Patent Application
20060135541
This application is a US National Stage under 35 U.S.C § 371 of International Application No. PCT/GB2004/002281 (filed May 27, 2004) which claims priority under 35 U.S.C. § 119(a)-(d) to Application No. EP 03291314.7 filed on Jun. 2, 2003.
The present invention relates to quinazoline derivatives for use in the treatment of disease, in particular proliferative diseases such as cancer and in the preparation of medicaments for use in the treatment of proliferative diseases, and to processes for their preparation, as well as pharmaceutical compositions containing them as active ingredient.
Cancer (and other hyperproliferative diseases) are characterised by uncontrolled cellular proliferation. This loss of the normal regulation of cell proliferation often appears to occur as the result of genetic damage to cellular pathways that control progress through the cell cycle.
In eukaryotes, an ordered cascade of protein phosphorylation is thought to control the cell cycle. Several families of protein kinases that play critical roles in this cascade have now been identified. The activity of many of these kinases is increased in human tumours when compared to normal tissue. This can occur by either increased levels of expression of the protein (as a result of gene amplification for example), or by changes in expression of co activators or inhibitory proteins.
The first identified, and most widely studied of these cell cycle regulators have been the cyclin dependent kinases (or CDKs). Activity of specific CDKs at specific times is essential for both initiation and coordinated progress through the cell cycle. For example, the CDK4 protein appears to control entry into the cell cycle (the G0-G1-S transition) by phosphorylating the retinoblastoma gene product pRb. This stimulates the release of the transcription factor E2F from pRb, which then acts to increase the transcription of genes necessary for entry into S phase. The catalytic activity of CDK4 is stimulated by binding to a partner protein, Cyclin D. One of the first demonstrations of a direct link between cancer and the cell cycle was made with the observation that the Cyclin D1 gene was amplified and cyclin D protein levels increased (and hence the activity of CDK4 increased) in many human tumours (reviewed in Sherr, 1996, Science 274: 1672-1677; Pines, 1995, Seminars in Cancer Biology 6: 63-72). Other studies (Loda et al., 1997, Nature Medicine 3(2): 231-234; Gemma et al., 1996, International Journal of Cancer 68(5): 605-11; Biledge et al. 1996, Trends in Cell Biology 6; 388-392) have shown that negative regulators of CDK function are frequently down regulated or deleted in human tumours again leading to inappropriate activation of these kinases.
More recently, protein kinases that are structuraly distinct from the CDK family have been identified which play critical roles in regulating the cell cycle and which also appear to be important in oncogenesis. They include the human homologues of the Drosophila aurora and S. cerevisiae Ipl1 proteins. The three human homologues of these genes Aurora-A, Aurora-B and Aurora-C (also known as aurora2, aurora1 and aurora3 respectively) encode cell cycle regulated serine-threonine protein kinases (summarised in Adams et al, 2001, Trends in Cell Biology. 11(2): 49-54). These show a peak of expression and kinase activity through G2 and mitosis. Several observations implicate the involvement of human aurora proteins in cancer. This evidence is strong for Aurora-A. The Aurora-A gene maps to chromosome 20q13, a region that is frequently amplified in human tumours including both breast and colon tumours. Aurora-A may be the major target gene of this amplicon, since Aurora-A DNA is amplified and mRNA overexpressed in greater than 50% of primary human colorectal cancers. In these tumours Aurora-A protein levels appear greatly elevated compared to adjacent normal tissue. In addition, transfection of rodent fibroblasts with human Aurora-A leads to transformation, conferring the ability to grow in soft agar and form tumours in nude mice (Bischoff et al., 1998, The EMBO Journal. 17(11): 3052-3065). Other work (Zhou et al., 1998, Nature Genetics. 20(2): 189-93) has shown that artificial overexpression of Aurora-A leads to an increase in centrosome number and an increase in aneuploidy, a known event in the development of cancer. Further work has shown an increase in expression of Aurora-B (Adams et al., 2001, Chromsoma. 110(2):65-74) and Aurora-C (Kimura et al., 1999, Journal of Biological Chemistry, 274(11): 733440) in tumour cells when compared to normal cells.
Importantly, it has also been demonstrated that abrogation of Aurora-A expression and function by antisense oligonucleotide treatment of human tumour cell lines (WO 97/22702 and WO 99/37788) leads to cell cycle arrest and exerts an antiproliferative effect in these tumour cell lines. Additionally, small molecule inhibitors of Aurora-A and Aurora-B have been demonstrated to have an antiproliferative effect in human tumour cells (Keen et al. 2001, Poster #2455, American Association of Cancer research annual meeting), as has selective abrogation of Aurora-B expression alone by siRNA treatment Ditchfield etal., 2003, Journal of Cell Biology, 161(2):267-280). This indicates that inhibition of the function of Aurora-A and/or Aurora-B will have an antiproliferative effect that may be useful in the treatment of human tumours and other hyperproliferative diseases. Further, inhibition of Aurora kinases as a therapeutic approach to these diseases may have significant advantages over targeting signalling pathways upstream of the cell cycle (e.g. those activated by growth factor receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) or other receptors). Since the cell cycle is ultimately downstream of all of these diverse signalling events, cell cycle directed therapies such as inhibition of Aurora Iinases would be predicted to be active across all proliferating tumour cells, whilst approaches directed at specific signalling molecules (e.g. EGFR) would be predicted to be active only in the subset of tumour cells which express those receptors. It is also believed that significant “cross taW” exists between these signalling pathways meaning that inhibition of one component may be compensated for by another.
A number of quinazoline derivatives have been proposed hitherto for use in the inhibition of Aurora kinases. For example, WO 01/21594, WO 01/21595 and WO 01/215968 describe the use of certain phenyl-quinazoline compounds as Aurora-A kinase inhibitors, which may be useful in the treatment of proliferative diseases and WO 01/21597 discloses other quinazoline derivatives as inhibitors of Aurora-A kinase. Additionally, WO 02/00649 discloses quinazoline derivative bearing a 5-membered heteroaromatic ring where the ring is, in particular, substituted thiazole or substituted thiophene. However despite the compounds of WO 02/00649 there still exists a need for further compounds having Aurora kinase inhibitory properties.
The applicants have been successful in finding a novel series of compounds which inhibit the effects of the Aurora kinases and in particular Aurora-A kinase and/or Aurora-B kinase which are thus of use in the treatment of proliferative diseases such as cancer. In particular, the compounds may be used to treat either solid or haematological tumours and more particularly colorectal, breast, lung, prostate, bladder, renal or pancreatic cancer or leukaemia or lymphoma. In addition certain aspects of the invention make them useful in the formulation of medicaments for the treatment of disease.
According to one aspect of the invention there is provided a compound of formula (1)
or a salt, ester or prodrcug thereof;
As a further aspect a compound of formula (I) or a pharmaceutically acceptable salt thereof is provided.
In another aspect the invention provides a compound of formula (IA)
or a salt or ester thereof;
In a preferred embodiment a compound of formula (IA) contains only one phosphonooxy group.
As a further aspect a compound of formula (IA) or a pharmaceutically acceptable salt thereof is provided.
In this specification the term alkyl when used either alone or as a suffix or prefix or otherwise includes straight-chain and branched-chain saturated structures comprising carbon and hydrogen atoms. References to individual alkyl groups such as propyl are specific for the straight-chain version only and references to individual branched-chain alkyl groups such as tert-butyl are specific for the branched chain version only. An analogous convention applies to other generic terms such as alkenyl and alkynyl.
Cycloalkyl is a monocyclic alkyl group, and cycloalkenyl and cycloalkyhyl are monocyclic alkenyl and alkynyl groups respectively.
The prefix Cm-n in Cm-nalkyl and other terms (where m and n are integers) indicates the range of carbon atoms that are present in the group, for example C1-3alkyl includes C1alkyl (methyl), C2alkyl (ethyl) and C3alkyl (propyl or isopropyl).
The term Cm-nalkoxy comprises —O—Cm-nalkyl groups.
The term halo includes fluoro, chloro, bromo and iodo.
Aryl groups are aromatic carbocyclic groups which may be monocyclic or bicyclic.
Unless otherwise stated heteroaryl groups are monocyclic or bicyclic aromatic rings containing 5 to 10 ring atoms of which 1, 2, 3 or 4 ring atoms are chosen from nitrogen, sulphur or oxygen where a ring nitrogen or sulphur may be oxidised.
Heterocyclyl is a saturated, unsaturated or partially saturated, monocyclic or bicyclic ring containing 4 to 7 ring atoms, of which 1, 2 or 3 ring atoms are selected from nitrogen, sulphur or oxygen, which ring may be carbon or nitrogen linked, wherein a —CH2— group can optionally be replaced by a —C(O)— group; wherein a ring nitrogen or sulphur atom is optionally oxidised to form the N-oxide or S-oxide(s); and wherein a ring —NH— is optionally substituted by acetyl, formyl, methyl or mesyl. When the term heterocyclyl is used within the definition of groups of formula (I), this heterocyclyl ring is optionally substituted by 1 or 2 groups selected from C1-4alkyl, C1-4alkoxy, hydroxyC1-4alkyl, hydroxy and haloC1-4alkyl, and preferably the ring is so substituted. When the term heterocyclyl is used within the definition of groups of formula (IA), this heterocyclyl ring is optionally substituted by 1 or 2 groups selected from C1-4alkyl, C1-4alkoxy, hydroxyC1-4alkyl, phosphonooxyC1-4alkyl, hydroxy, phosphonooxy and haloC1-4alkyl, and preferably the ring is so substituted. When the term heterocyclyl is used within the definition of R3 in formula (I), in one aspect of the invention it is a saturated monocyclic ring containing 4 to 7 ring atoms of which 1 or 2 are nitrogen and which ring is optionally substituted by C1-4alkyl, hydroxyC1-4alkyl and hydroxy. When the term heterocyclyl is used within the definition of R3′ in formula (IA), in one aspect of the invention it is a saturated monocyclic ring containing 4 to 7 ring atoms of which 1 or 2 are nitrogen and which ring is optionally substituted by C1-4alkyl, hydroxyC1-4alkyl, phosphonoooxyC1-4alkyl, hydroxy and phosphonooxy.
Phosphonooxy is in one aspect a group of formula —OP(O)(OH)2. However the term phosphonooxy also includes salts such as those formed with alkali metal ions such as sodium or potassium ions or alkaline earth metal ions, for example calcium or magnesium ions.
