gradually warmed to r.t. The reaction mixture was then filtered through a phase separator and the residue was washed with 1:1 DCM/hexane (2×10 mL). The filtrate was run through a 4 cm silica plug with 1:1 DCM/hexane solution (300 mL). The solvent was removed in vacuo to give the title compound as a brown oil (21.2 g, 87%). δH(CDCl3) 4.76 (2H, t, J 6.4 Hz), 3.62 (2H, t, J 6.4 Hz).

A solution of Intermediate 73 (21.1 g, 82.3 mmol) in anhydrous toluene (70 mL) was treated with phenyl sulfide (18.3 g, 98.7 mmol) at r.t. under nitrogen with stirring. The reaction mixture was heated to 100° C. under nitrogen for 5 h. The solution was allowed to cool to ambient temperature and diethyl ether (130 mL) added to precipitate the product which was isolated by filtration as a white powder (22.5 g, 62%). δH(CDCl3) 8.14-8.10 (4H, m), 7.86-7.62 (6H, m), 4.91 (2H, t, J 5.8 Hz), 3.71 (2H, t, J 5.8 Hz).

Intermediate 74 (11.4 g, 25.7 mmol) was dissolved in THF/H2O (2:1) (42 mL). KHCO3(3.1 g, 30.9 mmol) was added and the reaction mixture was stirred for 20 minutes at r.t. The solvent was evaporated immediately under reduced pressure (using a rotary evaporator connected to a high vacuum pump and keeping the water-bath temperature below 20° C.). The reaction mixture was then redissolved in DCM (40 mL), dried over MgSO4, filtered and evaporated. The residue was redissolved in DCM (10 mL) and loaded onto a silica bed (4 cm depth and 2.5 cm diameter). The resulting band was covered with 1 cm sand and eluted with DCM (400 mL), followed by 10% MeOH in DCM (200 mL). The product was isolated as a light brown oil (9.53 g, quantitative). δH(CDCl3) 7.90-7.87 (4H, m), 7.79-7.66 (6H, m), 7.53 (1H, dd, J 8.8, 8.8 Hz), 6.69 (1H, dd, J 9.5, 2.5 Hz), 6.55 (1H, dd, J 16.5, 2.5 Hz).

(−)-cis-(3R,4S)-4-Amino-1-benzylpyrrolidin-3-ol (5 g, 26 mmol) was dissolved in dry DMF (10 mL), cooled to 0° C., and triethylamine (3.6 mL, 26 mmol) was added under nitrogen. The reaction mixture was then treated with a solution of 2-(trimethylsilyl)-ethanesulfonyl chloride (4.93 g, 26 mmol) in DMF (2 mL). After stirring for 24 h at 0° C. to r.t. the solvent was removed in vacuo leaving a crude off-white semisolid. Purification by chromatography (SiO2; 6:4 hexane/EtOAc) gave the title compound (2 g, 22%) as a pale orange oil. The material was used as such for the subsequent step. LCMS (ES+) 357 (MH)+, RT 1.30 minutes (pH 10).

Intermediate 77 (0.55 g, 1.43 mmol) was hydrogenated using Pd(OH)2(100 mg) under 50 psi of hydrogen at 50° C. for 18 h. The catalyst was filtered off and the filtrate concentrated under vacuum to give the title compound (0.24 g, 57%) as a clear glass.1H NMR analysis indicated loss of the benzyl protecting group and the material was used in the subsequent step without further purification.

A degassed solution of cis-5-(benzyl)tetrahydropyrrolo[3,4-c]pyrrole-1,3-(2H,3H)-dione (250 mg, 1.30 mmol) in methanol (50 mL) was treated with Pd/C (25 mg) and a hydrogen balloon was attached. The mixture was stirred for 48 h before filtering through celite and removal of the solvents in vacuo gave the title compound as a brown solid (125 mg, 88%). δH(DMSO-d6) 11.01 (1H, s) 3.12 (4H, d, J 10.4 Hz), 2.75 (2H, m). LCMS RT 0.19 minutes, (ES+) 141 (M+H)+.