# Patent

## Title
Bicyclic heterocyclic compounds as protein tyrosine kinase inhibitors

## Abstract

The invention relates to new bicyclic heterocyclic derivative compounds of formula (I): wherein R1, q, A, B, X1, X2, X3, X4, X5 and R2 are as defined herein, to pharmaceutical compositions comprising said compounds and to the use of said compounds in the treatment of diseases, e.g. cancer.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 12/682,478, published on Nov. 11, 2010 as US 2010-0286113 A1, which is a national phase filing under 35 U.S.C. §371 of PCT International Application No. PCT/GB2008/003439, filed Oct. 10, 2008, and published under PCT Article 21(2) in English as WO2009/047522 on Apr. 16, 2009. PCT/GB2008/003439 claimed priority to U.S. Provisional Patent Application No. 60/979,589, which was filed on Oct. 12, 2007; U.S. Provisional Patent Application No. 61/061,172, which was filed on Jun. 13, 2008; and British Application No. 0720038.9, which was filed on Oct. 12, 2007. The entire contents of each of the prior applications are incorporated herein by reference.


FIELD OF THE INVENTION
The invention relates to new bicyclic heterocyclic derivative compounds, to pharmaceutical compositions comprising said compounds and to the use of said compounds in the treatment of diseases, e.g. cancer.
SUMMARY OF THE INVENTION
According to a first aspect of the invention there is provided a compound of formula (I):




wherein

X1, X2 and X3 are each independently selected from carbon or nitrogen, such that at least one of X1-X3 represents nitrogen and such that when X1 represents nitrogen, at least one of X2, X3, X4 and X5 is nitrogen;

X4 represents CR3, nitrogen, NH or C═O;

X5 represents CR6, nitrogen, NH or C═O;

provided that no more than three of X1-X5 represent nitrogen;

 represents a single or double bond, such that at least one bond within the 5 membered ring system is a double bond and such that the bond between X4 and X5 represents a single bond only when X4 or X5 represents C═O;

R3 represents hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C3-6 cycloalkyl, C3-6 cycloalkenyl, cyano, haloC1-6 alkyl or haloC1 alkoxy;

A represents an aromatic or non-aromatic carbocyclic or heterocyclic group which may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

B represents a —V-carbocyclic group or a —W-heterocyclyl group wherein said carbocyclic and heterocyclyl groups may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

R6 represents halogen, hydrogen, C1-6 alkyl, C1-6 alkoxy, C2-8 alkenyl, C2-5 alkynyl, —C≡N, C3-8 cycloalkyl, C3-8 cycloalkenyl, —NHSO2Rw, —CH═N—ORw, or a 3-6 membered monocyclic heterocyclyl group wherein said C1-6 alkyl, C2-C6 alkenyl, C2-6 alkynyl, C1-6 alkoxy and heterocyclyl groups may be optionally substituted by one or more Ra groups;

Re, Rf and Rw independently represent hydrogen or C1-6 alkyl;

Ra represents halogen, C1-6 alkyl, C2-8 alkenyl, C2-8 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, —ORx, —O—(CH2)n—ORx, haloC1-6 alkyl, haloC1-6 alkoxy, C1-6 alkanol, ═O, ═O, nitro, Si(Rx)4, —(CH2)s—CN, —S—Rx, —SO2—Rx, —CORx, —(CRxRy)s—COORz, —(CH2)s—CONRxRy, —(CH2)s—NRxRy, —(CH2)s—NRxCORy, —(CH2)s—NRxSO2—Ry, —(CH2)s—NH—SO2—NRxRy, —OCONRxRy, —(CH2)s—NRxCO2Ry, —O—(CH2)s—CRxRy—(CH2)t—ORz or —(CH2)s—SO2NRxRy groups;

Rx, Ry and Rz independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkanol, hydroxy, C1-6 alkoxy, haloC1-6 alkyl, —CO—(CH2)n—C1-6 alkoxy, C3-8 cycloalkyl or C3-8 cycloalkenyl;

R2 represents a —CONR7R8, —CORx or —COORz group;

R7 and R8 independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, aryl, heterocyclyl or R7 and R8 together with the nitrogen atom to which they are attached may form a nitrogen containing heterocyclyl ring, wherein said C1-6 alkyl, aryl and heterocyclyl may be optionally substituted by one or more (e.g. 1, 2 or 3) Rb groups;

R1 and Rb independently represent an Ra group or a —Y-carbocyclic or —Z-heterocyclyl group wherein said carbocyclic and heterocyclyl groups may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

V and W independently represent a bond or a —(CReRf)n— group;

Y and Z independently represent a bond, —CO—(CH2)s—, —COO—, —(CH2)n—, —NRx—(CH2)s—, —(CH2)s—NRx—, —CONRx—, —SO2NRx—, —NRxSO2—, —NRxCONRy—, —NRxCSNRy—, —O—(CH2)s—, —(CH2)s—O—, S—, —SO— or —(CH2)5—SO2—;

n represents an integer from 1-4;

s and t independently represent an integer from 0-4;

q represents an integer from 0-2;

or a pharmaceutically acceptable salt, solvate or derivative thereof.

WO 01/38326 (Merck), WO 2003/048132 (Merck), WO 02/080914 (Gruenenthal), WO 01/14375 (Astra Zeneca), WO 2004/052286 (Merck), WO 00/53605 (Merck), WO 03/101993 (Neogenesis), WO 2005/075470 (SmithKline Beecham), WO 2005/054230 (Cytopia), WO 2002/46168 (Astra Zeneca), WO 01/66098 (Aventis), WO 97/12613 (Warner Lambert), WO 2006/094235 (Sirtris Pharmaceuticals) and US 2006/0035921 (OSI Pharmaceuticals) each disclose a series of heterocyclic derivatives.


DETAILED DESCRIPTION OF THE INVENTION
According to a first aspect of the invention there is provided a compound of formula (I):




wherein

X1, X2 and X3 are each independently selected from carbon or nitrogen, such that at least one of X1-X3 represents nitrogen and such that when X1 represents nitrogen, at least one of X2, X3, X4 and X5 is nitrogen;

X4 represents CR3, nitrogen, NH or C═O;

X5 represents CR6, nitrogen, NH or C═O;

provided that no more than three of X1-X5 represent nitrogen;

 represents a single or double bond, such that at least one bond within the 5 membered ring system is a double bond and such that the bond between X4 and X5 represents a single bond only when X4 or X5 represents C═O;

R3 represents hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C3-6 cycloalkyl, C3-6 cycloalkenyl, cyano, haloC1-6 alkyl or haloC1-6 alkoxy;

A represents an aromatic or non-aromatic carbocyclic or heterocyclic group which may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

B represents a —V-carbocyclic group or a —W-heterocyclyl group wherein said carbocyclic and heterocyclyl groups may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

R6 represents halogen, hydrogen, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, —C≡N, C3-8 cycloalkyl, C3-8 cycloalkenyl, —NHSO2Rw, —CH═N—ORw, or a 3-6 membered monocyclic heterocyclyl group wherein said C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy and heterocyclyl groups may be optionally substituted by one or more Ra groups;

Re, Rf and Rw independently represent hydrogen or C1-6 alkyl;

Ra represents halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, —ORx, —O—(CH2)n—ORx, haloC1-6 alkyl, haloC1-6 alkoxy, C1-6 alkanol, ═O, ═S, nitro, Si(Rx)4, —(CH2)s—CN, —S—Rx, —SO—Rx, —SO2—Rx, —CORx, —(CRxRy)s—COORz, —(CH2)s—CONRxRy, —(CH2)s—NRxRy, —(CH2)s—NRxCORy, —(CH2)s—NRxSO2—Ry, —(CH2)s—NH—SO2—NRxRy, —OCONRxRy, —(CH2)s—NRxCO2Ry, —O—(CH2)s—CRxRy—(CH2)t—ORz or —(CH2)s—SO2NRxRy groups;

Rx, Ry and Rz independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkanol, hydroxy, C1-6 alkoxy, haloC1-6 alkyl, —CO—(CH2)n—C1-6 alkoxy, C3-8 cycloalkyl or C3-8 cycloalkenyl;

R2 represents a —CONR7R8, —CORx or —COORz group;
R7 and R8 independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, aryl, heterocyclyl or R7 and R8 together with the nitrogen atom to which they are attached may form a nitrogen containing heterocyclyl ring, wherein said C1-6 alkyl, aryl and heterocyclyl may be optionally substituted by one or more (e.g. 1, 2 or 3) Rb groups;
R1 and Rb independently represent an Ra group or a —Y-carbocyclic or —Z-heterocyclyl group wherein said carbocyclic and heterocyclyl groups may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;
V and W independently represent a bond or a —(CReRf)n— group;
Y and Z independently represent a bond, —CO—(CH2)s—, —COO—, —(CH2)n—, —NRx—(CH2)s, —(CH2)s—NRx—, —CONRx—, —NRxCO—, —SO2NRx—, —NRxCONRy—, —NRxCONRy—, —O—(CH2)s—, —(CH2)s—O—, S—, —SO— or —(CH2)s—SO2—;
n represents an integer from 1-4;
s and t independently represent an integer from 0-4;
q represents an integer from 0-2;
or a pharmaceutically acceptable salt, solvate or derivative thereof.
According to one particular aspect of the invention there is provided a compound of formula (Ia):




wherein

X1, X2 and X3 are each independently selected from carbon or nitrogen, such that at least one of X1-X3 represents nitrogen;

X4 represents CR3 or nitrogen;

X5 represents CR6, nitrogen or C═O;

provided that no more than three of X1-X5 represent nitrogen;

 represents a single or double bond, such that when X5 represents C═O, X4 and

X5 are joined by a single bond and such that at least one bond within the 5 membered ring system is a double bond;

R3 represents hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C3-6 cycloalkyl, C3-6 cycloalkenyl, cyano, haloC1-6 alkyl, haloC1-6 alkoxy or ═O;

A represents an aromatic or non-aromatic carbocyclic or heterocyclic group which may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

B represents a —V-carbocyclic group or a —W-heterocyclyl group wherein said carbocyclic and heterocyclyl groups may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

R6 represents halogen, hydrogen, C1-6 alkyl, C1-6 alkoxy, C2-6 alkenyl, C2-6 alkynyl, —C≡N, C3-8 cycloalkyl, C3-8 cycloalkenyl, —NHSO2Rw, —CH═N—ORw, or a 3-6 membered monocyclic heterocyclyl group wherein said C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy and heterocyclyl groups may be optionally substituted by one or more Ra groups;

Re, Rf and Rw independently represent hydrogen or C1-6 alkyl;

Ra represents halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-5 cycloalkyl, C3-5 cycloalkenyl, —ORx, —O—(CH2)n—ORx, haloC1-6 alkyl, haloC1-6 alkoxy, C1-6 alkanol, ═O, ═S, nitro, Si(Rx)4, —(CH2)s—CN, —S—Rx, —SO2—Rx, —CORx, —(CRxRy)s—COORz, —(CH2)s—CONRxRy, —(CH2)s—NRxRy, —(CH2)sNRxCORy, —(CH2)s—NRxSO2—Ry, —(CH2)s—NH—SO2—NRxRy, —OCONRxRy, —(CH2)s—NRxCO2Ry, —O—(CH2)s—CRxRy—(CH2)t—ORz or —(CH2)s—SO2NRxRy groups;

Rx, Ry and Rz independently represent hydrogen, C1-6, alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkanol, hydroxy, C1-6 alkoxy, haloC1-6 alkyl, —CO—(CH2)n—C1-6 alkoxy, C3-8 cycloalkyl or C3-8 cycloalkenyl;

R2 represents a —CONR7R8, —CORx or —COORz group;

R7 and R8 independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, aryl, heterocyclyl or R7 and R8 together with the nitrogen atom to which they are attached may form a nitrogen containing heterocyclyl ring, wherein said alkyl, aryl and heterocyclyl may be optionally substituted by one or more (e.g. 1, 2 or 3) Rb groups;

R1 and Rb independently represent an Ra group or a —Y-aryl or —Z-heterocyclyl group wherein said aryl and heterocyclyl groups may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

V and W independently represent a bond or a —(CReRf)n— group;

Y and Z independently represent a bond, —CO—(CH2)s—, —COO—, —(CH2)n—, —NRx—(CH2)n—, —(CH2)n—NRx—, —CONRx—, —NRxCO—, —SO2NRx—, —NRxSO2—, —NRxCONRy—, —NRxCSNRy—, —O—(CH2)s—, —(CH2)s—O—, S—, —SO— or —(CH2)s—SO2—;

n represents an integer from 1-4;

s and t independently represent an integer from 0-4;

q represents an integer from 0-2;

aryl represents a carbocyclic ring;

heterocyclyl represents a heterocyclic ring;

or a pharmaceutically acceptable salt, solvate or derivative thereof.

According to a further particular aspect of the invention there is provided a compound of formula (Ib):




wherein

X1, X2 and X3 are each independently selected from carbon or nitrogen, such that at least one of X1-X3 represents nitrogen and such that when X1 represents nitrogen, at least one of X2, X3, X4 and X5 is nitrogen;

X4 represents CR3 or nitrogen;

X5 represents CR6, nitrogen or C═O;

provided that no more than three of X1-X5 represent nitrogen;

 represents a single or double bond, such that at least one bond within the 5 membered ring system is a double bond;

R3 represents hydrogen, halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy, C3-6 cycloalkyl, C3-6 cycloalkenyl, cyano, haloC1-6 alkyl, haloC1-6 alkoxy or ═O;

A represents an aromatic or non-aromatic carbocyclic or heterocyclic group which may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

B represents a —V-carbocyclic group or a —W-heterocyclyl group wherein said carbocyclic and heterocyclyl groups may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

R6 represents halogen, hydrogen, C1-6 alkyl, C1-6, alkoxy, C2-6 alkenyl, C2-6 alkynyl, —C≡N, C3-8 cycloalkyl, C3-8 cycloalkenyl, —NHSO2Rw, —CH═N—ORw, or a 3-6 membered monocyclic heterocyclyl group wherein said C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkoxy and heterocyclyl groups may be optionally substituted by one or more Ra groups;

Re, Rf and Rw independently represent hydrogen or C1-6 alkyl;

Ra represents halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-6 cycloalkenyl, —ORx, —O—(CH2)n—ORx, haloC1-6 alkyl, haloC1-6 alkoxy, C1-6 alkanol, ═O, ═S, nitro, Si(Rx)4, —(CH2)s═CN, —S—Rx, —SO—Rx, —SO2—Rx, —CORx, —(CRxRy)s—COORz, —(CH2)s—CONRxRy, —(CH2)s—NRxRy, —(CH2)sNRxCORy, —(CH2)s—NRxSO2—Ry, —(CH2)s—NH—SO2—NRxRy, —OCONRxRy, —(CH2)s—NRxCO2Ry, —O—(CH2)s—CRxRy—(CH2)t—ORz or —(CH2)s—SO2NRxRy groups;

Rx, Ry and Rz independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkanol, hydroxy, C1-6 alkoxy, haloC1-6 alkyl, —CO—(CH2)n—C1-6 alkoxy, C3-8 cycloalkyl or C3-8 cycloalkenyl;

R2 represents a —CONR7R8, —CORx or —COORz group;

R7 and R8 independently represent hydrogen, C1-6 alkyl, C2-43 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, aryl, heterocyclyl or R7 and R8 together with the nitrogen atom to which they are attached may form a nitrogen containing heterocyclyl ring, wherein said alkyl, aryl and heterocyclyl may be optionally substituted by one or more (e.g. 1, 2 or 3) Rb groups;

R1 and Rb independently represent an Ra group or a —Y-carbocyclic or —Z-heterocyclyl group wherein said carbocyclic and heterocyclyl groups may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

V and W independently represent a bond or a —(CReRf)n— group;

Y and Z independently represent a bond, —CO—(CH2)s, —COO—, —(CH2)n, —NRx—(CH2)n—, —(CH2)s—NRx—, —CONRx—, —NRxCO, —SO2NRx—, —NRxSO2—, —NRxCONRy—, —NRxCSNRy—, —O—(CH2)s—, —(CH2)s—O—, S—, —SO— or —(CH2)s—SO2—;

n represents an integer from 1-4;

s and t independently represent an integer from 0-4;

q represents an integer from 0-2;

or a pharmaceutically acceptable salt, solvate or derivative thereof.