This specification also makes use of several composite terms to describe groups comprising more than one functionality. Such terms are to be interpreted as is understood in the art. For example Cm-ncycloalkylCm-nalkyl comprises Cm-nalkyl substituted by Cm-ncycloalkyl and Cm-nalkoxyCm-ncycloalkylCm-nalkyl comprises Cm-nalkyl substituted by Cm-ncycloalkyl, which Cm-ncycloalkyl is substituted by Cm-nalkoxy.
HaloCm-nalkyl is a Cm-nalkyl group that is substituted by 1, 2 or 3 halo substituents. Similarly other generic terms containing halo such as haloCm-ncycloalkyl and haloCm-ncycloalkylCm-nalkyl groups may contain 1, 2 or 3 halo substituents.
HydroxyCm-nalkyl is a Cm-nalkyl group that is substituted by 1, 2 or 3 hydroxy substituents. Similarly other generic terms containing hydroxy such as hydroxyCm-ncycloalkyl, hydroxyCm-nalkylCm-ncycloalkyl, hydroxyCm-ncycloalkylCm-nalkyl and hydroxyCm-nalkoxyCm-nalkyl groups may contain 1, 2 or 3 hydroxy substituents.
Cm-nalkoxyCm-nalkyl is a Cm-nalkyl group that is substituted by 1, 2 or 3 Cm-nalkoxy substituents. Similarly other generic terms containing Cm-nalkoxy such as Cm-nalkoxyCm-nalkoxy, Cm-nalkoxyCm-ncycloalkyl and Cm-nalkoxyCm-ncycloalkylCm-nalkyl groups may contain 1, 2 or 3 Cm-nalkoxy substituents.
Where optional substituents are chosen from, for example 1 or 2 or from 1, 2, or 3 groups or substituents it is to be understood that this definition includes all substituents being chosen from one of the specified groups i.e. all substituents being the same or the substituents being chosen from two or more of the specified groups i.e. the substituents not being the same.
Unless specifically stated the bonding atom of a group may be any atom of that group so for example propyl includes prop-1-yl and prop-2-yl.
Compounds of the present invention have been named with the aid of computer software (ACD/Name version 6.6 or ACD Name Batch version 6.0).
Suitable values for any R group or any part or substituent for such groups include:
Within the present invention, it is to be understood that, insofar as certain of compounds of formula (I) or formula (IA) herein defined may exist in optically active or racemic forms by virtue of one or more asymmetric carbon or sulphur atoms, the invention includes in its definition any such optically active or racemic form which possesses aurora kinase inhibitory activity and in particular Aurora-A and/or Aurora-B kinase inhibitory activity. The synthesis of optically active forms may be carried out by standard techniques of organic chemistry well known in the art, for example by synthesis from optically active starting materials or by resolution of a racemic form. Similarly, the above-mentioned activity may be evaluated using the standard laboratory techniques referred to herein.
Within the present invention it is to be understood that a compound of formula (I) or formula (IA) may exhibit the phenomenon of tautomerism and that the formulae drawings within this specification can represent only one of the possible tautomeric forms. It is to be understood that the invention encompasses any tautomeric form which has Aurora kinase inhibitory activity and in particular Aurora-A and/or Aurora-B ldnase inhibitory activity and is not to be limited merely to any one tautomeric form utilised within the formulae drawings.
It is also to be understood that certain compounds of formula (I) or formula (IA) can exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms which have Aurora kinase inhibitory activity and in particular Aurora-A and/or Aurora-B kinase inhibitory activity.
The present invention relates to the compounds of formula (I) or formula (IA) as herein defined as well as to the salts thereof. Salts for use in pharmaceutical compositions will be pharmaceutically acceptable salts, but other salts may be useful in the production of the compounds of formula (I) or formula (IA) and their pharmaceutically acceptable salts. Pharmaceutically acceptable salts of the invention may, for example, include acid addition salts of compounds of formula (I) or formula (IA) as herein defined which are sufficiently basic to form such salts. Such acid addition salts include but are not limited to furmarate, methanesulphonate, hydrochloride, hydrobromide, citrate and maleate salts and salts formed with phosphoric and sulphuric acid. In addition where compounds of formula (I) or formula (IA) are sufficiently acidic, salts are base salts and examples include but are not limited to, an alkali metal salt for example sodium or potassium, an alkaline earth metal salt for example calcium or magnesium, or organic amine salt for example triethylamine, ethanolamine, diethanolamine, triethanolamine, morpholine, N-methylpiperidine, N-ethylpiperidine, dibenzylamine or amino acids such as lysine.
The compounds of formula (I) or formula (IA) may also be provided as in vivo hydrolysable esters. An in vivo hydrolysable ester of a compound of formula (I) or formula (IA) containing carboxy or hydroxy group is, for example a pharmaceutically acceptable ester which is cleaved in the human or animal body to produce the parent acid or alcohol. Such esters can be identified by administering, for example, intravenously to a test animal, the compound under test and subsequently examining the test animal's body fluid.
Suitable pharmaceutically acceptable esters for carboxy include C1-6alkoxymethyl esters for example methoxymethyl, C1-6alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C3-8cycloalkoxycarbonyloxyC1-6alkyl esters for example 1-cyclohexylcarbonyloxyethyl; 1,3-dioxolen-2-onylmethyl esters for example 5-methyl-1,3-dioxolen-2-onylmethyl; and C1-6alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyloxyethyl and may be formed at any carboxy group in the compounds of this invention.
Suitable pharmaceutically acceptable esters for hydroxy include inorganic esters such as phosphate esters (including pliosphoramidic cyclic esters) and a-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown to give the parent hydroxy group/s. Examples of α-acyloxyalkyl ethers include acetoxymethoxy and 2,2-dimethylpropionyloxymethoxy. A selection of in vivo hydrolysable ester forming groups for hydroxy include C1-10alkanoyl, for example formyl, acetyl; benzoyl; phenylacetyl; substituted benzoyl and phenylacetyl, C1-10alkoxycarbonyl (to give alkyl carbonate esters), for example ethoxycarbonyl; di-C1-4alkylcarbamoyl and N-(di-C1-4alkylaminoethyl)-N-C1-4alkylcarbamoyl (to give carbamates); di-C1-4alkylaminoacetyl and carboxyacetyl. Examples of ring substituents on phenylacetyl and benzoyl include aminomethyl, C1-4alkylaminomethyl and di-(C1-4alkyl)aminomethyl, and morpholino or piperazino linked from a ring nitrogen atom via a methylene linking group to the 3- or 4-position of the benzoyl ring. Other interesting inl vivo hydrolysable esters include, for example, RA(O)OC1-6alkyl-CO—, wherein RA is for example, benzyloxy-C1-4alkyl, or phenyl). Suitable substituents on a phenyl group in such esters include, for example, 4-C1-4piperazino-C1-4alkyl, piperazino-C1-4alkyl and morpholino-C1-4alkyl.
The compounds of the formula (I) may be also be administered in the form of a prodrug which is broken down in the human or animal body to give a compound of the formula (I). Examples of prodrugs include in vivo hydrolysable esters of a compound of the formula (I). Various forms of prodrugs are known in the art. For examples of such prodrug derivatives, see:
Particular values of X, R1, R1′, R2, R2′, R3, R3′, R4 and R5 for compounds of formula (I) and formula (IA) are as follows. Such values may be used where appropriate with any of the definitions, claims or embodiments defined herein.
In one aspect of the invention X is NR6. In another aspect X is NH.
In one aspect of the invention R6 is hydrogen or methyl. In another aspect R6 is hydrogen.
In one aspect of the invention R1 is hydrogen or —OR11. In another aspect R1 is hydrogen.
In one aspect of the invention X1 is a direct bond or —O—. In another aspect X1 is a direct bond.
In one aspect of the invention R11 is hydrogen, heterocyclyl selected from piperidinyl and pyrrolidinyl or C1-4alkyl (optionally substituted by hydroxy, C1-4alkoxy, amino, C1-4alkylamino or bis(C1-4alkyl)amino). In another aspect R11 is hydrogen, C1-4alkyl or C1-4alkoxy. In another aspect R11 is hydrogen.
In one aspect of the invention R2 is hydrogen or —OR12. In another aspect R2 is hydrogen or methoxy. In a further aspect R2 is hydrogen. In yet a further aspect R2 is methoxy.
In one aspect of the invention X2 is a direct bond or —O—.
In one aspect of the invention R12 is hydrogen, C1-4alkyl (optionally substituted by heterocyclyl) or heterocyclyl. In another aspect R12 is hydrogen or C1-4alkyl. In another aspect of the invention R12 is hydrogen. In a further aspect of the invention R12 is methyl.
In one aspect of the invention R3 is —X3R13. In a further aspect R3 is 3-(2-hydroxymethylpyrrolidin-1-yl)propoxy or 3-[(2-hydroxyethyl)(propyl)amino]propoxy.
In one aspect of the invention X3 is —CH2═CH2—, —O— or —NH—. In another aspect X3 is —O—.
In one aspect of the invention R13 is C1-6alkyl substituted by —NR7R8, heterocyclyl or halo. In a further aspect of the invention R13 is ethyl or propyl, both of which are substituted by —NR7R8, heterocyclyl or halo. In yet a further aspect of the invention R13 is propyl substituted by chloro, —NR7R8 or a heterocyclyl selected from pyrrolidinyl, piperidinyl, piperazinyl, diazepanyl and azetidinyl which heterocyclyl is optionally substituted by hydroxy, methyl, hydroxymethyl or 2-hydroxyethyl. In another aspect R13 is propyl substituted by chloro or —NR7R8. In a further aspect R13 is propyl substituted by —NR7R8.
In one aspect of the invention R7 and R8 are independently selected from hydrogen, heterocyclyl, C1-6alkyl, hydroxyC1-6alkynyl, hydroxyC1-4alkylC3-6cycloalkyl, C1-3alkoxyC1-4alkyl, C3-6cycloalkyl, C3-6cycloalkylC1-3alkyl, haloC1-6alkyl, C2-6alkenyl, C2-6alkynyl, cyanoC1-4alkyl and bis(C1-3alkyl)aminoC1-6alkyl; or R7 and R8 together with the nitrogen to which they are attached form a heterocyclic ring which ring comprises 4 to 7 ring atoms of which one is nitrogen and of which another is optionally NH and which ring is optionally substituted on carbon or nitrogen by a group selected from C1-4alkyl, hydroxy, hydroxyC1-4alkyl and hydroxyC1-4alkoxyC1-4alkyl, and where a ring —CH2— is optionally replaced with —C(O)—. In a further aspect R7 and R8 are independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hydroxymethyl, 2-hydroxyethyl, 3-hydroxy-1,1-dimethylpropyl, methoxymethyl, 2-methoxyethyl, 2-ethoxyethyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 3,3,3-trifluoropropyl, alkyl, propargyl, 2-(dimethylamino)ethyl and 2-(diethylamino)ethyl; or R7 and R8 together with the nitrogen to which they are attached form a heterocyclic ring selected from pyrrolidinyl, piperidinyl, piperazinyl, diazepanyl and azetidinyl where the ring is optionally substituted by hydroxy, methyl, hydroxymethyl or 2-hydroxyethyl. In yet another aspect R7 and R8 are independently selected from hydrogen, methyl, ethyl, propyl, hydroxymethyl, 2-hydroxyethyl or 3-hydroxypropyl; or R7 and R8 together with the nitrogen to which they are attached form a heterocyclic ring selected from pyrrolidinyl, piperidinyl and piperazinyl, where the ring is optionally substituted by hydroxymethyl or 2-hydroxyethyl.