The term ‘C1-6 alkyl’ as used herein as a group or a part of the group refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, ten butyl, n-pentyl, isopentyl, neopentyl or hexyl and the like.
The term ‘C2-6 alkenyl’ as used herein as a group or a part of the group refers to a linear or branched hydrocarbon group containing a C═C bond.
The term ‘C1-6 alkoxy’ as used herein refers to an —O—C1-6 alkyl group wherein C1-6 alkyl is as defined herein. Examples of such groups include methoxy, ethoxy, propoxy, butoxy, pentoxy or hexoxy and the like.
The term ‘C1-6 alkanol’ as used herein refers to a C1-6 alkyl group substituted by one or more hydroxy groups, wherein C1-6 alkyl is as defined herein. Examples of such groups include hydroxymethyl, hydroxyethyl, hydroxypropyl and the like.
The term ‘C3-8 cycloalkyl’ as used herein refers to a saturated monocyclic hydrocarbon ring of 3 to 8 carbon atoms. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl and the like.
The term ‘C3-6 cycloalkyl’ as used herein refers to a saturated monocyclic hydrocarbon ring of 3 to 6 carbon atoms. Examples of such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
The term ‘halogen’ as used herein refers to a fluorine, chlorine, bromine or iodine atom.
The term ‘haloC1-6 alkyl’ as used herein refers to a C1-6 alkyl group as defined herein wherein at least one hydrogen atom is replaced with halogen. Examples of such groups include fluoroethyl, trifluoromethyl or trifluoroethyl and the like.
The term ‘haloC1-6 alkoxy’ as used herein refers to a C1-6 alkoxy group as herein defined wherein at least one hydrogen atom is replaced with halogen. Examples of such groups include difluoromethoxy or trifluoromethoxy and the like.
References to “carbocyclic” and “heterocyclic” groups as used herein shall, unless the context indicates otherwise, include both aromatic and non-aromatic ring systems. Thus, for example, the term “carbocyclic and heterocyclic groups” includes within its scope aromatic, non-aromatic, unsaturated, partially saturated and fully saturated carbocyclic and heterocyclic ring systems. In general, such groups may be monocyclic or bicyclic and may contain, for example, 3 to 12 ring members, more usually 5 to 10 ring members. Examples of monocyclic groups are groups containing 3, 4, 5, 6, 7, and 8 ring members, more usually 3 to 7, and preferably 5 or 6 ring members. Examples of bicyclic groups are those containing 8, 9, 10, 11 and 12 ring members, and more usually 9 or 10 ring members. Where reference is made herein to carbocyclic and heterocyclic groups, the carbocyclic or heterocyclic ring can, unless the context indicates otherwise, be unsubstituted or substituted by one or more substituents for example molecular fragments, molecular scaffolds or functional groups as discussed herein. It will be appreciated that references to “carbocyclic” and “heterocyclic” groups include reference to carbocyclic and heterocyclic groups which may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra or Rb groups.
The carbocyclic or heterocyclic groups can be aryl or heteroaryl groups having from 5 to 12 ring members, more usually from 5 to 10 ring members. The term “aryl” as used herein refers to a carbocyclic group having aromatic character and the term “heteroaryl” is used herein to denote a heterocyclic group having aromatic character. The terms “aryl” and “heteroaryl” embrace polycyclic (e.g. bicyclic) ring systems wherein one or more rings are non-aromatic, provided that at least one ring is aromatic. In such polycyclic systems, the group may be attached by the aromatic ring, or by a non-aromatic ring. It will be appreciated that the term “aryl” has the definition as defined herein except for compounds of formula (Ia), (Ic) and (Id) wherein aryl represents a carbocyclic ring as defined herein. In one embodiment of compounds of formula (Ia), (Ic) and (Id), aryl represents an aromatic ring.
The term “non-aromatic group” embraces unsaturated ring systems without aromatic character, partially saturated and fully saturated carbocyclic and heterocyclic ring systems. The terms “unsaturated” and “partially saturated” refer to rings wherein the ring structure(s) contains atoms sharing more than one valence bond i.e. the ring contains at least one multiple bond e.g. a C═C, C≡C or N═C bond. The term “fully saturated” refers to rings where there are no multiple bonds between ring atoms. Saturated carbocyclic groups include cycloalkyl groups as defined below. Partially saturated carbocyclic groups include cycloalkenyl groups as defined below, for example cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl. Saturated heterocyclic groups include piperidine, morpholine, thiomorpholine. Partially saturated heterocyclic groups include pyrazolines, for example 2-pyrazoline and 3-pyrazoline.
Examples of heteroaryl groups are monocyclic and bicyclic groups containing from five to twelve ring members, and more usually from five to ten ring members. The heteroaryl group can be, for example, a five membered or six membered monocyclic ring or a bicyclic structure formed from fused five and six membered rings or two fused six membered rings, or two fused five membered rings. Each ring may contain up to about five heteroatoms typically selected from nitrogen, sulphur and oxygen. Typically the heteroaryl ring will contain up to 4 heteroatoms, more typically up to 3 heteroatoms, more usually up to 2, for example a single heteroatom. In one embodiment, the heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atoms in the heteroaryl rings can be basic, as in the case of an imidazole or pyridine, or essentially non-basic as in the case of an indole or pyrrole nitrogen. In general the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.
Examples of five membered heteroaryl groups include but are not limited to pyrrole, furan, thiophene, imidazole, furazan, oxazole, oxadiazole, oxatriazole, isoxazole, thiazole, thiadiazole, isothiazole, pyrazole, triazole and tetrazole groups.
Examples of six membered heteroaryl groups include but are not limited to pyridine, pyrazine, pyridazine, pyrimidine and triazine.
A bicyclic heteroaryl group may be, for example, a group selected from:

    
    
        a) a benzene ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms;
        b) a pyridine ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms;
        c) a pyrimidine ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;
        d) a pyrrole ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms;
        e) a pyrazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;
        f) an imidazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;
        g) an oxazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;
        h) an isoxazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;
        i) a thiazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;
        j) an isothiazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms;
        k) a thiophene ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms;
        l) a furan ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms;
        m) a cyclohexyl ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms; and
        n) a cyclopentyl ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms.
    
    


Particular examples of bicyclic heteroaryl groups containing a five membered ring fused to another five membered ring include but are not limited to imidazothiazole (e.g. imidazo[2,1-b]thiazole) and imidazoimidazole (e.g. imidazo[1,2-a]imidazole).
Particular examples of bicyclic heteroaryl groups containing a six membered ring fused to a five membered ring include but are not limited to benzofuran, benzothiophene, benzimidazole, benzoxazole, isobenzoxazole, benzisoxazole, benzthiazole, benzisothiazole, isobenzofuran, indole, isoindole, indolizine, indoline, isoindoline, purine (e.g., adenine, guanine), indazole, pyrazolopyrimidine (e.g. pyrazolo[1,5-a]pyrimidine), triazolopyrimidine (e.g. [1,2,4]triazolo[1,5-a]pyrimidine), benzodioxole, imidazopyridine and pyrazolopyridine (e.g. pyrazolo[1,5-a]pyridine) groups.
Particular examples of bicyclic heteroaryl groups containing two fused six membered rings include but are not limited to quinoline, isoquinoline, chroman, thiochroman, chromene, isochromene, chroman, isochroman, benzodioxan, quinolizine, benzoxazine, benzodiazine, pyridopyridine, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine and pteridine groups.
Examples of polycyclic aryl and heteroaryl groups containing an aromatic ring and a non-aromatic ring include tetrahydronaphthalene, tetrahydroisoquinoline, tetrahydroquinoline, dihydrobenzthiene, dihydrobenzfuran, 2,3-dihydro-benzo[1,4]dioxine, benzo[1,3]dioxole, 4,5,6,7-tetrahydrobenzofuran, tetrahydrotriazolopyrazine (e.g. 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazine), indoline and indane groups.
A nitrogen-containing heteroaryl ring must contain at least one ring nitrogen atom. Each ring may, in addition, contain up to about four other heteroatoms typically selected from nitrogen, sulphur and oxygen. Typically the heteroaryl ring will contain up to 3 heteroatoms, for example 1, 2 or 3, more usually up to 2 nitrogens, for example a single nitrogen. The nitrogen atoms in the heteroaryl rings can be basic, as in the case of an imidazole or pyridine, or essentially non-basic as in the case of an indole or pyrrole nitrogen. In general the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.
Examples of nitrogen-containing heteroaryl groups include, but are not limited to, pyridyl, pyrrolyl, imidazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, furazanyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl (e.g., 1,2,3-triazolyl, 1,2,4-triazolyl), tetrazolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, benzisoxazole, benzthiazolyl and benzisothiazole, indolyl, 3H-indolyl, isoindolyl, indolizinyl, isoindolinyl, purinyl (e.g., adenine [6-aminopurine], guanine[2-amino-6-hydroxypurine]), indazolyl, quinolizinyl, benzoxazinyl, benzodiazinyl, pyridopyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl and pteridinyl.
Examples of nitrogen-containing polycyclic heteroaryl groups containing an aromatic ring and a non-aromatic ring include tetrahydroisoquinolinyl, tetrahydroquinolinyl, and indolinyl.
Examples of carbocyclic aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl groups.
Examples of non-aromatic heterocyclic groups are groups having from 3 to 12 ring members, more usually 5 to 10 ring members. Such groups can be monocyclic or bicyclic, for example, and typically have from 1 to 5 heteroatom ring members (more usually 1, 2, 3 or 4 heteroatom ring members), usually selected from nitrogen, oxygen and sulphur. The heterocyclic groups can contain, for example, cyclic ether moieties (e.g. as in tetrahydrofuran and dioxane), cyclic thioether moieties (e.g. as in tetrahydrothiophene and dithiane), cyclic amine moieties (e.g. as in pyrrolidine), cyclic amide moieties (e.g. as in pyrrolidone), cyclic thioamides, cyclic thioesters, cyclic ureas (e.g. as in imidazolidin-2-one) cyclic ester moieties (e.g. as in butyrolactone), cyclic sulphones (e.g. as in sulpholane and sulpholene), cyclic sulphoxides, cyclic sulphonamides and combinations thereof (e.g. thiomorpholine).
Particular examples include morpholine, piperidine (e.g. 1-piperidinyl, 2-piperidinyl, 3-piperidinyl and 4-piperidinyl), piperidone, pyrrolidine (e.g. 1-pyrrolidinyl, 2-pyrrolidinyl and 3-pyrrolidinyl), pyrrolidone, azetidine, pyran (2H-pyran or 4H-pyran), dihydrothiophene, dihydropyran, dihydrofuran, dihydrothiazole, tetrahydrofuran, tetrahydrothiophene, dioxane, tetrahydropyran (e.g. 4-tetrahydro pyranyl), imidazoline, imidazolidinone, oxazoline, thiazoline, 2-pyrazoline, pyrazolidine, piperazone, piperazine, and N-alkyl piperazines such as N-methyl piperazine. In general, preferred non-aromatic heterocyclic groups include saturated groups such as piperidine, pyrrolidine, azetidine, morpholine, piperazine and N-alkyl piperazines.
In a nitrogen-containing non-aromatic heterocyclic ring the ring must contain at least one ring nitrogen atom. The heterocylic groups can contain, for example cyclic amine moieties (e.g. as in pyrrolidine), cyclic amides (such as a pyrrolidinone, piperidone or caprolactam), cyclic sulphonamides (such as an isothiazolidine 1,1-dioxide, [1,2]thiazinane 1,1-dioxide or [1,2]thiazepane 1,1-dioxide) and combinations thereof. Particular examples of nitrogen-containing non-aromatic heterocyclic groups include aziridine, morpholine, thiomorpholine, piperidine (e.g. 1-piperidinyl, 2-piperidinyl, 3-piperidinyl and 4-piperidinyl), pyrrolidine (e.g. 1-pyrrolidinyl, 2-pyrrolidinyl and 3-pyrrolidinyl), pyrrolidone, dihydrothiazole, imidazoline, imidazolidinone, oxazoline, thiazoline, 6H-1,2,5-thiadiazine, 2-pyrazoline, 3-pyrazoline, pyrazolidine, piperazine, and N-alkyl piperazines such as N-methyl piperazine.
The carbocyclic and heterocyclic groups can be polycyclic fused ring systems or bridged ring systems such as bicycloalkanes, tricycloalkanes and their oxa- and aza analogues (e.g. adamantane and oxa-adamantane). For an explanation of the distinction between fused and bridged ring systems, see Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience, pages 131-133, 1992.
Examples of non-aromatic carbocyclic groups include cycloalkane groups such as cyclohexyl and cyclopentyl, cycloalkenyl groups such as cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl, as well as cyclohexadienyl, cyclooctatetraene, tetrahydronaphthenyl and decalinyl.
The heterocyclic groups can each be unsubstituted or substituted by one or more substituent groups. For example, heterocyclic groups can be unsubstituted or substituted by 1, 2, 3 or 4 substituents. Where the heterocyclic group is monocyclic or bicyclic, typically it is unsubstituted or has 1, 2 or 3 substituents.
Examples of ring systems encompassed by the definitions of X1-X5 are shown in the following formulae (a)-(p) and (r)-(t):







Further examples of ring systems encompassed by the definitions of X1-X5 are shown in the following formulae (u)-(v):




As mentioned above,  represents a single or double bond. It will be clear to the skilled person that when X4 or X5 represents C═O, X4 and X5 are joined by a single bond. In one embodiment X4 and X5 are joined by a double bond.
In one embodiment, two bonds within the 5 membered ring system are double bonds.
In one embodiment, X1 represents C.
In one embodiment, X1 and X3 represent C, X5 represents CH and X2 and X4 represent nitrogen (i.e. a ring system of formula (a)).
In an alternative embodiment, X1 and X3 represent C, X4 and X5 represent CH and X2 represents nitrogen (i.e. a ring system of formula (e)).
In an alternative embodiment, X1 and X3 represent C, X4 represents CH and X2 and X5 represent nitrogen (i.e. a ring system of formula (f)).
In an alternative embodiment, X1 and X2 represent C, X3 represents nitrogen, X4 represents CR3 (e.g. CH) and X5 represents CR3 (e.g. C-Me) (i.e. an example of a ring system of formula (h)).
In an alternative embodiment, X1 and X2 represent C, X4 and X5 represent CH and X3 represents nitrogen (i.e. a ring system of formula (j)).
In an alternative embodiment, X1 and X2 represent C, X4 represents CH and X3 and X5 represent nitrogen (i.e. a ring system of formula (k)).
In an alternative embodiment, X2 and X3 represent C, X5 represents CH and X1 and X4 represent nitrogen (i.e. a ring system of formula (r)).
In one embodiment, X1, X3 and X5 represent C and X2 and X4 represent nitrogen (i.e. an example of a ring system of formula (a)).
In an alternative embodiment, X1, X3, X4 and X5 represent C and X2 represents nitrogen (i.e. an example a ring system of formula (e)).
In an alternative embodiment, X1, X3 and X4 represent C and X2 and X5 represent nitrogen (i.e. an example a ring system of formula (f)).
In an alternative embodiment, X1 and X2 represent C, X3 represents nitrogen, X4 represents CR3 (e.g. CH) and X5 represents CR6 (e.g. C-Me) (i.e. an example a ring system of formula (h)).
In an alternative embodiment, X1, X2, X4 and X5 represent C and X3 represents nitrogen (i.e. an example a ring system of formula (j)).
In an alternative embodiment, X1, X2 and X4 represent C and X3 and X5 represent nitrogen (i.e. an example a ring system of formula (k)).
In an alternative embodiment, X2, X3 and X5 represent C and X1 and X4 represent nitrogen (i.e. an example a ring system of formula (r)).
In one embodiment, X2 represents C.
In one embodiment, X3 represents N.
In one embodiment, X4 represents CH or CR3.
In one embodiment, X5 represents CH or CR6.
In one embodiment, X1-X5 represent a ring system of formulae (a), (e), (f), (j), (k) or (r). In a further embodiment, X1-X5 represent a ring system of formulae (a), (e) or (j). In a further embodiment, X1-X5 represent a ring system of formula (a) or (j) In a further embodiment, X1-X5 represent a ring system of formula (j).
In one embodiment, when X1, X2 and X5 represents C, X3 represents nitrogen and A represents phenyl, B is a group other than a heterocyclic group.
In one embodiment, when X1, X2, X4 and X5 represents C, X3 represents nitrogen and A represents pyrimidinyl, B represents a group other than a heterocyclic group.
In one embodiment, when X1, X3, X4 and X5 represents C, X2 represents nitrogen and A represents pyrimidinyl, B represents a group other than a heterocyclic group.
In one embodiment, when X1, X3 and X5 represent C and X2 and X4 represent nitrogen, Ra is a group other than ═O.
In one embodiment of compounds of formula (Ia), when X2, X3, X4 and X5 represent C, X1 represents nitrogen, A represents thiazolyl, Ra represents a group other than —CONRy.
In one embodiment, when X2 and X3 represents C and X1 represents nitrogen, A represents a group other than pyrazinyl.
In one embodiment, when X2, X3, X4 and X5 represent C and X1 represents nitrogen, B represents a group other than phenyl.
In one embodiment, when X4 represents nitrogen, X1 represents a group other than nitrogen.
Examples of ring systems encompassed by the definition A are shown in the following formulae A1-A15, wherein B can be optionally substituted by one or more R1 as shown in formula (I):







The group A12 can be any tautomer of imidazole e.g. A12a.
In one embodiment, A is a group other than pyrazolyl. In one embodiment, A is a group other than imidazolyl.
In one embodiment, A represents a group selected from any one of formulae A1 to A10 and A12-A15. In a further embodiment, A is selected from A2, A14 and A15. In a further embodiment, A is selected from A2.
In one embodiment, A represents a 5- or 6-membered aromatic group.
In one embodiment, A represents a 5-membered aromatic group.
In one embodiment, A represents a non-aromatic group.
In one embodiment, A represents a 6-membered aromatic group.
In one embodiment, A represents pyridin-3-yl or phenyl.
In one embodiment, A represents a monocyclic aromatic carbocyclic or heterocyclic ring system having for example a 5, 6 or 7 membered ring. In a further embodiment, A represents a 6 membered carbocyclic ring. In a yet further embodiment, A represents a phenyl group (i.e. a ring system of formula A1) optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups. In one embodiment, A represents unsubstituted phenyl or phenyl substituted with an —(CH2)s—CONRxRy (e.g. —CONH2), —(CH2)s—CN (e.g. —CN), C1-6 alkyl (e.g. methyl) or C1-6 alkoxy (e.g. methoxy) group.
In one embodiment, A represents a monocyclic aromatic carbocyclic or heterocyclic ring system having for example a 5, 6 or 7 membered ring. In a further embodiment, A represents a 6 membered carbocyclic ring. In a yet further embodiment, A represents a phenyl group (i.e. a ring system of formula A1) or a pyridyl group (i.e. a ring system of formula A2 or A3) optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups. In one embodiment, A represents unsubstituted phenyl or phenyl substituted with an —(CH2)s—CONRxRy (e.g. —CONH2), —(CH2)s—CN (e.g. —CN), halogen (e.g. fluorine), C1-6 alkyl (e.g. methyl), C1-6 alkanol (e.g. —CH2OH) or —ORx(e.g. methoxy or —OCH(Me)2) group.
In one embodiment, A represents a group other than pyridinyl or pyrazinyl when B represents phenyl, pyridyl or pyrazinyl.
In one embodiment, A represents a group other than pyrazinyl. In one embodiment, A represents a group other than pyrimidinyl. In one embodiment, A represents a group other than pyridinyl or pyrimidinyl. In a further embodiment, A represents unsubstituted phenyl.
In one embodiment, A represents a 6 membered monocyclic aromatic carbocyclic or heterocyclic ring system (e.g. phenyl or pyridyl), substituted by NH—B—(R1)q at the 3-position or 5-position. When A represents phenyl, in one embodiment NH—B—(R1)q is present at the 3-position of the phenyl with respect to the position of attachment to X1.
In one embodiment, A represents a 6 membered monocyclic aromatic carbocyclic or heterocyclic ring system (e.g. phenyl or pyridyl), substituted by NH—B—(R1)q at the 5-position and further optionally substituted by a single Ra group at the 3-position.
When V and W represent a bond, examples of aromatic ring systems encompassed by the definition B—NH— are shown in the following formulae B1-B47, in particular B1-B45:



















When V and W represent a bond, particular examples of B rings include B1, B4 and B9. Further particular examples of B rings include B19-21, B22, B24, B25, B27-36, B38-40, B42 and B44.
When V represents CH2, one example of an aromatic ring system encompassed by the definition B—NH— is shown in the following formula B48:




When V and W represent a bond, examples of saturated or partially saturated ring systems encompassed by the definition B—NH— are shown in the following Table 1:










TABLE 1




















 














 














 














 














 














 





















In one embodiment, B represents —V-aryl. In one embodiment, V represents a group other than —C(H)(Me)-. In one embodiment, V represents a bond or CH2. In a further embodiment, V represents a bond. In one embodiment, the aryl group of B represents a phenyl group.
In one embodiment, B represents —W-heterocyclyl.
In one embodiment, W represents a group other than —C(H)(Me)-. In a further embodiment, W represents a bond.
In one embodiment, when B represents a —W-heterocyclyl group, W represents a bond.
In one embodiment, the aryl or heterocyclyl group of B represents a monocyclic aromatic carbocyclic or heterocyclic ring system having for example a 5, 6 or 7 membered ring (e.g. phenyl, pyridyl, pyrazinyl, triazolyl or thiadiazolyl). In a further embodiment, the heterocyclyl group of B represents a 5 or 6 membered heterocyclic ring (e.g. pyridyl, pyrazinyl, triazolyl or thiadiazolyl). In a further embodiment, the heterocyclyl group of B represents a 5 or 6 membered heterocyclic ring (e.g. pyridyl, pyrazinyl, triazolyl, oxadiazolyl, imidazolyl or thiadiazolyl). In a yet further embodiment, the heterocyclyl group of B represents a 5 membered heterocyclic ring group selected from compounds of formula Ba, Bb and Bc:




wherein Xa is selected from NH, CH, and S; Xb is selected from C, N, O, and S; Xc is selected from N, and O; Xd is selected from C, N, O, and S; Xe is selected from C and N and  represents the point of attachment to NH;





wherein the dotted line  can represent a single, or double bond;

Xa is selected from NH, CH, and S; Xb is selected from C, N, O, and S; Xc is selected from C, S and N; Xd is selected from C, N, O, and S; Xe is selected from C and N; and  represents the point of attachment to NH;





Bc 

wherein the dotted line  can represent a single, or double bond; Xa is selected from NH, CH, and S; Xb is selected from C, N, O, and S; Xc is selected from C, N, O, and S; Xd is selected from C, N, O, and S; Xe is selected from C and N; and  represents the point of attachment to NH.

In a yet still further embodiment, the heterocyclyl group of B represents oxadiazolyl, imidazolyl, triazolyl or thiadiazolyl. In a further embodiment, the heterocyclyl group of B represents triazolyl or thiadiazolyl. In a still yet further embodiment, the heterocyclyl group of B represents thiadiazolyl.
In one embodiment, q represents 0 or 1. When q represents 1, in one embodiment, R1 represents C1-6 alkyl (e.g. methyl). When q represents 1, in an alternative embodiment, R1 represents or —(CH2)s—NRxRy (e.g. —NH2). In a further embodiment, q represents 0.
In one embodiment, X5 represents CH or nitrogen.
In one embodiment, X5 represents CH, nitrogen or C═O.
In one embodiment, R2 represents a —CONR7R8 or —COORz group (e.g. —COOH). In a further embodiment, R2 represents a —CONR7R8 group.
In one embodiment, R2 represents a —COR1 group.
In one embodiment, Rx represents C1-6 alkyl (e.g. methyl, ethyl or isopropyl) or C3-8 cycloalkyl (e.g. cyclopropyl, cyclobutyl or cyclopentyl).
When R2 represents a —CORx group, in one embodiment Rx represents C1-6 alkyl (e.g. methyl, ethyl or isopropyl) or C3-8 cycloalkyl (e.g. cyclopropyl, cyclobutyl or cyclopentyl).
In one embodiment, R6 represents hydrogen.
In one embodiment, R6 represents C1-6 alkoxy (e.g. unsubstituted C1-43 alkoxy).
In one embodiment, R7 and R8 both represent hydrogen or C1-6 alkyl (e.g. methyl).
In a further embodiment, one of R7 and R8 represents hydrogen and the other represents C1-6 alkyl (e.g. methyl, ethyl or isopropyl) optionally substituted by an —ORx group (e.g. —(CH2)2—O-Me), C3-6 cycloalkyl (e.g. cyclobutyl) or heterocyclyl (e.g. thiophenyl). In a further embodiment, one of R7 and R8 represents hydrogen and the other represents C1-6 alkyl (e.g. methyl).
In a further embodiment, R7 and R8 together with the nitrogen atom to which they are attached form a nitrogen containing heterocyclyl ring optionally substituted by one or more (e.g. 1, 2 or 3) Rb groups.
In a further embodiment, R7 and R8 together with the nitrogen atom to which they are attached form a nitrogen containing heterocyclyl ring optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups.
In a further embodiment, R7 and R8 together with the nitrogen atom to which they are attached form a nitrogen containing heterocyclyl ring (e.g. azetidinyl or pyrrolidinyl) optionally substituted by one or more (e.g. 1, 2 or 3) Rb groups (e.g. —ORx (e.g. —OH), halogen (e.g. fluorine) or —Y-aryl (e.g. -phenyl)). In a further embodiment, R7 and R8 together with the nitrogen atom to which they are attached form a nitrogen containing heterocyclyl ring (e.g. azetidinyl).
In one embodiment, Y represents —O—(CH2)s—(e.g. —O—CH2—).
In one embodiment, Y and Z independently represent a bond, —CO—, (CH2)s—, —COO—, —(CH2)n—, —NRx—(CH2)n—, —(CH2)n—NRx—, —CONRx—, —NRxCO—, —SO2NRx—, —NRxSO2—, —NRxCONRy—, —NRxCSNRy—, —O—(CH2)s—, —(CH2)s—O—, S—, —SO— or —(CH2)s—SO2—.
In one embodiment, Y and Z independently represent —CO—, —O—(CH2)s— or —NH—(CH2)s— (e.g. NH).
In one embodiment, Y and Z independently represent —CO—, —O—(CH2)s or —NH—(CH2)n 
In one embodiment, Z represents a bond, CO, —(CH2)n—(e.g. —CH2—, —(CH2)2 or —(CH2)3) or —O—. In a further embodiment, Z represents —O—. CO or —(CH2)n— (e.g. —CH2—). In a yet further embodiment, Z represents —(CH2)n— (e.g. —CH2—).
In one embodiment, Y and Z independently represent a bond.
In one embodiment, Rb independently represents an Ra group or a —Y-aryl or —Z-heterocyclyl group wherein said aryl and heterocyclyl groups may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups.
In one embodiment, Re, Rf and Rw independently represent hydrogen or methyl. In a further embodiment, Re, Rf and Rw represent hydrogen.
In one embodiment, n represents 1.
In one embodiment, the compound of formula (I) is a compound of formula (Ic) or (Id):




wherein

A represents an aromatic carbocyclic or heterocyclic group which may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

B represents an aromatic or non-aromatic carbocyclic or heterocyclic group;

R4 and R5 independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, C1-6 alkanol, haloC1-6 alkyl, —(CH2)n—NRxRy, —(CH2)s—COORz, —(CH2)n—O—(CH2)m—OH, —(CH2)n-aryl, —(CH2)n—O-aryl, —(CH2)n-heterocyclyl or —(CH2)n—O-heterocyclyl wherein said C1-6 alkyl, C2-8 alkenyl, C2-6 alkynyl, C3-5 cycloalkyl,

C3-8 cycloalkenyl, aryl and heterocyclyl groups may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

Rx, Ry and Rz independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkanol, hydroxy, C1-6 alkoxy, haloC1-6 alkyl, —CO—(CH2)n—C1-6 alkoxy, C3-8 cycloalkyl or C3-6 cycloalkenyl;

R2 represents a —CONR7R8, —CORx or —COORz group;

R7 and R8 independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, aryl, heterocyclyl or R7 and R8 together with the nitrogen atom to which they are attached may form a nitrogen containing heterocyclyl ring, wherein said C1-6 alkyl, aryl and heterocyclyl may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

Ra represents halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, —ORx, —O—(CH2)n—ORx, haloC1-6 alkyl, haloC1-6 alkoxy, C1-6 alkanol, ═O, ═S, nitro, —(CH2)s—CN, —S—Rx, —SO—Rx, SO2—Rx, —CORx, —(CRxRy)s—COORz, —(CH2)s—CONRxRy, —(CH2)s—NRxRy, —(CH2)s—NRxCORy, —(CH2), NRxSO2—Ry, —OCONRxRy, —(CH2)s—NRxCO2Ry, —O—(CH2)s—CRxRy—(CH2)t—ORz or —(CH2)sSO2NRxRy groups;

R1 and Rb represents an Ra group or a —Y-aryl or —Z-heterocyclyl group wherein said aryl and heterocyclyl groups may be optionally substituted by one or more (e.g. 1, 2 or 3) Ra groups;

Y and Z independently represent a bond, —CO—(CH2)s, —COO—, —(CH2)n—, —NRx—(CH2)n—, —(CH2)n—NRx—, —CONRx—, —NRxCO—, —SO2NRx—, —NRxCONRy—, —NRxCSNRy—, —O—(CH2)s—, —(CH2)s—O—, S—, —SO— or —(CH2)5SO2—;

m and n independently represent an integer from 1-4;

s and t independently represent an integer from 0-4;

q represents an integer from 0-2;

aryl represents a carbocyclic ring;

heterocyclyl represents a heterocyclic ring;

or a pharmaceutically acceptable salt, solvate or derivative thereof.

In one embodiment of compounds of formula (Ic) and (Id), Y and Z independently represent a bond, —CO—(CH2)s—, —COO—, —(CH2)n—, —NRx—, (CH2)s—, —(CH2)s—NRx—, —CONRx—, —NRxCO—, —SO2NRx—, —NRxSO2—, —NRxCONRy—, —NRxCSNRy—, —O—(CH2)s—, —(CH2)s—O—, S—, —SO— or —(CH2)s—SO2—.
In one embodiment, the compound of formula (I) is a compound selected from Examples 1-4. In a further embodiment, the compound of formula (I) is a compound of Example 2.
In the specification, references to formula (I) include formulas such as (Ia), and (Ib), and sub-groups, examples or embodiments of formulae (I), (Ia), (Ib), (Ic) and (Id) unless the context indicates otherwise.
Thus for example, references to inter alia therapeutic uses, pharmaceutical formulations and processes for making compounds, where they refer to formula (I), are also to be taken as referring to formulae (I), (Ia), (Ib), (Ic) and (Id), and sub-groups, examples or embodiments of formulae (I), (Ia), (Ib), (Ic) and (Id).
Similarly, where preferences, embodiments and examples are given for compounds of the formula (I), they are also applicable to formulae (I), (Ia), (Ib), (Ic) and (Id), and sub-groups, examples or embodiments of formulae (I), (Ia), (Ib), (Ic) and (Id), unless the context requires otherwise.
Methods for the Preparation of Compounds of Formula (I)
In this section, as in all other sections of this application unless the context indicates otherwise, references to formula (I) also include all other sub-groups and examples thereof as defined herein.
Compounds of the formula (I) can be prepared in accordance with synthetic methods well known to the skilled person. In particular compounds of formula (I) are readily prepared by palladium mediated coupling chemistries between aromatic chloro, bromo, iodo, or pseudo-halogens such as a trifluoromethanesulphonate (triflate) or tosylate compounds, and aromatic boronic acids or stannane derivatives. In particular, Suzuki coupling chemistry is broadly applicable to synthesis of these compounds. The Suzuki reaction can be carried out under typical conditions in the presence of a palladium catalyst such as bis(tri-t-butylphosphine)palladium, tetrakis-(triphenylphosphine)palladium or a palladacycle catalyst (e.g. the palladacycle catalyst described in Bedford, R. B. and Cazin, C. S. J. (2001) Chem. Commun., 1540-1541) and a base (e.g. a carbonate such as potassium carbonate) as discussed in more detail below. The reaction may be carried out in polar solvent, for example an aqueous solvent system, including aqueous ethanol, or an ether such as dimethoxyethane or dioxane, and the reaction mixture is typically subjected to heating, for example to a temperature of 80° C. or more, e.g. a temperature in excess of 100° C.
In the process sections references to R and R′ are used to indicate groups as defined in R7 and R8, or protected forms thereof.
As illustrated in Scheme 1A, the imidazo[1,2-a]pyridine core can be synthesised from commercially available starting materials as outlined below to give a 3,7-disubstituted ring.