In one aspect of the invention R4 is hydrogen.
In one aspect of the invention R5 is aryl optionally substituted by 1 or 2 halo. In another aspect R5 is phenyl optionally substituted by 1 or 2 fluoro or chloro. In a further aspect R5 is phenyl optionally substituted by 1 or 2 fluoro. In yet another aspect R5 is 2,3-difluorophenyl or 3-fluorophenyl.
In one aspect of the invention R1′ is hydrogen or —OR11′. In another aspect R1′ is hydrogen.
In one aspect of the invention R11′ is hydrogen, heterocyclyl selected from piperidinyl or pyrrolidinyl or C1-4alkyl (optionally substituted by hydroxy, phosphonooxy, C1-4allcoxy, amino, C1-4alkylamino or bis(C1-4alkyl)amino). In another aspect R11′ is hydrogen, C1-4alkyl or C1-4alkoxy. In another aspect R11′ is hydrogen.
In one aspect of the invention R2′ is hydrogen or —OR12′. In another aspect R2′ is hydrogen or methoxy. In a further aspect R2′ is hydrogen. In yet a further aspect R2′ is methoxy.
In one aspect of the invention R12′ is hydrogen, C1-4alkyl (optionally substituted by eterocyclyl) or heterocyclyl. In another aspect R12′ is hydrogen or C1-4alkyl. In another aspect of the invention R12′ is hydrogen. In a further aspect of the invention R12′ is methyl.
In one aspect of the invention R3′ is —X3R13′.
In one aspect of the invention R13′ is C1-6alkyl substituted by —NR7′R8′. In a further aspect of the invention R13′ is propyl substituted by —NR7′R8′.
In one aspect of the invention R7′is selected from hydrogen, heterocyclyl, C1-6alkyl, C1-3alkoxyC1-6alkyl, cyanoC1-4alkyl and C3-6cycloalkyl. In another aspect R7′ is ethyl, propyl, cyclobutyl or 2-methoxyethyl.
In one aspect of the invention R8′ is phosphonooxyC1-4alkyl or phosphonooxyC1-4alkylC3-6cycloalkyl. In another aspect R8′ is 2-phosphonooxyethyl or 1,1-dimethyl-3-phosphonooxypropyl.
In one aspect of the invention R7′ and R8′ together with the nitrogen to which they are attached form a heterocyclic ring selected from pyrrolidinyl, piperidinyl and piperazinyl which ring is substituted on carbon or nitrogen by a group selected from phosphonooxy, phosponooxymethyl and 2-phoshonooxyethyl. In a further aspect R7′and R8′ together with the nitrogen to which they are attached form a pyrrolidinyl ring which ring is substituted on carbon by phosponooxymethyl.
A particular class of compounds is of formula (I) wherein:
The present invention also provides a process for the preparation of a compound of formula (I) or a salt, ester or prodrug thereof, which process comprises reacting a compound of formula (II)
where L is a suitable leaving group such as chloro, bromo, SMe etc.
The process may further comprise a process for the preparation of a compound of formula (II) from a compound of formula (IV)
by reacting it with a reagent such as formamidine acetate in a solvent such as 2-methoxyethanol under reflux to yield a compound of formula (V)
which may then be reacted with a reagent such as thionyl chloride in a solvent such as dimethyl formamide at elevated temperatures to yield a compound of formula (II) where L is, for example chloro.
Compounds of formula (IV) are either known in the art or can be derived from other compounds known in the art by conventional methods which would be apparent from the literature.
The process may further comprise a process for the preparation of a compound of formula (III) which process comprises the reaction of a compound of formula (VI)
with a compound of formula (VII)
R5—NH2 (VII)
The reaction is suitably effected in an organic solvent such as tetrahydrofuran (THF) or dichloromethane (DCM) at temperatures from 0° C. to 25° C. in the presence of a base such as sodium hydroxide or pyridine under an inert atmosphere for 1 to 5 hours.
Further provided is a process for the preparation of a compound of formula (IA) or a salt or ester thereof, which process comprises phosphorylation of a suitable compound of formula (I) by reacting a compound of formula (I) and tetrazole with di-tert-butyl diethylphosphoranmidite in an appropriate organic solvent such as dimethylformamide or dimethylacetamide under an inert atmosphere, followed by (after 1 to 5 hours) the addition of hydrogen peroxide and sodium metabisulphite. Deprotection of the phosphate group then yields a compound of formula (IA). Deprotection is suitably effected with hydrochloric acid in dioxane or dichloromethane (DCM) at ambient temperature for 6 to 30 hours.
Suitable reaction conditions are illustrated herein.
It will be appreciated that certain of the various ring substituents in the compounds of the present invention may be introduced by standard aromatic substitution reactions or generated by conventional functional group modifications either prior to or immediately following the processes mentioned above, and as such are included in the process aspect of the invention. Such reactions and modifications include, for example, introduction of a substituent by means of an aromatic substitution reaction, reduction of substituents, alkylation of substituents and oxidation of substituents. The reagents and reaction conditions for such procedures are well known in the chemical art. Particular examples of aromatic substitution reactions include the introduction of a nitro group using concentrated nitric acid, the introduction of an acyl group using, for example, an acyl halide and Lewis acid (such as aluminium trichloride) under Friedel Crafts conditions; the introduction of an alkyl group using an alkyl halide and Lewis acid (such as aluminium trichloride) under Friedel Crafts conditions; and the introduction of a halogen group. Particular examples of modifications include the reduction of a nitro group to an amino group by for example, catalytic hydrogenation with a nickel catalyst or treatment with iron in the presence of hydrochloric acid with heating; oxidation of alkylthio to alkylsulphinyl or alkylsulphonyl.
It will also be appreciated that in some of the reactions mentioned herein it may be pecessary/desirable to protect any sensitive groups in the compounds. The instances where protection is necessary or desirable and suitable methods for protection are known to those skilled in the art. Conventional protecting groups may be used in accordance with standard practice (for illustration see T.W. Green, Protective Groups in Organic Synthesis, John Wiley and Sons, 1991). Thus, if reactants include groups such as amino, carboxy or hydroxy it may be desirable to protect the group in some of the reactions mentioned herein.
A suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or tert-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an acyl group such as a tert-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulphuric or phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine.
A suitable protecting group for a hydroxy group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an aroyl group, for example benzoyl, or an arylmethyl group, for example benzyl. The deprotection conditions for the above protecting groups will necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or an aroyl group may be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an arylmethyl group such as a benzyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.
A suitable protecting group for a carboxy group is, for example, an esterifying group, for example a methyl or an ethyl group which may be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a tert-butyl group which may be removed, for example, by treatment with an acid, for example an organic acid such as trifluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.
The protecting groups may be removed at any convenient stage in the synthesis using conventional techniques well known in the chemical art.
According to a further aspect of the invention there is provided a pharmaceutical composition which comprises a compound formula (I), or a pharmaceutically acceptable salt, ester or prodrug thereof, as defined herein in association with a pharmaceutically acceptable diluent or carrier.
Also provided is a pharmaceutical composition which comprises a compound of formula (IA), or a pharmaceutically acceptable salt or ester thereof, as defined herein in association with a pharmaceutically acceptable diluent or carrier.
The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing).
The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
Suitable pharmaceutically acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal track, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art.
Compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, soya bean oil, coconut oil, or preferably olive oil, or any other acceptable vehicle.
Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame).
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible or lyophilised powders and granules suitable for preparation of an aqueous suspension or solution by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavouring and colouring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum ragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and ondensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring and preservative agents.
Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavouring and/or colouring agent.
The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, solutions, emulsions or particular systems, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in polyethylene glycol.
Suppository formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.
Topical formulations, such as creams, ointments, gels and aqueous or oily solutions or suspensions, may generally be obtained by formulating an active ingredient with a conventional, topically acceptable, vehicle or diluent using conventional procedure well known in the art.
Compositions for administration by insufflation may be in the form of a fmely divided powder containing particles of average diameter of, for example, 30 μm or much less preferably 5 μm or less and more preferably between 5 μm and 1 μm, the powder itself comprising either active ingredient alone or diluted with one or more physiologically acceptable carriers such as lactose. The powder for insufflation is then conveniently retained in a capsule containing, for example, 1 to 50mg of active ingredient for use with a turbo-inhaler device, such as is used for insufflation of the known agent sodium cromoglycate.
Compositions for administration by inhalation may be in the form of a conventional pressurised aerosol arranged to dispense the `active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.
For further information on formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
Therefore in a further aspect of the invention there is provided a compound of formula (I), or a pharmaceutically acceptable salt, ester or prodrug thereof, for use in therapy. In addition a compound of formula (IA) or a pharmaceutically acceptable salt or ester thereof is provided for use in therapy.
Further provided is a compound of formula (I), or a pharmaceutically acceptable salt, ester or prodrug thereof, for use as a medicament and also provided is a compound of formula (IA), or a pharmaceutically acceptable salt or ester thereof, for use as a medicament. Another aspect of the invention provides a compound of formula (I), or a pharmaceutically acceptable salt, ester or prodrug thereof, for use as a medicament for the treatment of a hyperproliferative disease such as cancer and in particular colorectal, breast, lung, prostate, bladder, renal or pancreatic cancer or leukaemia or lymphoma. Also provided is a compound of formula (IA), or a pharmaceutically acceptable salt or ester thereof, for use as a medicament for the treatment of a hyperproliferative disease such as cancer and in particular colorectal, breast, lung, prostate, bladder, renal or pancreatic cancer or leukaemia or lymphoma.