2-amino-isonicotinic acid methyl ester in an appropriate solvent and base can be cyclised under reflux with chloroacetaldehyde to give the imidazopyridine ring.
For synthesis of the R2 group of compounds of formula (I) the carboxylic ester is hydrolysed, for example using standard ester hydrolysis conditions such as aqueous base and heating. The carboxylic acid or an activated derivative thereof can then be reacted with the appropriate amine to form the amide (Scheme 1A).
The coupling reaction between the carboxylic acid and the amine is preferably carried out in the presence of a reagent of the type commonly used in the formation of peptide linkages. Examples of such reagents include 1,3-dicyclohexylcarbodiimide (DCC) (Sheehan et al, J. Amer. Chem. Soc., 1955, 77, 1067), 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide (referred to herein either as EDC or EDAC but also known in the art as EDCI and WSCDI) (Sheehan et al, J. Org. Chem., 1961, 26, 2525), uronium-based coupling agents such as O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) or O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) and phosphonium-based coupling agents such as 1-benzo-triazolyloxytris-(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) (Castro et al, Tetrahedron Letters, 1990, 31, 205). Carbodiimide-based coupling agents are advantageously used in combination with 1-hydroxy-7-azabenzotriazole (HOAt) (L. A. Carpino, J. Amer. Chem. Soc., 1993, 115, 4397) or 1-hydroxybenzotriazole (HOBt) (Konig et al, Chem. Ber., 103, 708, 2024-2034). Preferred coupling reagents include TBTU, EDC (EDAC) or DCC in combination with HOAt or HOBt.
The coupling reaction is typically carried out in a non-aqueous, non-protic solvent such as acetonitrile, 1,4-dioxane, dimethylsulphoxide, dichloromethane, dimethylformamide or N-methylpyrrolidine, or in an aqueous solvent optionally together with one or more miscible co-solvents. The reaction can be carried out at room temperature or, where the reactants are less reactive (for example in the case of electron-poor anilines bearing electron withdrawing groups such as sulphonamide groups) at an appropriately elevated temperature. The reaction may be carried out in the presence of a non-interfering base, for example a tertiary amine such as triethylamine or N,N-diisopropylethylamine.
As an alternative, a reactive derivative of the carboxylic acid, e.g. an anhydride or acid chloride, may be used. Reaction with a reactive derivative such an anhydride, is typically accomplished by stirring the amine and anhydride at room temperature in the presence of a base such as pyridine.
Amines for use in the reaction can be obtained from commercial sources or can be prepared by any of a large number of standard synthetic methods well known by those skilled in the art, see for example Advanced Organic Chemistry by Jerry March, 4th Edition, John Wiley & Sons, 1992, and Organic Syntheses, Volumes 1-8, John Wiley, edited by Jeremiah P. Freeman (ISBN: 0-471-31192-8), 1995, and see also the methods described in the experimental section below. For example the appropriate nitro-compound may be reduced to give the corresponding amino-compound. The reduction may be carried out by standard methods such as catalytic hydrogenation, for example in the presence of palladium on carbon in a polar solvent such as ethanol or dimethylformamide at room temperature. As an alternative, reduction may be effected using a reducing agent such as tin (II) chloride in ethanol, typically with heating, for example to the reflux temperature of the solvent.
The imidazo[1,2-a]pyridine-7-derivative, for example the imidazo[1,2-a]pyridine-7-carboxylic acid methyl ester or amide, in an appropriate solvent can then be iodinated, for example using N-iodosuccinimide at room temperature.
Appropriate functionality can then be added at the halogenated position, for example using a range of metal-catalysed reactions. In particular, appropriately functionalised boronic acids, trifluoroboronates, or their boronate esters may react with the aryl halide. This transformation, commonly known as the Suzuki reaction, has been reviewed by Rossi et al (2004), Synthesis 15, 2419.
The Suzuki reaction is often carried out in mixtures of water and organic solvents. Examples of suitable organic solvents include toluene, tetrahydrofuran, 1,4-dioxane, 1,2-dimethoxyethane, acetonitrile, N-methylpyrrolidinone, ethanol, methanol and dimethylformamide. The reaction mixture is typically subjected to heating, for example to a temperature in excess of 100° C. The reaction is carried out in the presence of a base. Examples of suitable bases include sodium carbonate, potassium carbonate, cesium carbonate and potassium phosphate. Examples of suitable catalysts include bis(tri-t-butylphosphine)palladium(0), tris(dibenzylideneacetone)dipalladium(0), bis(triphenylphosphine)palladium(II) chloride, palladium(II) acetate, tetrakis(triphenylphosphine)palladium(0), bis(tricyclohexylphosphine) palladium(0), [1,1′-bis(diphenylphosphino)-ferrocene]dichloropalladium(II), dichlorobis(tri-o-tolylphosphine)palladium(II), 2′-(dimethylamino)-2-biphenylyl-palladium(II) chloride dinorbornylphosphine complex and 2-(dimethylamino)ferrocen-1-yl-palladium(II) chloride dinorbornylphosphine complex. In some cases additional ligands may be added to facilitate the coupling reaction. Examples of suitable ligands include tri-t-butylphosphine, 2,2-bis(diphenylphosphino)-1,1-binaphthyl, triphenylphosphine, 1,2-bis(diphenylphosphino)ethane, 1,1′-bis(diphenylphosphino)ferrocene, tricyclohexylphosphine, 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene, 1,3-bis(diphenylphosphino)propane, 2-(di-t-butylphosphino)biphenyl, 2-dicyclohexylphosphino-2′-(n,n-dimethylamino)-biphenyl, tri-o-tolylphosphine, 2-(dicyclohexylphosphino)biphenyl, 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl, tri(2-furyl)phosphine, 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl and 2-di-tert-butylphosphino-2′,4′,6′-triisopropylbiphenyl.
Other examples of possible metal catalysed functionalisations of the halide are reactions with organo-tin reagents (the Stille reaction), with Grignard reagents and reaction with nitrogen nucleophiles. A general overview, and further leading references, of these transformations is presented in ‘Palladium Reagents and Catalysts’ [Jiro Tsuji, Wiley, ISBN 0-470-85032-9] and Handbook of OrganoPalladium Chemistry for Organic Synthesis [Volume 1, Edited by Ei-ichi Negishi, Wiley, ISBN 0-471-31506-0].
In particular, one reaction which can be utilised is the Buchwald-Hartwig type reaction (see Review: J. F. Hartwig (1998), Angew. Chem. Int. Ed. 37, 2046-2067) which provides a means for palladium-catalyzed synthesis of aryl amines. The starting materials are aryl halides or pseudohalides (for example triflates) and primary or secondary amines, in the presence of a strong base such as sodium tert-butoxide and a palladium catalyst such as tris-(dibenzylideneacetone)-di-palladium (Pd2(dba)3), or 2,2′-bis(diphenylphosphino)-1′1-binaphthyl (BINAP).
In particular, for synthesis compounds of formula (I) the aryl halide can be reacted with 3-aminobenzeneboronic acid using an appropriate metal catalyst e.g. bis(triphenylphosphine)palladium(II) chloride to form the amino precursor for secondary amine bond formations.
This sequence of reactions outlined in Scheme 1A can be alternated as outlined in Scheme 1B or 1C.




In Scheme 1B, the imidazo[1,2-a]pyridine-7-carboxylic acid methyl ester is iodinated first and the metal-catalysed coupling reaction performed, before conversion of the methyl ester to the amide group R2.




In Scheme 1C, the imidazo[1,2-a]pyridine-7-amide is synthesized directly from the 4-amide-pyridin-2-ylamine and is then iodinated and used in the metal-catalysed coupling reaction. This reaction scheme is particularly suitable for synthesizing compounds where R2 is CONH2.
Alternatively the 4-chloro-pyridin-2-ylamine or 4-bromo-pyridin-2-ylamine in an appropriate solvent and base can be cyclised under reflux with chloroacetaldehyde to give the 7-halo-imidazopyridine ring (as shown in Scheme 2). The halogen functionality at the 7-position of the imidazo[1,2-a]pyridine can then be converted to an amide by either of the two routes outlined in Scheme 2.




The halide can be converted to the nitrile using CuCN in N-methylpyrrolidine at reflux (for example as described in Funhoff, D. J. H et al, Angew. Chem. Int. Ed. 1986, 25(8), 724) or CuCN in DMF, which is then hydrolysed with an alkali metal hydroxide such as potassium hydroxide to give the acid and/or the amide. Where a mixture of the acid and amide are formed, they may be separated according to standard methods such as chromatography. The acid can then be coupled with an amine of the formula under typical amide coupling conditions of the type described above to give the compound of the formula (I).
Alternatively the halide can be converted to the acid using n-butyllithium or magnesium and subsequent reaction of the intermediate with a carbonylating agent such as CO2 to produce the carboxylic acid for use as a compound of formula (I) or for conversion to the amide or ester. The amide can be accessed directly from the halide either through transmetallation with nBuLi and subsequent quenching with an appropriate isocyanate (Pansegran, P. D. et al, JACS, 1988, 110, 7178) or via carbonylation with carbon monoxide and in the presence of the appropriate amine and catalytic [P,P′-1,3-bis(di-1-propylphosphino)propane][P-1,3-bis(di-iso-propylphosphino)propane]palladium (0) in a solvent such as xylene at heating (e.g. to 150° C.) (for example as described in Ben-David, Y. et al, JACS, 1989, 111(23), 8742).
In addition, the halide can be converted using carbon monoxide and palladium catalyst to the aldehyde, which can then be oxidised to the carboxylic acid using an oxidising agent, such as permanganate or chromic acid, and then undergo conversion to the amide using standard coupling conditions described previously or esterified to the ester. The halide can also be converted directly to the ester using carbon monoxide, palladium catalyst and the appropriate alcohol. This can then be a compound of formula (I) or hydrolysed to the acid, or then hydrolysed to the acid and converted to the amide, or converted directly to the amide.
The halide could also be converted directly to the dimethylamide using trimethylsilyl-dimethyl amide and reacting with bis(tri-tbutylphosphine)palladium and heating to 100° C. as described in Cunico, R. F., Organic Letters, 2002, 4 (24), 4357.
Other conversions of aromatic bromides to aromatic aldehydes can take place using the Stille carbonyl synthesis (Stille, JACS, 1983, 105, 7175), or the Bodroux-Chichibabin-aldehyde synthesis described in Einchorn, J, Tetrahedron Lett., 1983, 27, 1791. The aldehyde can then be oxidised to the acid and converted to an amide as described above.
Polyfunctional 2-amino-5-bromopyridines or the aromatic bromides can be converted to aldehyde via Grignard type formation and quenching with DMF (Misra, Bioorg. Med. Chem. Lett., 2004, 14(11), 2973) or they can be converted to ethyl esters via standard palladium carbonylation in the presence of alcohol (Cheung, M. Heterocycles, 2001, 55, 1583).
Alternatively the 4-methyl-pyridin-2-ylamine can be used in the cyclisation reaction to give the 7-methyl-imidazo[1,2-a]pyridine ring, which alternatively is commercially available. The methyl can then be oxidised to the aldehyde using the Étard reaction or carboxylic acid using an oxidising agent such as permanganate. The Étard reaction involves the direct oxidation of an aromatic or heterocyclic bound methyl group to an aldehyde using chromyl chloride.
Alternatively the ethyl imidazo[1,2-a]pyridine-7-carboxylate, which is also commercially available, can be used as the startpoint for the amide conversion or iodinations and metal-catalysed reactions.
Ketones, where R2 is CORx, can be synthesized from the corresponding carboxylic acid via the N,O-dimethylhydroxamic acid (Weinreb Amide) or the N-methyl, O-t-butyl hydroxamic acid (Weinreb type Amide) intermediate and subsequent reaction with the appropriate Grignard reaction (Labeeuw, O. et al Tetrahedron Lett 2004, 45 (38), 7107-7110.). Derivatisation to the corresponding Weinreb Amide uses N,O-dimethylhydroxylamine hydrochloride as described in L. De Luca, G. Giacomelli, M. Taddei, J. Org. Chem., 2001, 66, 2534-2537. Conversion of the standard aromatic Weireb Amide to a methyl ketone requires methylene-triphenyl-lambda*5*-phosphane in a solvent such at tetrahydrofuran as reported in Murphy, J. A. et al Org Lett 2005, 7 (7), 1427-1429.
Alternatively ketones can be prepared from the chloride using vinylethertin (Stille type) coupling with haloaromatic or haloheteroaromatic. As an example the acetyl ketone can be prepared by heating tributyl-(1-ethoxy-vinyl)-stannane, lithium chloride and tetrakis(triphenylphosphine)-palladium(0) in solvent such as acetonitrile or via a Heck type reaction reported in Mo, J. Angew Chem, Int Ed, 2006, 45(25), 4152.




A range of compounds of formula (I) can be accessed by use of 3-aminobenezeboronic acid in the Suzuki reaction and subsequent derivatisation. In particular, as outlined Scheme 3, the amine functionality introduced can be used to synthesise secondary amine compounds.
Primary amines can be prepared by reduction of the corresponding nitro-compound under standard conditions. The reduction may be effected, for example by catalytic hydrogenation in the presence of a catalyst such as palladium on carbon in a polar solvent such as ethanol or dimethylformamide at room temperature.
Compounds of the formula (I) containing a secondary amine group, can be prepared from the amino compounds by a number of methods. Reductive amination with an appropriately substituted aldehyde or ketone can be carried out in the presence of a variety of reducing agents (see Advanced Organic Chemistry by Jerry March, 4th Edition, John Wiley & Sons, 1992, p898-900). For example, reductive amination can be carried out in the presence of sodium triacetoxyborohydride in the presence of an aprotic solvent, such as dichloromethane, at or near ambient temperatures. They can also be prepared by the reaction of the amino compound in a nucleophilic displacement reaction, where the reagent contains a leaving group such as a halogen.
In addition the thiadiazolylamino compound can be synthesised by use of the appropriate substituted boronic acid e.g. 3-([1,3,4]Thiadiazol-2-ylamino)-phenyl boronic acid pinacol ester or 3-(5-Methyl-[1,3,4]thiadiazol-2-ylamino)-phenyl boronic acid pinacol ester in the Suzuki reaction with an appropriately substituted imidazo[1,2-a]pyrimidine. These can be synthesised as described herein.
Alternatively the secondary amine can be formed by cyclisation of an appropriate group to form a ring. Amino-thiadiazole compounds can be synthesised as described in Scheme 4.




This involves reacting the amino compound in anhydrous solvent e.g. toluene, with 1,1′-thiocarbonyldi-2(1H)-pyridone. Typical reaction conditions are heating for 1 hour, work up and then treatment with hydrazine hydrate to form the thiosemicarbazide. This is then cyclised under conditions, such as through addition of diethyl chlorophosphate dropwise. This may also generate an alternate cyclisation product and hence separation may be required.
Alternate amino-heterocyclic groups can be formed by known heterocyclic ring formation reactions. For example the amino-triazole (e.g. 3H-[1,2,3]triazol-4-yl)-amine) can be formed by reaction of sodium nitrite in H2O with the amine in acid e.g. 2N HCl, followed by addition of aminoacetonitrile hydrogen sulphate in H2O. After an appropriate period of time NaOAc is added and rearrangement to the desired heterocycle is achieved by heating in solvent e.g. ethanol, for 16 hours.
Appropriate starting material and reagents for these reactions can be obtained commercially or by any of a large number of standard synthetic methods well known to those skilled in the art, for example see Advanced Organic Chemistry by Jerry March, 4th Edition, John Wiley & Sons, 1992, and Organic Syntheses, Volumes 1-8, John Wiley, edited by Jeremiah P. Freeman (ISBN: 0-471-31192-8), 1995, and see also the methods described in the experimental section below. For example a range of appropriate functionalized aniline and amino pyridine starting materials, and metal catalysts are commercially available.
Many boronates, for example boronic acids, esters or trifluoroborates, suitable for use in preparing compounds of the invention are commercially available, for example from Boron Molecular Limited of Noble Park, Australia, or from Combi-Blocks Inc. of San Diego, USA. Where the appropriately substituted boronate is not commercially available, they can be prepared by methods known in the art, for example as described in the review article by Miyaura, N. and Suzuki, A. (1995) Chem. Rev. 95, 2457. Thus, boronates can be prepared by reacting the corresponding bromo-compound with an alkyl lithium such as butyl lithium and then reacting with a borate ester e.g. (iPrO)3B. The reaction is typically carried out in a dry polar solvent such as tetrahydrofuran at a reduced temperature (for example −78° C.). Boronate esters (for example a pinacolatoboronate) can also be prepared from a bromo-compound by reaction with a diboronate ester such as bis(pinacolato)diboron in the presence of a phosphine such as tricyclohexyl-phosphine and a palladium (0) reagent such as tris(dibenzylideneacetone)-dipalladium (0). The formation of the boronate ester is typically carried out in a dry polar aprotic solvent such as dioxane or DMSO with heating to a temperature of up to 100° C., for example around 80° C. The resulting boronate ester derivative can, if desired, be hydrolysed to give the corresponding boronic acid or converted into the trifluoroborate.
All of the reactions described above can be used to functionalise alternative heterocyclic templates of formula (I), whose synthesis is outlined below.
Once synthesised, a range of functional group conversions can be employed on the substituted imidazopyridine compounds to produce further compounds of formula (I). For example, some of the following reactions can be used hydrogenation, hydrolysis, deprotection, and oxidation, to convert one compound of formula (I) into an alternative compound of formula (I).
Pyrazolo[1,5-a]pyrimidines
The pyrazolo[1,5-a]pyrimidine template can be synthesised from the appropriately substituted aminopyrazole (VI) and fragments (VII) as shown in Scheme 5A, where Ra can be hydrogen or A-NH—B—H(R1)q. This may occur by a one step or two step process, where Xa and Xb are electrophilic carbons (i.e. carbonyl, masked carbonyl i.e. acetal, enamine, conjugated alkenes or alkynes) (Perkin I, J. C. S. (1979), 3085-3094). Xc is an appropriate substituent, either a group R2 or groups such as halogen or pseudo halogens or methyl, which will allow reaction to introduce R2 as described herein. Cyclisation of the pyrazole (VI) with an appropriately substituted free or masked 1,3-dicarbonyl derivative can be used to prepare substituted pyrazolo[1,5-a]pyrimidines. Cyclisation occurs typically in an alcohol solvent or in toluene or in acetic acid, and may have additives such as piperidine, sodium ethoxide, HCl, AcOH, pTsOH, or ZnCl2 present (J. Med. Chem. (2001), 44 (3), 350-361; Bull. Korean Chem. Soc. (2002), 23 (4), 610-612; Australian Journal of Chemistry (1985), 38(1), 221-30).




A particular synthetic scheme for the preparation of disubstituted pyrazolo[1,5-a]pyrimidines is outlined in Scheme 5B. The pyrazolopyrimidine ring is formed by reaction of a substituted malonaldehyde as fragment VII with aminopyrazole. The substituted malonaldehyde can be substituted with methyl, or with a latent functionality e.g. a halogen as in 2-bromo-malonaldehyde, which allows further derivatisation at this position as in the scheme shown below using the reactions outlined herein.