Additionally a compound of formula (I), or a pharmaceutically acceptable salt, ester or prodrug thereof is provided for use in a method of treatment of a warm-blooded animal such as man by therapy. A compound of formula (IA) or a pharmaceutically acceptable salt or ester thereof is also provided for use in a method of treatment of a warm-blooded animal such as man by therapy. Another aspect of the invention provides a compound of formula (I), or a pharmaceutically acceptable salt, ester or prodrug thereof, for use in a method of treatment of a hyperproliferative disease such as cancer and in particular colorectal, breast, lung, prostate, bladder, renal or pancreatic cancer or leukaemia or lymphoma. Also provided is a compound of formula (IA), or a pharmaceutically acceptable salt or ester thereof, for use in a method of treatment of a hyperproliferative disease such as cancer and in particular colorectal, breast, lung, prostate, bladder, renal or pancreatic cancer or leukaemia or lymphoma.
In another aspect of the invention, there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof, in the preparation of a medicament for the treatment of a disease where the inhibition of one or more Aurora kinase(s) is beneficial. The use of a compound of formula (IA) or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for the treatment of a disease where the inhibition of one or more Aurora kinase(s) is beneficial is also provided. In particular it is envisaged that inhibition of Aurora-A kinase and/or Aurora-B kinase may be beneficial. Preferably inhibition of Aurora-B kinase is beneficial.
In another aspect of the invention, there is provided the use of a compound of formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof, in the preparation of a medicament for the treatment of a hyperproliferative disease such as cancer and in particular colorectal, breast, lung, prostate, bladder, renal or pancreatic cancer or leukaemia or lymphoma. Also provided is the use of a compound of formula (IA) or a pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for the treatment of a hyperproliferative disease such as cancer and in particular colorectal, breast, lung, prostate, bladder, renal or pancreatic cancer or leukaemia or lymphoma.
According to yet another aspect, there is provided a compound of formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof for use in the method of treating a human suffering from a disease in which the inhibition of one or more Aurora linases is beneficial, comprising the steps of administering to a person in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof. Further provided is a compound of formula (IA) or a pharmaceutically acceptable salt thereof for use in the method of treating a human suffering from a disease in which the inhibition of one or more Aurora kinases is beneficial, comprising the steps of administering to a person in need thereof a therapeutically effective amount of a compound of formula (IA) or a pharmaceutically acceptable salt thereof. In particular it is envisaged that inhibition of Aurora-A kinase and/or Aurora-B kinase may be beneficial. Preferably inhibition of Aurora-B kinase is beneficial. Further provided is a compound of formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof for use in the method of treating a human suffering from a hyperproliferative disease such as cancer and in particular colorectal, breast, lung, prostate, bladder, renal or pancreatic cancer or leukaemia or lymphoma, comprising the steps of adrninstering to a person in need thereof a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof. A compound of formula (IA) is also provided for use in the method of treating a human suffering from a hyperproliferative disease such as cancer and in particular colorectal, breast, lung, prostate, bladder, renal or pancreatic cancer or leukaemia or lymphoma, comprising the steps of administering to a person in need thereof a therapeutically effective amount of a compound of formula (IA) or a pharmaceutically acceptable salt or ester thereof. The use of a compound of formula (I) or a pharmaceutically acceptable salt, ester or prodrug thereof in any of the methods of treating a human described above also form aspects of this invention. Additionally the use of a compound of formula (IA) or a pharmaceutically acceptable salt or ester thereof in any of the methods of treating a human described above is another aspects of this invention.
For the above mentioned therapeutic uses the dose administered will vary with the compound employed, the mode of administration, the treatment desired, the disorder indicated and the age and sex of the animal or patient. The size of the dose would thus be calculated according to well known principles of medicine.
In using a compound of formula (I) or formula (IA) for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.05 mg/kg to 50 mg/kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous administration, a dose in the range, for example, 0.05 mg/kg to 25 mg/kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.05 mg/kg to 25 mg/kg body weight will be used.
The treatment defined herein may be applied as a sole therapy or may involve, in addition to the compound of the invention, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following categories of anti-tumour agents.
In addition a compound of the invention or a pharmaceutically acceptable salt, ester or prodrug thereof, may be used in combination with one or more cell cycle inhibitors. In particular with cell cycle inhibitors which inhibit bub1, bubR1 or CDK.
Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compounds of this invention within the dosage range described herein and the other pharmaceutically-active agent within its approved dosage range.
In addition to their use in therapeutic medicine, a compound of formula (I) and a pharmaceutically acceptable salt, ester or prodrug thereof are also useful as pharmacological tools in the development and standardisation of in vitro and in vivo test systems for the evaluation of the effects of inhibitors of cell cycle activity in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents.
In the above other pharmaceutical composition, process, method, use and medicament manufacture features, the alternative and preferred embodiments of the compounds of the invention described herein also apply.
The compounds of the invention inhibit the serine-threonine kinase activity of the Aurora kinases, in particular Aurora-A Idnase and/or Aurora-B kinase and thus inhibit the cell cycle and cell proliferation. These properties may be assessed for example, using one or more of the procedures set out below.
(a) In Vitro Aurora-A Kinase Inhibition Test
This assay determines the ability of a test compound to inhibit serine-threonine kinase activity. DNA encoding Aurora-A may be obtained by total gene synthesis or by cloning. This DNA may then be expressed in a suitable expression system to obtain polypeptide with serine-threonine kinase activity. In the case of Aurora-A, the coding sequence was isolated from cDNA by polymerase chain reaction (PCR) and cloned into the BamH1 and Not1 restriction endonuclease sites of the baculovirus expression vector pFastBac HTc (GibcoBRL/Life technologies). The 5′ PCR primer contained a recognition sequence for the restriction endonuclease BamnH1 5′ to the Aurora-A coding sequence. This allowed the insertion of the Aurora-A gene in frame with the 6 histidine residues, spacer region and rTEV protease cleavage site encoded by the pFastBac HTc vector. The 3′ PCR primer replaced the Aurora-A stop codon with additional coding sequence followed by a stop codon and a recognition sequence for the restriction endonuclease Not1. This additional coding sequence (5′ TAC CCA TAC GAT GTT CCA GAT TAC GCT TCT TAA 3′) encoded for the polypeptide sequence YPYDVPDYAS. This sequence, derived from the influenza hemagglutin protein, is frequently used as a tag epitope sequence that can be identified using specific monoclonal antibodies. The recombinant pFastBac vector therefore encoded for an N-terminally 6 his tagged, C terminally influenza hemagglutin epitope tagged Aurora-A protein. Details of the methods for the assembly of recombinant DNA molecules can be found in standard texts, for example Sambrook et al. 1989, Molecular Cloning—A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press and Ausubel et al. 1999, Current Protocols in Molecular Biology, John Wiley and Sons Inc.
Production of recombinant virus can be performed following manufacturer's protocol from GibcoBRL. Briefly, the pFastBac-I vector carrying the Aurora-A gene was transformed into E. coli DH10Bac cells containing the baculovirus genome (bacmid DNA) and via a transposition event in the cells, a region of the pFastBac vector containing gentamycin resistance gene and the Aurora-A gene including the baculovirus polyhedrin promoter was transposed directly into the bacmid DNA. By selection on gentamycin, kanamycin, tetracycline and X-gal, resultant white colonies should contain recombinant bacmid DNA encoding Aurora-A. Bacmid DNA was extracted from a small scale culture of several BH10Bac white colonies and transfeccted into Spodoptera frugiperda Sf21 cells grown in TC100 medium (GibcoBRL) containing 10% serum using CellFECTIN reagent (GibcoBRL) following manufacturer's instructions. Virus particles were harvested by collecting cell culture medium 72 hrs post transfection. 0.5 mls of medium was used to infect 100 ml suspension culture of Sf21s containing 1×107 cells/ml. Cell culture medium was harvested 48 hrs post infection and virus titre determined using a standard plaque assay procedure. Virus stocks were used to infect Sf9 and “High 5” cells at a multiplicity of infection (MOI) of 3 to ascertain expression of recombinant Aurora-A protein.
For the large scale expression of Aurora-A kinase activity, Sf21 insect cells were grown at 28° C. in TC100 medium supplemented with 10% foetal calf serum (Viralex) and 0.2% F68 Pluronic (Sigma) on a Wheaton roller rig at 3 r.p.m. When the cell density reached 1.2×106 cells ml−1 they were infected with plaque-pure Aurora-A recombinant virus at a multiplicity of infection of 1 and harvested 48 hours later. All subsequent purification steps were performed at 4° C. Frozen insect cell pellets containing a total of 2.0×108 cells were thawed and diluted with lysis buffer (25 mM HEPES (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid]) pH7.4 at 4° C., 100 mM KCl, 25 mM NaF, 1 mM Na3VO4, 1 mM PMSF (phenylmethylsulphonyl fluoride), 2 mM 2-mercaptoethanol, 2 mM imidazole, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml leupeptin), using 1.0 ml per 3×107 cells. Lysis was achieved using a dounce homogeniser, following which the lysate was centrifuged at 41,000 g for 35 minutes. Aspirated supernatant was pumped onto a 5 mm diameter chromatography column containing 500 μl Ni NTA (nitrilo-tri-acetic acid) agarose (Qiagen, product no. 30250) which had been equilibrated in lysis buffer. A baseline level of UV absorbance for the eluent was reached after washing the column with 12 ml of lysis buffer followed by 7 ml of wash buffer (25 mM HEPES pH7.4 at 4° C., 100 mM KCl, 20 mM imidazole, 2 mM 2-mercaptoethanol). Bound Aurora-A protein was eluted from the column using elution buffer (25 mM HEPES pH7.4 at 4° C., 100 mM KCl, 400 mM irnidazole, 2 mM 2-mercaptoethanol). An elution fraction (2.5 ml) corresponding to the peak in UV absorbance was collected. The elution fraction, containing active Aurora-A kinase, was dialysed exhaustively against dialysis buffer (25 mM HEPES pH7.4 at 4° C., 45% glycerol (v/v), 100 mM KCl, 0.25% Nonidet P40 (vlv), 1 mM dithiothreitol).