In the cyclisation reaction, the malonaldehyde in solvent is added to 3-aminopyrazole followed by acid e.g. glacial acetic acid. The reagents are then cyclised upon heating under reflux. The compound of formula (I) can then be synthesised using the oxidative and coupling process outlined herein.
Compounds of formula (VI) and (VII) are known compounds or can be prepared by analogy to known methods. Many pyrazoles of formula (VI) are commercially available. Alternatively they can be obtained from known methods e.g. from ketones in a process described in EP308020 (Merck), or the methods discussed by Schmidt in Helv. Chim. Acta. (1956), 39, 986-991 and Helv. Chim. Acta. (1958), 41, 1052-1060 or by conversion of the pyrazoles of formula (VI) or the compound of formula (I) where Ra is hydrogen, halogen, nitro, ester, or amide to the desired R1 functionality by standard methods known to a person skilled in the art. For example, where R1 is halogen, coupling reactions with tin or palladium chemistry could be performed as described herein.
Alternatively the pyrazolo[1,5-a]pyrimidine-6-carboxylic acid or aldehyde are commercially available and can be used in the reactions described herein to synthesise di-substituted pyrazolo[1,5-a]pyrimidines.
Pyrazolo[1,5-a]pyrazines




Reaction of a mixture of 2-bromo-5-iodo-pyrazine and copper (I) iodide under inert conditions in an appropriate solvent and base e.g. DMF/Et3N with ethynyl-trimethyl-silane using a palladium catalyst e.g. Pd(PPh3)4 at room temperature gives 2-Bromo-5-trimethylsilanylethynyl-pyrazine. This material can be used without further purification and reacted to form 6-bromo-2-trimethylsilanyl-pyrazolo[1,5-a]pyrazine using O-(mesitylenesulfonyl)hydroxylamine to form the N-amino adduct. This can then be cyclised by reacting with base e.g. K2CO3 to form pyrazolopyrazine core (Scheme 6).
Appropriate groups can then be introduced by halogenation and reaction of the latent functionality in the metal catalysed reactions and the amide conversions at the other position as described herein.
Pyrazolo[1,5-a]pyridines
O-(Mesitylenesulfonyl)hydroxylamine is reacted with 3-substituted-pyridine under inert conditions to form the N-aminopyridine which can be used without further purification (Scheme 7). Cyclisation of the N-adduct using base (K2CO3) and 2-benzenesulfonyl-3-dimethylamino-acrylic acid methyl ester in an inert atmosphere gives the 3-carboxylic acid ester pyrazolo[1,5-a]pyridine. The carboxylic ester can be removed for example by saponification using sodium hydroxide to form the acid and then decarboxylation in polyphosphoric acid. The bromide can then be converted to the desired R2 group using the methods described herein.




Iodination with N-iodosuccinimide and metal catalysed reaction of aryl halides, can be used to introduced the required functionality as outlined herein.
Imidazo[4,5-b]pyridines
An imidazo[4,5-b]pyridine ring system may be constructed by reaction of an aniline with 2-chloro-3-amino pyridine as described in J. Heterocyclic Chemistry (1983), 20(5), 1339 (Scheme 8).




It will be appreciated that the resultant bicyclic ring in Scheme 8 can be functionalised by halogenation or alkylation and converted to R2 as described herein.
A more functionalized intermediate could be prepared for example as outlined in Scheme 9A based on methods described in U.S. Pat. No. 6,723,735.




As described herein the aryl halides similar to that shown above may undergo a range of metal catalysed reactions to generate the required compounds of formula (I).




Alternatively they could be synthesised as outlined above in Scheme 9B.
Imidazo[4,5-c]pyridines
A 3-aryl-3H-imidazo[4,5-c]pyridine ring system may be constructed by reaction of 3H-imidazo[4,5-c]pyridine with an aryl iodide as discussed in Biorg. Med. Chem. Lett. (2004), 14, 5263 (Scheme 10).




It is reported that the regioisomeric products may be separated by chromatography. A possible way to further elaborate this material to give the desired substitution pattern is illustrated below (Scheme 11).




Reaction with an oxidizing agent, such as 3-chloro perbenzoic acid, could be used to prepare the N-oxide which may be rearranged to the disubstituted 3H-imidazo[4,5-c]pyridine with several reagents e.g. POCl3, SOCl2. The regioisomeric products could then be separated by chromatography. Displacement of the halogen with potassium cyanide in DMSO or reaction with palladium and Zn(CN)2 (Bioorg. Med. Chem. Lett., 2003, 13 (9), 1591), produces the nitrile which can be converted to the acid as outlined previously.
An alternative strategy is shown in Scheme 12. The synthesis of 6-chloro-3H-imidazo[4,5-c]pyridine is described in J. Heterocyclic Chem (1965), 2(2), 196-201. The chloro group may be converted as outline herein. Subsequent elaboration to the N-aryl compounds could then be achieved according to the conditions shown in Scheme 10.




1,5-Diary)-1H-benzoimidazole




A synthesis of 1,5-diary)-1H-benzoimidazoles is reported in Bioorg. Med. Chem. Lett. (2003), 13, 2485-2488 (Scheme 13).




Displacement of fluorine from 4-bromo-1-fluoro-2-nitro-benzene with an appropriate aniline followed by reduction and cyclisation with triethyl orthoformate gives the bromo-benzoimidazole with the desired substitution pattern. The product may be further elaborated by reaction of the bromide as described herein to give 1,5-disubstituted benzoimidazoles.
1,5-disubstituted benzoimidazoles maybe synthesised using analogous chemistry to that described in Scheme 11.
Imidazo[1,2-c]pyrimidines
Di-substituted imidazo[1,2-c]pyrimidines can be prepared as outlined in Scheme 14.




This starts from 7-chloro-imidazo[1,2-c]pyrimidine, whose synthesis has been described in Yanai et al, Heterocyclic compounds. XVIII. Synthesis of imidazo[1,2-c]-pyrimidine derivatives, Yakugaku Zasshi (1974), 94(12), 1503-14. This material can then be further elaborated using any of the reactions described above.
Where the 3-position is an aryl or heteroaryl group the SNAr group can be replaced with a standard palladium cross coupling reaction using similar chemistries as described herein (Scheme 16).




Alternatively the 6-chloropyrimid-4-ylamine can be reacted to form the bicyclic ring system and then convert the chloro to the R2 group.
Alternatively the 6-amino-pyrimidine-4-carboxylic acid can be used as the starting material.
Imidazo[1,2-c]pyrimidin-5-one
3,7-disubstituted imidazo[1,2-c]pyrimidin-5-ones can be prepared from the 7-Chloro-6H-imidazo[1,2-c]pyrimidin-5-one (CAS number 56817-09-5) whose synthesis is described in Maggiali et al (1982), Acta Naturalia de l'Ateneo Parmense, 18(3), 93-101 and Bartholomew et al (1975) Journal of Organic Chemistry, 40(25), 3708-13.
7-Chloro-6H-imidazo[1,2-c]pyrimidin-5-one can be derivatised using nucleophilic substitution reactions such as SNAr to add functionality at the 7 position (Scheme 17). The SNAr reaction can be performed using potassium cyanide, and then converted to the amide. This compound can then be iodinated as described above before further functionalisation using the Suzuki reaction.




Alternatively 7-Chloro-6H-imidazo[1,2-c]pyrimidin-5-one could be directly iodinated to the intermediate below for use in the reactions described herein (Scheme 18).




In addition, other oxo-heterocycles could be synthesized from the appropriate chloro derivative by hydrolysis. The protected compound would be subjected to base hydrolysis to afford the pyridone. This could be performed with NaOH (or NaOH/H2O2) in H2O/MeOH or H2O/dioxane following procedures described in the literature for the hydrolysis of chloropyridines (e.g. Australian J. Chem. (1984), 37(12), 2469-2477).
Imidazo[1,2-b]pyridazine




The synthesis of the Imidazo[1,2-b]pyridazine core can be performed as described in Scheme 19 using a pyridazin-3-ylamine derivative.
Many methyl, carboxylic acid, carboxylic ester, or halide substituted bicyclic or monocyclic aromatic compounds are commercially available. Therefore, these and other heterocycles, may be synthesised directly from the methyl, carboxylic acid, carboxylic ester, or halide substituted bicyclic compounds or from the methyl, carboxylic acid, carboxylic ester, or halide substituted monocyclic aromatic compounds using the cyclisation reactions described herein.
Other heterocycles can be synthesised using well known reactions, for example as described in Comprehensive Heterocyclic Chemistry I (Edited by Katritzky, A. R. and Rees, C. W. (1982) Elsevier) and Comprehensive Heterocyclic Chemistry II (Edited by Katritzky, A. R., Rees, C. W. and Scriven, E. F. V. (1996) Elsevier, ISBN 0-08-042072-9).
In many of the reactions described above, it may be necessary to protect one or more groups to prevent reaction from taking place at an undesirable location on the molecule. Examples of protecting groups, and methods of protecting and deprotecting functional groups, can be found in Protective Groups in Organic Synthesis (Green, T. and Wuts, P. (1999); 3rd Edition; John Wiley and Sons).
A hydroxy group may be protected, for example, as an ether (—OR) or an ester (—OC(═O)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl)ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester (—OC(═O)CH3, —OAc). An aldehyde or ketone group may be protected, for example, as an acetal (R—CH(OR)2) or ketal (R2C(OR)2), respectively, in which the carbonyl group (>C═O) is converted to a diether (>C(OR)2), by reaction with, for example, a primary alcohol. The aldehyde or ketone group is readily regenerated by hydrolysis using a large excess of water in the presence of acid. An amine group may be protected, for example, as an amide (—NRCO—R) or a urethane (—NRCO—OR), for example, as: a methyl amide (—NHCO—CH3); a benzyloxy amide (—NHCO—OCH2C6H5, —NH-Cbz); as a t-butoxy amide (—NHCO—OC(CH3)3, —NH-Boc); a 2-biphenyl-2-propoxy amide (—NHCO—OC(CH3)2C6H4C6H5, —NH-Bpoc), as a 9-fluorenylmethoxy amide (—NH-Fmoc), as a 6-nitroveratryloxy amide (—NH-Nvoc), as a 2-trimethylsilylethyloxy amide (—NH-Teoc), as a 2,2,2-trichloroethyloxy amide (—NH-Troc), as an allyloxy amide (—NH-Alloc), or as a 2(-phenylsulphonyl)ethyloxy amide (—NH-Psec). Other protecting groups for amines, such as cyclic amines and heterocyclic N—H groups, include toluenesulphonyl (tosyl) and methanesulphonyl (mesyl) groups and benzyl groups such as a para-methoxybenzyl (PMB) group. A carboxylic acid group may be protected as an ester for example, as: an C1-7 alkyl ester (e.g., a methyl ester; a t-butyl ester); a C1-7 haloalkyl ester (e.g., a C1-7 trihaloalkyl ester); a triC1-7alkylsilyl-C1-7alkyl ester; or a C5-20 aryl-C1-7 alkyl ester (e.g., a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as a methyl amide. A thiol group may be protected, for example, as a thioether (—SR), for example, as: a benzyl thioether; an acetamidomethyl ether (—S—CH2NHC(═O)CH3).
Key intermediates in the preparation of the compounds of formula (I) are the compounds of formula (II) and (III). Novel chemical intermediates of the formula (II) and (III) form a further aspect of the invention.
A further aspect of the invention is a process for the preparation of a compound of formula (I) as defined herein, which process comprises:
(i) the reaction of a compound of the formula (II):




or a protected form thereof, with an appropriately substituted aldehyde or ketone; or

(ii) the reaction of a compound of the formula (II):





or a protected form thereof, with hydrazine hydrate and then cyclising; or

(iii) the reaction of a compound of the formula (III):





or a protected form thereof, wherein Y is a groups which can be converted to an amide e.g. methyl, carboxylic acid, carboxylic ester, halide;

    
    
        and then converting to an amide;
        and thereafter removing any protecting group present;
        wherein X1-5, A, B, R1 and R2 are as defined herein; and optionally thereafter converting one compound of the formula (I) into another compound of the formula (I).
    
    


According to a further aspect of the invention there is provided a novel intermediate as defined herein.
Pharmaceutically Acceptable Salts, Solvates or Derivatives Thereof
In this section, as in all other sections of this application, unless the context indicates otherwise, references to formula (I) include references to all other sub-groups, preferences and examples thereof as defined herein.
Unless otherwise specified, a reference to a particular compound also includes ionic forms, salts, solvates, isomers, tautomers, N-oxides, esters, prodrugs, isotopes and protected forms thereof, for example, as discussed below; preferably, the ionic forms, or salts or tautomers or isomers or N-oxides or solvates thereof; and more preferably, the ionic forms, or salts or tautomers or solvates or protected forms thereof. Many compounds of the formula (I) can exist in the form of salts, for example acid addition salts or, in certain cases salts of organic and inorganic bases such as carboxylate, sulphonate and phosphate salts. All such salts are within the scope of this invention, and references to compounds of the formula (I) include the salt forms of the compounds.
The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.
Acid addition salts may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulphonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)-(1S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (−)-L-malic, malonic, (±)-DL-mandelic, methanesulphonic, naphthalenesulphonic (e.g. naphthalene-2-sulphonic), naphthalene-1,5-disulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, (+)-L-tartaric, thiocyanic, toluenesuiphonic (e.g. p-toluenesulphonic), undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.
One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulphuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulphonic, toluenesuiphonic, methanesulphonic (mesylate), ethanesulphonic, naphthalenesulphonic, valeric, acetic, propanoic, butanoic, malonic, glucuronic and lactobionic acids.
Another group of acid addition salts includes salts formed from acetic, adipic, ascorbic, aspartic, citric, DL-Lactic, fumaric, gluconic, glucuronic, hippuric, hydrochloric, glutamic, DL-malic, methanesulphonic, sebacic, stearic, succinic and tartaric acids.
The compounds of the invention may exist as mono- or di-salts depending upon the pKa of the acid from which the salt is formed.
If the compound is anionic, or has a functional group which may be anionic (e.g., —COOH may be —COO−), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth metal cations such as Ca2+ and Mg2+, and other cations such as Al3+. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4+) and substituted ammonium ions (e.g., NH3R+, NH2R2+, NHR3+, NR4+).
Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+.
Where the compounds of the formula (I) contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of formula (I).
The salt forms of the compounds of the invention are typically pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are discussed in Berge et al. (1977), “Pharmaceutically Acceptable Salts,” J. Pharm. Sci., Vol. 66, pp. 1-19. However, salts that are not pharmaceutically acceptable may also be prepared as intermediate forms which may then be converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salts forms, which may be useful, for example, in the purification or separation of the compounds of the invention, also form part of the invention.
Compounds of the formula (I) containing an amine function may also form N-oxides. A reference herein to a compound of the formula (I) that contains an amine function also includes the N-oxide.
Where a compound contains several amine functions, one or more than one nitrogen atom may be oxidised to form an N-oxide. Particular examples of N-oxides are the N-oxides of a tertiary amine or a nitrogen atom of a nitrogen-containing heterocycle.
N-Oxides can be formed by treatment of the corresponding amine with an oxidizing agent such as hydrogen peroxide or a per-acid (e.g. a peroxycarboxylic acid), see for example Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience, pages. More particularly, N-oxides can be made by the procedure of Deady, L. W. (Syn. Comm. (1977), 7, 509-514) in which the amine compound is reacted with m-chloroperoxybenzoic acid (MCPBA), for example, in an inert solvent such as dichloromethane.
The compounds of the invention may form solvates, for example with water (i.e., hydrates) or common organic solvents. As used herein, the term “solvate” means a physical association of the compounds of the present invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. The term “solvate” is intended to encompass both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include compounds on the invention in combination with water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid or ethanolamine and the like. The compounds of the invention may exert their biological effects whilst they are in solution.
Solvates are well known in pharmaceutical chemistry. They can be important to the processes for the preparation of a substance (e.g. in relation to their purification, the storage of the substance (e.g. its stability) and the ease of handling of the substance and are often formed as part of the isolation or purification stages of a chemical synthesis. A person skilled in the art can determine by means of standard and long used techniques whether a hydrate or other solvate has formed by the isolation conditions or purification conditions used to prepare a given compound. Examples of such techniques include thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray crystallography (e.g. single crystal X-ray crystallography or X-ray powder diffraction) and Solid State NMR(SS-NMR, also known as Magic Angle Spinning NMR or MAS-NMR). Such techniques are as much a part of the standard analytical toolkit of the skilled chemist as NMR, IR, HPLC and MS.
Alternatively the skilled person can deliberately form a solvate using crystallisation using crystallisation conditions that include an amount of the solvent required for the particular solvate. Thereafter the standard methods described above, can be used to establish whether solvates had formed.
Furthermore, the compounds of the present invention may have one or more polymorph, amorphous or crystalline forms and as such are intended to be included in the scope of the invention.
Compounds of the formula (I) may exist in a number of different geometric isomeric, and tautomeric forms and references to compounds of the formula (I) include all such forms. For the avoidance of doubt, where a compound can exist in one of several geometric isomeric or tautomeric forms and only one is specifically described or shown, all others are nevertheless embraced by formula (I).
Other examples of tautomeric forms include, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, and nitro/aci-nitro.




Where compounds of the formula (I) contain one or more chiral centres, and can exist in the form of two or more optical isomers, references to compounds of the formula (I) include all optical isomeric forms thereof (e.g. enantiomers, epimers and diastereoisomers), either as individual optical isomers, or mixtures (e.g. racemic mixtures) or two or more optical isomers, unless the context requires otherwise.
The optical isomers may be characterised and identified by their optical activity (i.e. as + and − isomers, or d and l isomers) or they may be characterised in terms of their absolute stereochemistry using the “R and S” nomenclature developed by Cahn, Ingold and Prelog, see Advanced Organic Chemistry by Jerry March, 4th Edition, John Wiley & Sons, New York, 1992, pages 109-114, and see also Cahn, IngoId & Prelog (1966), Angew. Chem. Int. Ed. Engl., 5, 385-415.
Optical isomers can be separated by a number of techniques including chiral chromatography (chromatography on a chiral support) and such techniques are well known to the person skilled in the art.
As an alternative to chiral chromatography, optical isomers can be separated by forming diastereoisomeric salts with chiral acids such as (+)-tartaric acid, (−)-pyroglutamic acid, (−)-di-toluoyl-L-tartaric acid, (+)-mandelic acid, (−)-malic acid, and (−)-camphorsulphonic, separating the diastereoisomers by preferential crystallisation, and then dissociating the salts to give the individual enantiomer of the free base.
Where compounds of the formula (I) exist as two or more optical isomeric forms, one enantiomer in a pair of enantiomers may exhibit advantages over the other enantiomer, for example, in terms of biological activity. Thus, in certain circumstances, it may be desirable to use as a therapeutic agent only one of a pair of enantiomers, or only one of a plurality of diastereoisomers. Accordingly, the invention provides compositions containing a compound of the formula (I) having one or more chiral centres, wherein at least 55% (e.g. at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%) of the compound of the formula (I) is present as a single optical isomer (e.g. enantiomer or diastereoisomer). In one general embodiment, 99% or more (e.g. substantially all) of the total amount of the compound of the formula (I) may be present as a single optical isomer (e.g. enantiomer or diastereoisomer).
The compounds of the invention include compounds with one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element. For example, a reference to hydrogen includes within its scope 1H, 2H (D), and 3H (T). Similarly, references to carbon and oxygen include within their scope respectively 12C, 13C and 14C and 16O and 18O.
The isotopes may be radioactive or non-radioactive. In one embodiment of the invention, the compounds contain no radioactive isotopes. Such compounds are preferred for therapeutic use. In another embodiment, however, the compound may contain one or more radioisotopes. Compounds containing such radioisotopes may be useful in a diagnostic context.
Esters such as carboxylic acid esters and acyloxy esters of the compounds of formula (I) bearing a carboxylic acid group or a hydroxyl group are also embraced by formula (I). In one embodiment of the invention, formula (I) includes within its scope esters of compounds of the formula (I) bearing a carboxylic acid group or a hydroxyl group. In another embodiment of the invention, formula (I) does not include within its scope esters of compounds of the formula (I) bearing a carboxylic acid group or a hydroxyl group. Examples of esters are compounds containing the group —C(═O)OR, wherein R is an ester substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Particular examples of ester groups include, but are not limited to, —C(═O)OCH3, —C(═O)OCH2CH3, —C(═O)OC(CH3)3, and —C(═O)OPh. Examples of acyloxy (reverse ester) groups are represented by —OC(═O)R, wherein R is an acyloxy substituent, for example, a C1-7 alkyl group, a C3-20 heterocyclyl group, or a C5-20 aryl group, preferably a C1-7 alkyl group. Particular examples of acyloxy groups include, but are not limited to, —OC(═O)CH3 (acetoxy), —OC(═O)CH2CH3, —OC(═O)C(CH3)3, —OC(═O)Ph, and —OC(═O)CH2Ph.
Also encompassed by formula (I) are any polymorphic forms of the compounds, solvates (e.g. hydrates), complexes (e.g. inclusion complexes or clathrates with compounds such as cyclodextrins, or complexes with metals) of the compounds, and prodrugs of the compounds. By “prodrugs” is meant for example any compound that is converted in vivo into a biologically active compound of the formula (I).
For example, some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester). During metabolism, the ester group (—C(═O)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (—C(═O)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required.
Examples of such metabolically labile esters include those of the formula —C(═O)OR wherein R is:
C1-7alkyl (e.g., -Me, -Et, -nPr, -iPr, -nBu, -sBu, -iBu, -tBu);
C1-7-aminoalkyl (e.g., aminoethyl; 2-(N,N-diethylamino)ethyl; 2-(4-morpholino)ethyl); and
acyloxy-C1-7alkyl (e.g., acyloxymethyl; acyloxyethyl; pivaloyloxymethyl; acetoxymethyl;


    1-acetoxyethyl; 1-(1-methoxy-1-methyl)ethyl-carbonyloxyethyl; 1-(benzoyloxy)ethyl; isopropoxy-carbonyloxymethyl;
    1-isopropoxy-carbonyloxyethyl; cyclohexyl-carbonyloxymethyl; 1-cyclohexyl-carbonyloxyethyl; cyclohexyloxy-carbonyloxymethyl;
    1-cyclohexyloxy-carbonyloxyethyl; (4-tetrahydropyranyloxy) carbonyloxymethyl; 1-(4-tetrahydropyranyloxy)carbonyloxyethyl;
    (4-tetrahydropyranyl)carbonyloxymethyl; and 1-(4-tetrahydropyranyl)carbonyloxyethyl).


Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound (for example, as in antigen-directed enzyme pro-drug therapy (ADEPT), gene-directed enzyme pro-drug therapy (GDEPT) and ligand-directed enzyme pro-drug therapy (LIDEPT) etc.). For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.
It will be appreciated that references to “derivatives” include references to ionic forms, salts, solvates, isomers, tautomers, N-oxides, esters, prodrugs, isotopes and protected forms thereof.
According to one aspect of the invention there is provided a compound as defined herein or a salt, tautomer, N-oxide or solvate thereof.
According to a further aspect of the invention there is provided a compound as defined herein or a salt or solvate thereof.
References to compounds of the formula (I), (Ia), (Ib), (Ic) and (Id) and sub-groups thereof as defined herein include within their scope the salts or solvates or tautomers or N-oxides of the compounds.
Protein Tyrosine Kinases (PTK)
The compounds of the invention described herein inhibit or modulate the activity of certain tyrosine kinases, and thus the compounds will be useful in the treatment or prophylaxis of disease states or conditions mediated by those tyrosine kinases in particular FGFR.
FGFR
The fibroblast growth factor (FGF) family of protein tyrosine kinase (PTK) receptors regulates a diverse array of physiologic functions including mitogenesis, wound healing, cell differentiation and angiogenesis, and development. Both normal and malignant cell growth as well as proliferation are affected by changes in local concentration of FGFs, extracellular signalling molecules which act as autocrine as well as paracrine factors. Autocrine FGF signalling may be particularly important in the progression of steroid hormone-dependent cancers to a hormone independent state (Powers, et al. (2000), Endocr. Relat. Cancer, 7, 165-197).
FGFs and their receptors are expressed at increased levels in several tissues and cell lines and overexpression is believed to contribute to the malignant phenotype. Furthermore, a number of oncogenes are homologues of genes encoding growth factor receptors, and there is a potential for aberrant activation of FGF-dependent signalling in human pancreatic cancer (Ozawa, et al. (2001), Teratog. Carcinog. Mutagen., 21, 27-44).
The two prototypic members are acidic fibroblast growth factor (aFGF or FGF1) and basic fibroblast growth factor (bFGF or FGF2), and to date, at least twenty distinct FGF family members have been identified. The cellular response to FGFs is transmitted via four types of high affinity transmembrane protein tyrosine-kinase fibroblast growth factor receptors (FGFR) numbered 1 to 4 (FGFR1 to FGFR4). Upon ligand binding, the receptors dimerize and auto- or trans-phosphorylate specific cytoplasmic tyrosine residues to transmit an intracellular signal that ultimately regulates nuclear transcription factor effectors.
Disruption of the FGFR1 pathway should affect tumor cell proliferation since this kinase is activated in many tumor types in addition to proliferating endothelial cells. The over-expression and activation of FGFR1 in tumor-associated vasculature has suggested a role for these molecules in tumor angiogenesis.
Fibroblast growth factor receptor 2 has high affinity for the acidic and/or basic fibroblast growth factors, as well as the keratinocyte growth factor ligands. Fibroblast growth factor receptor 2 also propagates the potent osteogenic effects of FGFs during osteoblast growth and differentiation. Mutations in fibroblast growth factor receptor 2, leading to complex functional alterations, were shown to induce abnormal ossification of cranial sutures (craniosynostosis), implying a major role of FGFR signalling in intramembranous bone formation. For example, in Apert (AP) syndrome, characterized by premature cranial suture ossification, most cases are associated with point mutations engendering gain-of-function in fibroblast growth factor receptor 2 (Lemonnier, et al. (2001), J. Bone Miner. Res., 16, 832-845). In addition, mutation screening in patients with syndromic craniosynostoses indicates that a number of recurrent FGFR2 mutations accounts for severe forms of Pfeiffer syndrome (Lajeunie et al, European Journal of human Genetics (2006) 14, 289-298). Particular mutations of FGFR2 include W290C, D321A, Y340C, C342R, C342S, C342W, N549H, K641R in FGFR2.
Several severe abnormalities in human skeletal development, including Apert, Crouzon, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Pfeiffer syndromes are associated with the occurrence of mutations in fibroblast growth factor receptor 2. Most, if not all, cases of Pfeiffer Syndrome (PS) are also caused by de novo mutation of the fibroblast growth factor receptor 2 gene (Meyers, et al. (1996), Am. J. Hum. Genet., 58, 491-498; Plomp, et al. (1998), Am. J. Med. Genet., 75, 245-251), and it was recently shown that mutations in fibroblast growth factor receptor 2 break one of the cardinal rules governing ligand specificity. Namely, two mutant splice forms of fibroblast growth factor receptor, FGFR2c and FGFR2b, have acquired the ability to bind to and be activated by atypical FGF ligands. This loss of ligand specificity leads to aberrant signalling and suggests that the severe phenotypes of these disease syndromes result from ectopic ligand-dependent activation of fibroblast growth factor receptor 2 (Yu, et al., (2000), Proc. Natl. Acad. Sci. U.S.A., 97, 14536-14541).
Genetic aberrations of the FGFR3 receptor tyrosine kinase such as chromosomal translocations or point mutations result in ectopically expressed or deregulated, constitutively active, FGFR3 receptors. Such abnormalities are linked to a subset of multiple myelomas and in bladder, hepatocellular, oral squamous cell carcinoma and cervical carcinomas (Powers, C. J. (2000), et al., Endocr. Rel. Cancer, 7, 165; Qiu, W. et. al. (2005), World Journal Gastroenterol, 11(34)). Accordingly, FGFR3 inhibitors would be useful in the treatment of multiple myeloma, bladder and cervical carcinomas. FGFR3 is also over-expressed in bladder cancer, in particular invasive bladder cancer. FGFR3 is frequently activated by mutation in urothelial carcinoma (UC) (Journal of Pathology (2007), 213(1), 91-98). Increased expression was associated with mutation (85% of mutant tumors showed high-level expression) but also 42% of tumors with no detectable mutation showed over-expression, including many muscle-invasive tumors.
As such, the compounds which inhibit FGFR will be useful in providing a means of preventing the growth or inducing apoptosis in tumours, particularly by inhibiting angiogenesis. It is therefore anticipated that the compounds will prove useful in treating or preventing proliferative disorders such as cancers. In particular tumours with activating mutants of receptor tyrosine kinases or upregulation of receptor tyrosine kinases may be particularly sensitive to the inhibitors. Patients with activating mutants of any of the isoforms of the specific RTKs discussed herein may also find treatment with RTK inhibitors particularly beneficial.
Over expression of FGFR4 has been linked to poor prognosis in both prostate and thyroid carcinomas (Ezzat, S., et al., (2002), The Journal of Clinical Investigation, 109, 1; Wang et al., (2004), Clinical Cancer Research, 10). In addition a germline polymorphism (Gly388Arg) is associated with increased incidence of lung, breast, colon and prostate cancers (Wang et al. (2004), Clinical Cancer Research, 10). In addition, a truncated form of FGFR4 (including the kinase domain) has also been found to present in 40% of pituitary tumours but not present in normal tissue.
A recent study has shown a link between FGFR1 expression and tumorigenicity in Classic Lobular Carcinomas (CLC). CLCs account for 10-15% of all breast cancers and, in general, lack p53 and Her2 expression whilst retaining expression of the oestrogen receptor. A gene amplification of 8p12-p11.2 was demonstrated in  ̃50% of CLC cases and this was shown to be linked with an increased expression of FGFR1. Preliminary studies with siRNA directed against FGFR1, or a small molecule inhibitor of the receptor, showed cell lines harbouring this amplification to be particularly sensitive to inhibition of this signalling pathway (Reis-Filho et al. (2006), Clin Cancer Res. 12(22): 6652-6662.
Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma likely results from abnormal proliferation and differentiation during skeletal myogenesis. FGFR1 is over-expressed in primary rhabdomyosarcoma tumors and is associated with hypomethylation of a 5′ CpG island and abnormal expression of the AKT1, NOG, and BMP4 genes (Genes, Chromosomes & Cancer (2007), 46(11), 1028-1038).
Fibrotic conditions are a major medical problem resulting from abnormal or excessive deposition of fibrous tissue. This occurs in many diseases, including liver cirrhosis, glomerulonephritis, pulmonary fibrosis, systemic fibrosis, rheumatoid arthritis, as well as the natural process of wound healing. The mechanisms of pathological fibrosis are not fully understood but are thought to result from the actions of various cytokines (including tumor necrosis factor (TNF), fibroblast growth factors (FGF's), platelet derived growth factor (PDGF) and transforming growth factor beta. (TGFβ) involved in the proliferation of fibroblasts and the deposition of extracellular matrix proteins (including collagen and fibronectin). This results in alteration of tissue structure and function and subsequent pathology.
A number of preclinical studies have demonstrated the up-regulation of fibroblast growth factors in preclinical models of lung fibrosis (Inoue, et al., 1997 & 2002; Barrios, et al. 1997)). TGFβ1 and PDGF have been reported to be involved in the fibrogenic process (reviewed by Atamas & White, 2003) and further published work suggests the elevation of FGF's and consequent increase in fibroblast proliferation, may be in response to elevated TGFβ1 (Khalil, et al., 2005). The potential therapeutic relevance of this pathway in fibrotic conditions is suggested by the reported clinical effect of Pirfenidone (Arata, et al., 2005) in idiopathic pulmonary fibrosis (IPF).
Idiopathic pulmonary fibrosis (also referred to as Cryptogenic fibrosing alveolitis) is a progressive condition involving scarring of the lung. Gradually, the air sacs of the lungs become replaced by fibrotic tissue, which becomes thicker, causing an irreversible loss of the tissue's ability to transfer oxygen into the bloodstream. The symptoms of the condition include shortness of breath, chronic dry coughing, fatigue, chest pain and loss of appetite resulting in rapid weight loss. The condition is extremely serious with approximately 50% mortality after 5 years.
Vascular Endothelial Growth Factor (VEGFR)
Chronic proliferative diseases are often accompanied by profound angiogenesis, which can contribute to or maintain an inflammatory and/or proliferative state, or which leads to tissue destruction through the invasive proliferation of blood vessels. (Folkman (1997), 79, 1-81; Folkman (1995), Nature Medicine, 1, 27-31; Folkman and Shing (1992), J. Biol. Chem., 267, 10931).
Angiogenesis is generally used to describe the development of new or replacement blood vessels, or neovascularisation. It is a necessary and physiological normal process by which vasculature is established in the embryo. Angiogenesis does not occur, in general, in most normal adult tissues, exceptions being sites of ovulation, menses and wound healing. Many diseases, however, are characterized by persistent and unregulated angiogenesis. For instance, in arthritis, new capillary blood vessels invade the joint and destroy cartilage (Colville-Nash and Scott (1992), Ann. Rhum. Dis., 51, 919). In diabetes (and in many different eye diseases), new vessels invade the macula or retina or other ocular structures, and may cause blindness (Brooks, et al., (1994), Cell, 79, 1157). The process of atherosclerosis has been linked to angiogenesis (Kahlon, et al., (1992), Can. J. Cardiol., 8, 60). Tumor growth and metastasis have been found to be angiogenesis-dependent (Folkman (1992), Cancer Biol, 3, 65; Denekamp (1993), Br. J. Rad., 66, 181; Fidler and Ellis (1994), Cell, 79, 185).
The recognition of the involvement of angiogenesis in major diseases has been accompanied by research to identify and develop inhibitors of angiogenesis. These inhibitors are generally classified in response to discrete targets in the angiogenesis cascade, such as activation of endothelial cells by an angiogenic signal; synthesis and release of degradative enzymes; endothelial cell migration; proliferation of endothelial cells; and formation of capillary tubules. Therefore, angiogenesis occurs in many stages and attempts are underway to discover and develop compounds that work to block angiogenesis at these various stages.
There are publications that teach that inhibitors of angiogenesis, working by diverse mechanisms, are beneficial in diseases such as cancer and metastasis (O'Reilly, et al. (1994), Cell, 79, 315; Ingber, et al., (1990), Nature, 348, 555), ocular diseases (Friedlander, et al. (1995), Science, 270, 1500), arthritis (Peacock, et al. (1992), J. Exp. Med., 175, 1135; Peacock et al. (1995), Cell. Immun., 160, 178) and hemangioma (Taraboletti, et al. (1995), J. Natl. Cancer Inst., 87, 293).
Receptor tyrosine kinases (RTKs) are important in the transmission of biochemical signals across the plasma membrane of cells. These transmembrane molecules characteristically consist of an extracellular ligand-binding domain connected through a segment in the plasma membrane to an intracellular tyrosine kinase domain. Binding of ligand to the receptor results in stimulation of the receptor-associated tyrosine kinase activity that leads to phosphorylation of tyrosine residues on both the receptor and other intracellular proteins, leading to a variety of cellular responses. To date, at least nineteen distinct RTK subfamilies, defined by amino acid sequence homology, have been identified.
Vascular endothelial growth factor (VEGF), a polypeptide, is mitogenic for endothelial cells in vitro and stimulates angiogenic responses in vivo. VEGF has also been linked to inappropriate angiogenesis (Pinedo, H. M., et al. (2000), The Oncologist, 5(90001), 1-2). VEGFR(s) are protein tyrosine kinases (PTKs). PTKs catalyze the phosphorylation of specific tyrosine residues in proteins involved in cell function thus regulating cell growth, survival and differentiation. (Wilks, A. F. (1990), Progress in Growth Factor Research, 2, 97-111; Courtneidge, S. A. (1993), Dev. Supp. I, 57-64; Cooper, J. A. (1994), Semin. Cell Biol., 5(6), 377-387; Paulson, R. F. (1995), Semin. Immunol., 7(4), 267-277; Chan, A. C. (1996), Curr. Opin. Immunol., 8(3), 394-401).
Three PTK receptors for VEGF have been identified: VEGFR-1 (Flt-1); VEGFR-2 (Flk-1 or KDR) and VEGFR-3 (Flt-4). These receptors are involved in angiogenesis and participate in signal transduction (Mustonen, T., et al. (1995), J. Cell Biol., 129, 895-898).
Of particular interest is VEGFR-2, which is a transmembrane receptor PTK expressed primarily in endothelial cells. Activation of VEGFR-2 by VEGF is a critical step in the signal transduction pathway that initiates tumour angiogenesis. VEGF expression may be constitutive to tumour cells and can also be upregulated in response to certain stimuli. One such stimuli is hypoxia, where VEGF expression is upregulated in both tumour and associated host tissues. The VEGF ligand activates VEGFR-2 by binding with its extracellular VEGF binding site. This leads to receptor dimerization of VEGFRs and autophosphorylation of tyrosine residues at the intracellular kinase domain of VEGFR-2. The kinase domain operates to transfer a phosphate from ATP to the tyrosine residues, thus providing binding sites for signalling proteins downstream of VEGFR-2 leading ultimately to initiation of angiogenesis (McMahon, G. (2000), The Oncologist, 5(90001), 3-10).
Inhibition at the kinase domain binding site of VEGFR-2 would block phosphorylation of tyrosine residues and serve to disrupt initiation of angiogenesis.
Angiogenesis is a physiologic process of new blood vessel formation mediated by various cytokines called angiogenic factors. Although its potential pathophysiologic role in solid tumors has been extensively studied for more than 3 decades, enhancement of angiogenesis in chronic lymphocytic leukemia (CLL) and other malignant hematological disorders has been recognized more recently. An increased level of angiogenesis has been documented by various experimental methods both in bone marrow and lymph nodes of patients with CLL. Although the role of angiogenesis in the pathophysiology of this disease remains to be fully elucidated, experimental data suggest that several angiogenic factors play a role in the disease progression. Biologic markers of angiogenesis were also shown to be of prognostic relevance in CLL. This indicates that VEGFR inhibitors may also be of benefit for patients with leukemia's such as CLL.
In order for a tumour mass to get beyond a critical size, it must develop an associated vasculature. It has been proposed that targeting a tumor vasculature would limit tumor expansion and could be a useful cancer therapy. Observations of tumor growth have indicated that small tumour masses can persist in a tissue without any tumour-specific vasculature. The growth arrest of nonvascularized tumors has been attributed to the effects of hypoxia at the center of the tumor. More recently, a variety of proangiogenic and antiangiogenic factors have been identified and have led to the concept of the “angiogenic switch,” a process in which disruption of the normal ratio of angiogenic stimuli and inhibitors in a tumor mass allows for autonomous vascularization. The angiogenic switch appears to be governed by the same genetic alterations that drive malignant conversion: the activation of oncogenes and the loss of tumour suppressor genes. Several growth factors act as positive regulators of angiogenesis. Foremost among these are vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiogenin. Proteins such as thrombospondin (Tsp-1), angiostatin, and endostatin function as negative regulators of angiogenesis.
Inhibition of VEGFR2 but not VEGFR1 markedly disrupts angiogenic switching, persistent angiogenesis, and initial tumor growth in a mouse model. In late-stage tumors, phenotypic resistance to VEGFR2 blockade emerged, as tumors regrew during treatment after an initial period of growth suppression. This resistance to VEGF blockade involves reactivation of tumour angiogenesis, independent of VEGF and associated with hypoxia-mediated induction of other proangiogenic factors, including members of the FGF family. These other proangiogenic signals are functionally implicated in the revascularization and regrowth of tumours in the evasion phase, as FGF blockade impairs progression in the face of VEGF inhibition. Inhibition of VEGFR2 but not VEGFR1 markedly disrupted angiogenic switching, persistent angiogenesis, and initial tumor growth. In late-stage tumours, phenotypic resistance to VEGFR2 blockade emerged, as tumours regrew during treatment after an initial period of growth suppression. This resistance to VEGF blockade involves reactivation of tumour angiogenesis, independent of VEGF and associated with hypoxia-mediated induction of other proangiogenic factors, including members of the FGF family. These other proangiogenic signals are functionally implicated in the revascularization and regrowth of tumours in the evasion phase, as FGF blockade impairs progression in the face of VEGF inhibition.
A FGF-trap adenovirus has been previously reported to bind and block various ligands of the FGF family, including FGF1, FGF3, FGF7, and FGF10, thereby effectively inhibiting angiogenesis in vitro and in vivo. Indeed, adding the FGF-trap treatment in the regrowth phase of a mouse model produced a significant decrease in tumor growth compared to anti-VEGFR2 alone. This decrease in tumor burden was accompanied by a decrease in angiogenesis that was observed as decreased intratumoral vessel density.
Batchelor et al. (Batchelor et al., 2007, Cancer Cell, 11(1), 83-95) provide evidence for normalization of glioblastoma blood vessels in patients treated with a pan-VEGF receptor tyrosine kinase inhibitor, AZD2171, in a phase 2 study. The rationale for using AZD2171 was based partially on results showing a decrease in perfusion and vessel density in an in vivo breast cancer model (Miller et al., 2006, Clin. Cancer Res. 12, 281-288). Furthermore, using an orthotopic glioma model, it had previously been identified that the optimal window of time to deliver anti-VEGFR2 antibody to achieve a synergistic effect with radiation. During the window of normalization, there was improved oxygenation, increased pericyte coverage, and upregulation of angiopoietin-1 leading to a decrease in interstitial pressure and permeability within the tumour (Winkler et al., 2004, Cancer Cell 6, 553-563). The window of normalization can be quantified using magnetic resonance imaging (MRI) using MRI gradient echo, spin echo, and contrast enhancement to measure blood volume, relative vessel size, and vascular permeability.
The authors showed that progression on treatment with AZD2171 was associated with an increase in CECs, SDF1, and FGF2, while progression after drug interruptions correlated with increases in circulating progenitor cells (CPCs) and plasma FGF2 levels. The increase in plasma levels of SDF1 and FGF2 correlated with MRI measurements, demonstrated an increase in the relative vessel density and size. Thus, MRI determination of vessel normalization in combination with circulating biomarkers provides for an effective means to assess response to antiangiogenic agents.
PDGFR
A malignant tumour is the product of uncontrolled cell proliferation. Cell growth is controlled by a delicate balance between growth-promoting and growth-inhibiting factors. In normal tissue the production and activity of these factors results in differentiated cells growing in a controlled and regulated manner that maintains the normal integrity and functioning of the organ. The malignant cell has evaded this control; the natural balance is disturbed (via a variety of mechanisms) and unregulated, aberrant cell growth occurs. A growth factor of importance in tumour development is the platelet-derived growth factor (PDGF) that comprises a family of peptide growth factors that signal through cell surface tyrosine kinase receptors (PDGFR) and stimulate various cellular functions including growth, proliferation, and differentiation. PDGF expression has been demonstrated in a number of different solid tumours including glioblastomas and prostate carcinomas. The tyrosine kinase inhibitor imatinib mesylate, which has the chemical name 4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-ylpyridinyl]amino]-phenyl]benzamide methanesulfonate, blocks activity of the Bcr-Abl oncoprotein and the cell surface tyrosine kinase receptor c-Kit, and as such is approved for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumours. Imatinib mesylate is also a potent inhibitor of PDGFR kinase and is currently being evaluated for the treatment of chronic myelomonocytic leukemia and glioblastoma multiforme, based upon evidence in these diseases of activating mutations in PDGFR. In addition, sorafenib (BAY 43-9006) which has the chemical name 4-(4-(3-(4-chloro-3(trifluoromethyl)phenyl)ureido) phenoxy)-N2-methylpyridine-2-carboxamide, targets both the Raf signalling pathway to inhibit cell proliferation and the VEGFR/PDGFR signalling cascades to inhibit tumour angiogenesis. Sorafenib is being investigated for the treatment of a number of cancers including liver and kidney cancer.
There are conditions which are dependent on activation of PDGFR such as hypereosinophilic syndrome. PDGFR activation is also associated with other malignancies, which include chronic myelomonocytic leukemia (CMML). In another disorder, dermatofibrosarcoma protuberans, an infiltrative skin tumor, a reciprocal translocation involving the gene encoding the PDGF-B ligand results in constitutive secretion of the chimeric ligand and receptor activation. Imatinib has which is a known inhibitor of PDGFR has activity against all three of these diseases.
Advantages of a Selective Inhibitor
Development of FGFR kinase inhibitors with a differentiated selectivity profile provides a new opportunity to use these targeted agents in patient sub-groups whose disease is driven by FGFR deregulation. Compounds that exhibit reduced inhibitory action on additional kinases, particularly VEGFR2 and PDGFR-beta, offer the opportunity to have a differentiated side-effect or toxicity profile and as such allow for a more effective treatment of these indications. Inhibitors of VEGFR2 and PDGFR-beta are associated with toxicities such as hypertension or oedema respectively. In the case of VEGFR2 inhibitors this hypertensive effect is often dose limiting, may be contraindicated in certain patient populations and requires clinical management.
Biological Activity and Therapeutic Uses
The compounds of the invention, and subgroups thereof, have fibroblast growth factor receptor (FGFR) inhibiting or modulating activity and/or vascular endothelial growth factor receptor (VEGFR) inhibiting or modulating activity, and/or platelet derived growth factor receptor (PDGFR) inhibiting or modulating activity, and which will be useful in preventing or treating disease states or conditions described herein. In addition the compounds of the invention, and subgroups thereof, will be useful in preventing or treating diseases or condition mediated by the kinases. References to the preventing or prophylaxis or treatment of a disease state or condition such as cancer include within their scope alleviating or reducing the incidence of cancer.
As used herein, the term “modulation”, as applied to the activity of a kinase, is intended to define a change in the level of biological activity of the protein kinase. Thus, modulation encompasses physiological changes which effect an increase or decrease in the relevant protein kinase activity. In the latter case, the modulation may be described as “inhibition”. The modulation may arise directly or indirectly, and may be mediated by any mechanism and at any physiological level, including for example at the level of gene expression (including for example transcription, translation and/or post-translational modification), at the level of expression of genes encoding regulatory elements which act directly or indirectly on the levels of kinase activity. Thus, modulation may imply elevated/suppressed expression or over- or under-expression of a kinase, including gene amplification (i.e. multiple gene copies) and/or increased or decreased expression by a transcriptional effect, as well as hyper- (or hypo-)activity and (de)activation of the protein kinase(s) (including (de)activation) by mutation(s). The terms “modulated”, “modulating” and “modulate” are to be interpreted accordingly.