Each new batch of Aurora-A enzyme was titrated in the assay by dilution with enzyme diluent (25 mM Tris-HCl pH7.5, 12.5 mM KCl, 0.6 mM DTT). For a typical batch, stock enzyme is diluted 1 in 666 with enzyme diluent & 20 μl of dilute enzyme is used for each assay well. Test compounds (at 10 mM in dimethylsulphoxide (DMSO) were diluted with water & 10 μlof diluted compound was transferred to wells in the assay plates. “Total” & “blank” control wells contained 2.5% DMSO instead of compound. Twenty microlitres of freshly diluted enzyme was added to all wells, apart from “blank” wells. Twenty microlitres of enzyme diluent was added to “blank” wells. Twenty microlitres of reaction mix (25 mM Tris-HCl, 78.4 mM KCl, 2.5 mM NaF, 0.6 mM dithiothreitol, 6.25 mM MnCl2, 6.26 mM ATP, 7.5 μM peptide substrate [biotin-LRRWSLGLRRWSLGLRRWSLGLRRWSLG]) containing 0.2 μCi [γ33P]ATP (Amersham Pharmacia, specific activity ≧2500 Ci/mmol) was then added to all test wells to start the reaction. The plates were incubated at room temperature for 60 minutes. To stop the reaction 100 μl 20% v/v orthophosphoric acid was added to all wells. The peptide substrate was captured on positively-charged nitrocellulose P30 filtermat (Whatman) using a 96-well plate harvester (TomTek) & then assayed for incorporation of 33P with a Beta plate counter. “Blank” (no enzyme) and “total” (no compound) control values were used to determine the dilution range of test compound which gave 50% inhibition of enzyme activity. In this test, the compounds of the invention generally give 50% inhibition of enzyme activity at concentrations of 1 nM to 1000 nM and in particular compound 5 in Table 1 gave 50% inhibition of enzyme activity at a concentration of 5 μM and compound 8 in Table 1 gave 50% inhibition of enzyme activity at a concentration of 6 μM.
(b) In Vitro Aurora-B Kinase Inhibition Test
This assay determines the ability of a test compound to inhibit serine-threonine kinase activity. DNA encoding Aurora-B may be obtained by total gene synthesis or by cloning. This DNA may then be expressed in a suitable expression system to obtain polypeptide with serine-threonine kinase activity. In the case of Aurora-B, the coding sequence was isolated from cDNA by polymerase chain reaction (PCR) and cloned into the pFastBac system in a manner similar to that described above for Aurora-A (i.e. to direct expression of a 6-histidine tagged Aurora-B protein).
For the large scale expression of Aurora-B kinase activity, Sf21 insect cells were grown at 28° C. in TC100 medium supplemented with 10% foetal calf serum (Viralex) and 0.2% F68 Pluronic (Sigma) on a Wheaton roller rig at 3 r.p.m. When the cell density reached 1.2×106 cells ml−1 they were infected with plaque-pure Auora-B recombinant virus at a multiplicity of infection of 1 and harvested 48 hours later. All subsequent purification steps were performed at 4° C. Frozen insect cell pellets containing a total of 2.0×108 cells were thawed and diluted with lysis buffer (50 mM HEPES (N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulphonic acid]) pH7.5 at 4° C., 1 mM Na3VO4, 1 mM PMSF (phenylmethylsulphonyl fluoride), 1 mM dithiothreitol, 1 μg/tml aprotinin, 1 μg/ml pepstatin, 1 μg/tml leupeptin), using 1.0 ml per 2×107 cells. Lysis was achieved using a sonication homogeniser, following which the lysate was centrifuged at 41,000 g for 35 minutes. Aspirated supernatant was pumped onto a 5 mm diameter chromatography column containing 1.0 ml CM sepharose Past Flow (Amersham Pharmacia Biotech) which had been equilibrated in lysis buffer. A baseline level of UV absorbance for the eluent was reached after washing the column with 12 ml of lysis buffer followed by 7 ml of wash buffer (50 mM HEPES pH7.4 at 4° C., 1 mM dithiothreitol). Bound Aurora-B B protein was eluted from the column using a gradient of elution buffer (50 mM HEPES pH7.4 at 4° C. 0.6 M NaCl, 1 mM dithiothreitol, running from 0% elution buffer to 100% elution buffer over 15 minutes at a flowrate of 0.5 ml/min). Elution fractions (1.0 ml) corresponding to the peak in UV absorbance was collected. Elution fractions were dialysed exhaustively against dialysis buffer (25 mM HEPES pH7.4 at 4° C., 45% glycerol (v/v), 100 mM KCl, 0.05% (v/v) IGEPAL CA630 (Sigrna Aldrich), 1 mM dithiothreitol). Dialysed fractions were assayed for Aurora-B kinase activity.
Each new batch of Aurora-B enzyme was titrated in the assay by dilution with enzyme diluent (25 mM Tris-HCl pH7.5, 12.5 mM KCl, 0.6 mM DIT). For a typical batch, stock enzyme is diluted 1 in 40 with enzyme diluent & 20 μl of dilute enzyme is used for each assay well. Test compounds (at 10 mM in dimethylsulphoxide (DMSO) were diluted with water & 10 μl of diluted compound was transferred to wells in the assay plates. “Total” & “blank” control wells contained 2.5% DMSO instead of compound. Twenty microlitres of freshly diluted enzyme was added to all wells, apart from “blank” wells. Twenty microlitres of enzyme diluent was added to “blank” wells. Twenty microlitres of reaction mix (25 mM Tris-HCl, 78.4 mM KCl, 2.5 mM NaF, 0.6 mM dithiothreitol, 6.26 mM MnCl2, 37.5 mM ATP, 25 μM peptide substrate [biotin-LRRWSLGLRRWSLGLRRWSLGLRRWSLG]) containing 0.2 μCi [γ33P]ATP (Amersham Pharmacia, specific activity ≧2500 Ci/mmol) was then added to all test wells to start the reaction. The plates were incubated at room temperature for 60 minutes. To stop the reaction 100 μl 20% v/v orthophosphoric acid was added to all wells. The peptide substrate was captured on positively-charged nitrocellulose P30 filtermat (Whatman) using a 96-well plate harvester (TomTek) & then assayed for incorporation of 33P with a Beta plate counter. “Blank” (no enzyme) and “total” (no compound) control values were used to determine the dilution range of test compound which gave 50% inhibition of enzyme activity. In this test, the compounds of the invention generally give 50% inhibition of enzyme activity at concentrations of 1 nM to 1000 nM and in particular compound 5 in Table 1 gave 50% inhibition of enzyme activity at a concentration of 2 μM and compound 8 in Table 1 gave 50% inhibition of enzyme activity at a concentration of 1 μM.
(c) In Vitro Cell Proliferation Assay
This and other assays can be used to determine the ability of a test compound to inhibit the growth of adherent mammalian cell lines, for example the human tumour cell line SW620 (ATCC CCL-227). This assay determines the ability of at test compound to inhibit the incorporation of the thymidine analogue, 5′-bromo-2′-deoxy-uridine (BrdU) into cellular DNA. SW620 or other adherent cells were typically seeded at 1×105 cells per well in L-15 media (GIBCO) plus 5% foetal calf serum, 1% L-glutamine (100 μl/well) in 96 well tissue culture treated 96 well plates (Costar) and allowed to adhere overnight. The following day the cells were dosed with compound (diluted from 10 mM stock in DMSO using L-15 (with 5% FCS, 1% L-glutamine). Untreated control wells and wells containing a compound known to give 100% inhibition of BrdU incorporation were included on each plate. After 48 hours in the presence/absence of test compound the ability of the cells to incorporate BrdU over a 2 hour labelling period was determined using a Boehringer (Roche) Cell Proliferation BrdU ELISA kit (cat. No. 1 647 229) according to manufacturers directions. Briefly, 15 μl of BrdU labelling reagent (diluted 1:100 in media—L-15, 5% FCS, 1% L-glutamine) was added to each well and the plate returned to a humidified (+5% CO2) 37° C. incubator for 2 hours. After 2 hours the labelling reagent was removed by decanting and tapping the plate on a paper towel. FixDenat solution (50 μl per well) was added and the plates incubated at room temperature for 45 mins with shaking. The FixDenat solution was removed by decanting and tapping the inverted plate on a paper towel. The plate was then washed once with phosphate buffered saline (PBS) and 100 μl/well of Anti-BrdU-POD antibody solution (diluted 1:100 in antibody dilution buffer) added. The plate was then incubated at room temperature with shaking for 90 min. Unbound Anti-BrdU-POD antibody was removed by decanting and washing the-plate 4 times with PBS before being blotted dry. TMB substrate solution was added (100 μl/well) and incubated for approximately 10 minutes at room temperature with shaking until a colour change was apparent. The optical density of the wells was then determined at 690 nm wavelength using a Titertek Multiscan plate reader. The values from compound treated, untreated and 100% inhibition controls were-used to determine the dilution range of a test compound that gave 50% inhibition of BrdU incorporation. The compounds of the invention are generally active at 1 nM to 100 μM in this test.
(d) In Vitro Cell Cycle Analysis Assay
This assay determines the ability of a test compound to arrest cells in specific phases of the cell cycle. Many different mammalian cell lines could be used in this assay and SW620 cells are included here as an example. SW620 cells were seeded at 7×105 cells per T25 flask (Costar) in 5 ml L-15 (5% FCS, 1% L-glutamine). Flasks were then incubated overnight in a humidified 37° C. incubator with 5% CO2. The following day, 5 μl of L-15 (5% FCS, 1% L-glutamine) carrying the appropriate concentration of test compound solubilised in DMSO was added to the flask. A no compound control treatment was also included (0.5% DMSO). The cells were then incubated for a defined time (24 hours) with compound. After this time the media was aspirated from the cells and they were washed with 5 ml of prewarmed (37° C.) sterile PBSA, then detached from the flask by brief incubation with trypsin and followed by resuspension in 5 ml of 1% Bovine Serum Albumin (BSA, Sigma-Aldrich Co.) in sterile PBSA. The samples were then centrifuged at 2200 rpm for 10 min. The supernatant was aspirated to leave 200 μl of the PBSFBSA solution. The pellet was resuspended in this 200 μl of solution by pipetting 10 times to create a single cell suspension. One ml of ice-cold 80% ethanol was slowly added to each cell suspension and the samples stored at −20° C. overnight or until required for staining. Cells were pelleted by centrifugation, ethanol aspirated off and pellets resuspended in 200 μl PBS containing 100 μg/ml RNAse (Sigma Aldrich) & 10 μg/ml Propidium Iodide (Sigma Aldrich). Cell suspensions were incubated at 37° C. for 30 min, a further 200 μl PBS added and samples stored in the dark at 4° C. overnight.
Each sample was then syringed 10 times using 21-guage needle. The samples were then transferred to LPS tubes and DNA content per cell analysed by Fluorescence activated cell sorting FACS) using a FACScan flow cytometer (Becton Dickinson). Typically 30,000 events were counted and recorded using CellQuest v1.1 software (Verity Software). Cell cycle distribution of the population was calculated using Modfit software (Verity Software) and expressed as percentage of cells with 2N (G0/G1), 2N-4N (S phase) and with 4N (G2/M) DNA content.
The compounds of the invention are generally active in this test at 1 nM to 10 μM.
The invention will now be illustrated in the following examples, in which standard techniques known to the skilled chemist and techniques analogous to those described in these examples may be used where appropriate, and in which, unless otherwise stated:
(vii) work up and purification of reaction mixtures from robotic synthesis was carried out as follows: evaporations were carried out in vacuo using a Genevac HT 4; column chromatography was performed using either an Anachem Sympur MPLC system on silica using 27 mm diameter columns filled with Merck silica (60 μm, 25 g); the structures of the final products were confirmed by LCMS on a Waters 2890/ZMD micromass system using the following and are quoted as retention time (RT) in minutes:
(viii) Analytical LCMS for compounds which had not been prepared by robotic synthesis was performed on a Waters Alliance HT system using the following and are quoted as retention time (RT) in minutes:
Waters preparative LCMS instrument, with retention time (RT) measured in minutes:
Gilson preparative HPLC instrument, with retention time (RT) measured in minutes:
(x) intermediates were not generally fully characterised and purity was assessed by thin layer chromatography (TLC), HPLC, infra-red (IR), MS or NMR analysis.
Potassium iodide (100 mg, 0.6 mmol), 2-(3-{[7-(3-chloropropoxy)quinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide hydrochloride (147 mg, 0.30 mmol) and D-prolinol (152 mg, 1.5 mmol) were heated in dimethylacetamide (1 ml) for 2.5 hours at 90° C. The reaction was cooled to ambient temperature, diluted with dichloromethane (10 ml) and purified by flash chromatography on silica gel, eluting with dichloromethane: methanol: aqueous ammonia (100:5:0.5) to (100:25:2). Evaporation of the fractions in vacuo afforded a clear oil which was crystallised by trituration with acetonitrile:dichloromethane:isohexane to yield compound 1 in table 1 (83 mg, 53% yield) as a cream coloured solid:
1H-NMR (DMSO d6): 10.50 (br s, 1H), 10.27 (s, 1H), 8.53 (m, 2H), 7.75 (s, 1H), 7.59 (m, 1H, 7.34 (m, 2H), 7.14 (m, 2H), 6.92 (m, 1H), 6.86 (s, 1H), 4.98 (s, 2H), 4.30 (br s, 1H), 4.17 (t, 2H), 3.38 (m, 1H), 3.19 (m, 1H), 3.06 (m, 1H), 2.96 (m, 1H), 2.42 (m, 2H), 2.17 (m, 1H), 1.92 (m, 2H), 1.80 (m, 1H), 1.64 (m, 2H), 1.54 (m, 1H): MS (+ve ESI): 520 (M+H)+, MS (−ve ESI): 518 (M−H)−.
2-(3-{[7-(3-chloropropoxy)quinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide hydrochloride, used as the starting material, was obtained as follows:
a) 2-Amino-4-fluorobenzoic acid (15 g, 96 mmol) was dissolved in 2-methoxyethanol (97 ml). Formamidine acetate (20.13 g, 193.4 mmol) was added and the mixture heated to reflux for 18 hours. The reaction was cooled, concentrated and the residue stirred in aqueous ammonium hydroxide (0.01 N, 250 ml) for 1 hour. The suspension was filtered, washed with water and dried over phosphorus pentoxide to yield 7-fluoroquinazolin-4(3H)-one as an off-white solid (10.35 g, 65% yield):
1H-NMR (DMSO d6): 12.32 (br s, 1H), 8.19 (dd, 1H), 8.14 (s, 1H), 7.45 (dd, 1H), 7.39 (m, 1H): MS (−ve ESI): 163 (M−H)−, MS (+ve ESI): 165 (M+H)+.
b) Sodium hydride (14.6 g, 365 mmol) was added at 0° C. to a solution of 1,3-propanediol (27.8 g, 365 rnmol) in dimethylformamide (70 ml). 7-Fluoroquinazolin-4(3H)-one (10 g, 60.9 mmol) was added portionwise and the reaction mixture heated at 60° C., then at 110° C. for 3 hours. The reaction was cooled to 0° C. quenched with water (280 ml) and adjusted to pH 5.9. The resulting suspension was filtered, washed with water then diethyl ether and dried over phosphorus pentoxide to afford 7-(3-hydroxypropoxy)quinazolin-4(3H)-one as a white powder (12.41 g, 92% yield):
1H-NMR (DMSO d6): 11.90 (br s, 1H), 8.04 (s, 1H), 8.00 (d, 1H), 7.10 (m, 2H), 4.17 (t, 2H), 3.58 (t, 2H), 1.92 (m, 2H): MS (+ve ESI): 221 (M+H)+.
1H-NMR (DMSO d6): 13.25 (br s, 1H), 8.34 (s, 1H), 8.06 (d, 1H), 7.17 (m, 2H), 4.21 (t, 2H), 3.83 (t, 2H), 2.23 (m, 211): MS (+ve ESI): 257, 259 (M+H)+.
d) 4.0 N Hydrochloric acid in dioxane (0.46 ml, 1.85 mmol) was added dropwise to a stirred solution of 4-chloro-7-(3-chloropropoxy)quinazoline (475 mg, 1.85 mmol) and 2-(3-amino-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide (430 mg, 1.85 mmol) in dimethylacetamide (5 ml) at 50° C. The reaction was heated at 70° C. for 1 hour, allowed to cool to ambient temperature and then stirred with diethyl ether (5 ml) at ambient temperature for 1 hour. Collection of the solid which precipitated by suction filtration, followed by washing with diethyl ether and acetonitrile and prolonged drying in vacuo, yielded 2-(3-{[7-(3-chloropropoxy)quinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide hydrochloride (919 mg, 99% yield) as a pale yellow solid:
1H-NMR (DMSO d6): 11.93 (br s, 1H), 10.96 (br s, 1H), 8.96 (s, 1H), 8.79 (d, 1H), 7.90 (d, 1H), 7.62 (m, 1H), 7.45 (m, 1H), 7.38 (m, 3H), 6.90 (m, 1H), 6.85 (d, 1H), 5.12 (s, 2H), 4.31 (t, 2H), 3.84 (t, 2H), 2.26 (m, 2H): MS (+ve ESI) 455 (M+H)+, MS (−ve ESI): 453 (M−H)−.
2-(3-amino-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide, used as the starting material in reaction 1d), was obtained as follows:
e) 1.0 N Aqueous sodium hydroxide solution (54 ml, 54 mmol) was added to a stirred solution of 3-fluoroaniline (11.1 g, 100 mmol) in diethyl ether (100 ml) and the reaction cooled to ° C. A solution of bromoacetyl bromide (10.2 ml, 117 mmol) in diethyl ether (100 ml) was added with vigorous stirring over 20 minutes keeping the temperature below 0° C. The reaction was allowed to warm to ambient temperature over 10 minutes before the diethyl ether layer was separated, dried over magnesium sulphate and evaporated in vacuo. The product was purified by flash chromatography on a small pad of silica gel, eluting with dichloromethane: diethyl ether (95:5). Addition of isohexane to the fractions from the column, followed by concentration in vacuo caused precipitation of a white solid which was collected by suction filtration and dried in vacuo to yield 2-bromo-N-(3-fluorophenyl)-acetamide (21.7 g, 94% yield) as a white crystalline solid:
1H-NMR (DMSO d6): 7.58 (m, 1H), 7.35 (m, 1H), 7.28 (m, 1H), 6.90 (m, 1H), 4.05 (s, 2H: MS (−ve ESI): 230, 232 (M−H)−.
f) A solution of 2-bromo-N-(3-fluorophenyl)-acetamide (2.78 g, 12.0 mmol) in dichloromethane was added dropwise over 5 minutes to a stirred suspension of 1.0N aqueous sodium hydroxide (28.8 ml, 28.8 mmol), 3-nitro-1H-pyrazole (1.50 g, 13.2 mmol) and tetra-n-butylammonium sulphate (4.88 g, 14.4 mmol) in dichloromethane (10 ml) at 2° C. The reaction was allowed to warm to ambient temperature over 4 hours after which time the organic layer was separated and passed through a short plug of silica gel (washed with diethyl ether). Evaporation of the combined organic layers in vacuo yielded N-(3-fluorophenyl)-2-(3-nitro-1H-pyrazol-1-yl)acetamide (1.41 g, 45% yield) as a white solid:
1H-NMR (DMSO d6): 10.62 (s, 1H), 8.08 (d, 1H), 7.55 (m, 1H), 7.38 (m, 1H), 7.31 (m, 1H), 7.10 (d, 1H), 6.93 (m, 1H), 5.23 (s, 2H): MS (−ve ESI): 263 (M−H)−.
g) A solution of N-(3-fluorophenyl)-2-(3-nitro-1H-pyrazol-1-yl)acetamide (1.33 g, 50.5 mmol) in ethyl acetate (50 ml) and ethanol (12.5 ml) was hydrogenated under an atmosphere of hydrogen in the presence of 10% palladium on carbon (130 mg) for 4 hours. The catalyst was removed by filtration through Celite and evaporation of the solvents in vacuo yielded 2-(3-amino-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide (898 mg, 76% yield) as a white crystalline solid:
1H-NMR (DMSO d6): 10.36 (br s, 1H), 7.58 (m, 1H), 7.38 (m, 2H), 7.32 (m, 1H), 5.45 (d, 1H), 4.69 (s, 2H), 4.57 (br s, 2H): MS (+ve ESI): 235 (M+H)+, MS (−ve ESI): 233 (M−H)−.
An analogous reaction to that described in example 1, but starting with 2-(3-{[7-(3-chloropropoxy)quinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide hydrochloride (147 mg, 0.30 mmol) and 2-(n-propylamino)ethanol (155 mg, 1.5 mmol) yielded compound 2 in table 1 (96 mg, 61% yield) as a pale yellow solid:
1H-NMR (DMSO d6): 10.50 (s, 1H), 10.27 (s, 1H), 8.53 (m, 2H), 7.75 (m, 1H), 7.60 (m, 1H), 7.34 (m, 2H), 7.14 (m, 2H), 6.91 (m, 1H), 6.87 (s, 1H), 4.99 (s, 2H), 4.32 (br s, 1H), 4.18 (t, 2H), 3.46 (m, 2H), 2.62 (m, 2H), 2.53 (m, 2H), 2.32 (m, 2H), 1.91 (m, 2H), 1.42 (m, 2H), 0.84 (t, 3H): MS (+ve ESI): 522 (M+H)+, MS (−ve ESI): 520 (M−H)−.
An analogous reaction to that described in example 1, but starting with 2-(3-{[7-(3-chloropropoxy)quinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(2,3-difluorophenyl)acetamide hydrochloride (153 mg, 0.30 mmol) and D-prolinol (152 mg, 1.5 mmol) yielded compound 3 in table 1 (123 mg, 76% yield) as a pale yellow solid:
1H-NMR (DMSO d6): 10.29 (br s, 2H), 8.52 (m, 2H), 7.74 (m, 2H), 7.18 (m, 4H), 6.87 (s, 1H), 5.09 (s, 2H), 4.28 (br s, 1H), 4.18 (t, 2H), 3.39 (m, 1H), 3.19 (m, 1H), 3.06 (m, 1H), 2.97 (m, 1H), 2.44 (m, 2H), 2.17 (m, 1H), 1.91 (m, 2H), 1.80 (m, 1H), 1.64 (m, 2H), 1.52 (m, 1H): MS (+ve ESI): 538 (M+H)+, MS (−ve ESI): 536 (M−H)−.
2-(3-{[7-(3-chloropropoxy)quinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(2,3-difluorophenyl)acetamide hydrochloride, used as the starting material, was obtained as follows:
a) An analogous reaction to that described in example 1e), but starting with 2,3-difluoroaniline (12.9 g, 100 mmol) yielded 2-bromo-N-(2,3-difluorophenyl)acetamide (23.9 g, 96% yield) as a white solid:
1H-NMR (DMSO d6): 7.68 (m, 1H), 7.20 (m, 2H), 4.17 (s, 2H): MS (−ve ESI): 248, 250 (M−H)−.
b) An analogous reaction to that described in example 1f), but starting with 2-bromo-N-(2,3-difluorophenyl)acetamide (2.00 g, 8.00 mmol) and 3-nitro-1H-pyrazole (1.00 g, 8.85 mmol) yielded N-(2,3-difluorophenyl)-2-(3-nitro-1H-pyrazol-1-yl)acetamide (1.15 g, 51% yield) as a white crystalline solid:
1H-NMR (DMSO d6): 8.05 (d, 1H), 7.69 (m, 1H), 7.18 (m, 2H), 7.08 (d, 1H), 5.31 (s, 2H): MS (−ve ESI): 281 (M−H)−.
c) An analogous reaction to that described in example 1g), but starting with N-(2,3-difluorophenyl)-2-(3-nitro-1H-pyrazol-1-yl)acetamide (1.13 g, 4.00 mmol) yielded 2-(3-amino-1H-pyrazol-1-yl)-N-(2,3-difluorophenyl)acetamide (934 mg, 93% yield) as a white crystalline solid:
1H-NMR (DMSO d6): 10.08 (br s, 1H), 7.73 (m, 1H), 7.38 (m, 1H), 7.17 (m, 2H), 5.45 (s, 1H), 4.79 (s, 2H), 4.62 (br s, 2H): MS (+ve ESI): 253 (M+H)+, MS (−ve ESI): 251 (M−H)−.
d) An analogous reaction to that described in example 1d), but starting with 4-chloro-7-(3-chloroxypropoxy)quinazoline (450 mg, 1.75 mmol) and 2-(3-amino-1H-pyrazol-1-yl)-N-(2,3-difluorophenyl)acetamide (440 mg, 1.75 mmol) yielded 2-(3-{[7-(3-chloropropoxy)quinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(2,3-difluorophenyl)acetamide hydrochloride (800 mg, 90% yield) as a white solid:
1H-NMR (DMSO d6): 11.94 (br s, 1H), 10.50 (br s, 1H), 8.95 (s, 1H), 8.80 (d, 1H), 7.90 (s, 1H), 7.68 (m, 1H), 7.34 (m, 2H), 7.21 (m, 2H), 6.96 (s, 1H), 5.20 (s, 2H), 4.32 (t, 2H), 3.94 (t, 2H), 2.26 (m, 2H): MS (+ve ESI): 473 (M+H)+, MS (−ve ESI) 471 (M−H)−.
An analogous reaction to that described in example 1, but starting with 2-(3-{[7-(3-chloropropoxy)quinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(2,3-difluorophenyl)acetamide hydrochloride (153 mg, 0.30 mmol) and 2-(n-propylamino)ethanol (155 mg, 1.5 mmol) yielded compound 4 in table 1 (112 mg, 69% yield) as a pale yellow solid:
1H-NMR (DMSO d6): 10.29 (br s, 2H), 8.54 (m, 2H), 7.74 (m, 2H), 7.20 (m, 4H), 6.88 (s, 1H), 5.09 (s, 2H), 4.25 (br s, 1H), 4.18 (t, 2H), 3.46 (m, 2H), 2.56 (m, 6H), 1.91 (m, 2H), 1.40 (m, 2H), 0.83 (t, 3H): MS (+ve ESI): 540 (M+H)+, MS (−ve ESI): 538 (M−H)−.
An analogous reaction to that described in example 1, but starting with 2-(3-{[7-(3-chloropropoxy)-6-methoxyquinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide hydrochloride (156 mg, 0.30 mmol) and D-prolinol (152 mg, 1.5 mmol) yielded compound 5 in table 1 (129 mg, 78% yield) as a pale yellow solid:
1H-NMR (DMSO d6): 10.50 (br s, 1H), 10.19 (s, 1H), 8.48 (s, 1H), 7.98 (s, 1H), 7.74 (m, 2H), 7.59 (m, 1H), 7.35 (m, 2H), 7.16 (s, 1H), 6.90 (m, 2H), 4.98 (s, 2H), 4.32 (br s, 1H), 4.18 (t, 2H), 3.91 (s, 3H), 3.40 (m, 1H), 3.23 (m, 1H), 3.10 (m, 1H), 3.00 (m, 1H), 2.46 (m, 2H), 2.20 (m, 1H), 1.96 (m, 2H), 1.81 (m, 1H), 1.67 (m, 2H), 1.56 (m, 1H): MS (+ve ESI): 550 (M+H)+, MS (−ve ESI): 548 (M−H)−.
2-(3-{[7-(3-chloropropoxy)-6-methoxyquinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide, used as the starting material, was obtained as follows:
a) A mixture of 2-amino-4-benzyloxy-5-methoxybenzamide (10 g, 0.04 mol), (prepared according to J. Med. Chem. 1977, 20, 146-149), and Gold's reagent (7.4 g, 0.05 mol) in dioxane (100 ml) was stirred and heated at reflux for 24 hours. Sodium acetate (3.02 g, 0.037 mol) and acetic acid (1.65 ml, 0.029 mol) were added to the reaction mixture and it was heated for a further 3 hours. The volatiles were removed by evaporation, water was added to the residue, the solid was collected by filtration, washed with water and dried. Recrystallisation from acetic acid yielded 7-(benzyloxy)-6-methoxyquinazolin-4(3H)-one (8.7 g, 84% yield) as a white solid.
b) Chloromethyl pivalate (225 ml, 1.56 mol) was added dropwise to a stirred mixture of 7-(benzyloxy)-6-methoxyquinazolin-4(3H)-one (400 g, 1.42 mol) and potassium carbonate (783 g, 5.67 mol) in dimethylacetamide (5500 ml). The reaction was heated to 90° C. for 4 hours. The reaction was cooled and filtered to remove inorganic salts. The filtrate was concentrated in vacuo to yield, crude tert-butyl 2-[7-(enzyloxy)-6-methoxy-4-oxo-3(4H)-quinazolinyl]acetate (562 g, 100% yield):
1H-NMR (DMSO-d6): 8.33 (s, 1H), 7.30-7.50 (m, 6H), 7.25 (s, 1H), 5.90 (s, 2H), 5.25 (s, 2H), 3.88 (s, 3H), 1.10 (s, 9H): MS (+ve ESI): 397 (M+H)+
c) 10% palladium on carbon (56 g, 53 mmol) was added to a solution of tert-butyl 2-[7-(benzyloxy)-6-methoxy-4-oxo-3(4H)-quinazolinyl]acetate (562 g, 1.42 mmol) in dimethylacetamide (3500 ml) at ambient temperature and stirred for 3 hours under an atmosphere of hydrogen (1 bar). The reaction was filtered through a pad of celite and the solvent evaporated in vacuo. The residual solid was dissolved in 20% methanol in dichloromethane and passed through a pad of silica gel. Evaporation of the solvent in vacuo followed by trituration with methanol yielded, tert-butyl
2-[7-hydroxy-6-methoxy-4-oxo-3(4H)-quinazolinyl]acetate (188 g, 43% yield):
1H-NMR (DMSO-d6): 8.25 (s, 1H), 7.45 (s, 1H), 6.97 (s, 1H), 5.85 (s, 2H), 4.04 (s, 1H), 3.87 (s, 3H), 1.10 (s, 9H): MS (+ve ESI): 307 (M+H)+
d) A mixture of tert-butyl 2-[7-hydroxy-6-methoxy-4oxo-3(4H)-quinazolinyl]-acetate (100 g, 0.327 mol), 3-bromopropanol (49.3 g, 0.355 mol) and potassium carbonate (133 g, 0.967 mol) in dimethylformamide (500 ml) was stirred at 80° C. for 20 hours. The reaction was cooled and concentrated to quarter volume in vacuo. The residue was poured into ice/water (1500 ml) and the resulting solid collected by suction filtration. Purification by crystallisation from ethanol, yielded crude tert-butyl
2-[7-(3-hydroxypropoxy)-6-methoxyoxo-3(4H)-quinazolinyl]acetate (33.8 g, 41% yield) as a beige solid:
1H-NMR (DMSO-d6): 7.95 (s, 1H), 7.43 (s, 1H), 7.1 (s, 1H), 4.16 (t, 2H), 3.86 (m, 5H), 2.08 (t, 2H), 1.12 (s, 9H): MS (+ve ESI): 365 (M+H)+
e) Aqueous sodium hydroxide solution (100 ml, 0.2 mol) was added to a solution of tert-butyl 2-[7-(3-hydroxypropoxy)-6-methoxy-4-oxo-3(4H)-quinazolinyl]acetate (33.8 g, 93 mmol) in methanol (300 ml) and the solution heated to reflux for 1 hour. The methanol was evaporated in vacuo and the aqueous residue acidified with aqueous hydrochloric acid then sodium bicarbonate was added. Collection of the solid by suction filtration, washing with water and drying gave 7-(3-hydroxypropoxy)-6-methoxy-4-quinazolone (26 g, 95% yield):
1H-NMR (DMSO-d6): 7.96 (s, 1H), 7.41 (s, 1H), 7.07 (s, 1H), 4.14 (t, 2H), 3.84 (s, 3H), 3.55 (t, 2H), 1.90 (t, 2H): MS (+ve ESI): 251 (M+H)+
f) 7-(3-hydroxypropoxy)-6-methoxy-4-quinazolone (25 g, 100 mmol) was added slowly to a solution of dimethylformamide (1 ml) in thionyl chloride (250 ml). The mixture was heated to reflux for 4 hours then cooled and the solvents evaporated in vacuo. The residue was dissolved in dichloromethane and washed with aqueous sodium bicarbonate, brine, dried over magnesium sulphate and evaporated. Trituration and collection of the solid by suction filtration yielded, 4-chloro-6-methoxy-7-(3-chloroxypropoxy)quinazoline (19.5 g, 68% yield) as a yellow solid:
1H-NMR (CDCl3): 8.85 (s, 1H), 7.40 (s, 1H), 7.38 (s, 1H), 4.38 (t, 2H), 4.03 (s, 3H), 3.8 (t, 2H), 2.40 (m, 2H): MS (+ve ESI): 287 (M+H)+
g) An analogous reaction to that described in example 1d), but starting with 4-chloro-6-methoxy-7-(3-chloroxypropoxy)quinazoline (531 mg, 1.85 mmol) and 2-(3-amino-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide (430 mg, 1.85 mmol) yielded 2-(3-{[7-(3-chloropropoxy)-6-methoxyquinazolinyl]amino}-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide hydrochloride (841 mg, 86% yield) as a white solid:
1H-NMR (DMSO d6): 11.80 (br s, 1H), 10.94 (br s, 1H), 8.92 (s, 1H), 8.26 (s, 1H), 7.89 (s, 1H), 7.61 (m, 1H), 7.45 (s, 1H), 7.38 (m, 2H), 6.90 (m, 2H), 5.12 (s, 2H), 4.30 (t, 2H), 3.97 (s, 3H), 3.82 (t, 2H), 2.30 (m, 2H): MS (+ve ESI): 485 (M+H)+, MS (−ve ESI): 483 (M−H)−.
An analogous reaction to that described in example 1, but starting with 2-(3-{[7-(3-chloropropoxy)-6-methoxyquinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(3-fluorophenyl)acetamide hydrochloride (156 mg, 0.30 mmol) and 2-(n-propylamino)ethanol (155 mg, 1.5 mmol) yielded compound 6 in table 1 (111 mg, 67% yield) as a pale yellow solid:
1H-NMR (DMSO d6): 10.50 (s, 1H), 10.19 (s, 1H), 8.47 (s, 1H), 7.97 (s, 1H), 7.74 (s, 1H), 7.57 (m, 1H), 7.35 (m, 2H), 7.14 (s, 1H), 6.91 (m, 2H), 4.99 (s, 2H), 4.26 (m, 1H), 4.18 (t, 2H), 3.92 (s, 3H), 3.44 (m, 2H), 2.58 (t, 2H), 2.49 (m, 2H), 2.39 (t, 2H), 1.89 (m, 2H), 1.29 (m, 2H), 0.81 (t,3H): MS (+ve ESI): 552 (M+H)+, MS (−ve ESI): 550 (M−H)−.
An analogous reaction to that described in example 1, but starting with 2-(3-{[7-(3-chloropropoxy)-6-methoxyquinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(2,3-difluorophenyl)acetamide hydrochloride (162 mg, 0.30 mmol) and D-prolinol (152 mg, 1.5 mmol) yielded compound 7 in table 1 (132 mg, 78% yield) as a pale yellow solid:
1H-NMR (DMSO d6): 10.29 (br s, 1H), 10.21 (s, 1H), 8.47 (s, 1H), 7.97 (s, 1H), 7.72 (m, 2H), 7.19 (m, 3H), 6.90 (s, 1H), 5.09 (s, 2H), 4.29 (br s, 1H), 4.16 (t, 2H), 3.93 (s, 3H), 3.40 (m, 1H), 3.18 (m, 1H), 3.08 (m, 1H), 2.96 (m, 1H), 2.42 (m, 2H), 2.16 (m, 1H), 1.95 (m, 2H), 1.80 (m, 1H), 1.64 (m, 2H), 1.55 (m, 1H): MS (+ve ESI): 568 (M+H)+, MS (−ve ESI): 566 (M−H)−.
2-(3-{[7-(3-chloropropoxy)-6-methoxyquinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(2,3-difluorophenyl)acetamide hydrochloride, used as the starting material, was obtained as follows:
a) An analogous reaction to that described in example 1d), but starting with 4-chloro-6-methoxy-7-(3-chloroxypropoxy)quinazoline (502 mg, 1.75 mmol) and 2-(3-amino-1H-pyrazol-1-yl)-N-(2,3-difluorophenyl)acetamide (440 mg, 1.75 mmol) yielded 2-(3-{[7-(3-chloropropoxy)-6-methoxyquinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(2,3-difluorophenyl)acetamide (650 mg, 69% yield) as a white solid:
1H-NMR (MSO d6): 11.80 (br s, 1H), 10.50 (br s, 1H), 8.91 (s, 1H), 8.26 (s, 1H), 7.90 (s, 1H), 7.59 (m, 1H), 7.47 (s, 1H), 7.20 (m, 2H), 6.90 (s, 1H), 5.20 (s, 2H), 4.29 (t, 2H), 3.97 (s, 3H), 3.84 (t, 2H), 2.31 (m, 2H): MS (+ve ESI): 503 (M+H)+, MS (−ve ESI): 501 (M−H)−.
An analogous reaction to that described in example 1, but starting with 2-(3-{[7-(3-chloropropoxy)-6-methoxyquinazolin-4-yl]amino}-1H-pyrazol-1-yl)-N-(2,3-difluorophenyl)acetamide (162 mg, 0.30 mmol) and 2-(n-propylamino)ethanol (155 mg, 1.5 mmol) yielded compound 8 in table 1 (127 mg, 75% yield) as a pale yellow solid:
1H-NMR (DMSO d6): 10.28 (br s, 1H), 10.21 (br s, 1H), 8.48 (s, 1H), 7.98 (s, 1H), 7.74 (m, 2H), 7.20 (m, 2H), 7.15 (s, 1H), 6.92 (s, 1H), 5.09 (s, 2H), 4.27 (t, 1H), 4.17 (t, 2H), 3.93 (s, 3H), 3.44 (m, 2H), 2.59 (m, 2H), 2.50 (m, 2H), 2.39 (t, 2H), 1.89 (m, 2H), 1.39 (m, 2H), 0.82 (t, 3H): MS (+ve ESI): 570 (M+H)+, MS (−ve ESI): 568 (M−H)−.
1H-NMR (DMSO d6): 8.96 (s, 1H), 8.80 (d, 1H), 7.90 (s, 1H), 7.61 (m, 1H), 7.40 (m, 4H), 6.92 (m, 1H), 6.86 (s, 1H), 5.12 (s, 2H), 4.32 (t, 2H), 4.21 (m, 2H), 3.77 (m, 1H), 3.64 (m, 2H), 3.29 (m, 1H), 3.19 (m, 1H), 2.32 (m, 2H), 2.19 (m, 1H), 1.97 (m, 2H), 1.81 (m, 1H): MS (+ve ESI): 600 (M+H)+, MS (−ve ESI): 598 (M−H)−
An analogous reaction to that described in example 9, but starting with N-(2,3-difluorophenyl)-2-{3-[(7-{3-[(2R)-2-(hydroxymethyl)pyrrolidin-1-yl]propoxy}quinazolin-4-yl)amino]-1H-pyrazol-1-yl}acetamide (243 mg, 0.452 mmol), yielded compound 10 in table 2 (239 mg, 74% yield):
1H-NM (DMSO d6): 8.94 (s, 1H), 8.80 (d, 1H), 7.89 (s, 1H), 7.68 (m, 1H), 7.45 (m, 2H), 7.21 (m, 2H), 6.86 (m, 2H), 5.20 (s, 2H), 4.33 (t, 2H), 4.21 (m, 2H), 3.77 (m, 1H), 3.62 (m, 2H), 3.30 (m, 1H), 3.18 (m, 1H), 2.33 (m, 2H), 2.18 (m, 1H), 1.98 (m, 2H), 1.81 (m, 1H): MS (+ve ESI): 618 (M+H)+, MS (−ve ESI): 616 (M−H)−
An analogous reaction to that described in example 9 but starting with N-(3-fluorophenyl)-2-{3-[(7-{3-[(2R)-2-(hydroxymethyl)pyrrolidin-1-yl]propoxy}-6-methoxyquinazolin-4-yl])amino]-1H-pyrazol-1-yl}acetamide (278 mg, 0.506 mmol), yielded compound 11 in table 2 (295 mg, 79% yield):
1H-NMR (DMSO d6): 8.92 (s, 1H), 8.28 (s, 1H), 7.90 (s, 1H), 7.62 (m, 1H), 7.49 (s, 1H), 7.38 (m, 2H), 6.91 (m, 2H), 5.12 (s, 2H), 4.31 (t, 2H), 4.22 (m, 2H), 3.98 (s, 3H), 3.78 (m, 1H), 3.68 (m, 1H), 3.58 (m, 1H), 3.27 (m, 1H), 3.20 (m, 1H), 2.34 (m, 2H), 2.19 (m, 1H), 2.03 (m, 1H), 1.96 (m, 1H), 1.82 (m, 1H): MS (+ve ESI): 630 (M+H)+, MS (−ve ES]): 628 (M−H)−
An analogous reaction to that described in example 9 but starting with N-(2,3-difluorophenyl)-2-{3-[(7-{3-[(2R)-2-(hydroxymethyl)pyrrolidin-1-yl]propoxy}-6-methoxyquinazolin-4-yl)amino]-1H-pyrazol-1-yl}acetamide (241 mg, 0.439 mmol), yielded compound 12 in table 2 (161 mg, 49% yield):
1H-NMR (DMSO d6): 8.93 (s, 1H), 8.30 (s, 1H), 7.90 (s, 1H), 7.68 (m, 1H), 7.51 (s, 1H), 7.20 (m, 2H), 6.90 (s, 1H), 5.20 (s, 2H), 4.31 (t, 2H), 4.20 (m, 2H), 3.99 (s, 3H), 3.78 (m, 1H), 3.68 (m, 1H), 3.59 (m, 1H), 3.27 (m, 1H), 3.20 (m, 1H), 2.34 (m, 2H), 2.19 (m, 1H), 2.02 (m, 1H), 1.95 (m, 1H), 1.82 (m, 1H): MS (+ve ESI): 648 (M+H)+, MS (−ve ESI): 646 (M−H)−
| Number | Date | Country | Kind |
|---|---|---|---|
| 03291314.7 | Jun 2003 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB04/02281 | 5/27/2004 | WO | 12/1/2005 |