As used herein, the term “mediated”, as used e.g. in conjunction with a kinase as described herein (and applied for example to various physiological processes, diseases, states, conditions, therapies, treatments or interventions) is intended to operate limitatively so that the various processes, diseases, states, conditions, treatments and interventions to which the term is applied are those in which the kinase plays a biological role. In cases where the term is applied to a disease, state or condition, the biological role played by a kinase may be direct or indirect and may be necessary and/or sufficient for the manifestation of the symptoms of the disease, state or condition (or its aetiology or progression). Thus, kinase activity (and in particular aberrant levels of kinase activity, e.g. kinase over-expression) need not necessarily be the proximal cause of the disease, state or condition: rather, it is contemplated that the kinase mediated diseases, states or conditions include those having multifactorial aetiologies and complex progressions in which the kinase in question is only partially involved. In cases where the term is applied to treatment, prophylaxis or intervention, the role played by the kinase may be direct or indirect and may be necessary and/or sufficient for the operation of the treatment, prophylaxis or outcome of the intervention. Thus, a disease state or condition mediated by a kinase includes the development of resistance to any particular cancer drug or treatment.
Thus, for example, it is envisaged that the compounds of the invention will be useful in alleviating or reducing the incidence of cancer.
More particularly, the compounds of the formulae (I) and sub-groups thereof are inhibitors of FGFRs. For example, compounds of the invention have activity against FGFR1, FGFR2, FGFR3, and/or FGFR4, and in particular FGFRs selected from FGFR1, FGFR2 and FGFR3.
Preferred compounds are compounds that inhibit one or more FGFR selected from FGFR1, FGFR2 and FGFR3, and also FGFR4. Preferred compounds of the invention are those having IC50 values of less than 0.1 μM.
Compounds of the invention also have activity against VEGFR.
Compounds of the invention also have activity against PDGFR kinases. In particular, the compounds are inhibitors of PDGFR and, for example, inhibit PDGFR A and/or PDGFR B.
In addition many of the compounds of the invention exhibit selectivity for the FGFR 1, 2, and/or 3 kinase, and/or FGFR4 compared to VEGFR (in particular VEGFR2) and/or PDGFR and such compounds represent one preferred embodiment of the invention. In particular, the compounds exhibit selectivity over VEGFR2. For example, many compounds of the invention have IC50 values against FGFR1, 2 and/or 3 and/or FGFR4 that are between a tenth and a hundredth of the IC50 against VEGFR (in particular VEGFR2) and/or PDGFR B. In particular preferred compounds of the invention have at least 10 times greater activity against or inhibition of FGFR in particular FGFR1, FGFR2, FGFR3 and/or FGFR4 than VEGFR2. More preferably the compounds of the invention have at least 100 times greater activity against or inhibition of FGFR in particular FGFR1, FGFR2, FGFR3 and/or FGFR4 than VEGFR2. This can be determined using the methods described herein.
As a consequence of their activity in modulating or inhibiting FGFR, VEGFR and/or PDGFR kinases, the compounds will be useful in providing a means of preventing the growth or inducing apoptosis of neoplasias, particularly by inhibiting angiogenesis. It is therefore anticipated that the compounds will prove useful in treating or preventing proliferative disorders such as cancers. In addition, the compounds of the invention could be useful in the treatment of diseases in which there is a disorder of proliferation, apoptosis or differentiation.
In particular tumours with activating mutants of VEGFR or upregulation of VEGFR and patients with elevated levels of serum lactate dehydrogenase may be particularly sensitive to the compounds of the invention. Patients with activating mutants of any of the isoforms of the specific RTKs discussed herein may also find treatment with the compounds of the invention particularly beneficial. For example, VEGFR overexpression in acute leukemia cells where the clonal progenitor may express VEGFR. Also, particular tumours with activating mutants or upregulation or overexpression of any of the isoforms of FGFR such as FGFR1, FGFR2 or FGFR3 or FGFR4 may be particularly sensitive to the compounds of the invention and thus patients as discussed herein with such particular tumours may also find treatment with the compounds of the invention particularly beneficial. It may be preferred that the treatment is related to or directed at a mutated form of one of the receptor tyrosine kinases, such as discussed herein. Diagnosis of tumours with such mutations could be performed using techniques known to a person skilled in the art and as described herein such as RTPCR and FISH.
Examples of cancers which may be treated (or inhibited) include, but are not limited to, a carcinoma, for example a carcinoma of the bladder, breast, colon (e.g. colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermis, liver, lung, for example adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas, oesophagus, gall bladder, ovary, pancreas e.g. exocrine pancreatic carcinoma, stomach, cervix, endometrium, thyroid, prostate, or skin, for example squamous cell carcinoma; a hematopoietic tumour of lymphoid lineage, for example leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, or Burkett's lymphoma; a hematopoietic tumour of myeloid lineage, for example leukemias, acute and chronic myelogenous leukemias, myeloproliferative syndrome, myelodysplastic syndrome, or promyelocytic leukemia; multiple myeloma; thyroid follicular cancer; a tumour of mesenchymal origin, for example fibrosarcoma or rhabdomyosarcoma; a tumour of the central or peripheral nervous system, for example astrocytoma, neuroblastoma, glioma or schwannoma; melanoma; seminoma; teratocarcinoma; osteosarcoma; xeroderma pigmentosum; keratoctanthoma; thyroid follicular cancer; or Kaposi's sarcoma.
Certain cancers are resistant to treatment with particular drugs. This can be due to the type of the tumour or can arise due to treatment with the compound. In this regard, references to multiple myeloma includes bortezomib sensitive multiple myeloma or refractory multiple myeloma. Similarly, references to chronic myelogenous leukemia includes imitanib sensitive chronic myelogenous leukemia and refractory chronic myelogenous leukemia. Chronic myelogenous leukemia is also known as chronic myeloid leukemia, chronic granulocytic leukemia or CML. Likewise, acute myelogenous leukemia, is also called acute myeloblastic leukemia, acute granulocytic leukemia, acute nonlymphocytic leukaemia or AML.
The compounds of the invention can also be used in the treatment of hematopoetic diseases of abnormal cell proliferation whether pre-malignant or stable such as myeloproliferative diseases. Myeloproliferative diseases (“MPD”s) are a group of diseases of the bone marrow in which excess cells are produced. They are related to, and may evolve into, myelodysplastic syndrome. Myeloproliferative diseases include polycythemia vera, essential thrombocythemia and primary myelofibrosis.
Thus, in the pharmaceutical compositions, uses or methods of this invention for treating a disease or condition comprising abnormal cell growth, the disease or condition comprising abnormal cell growth in one embodiment is a cancer.
Further T-cell lymphoproliferative diseases include those derived from natural Killer cells. The term B-cell lymphoma includes diffuse large B-cell lymphoma.
In addition the compounds of the invention can be used to gastrointestinal (also known as gastric) cancer e.g. gastrointestinal stromal tumours. Gastrointestinal cancer refers to malignant conditions of the gastrointestinal tract, including the esophagus, stomach, liver, biliary system, pancreas, bowels, and anus.
A further example of a tumour of mesenchymal origin is Ewing's sarcoma.
Thus, in the pharmaceutical compositions, uses or methods of this invention for treating a disease or condition comprising abnormal cell growth, the disease or condition comprising abnormal cell growth in one embodiment is a cancer.
Particular subsets of cancers include multiple myeloma, bladder, cervical, prostate and thyroid carcinomas, lung, breast, and colon cancers.
A further subset of cancers includes multiple myeloma, bladder, hepatocellular, oral squamous cell carcinoma and cervical carcinomas.
It is further envisaged that the compound of the invention having FGFR such as FGFR1 inhibitory activity, will be particularly useful in the treatment or prevention of breast cancer in particular Classic Lobular Carcinomas (CLC).
As the compounds of the invention have FGFR4 activity they will also be useful in the treatment of prostate or pituitary cancers.
In particular the compounds of the invention as FGFR inhibitors, are useful in the treatment of multiple myeloma, myeloproliferatoive disorders, endometrial cancer, prostate cancer, bladder cancer, lung cancer, ovarian cancer, breast cancer, gastric cancer, colorectal cancer, and oral squamous cell carcinoma.
Further subsets of cancer are multiple myeloma, endometrial cancer, bladder cancer, cervical cancer, prostate cancer, lung cancer, breast cancer, colorectal cancer and thyroid carcinomas.
In particular the compounds of the invention are in the treatment of multiple myeloma (in particular multiple myeloma with t(4; 14) translocation or overexpressing FGFR3), prostate cancer (hormone refractory prostrate carcinomas), endometrial cancer (in particular endometrial tumours with activating mutations in FGFR2) and breast cancer (in particular lobular breast cancer).
In particular the compounds are useful for the treatment of lobular carcinomas such as CLC(Classic lobular carcinoma).
As the compounds have activity against FGFR3 they will be useful in the treatment of multiple myeloma and bladder.
In particular the compounds are useful for the treatment of t(4; 14) translocation positive multiple myeloma.
As the compounds have activity against FGFR2 they will be useful in the treatment of endometrial, ovarian, gastric and colorectal cancers. FGFR2 is also overexpressed in epithelial ovarian cancer, therefore the compounds of the invention may be specifically useful in treating ovarian cancer such as epithelial ovarian cancer.
Compounds of the invention may also be useful in the treatment of tumours pre-treated with VEGFR2 inhibitor or VEGFR2 antibody (e.g. Avastin).
In particular the compounds of the invention may be useful in the treatment of VEGFR2-resistant tumours. VEGFR2 inhibitors and antibodies are used in the treatment of thyroid and renal cell carcinomas, therefore the compounds of the invention may be useful in the treatment of VEGFR2-resistant thyroid and renal cell carcinomas.
The cancers may be cancers which are sensitive to inhibition of any one or more FGFRs selected from FGFR1, FGFR2, FGFR3, FGFR4, for example, one or more FGFRs selected from FGFR1, FGFR2 or FGFR3.
Whether or not a particular cancer is one which is sensitive to inhibition of FGFR, VEGFR or PDGFR signalling may be determined by means of a cell growth assay as set out below or by a method as set out in the section headed “Methods of Diagnosis”.
It is further envisaged that the compounds of the invention, and in particular those compounds having FGFR, VEGFR or PDGFR inhibitory activity, will be particularly useful in the treatment or prevention of cancers of a type associated with or characterised by the presence of elevated levels of FGFR, VEGFR or PDGFR, for example the cancers referred to in this context in the introductory section of this application.
It has been discovered that some FGFR inhibitors can be used in combination with other anticancer agents. For example, it may be beneficial to combine an inhibitor that induces apoptosis with another agent which acts via a different mechanism to regulate cell growth thus treating two of the characteristic features of cancer development. Examples of such combinations are set out below.
It is also envisaged that the compounds of the invention will be useful in treating other conditions which result from disorders in proliferation such as type II or non-insulin dependent diabetes mellitus, autoimmune diseases, head trauma, stroke, epilepsy, neurodegenerative diseases such as Alzheimer's, motor neurone disease, progressive supranuclear palsy, corticobasal degeneration and Pick's disease for example autoimmune diseases and neurodegenerative diseases.
One sub-group of disease states and conditions where it is envisaged that the compounds of the invention will be useful consists of inflammatory diseases, cardiovascular diseases and wound healing.
FGFR, VEGFR and PDGFR are also known to play a role in apoptosis, angiogenesis, proliferation, differentiation and transcription and therefore the compounds of the invention could also be useful in the treatment of the following diseases other than cancer; chronic inflammatory diseases, for example systemic lupus erythematosus, autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, autoimmune diabetes mellitus, Eczema hypersensitivity reactions, asthma, COPD, rhinitis, and upper respiratory tract disease; cardiovascular diseases for example cardiac hypertrophy, restenosis, atherosclerosis; neurodegenerative disorders, for example Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotropic lateral sclerosis, retinitis pigmentosa, spinal muscular atropy and cerebellar degeneration; glomerulonephritis; myelodysplastic syndromes, ischemic injury associated myocardial infarctions, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol related liver diseases, haematological diseases, for example, chronic anemia and aplastic anemia; degenerative diseases of the musculoskeletal system, for example, osteoporosis and arthritis, aspirin-sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis, kidney diseases and cancer pain.
In addition, mutations of FGFR2 are associated with several severe abnormalities in human skeletal development and thus the compounds of invention could be useful in the treatment of abnormalities in human skeletal development, including abnormal ossification of cranial sutures (craniosynostosis), Apert (AP) syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Beare-Stevenson cutis gyrate syndrome, and Pfeiffer syndrome.
It is further envisaged that the compound of the invention having FGFR such as FGFR2 or FGFR3 inhibitory activity, will be particularly useful in the treatment or prevention of the skeletal diseases. Particular skeletal diseases are achondroplasia or thanatophoric dwarfism (also known as thanatophoric dysplasia).
It is further envisaged that the compound of the invention having FGFR such as FGFR1, FGFR2 or FGFR3 inhibitory activity, will be particularly useful in the treatment or prevention in pathologies in which progressive fibrosis is a symptom. Fibrotic conditions in which the compounds of the inventions may be useful in the treatment of in include diseases exhibiting abnormal or excessive deposition of fibrous tissue for example in liver cirrhosis, glomerulonephritis, pulmonary fibrosis, systemic fibrosis, rheumatoid arthritis, as well as the natural process of wound healing. In particular the compounds of the inventions may also be useful in the treatment of lung fibrosis in particular in idiopathic pulmonary fibrosis.
The over-expression and activation of FGFR and VEGFR in tumor-associated vasculature has also suggested a role for compounds of the invention in preventing and disrupting initiation of tumor angiogenesis. In particular the compounds of the invention may be useful in the treatment of cancer, metastasis, leukemia's such as CLL, ocular diseases such as age-related macular degeneration in particular wet form of age-related macular degeneration, ischemic proliferative retinopathies such as retinopathy of prematurity (ROP) and diabetic retinopathy, rheumatoid arthritis and hemangioma.
Since compounds of the invention inhibit PDGFR they may also be useful in the treatment of a number of tumour and leukemia types including glioblastomas such as glioblastoma multiforme, prostate carcinomas, gastrointestinal stromal tumours, liver cancer, kidney cancer, chronic myeloid leukemia, chronic myelomonocytic leukemia (CMML) as well as hypereosinophilic syndrome, a rare proliferative hematological disorder and dermatofibrosarcoma protuberans, an infiltrative skin tumour.
The activity of the compounds of the invention as inhibitors of FGFR1-4, VEGFR and/or PDGFR NB can be measured using the assays set forth in the examples below and the level of activity exhibited by a given compound can be defined in terms of the IC50 value. Preferred compounds of the present invention are compounds having an IC50 value of less than 1 μM, more preferably less than 0.1 μM.
The invention provides compounds that have FGFR inhibiting or modulating activity, and which it is envisaged will be useful in preventing or treating disease states or conditions mediated by FGFR kinases.
In one embodiment, there is provided a compound as defined herein for use in therapy. In a further embodiment, there is provided a compound as defined herein for use in the prophylaxis or treatment of a disease state or condition mediated by a FGFR kinase.
Thus, for example, it is envisaged that the compounds of the invention will be useful in alleviating or reducing the incidence of cancer. Therefore, in a further embodiment, there is provided a compound as defined herein for use in the prophylaxis or treatment of cancer.
Accordingly, in one aspect, the invention provides the use of a compound for the manufacture of a medicament for the prophylaxis or treatment of a disease state or condition mediated by a FGFR kinase, the compound having the formula (I) as defined herein.
In one embodiment, there is provided the use of a compound as defined herein for the manufacture of a medicament for the prophylaxis or treatment of a disease state or condition as described herein.
In a further embodiment, there is provided the use of a compound as defined herein for the manufacture of a medicament for the prophylaxis or treatment of cancer.
Accordingly, the invention provides inter alia:
A method for the prophylaxis or treatment of a disease state or condition mediated by a FGFR kinase, which method comprises administering to a subject in need thereof a compound of the formula (I) as defined herein.
In one embodiment, there is provided a method for the prophylaxis or treatment of a disease state or condition as described herein, which method comprises administering to a subject in need thereof a compound of the formula (I) as defined herein.
In a further embodiment, there is provided a method for the prophylaxis or treatment of cancer, which method comprises administering to a subject in need thereof a compound of the formula (I) as defined herein.
A method for alleviating or reducing the incidence of a disease state or condition mediated by a FGFR kinase, which method comprises administering to a subject in need thereof a compound of the formula (I) as defined herein.
A method of inhibiting a FGFR kinase, which method comprises contacting the kinase with a kinase-inhibiting compound of the formula (I) as defined herein.
A method of modulating a cellular process (for example cell division) by inhibiting the activity of a FGFR kinase using a compound of the formula (I) as defined herein.
A compound of formula (I) as defined herein for use as a modulator of a cellular process (for example cell division) by inhibiting the activity of a FGFR kinase.
A compound of formula (I) as defined herein for use as a modulator (e.g. inhibitor) of FGFR.
The use of a compound of formula (I) as defined herein for the manufacture of a medicament for modulating (e.g. inhibiting) the activity of FGFR.
Use of a compound of formula (I) as defined herein in the manufacture of a medicament for modulating a cellular process (for example cell division) by inhibiting the activity of a FGFR kinase.
The use of a compound of the formula (I) as defined herein for the manufacture of a medicament for prophylaxis or treatment of a disease or condition characterised by up-regulation of a FGFR kinase (e.g. FGFR1 or FGFR2 or FGFR3 or FGFR4).
The use of a compound of the formula (I) as defined herein for the manufacture of a medicament for the prophylaxis or treatment of a cancer, the cancer being one which is characterised by up-regulation of a FGFR kinase (e.g. FGFR1 or FGFR2 or FGFR3 or FGFR4).
The use of a compound of the formula (I) as defined herein for the manufacture of a medicament for the prophylaxis or treatment of cancer in a patient selected from a sub-population possessing a genetic aberrations of FGFR3 kinase.
The use of a compound of the formula (I) as defined herein for the manufacture of a medicament for the prophylaxis or treatment of cancer in a patient who has been diagnosed as forming part of a sub-population possessing a genetic aberrations of FGFR3 kinase.
A method for the prophylaxis or treatment of a disease or condition characterised by up-regulation of a FGFR kinase (e.g. FGFR1 or FGFR2 or FGFR3 or FGFR4), the method comprising administering a compound of the formula (I) as defined herein.
A method for alleviating or reducing the incidence of a disease or condition characterised by up-regulation of a FGFR kinase (e.g. FGFR1 or FGFR2 or FGFR3 or FGFR4), the method comprising administering a compound of the formula (I) as defined herein.
A method for the prophylaxis or treatment of (or alleviating or reducing the incidence of) cancer in a patient suffering from or suspected of suffering from cancer; which method comprises (i) subjecting a patient to a diagnostic test to determine whether the patient possesses a genetic aberrations of FGFR3 gene; and (ii) where the patient does possess the said variant, thereafter administering to the patient a compound of the formula (I) as defined herein having FGFR3 kinase inhibiting activity.
A method for the prophylaxis or treatment of (or alleviating or reducing the incidence of) a disease state or condition characterised by up-regulation of an FGFR kinase (e.g. e.g. FGFR1 or FGFR2 or FGFR3 or FGFR4); which method comprises (i) subjecting a patient to a diagnostic test to detect a marker characteristic of up-regulation of a FGFR kinase (e.g. FGFR1 or FGFR2 or FGFR3 or FGFR4) and (ii) where the diagnostic test is indicative of up-regulation of FGFR kinase, thereafter administering to the patient a compound of the formula (I) as defined herein having FGFR kinase inhibiting activity.
In one embodiment, the disease mediated by FGFR kinases is a oncology related disease (e.g. cancer). In one embodiment, the disease mediated by FGFR kinases is a non-oncology related disease (e.g. any disease disclosed herein excluding cancer). In one embodiment the disease mediated by FGFR kinases is a condition described herein. In one embodiment the disease mediated by FGFR kinases is a skeletal condition described herein. Particular abnormalities in human skeletal development, include abnormal ossification of cranial sutures (craniosynostosis), Apert (AP) syndrome, Crouzon syndrome, Jackson-Weiss syndrome, Beare-Stevenson cutis gyrate syndrome, Pfeiffer syndrome, achondroplasia and thanatophoric dwarfism (also known as thanatophoric dysplasia).
Mutated Kinases
Drug resistant kinase mutations can arise in patient populations treated with kinase inhibitors. These occur, in part, in the regions of the protein that bind to or interact with the particular inhibitor used in therapy. Such mutations reduce or increase the capacity of the inhibitor to bind to and inhibit the kinase in question. This can occur at any of the amino acid residues which interact with the inhibitor or are important for supporting the binding of said inhibitor to the target. An inhibitor that binds to a target kinase without requiring the interaction with the mutated amino acid residue will likely be unaffected by the mutation and will remain an effective inhibitor of the enzyme (Carter et al (2005), PNAS, 102(31), 11011-110116).
A study in gastric cancer patient samples showed the presence of two mutations in FGFR2, Ser167Pro in exon IIIa and a splice site mutation 940-2A-G in exon IIIc. These mutations are identical to the germline activating mutations that cause craniosynotosis syndromes and were observed in 13% of primary gastric cancer tissues studied. In addition activating mutations in FGFR3 were observed in 5% of the patient samples tested and overexpression of FGFRs has been correlated with a poor prognosis in this patient group (Jang et. al. (2001) Cancer Research 61 3541-3543.
There are mutations that have been observed in PDGFR in imatinib-treated patients, in particular the T674I mutation. The clinical importance of these mutations may grow considerably, as to date it appears to represent the primary mechanism of resistance to src/Abl inhibitors in patients.
In addition there are chromosomal translocations or point mutations that have been observed in FGFR which give rise to gain-of-function, over-expressed, or constitutively active biological states.
The compounds of the invention would therefore find particular application in relation to cancers which express a mutated molecular target such as FGFR or PDGFR including PDGFR-beta and PDGFR-alpha in particular the T674I mutation of PDGFR.
Diagnosis of tumours with such mutations could be performed using techniques known to a person skilled in the art and as described herein such as RTPCR and FISH.
It has been suggested that mutations of a conserved threonine residue at the ATP binding site of FGFR would result in inhibitor resistance. The amino acid valine 561 has been mutated to a methionine in FGFR1 which corresponds to previously reported mutations found in Abl (T315) and EGFR (1766) that have been shown to confer resistance to selective inhibitors. Assay data for FGFR1V561 M showed that this mutation conferred resistance to a tyrosine kinase inhibitor compared to that of the wild type.
Advantages of the Compositions of the Invention
The compounds of the formula (I) have a number of advantages over prior art compounds.
For example, the compounds of formula (I) have advantageous ADMET and physiochemical properties over prior art compounds.
Pharmaceutical Formulations
While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation) comprising at least one active compound of the invention together with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents.
Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one active compound, as defined above, together with one or more pharmaceutically acceptable carriers, excipients, buffers, adjuvants, stabilizers, or other materials, as described herein.
The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
Pharmaceutical compositions containing compounds of the formula (I) can be formulated in accordance with known techniques, see for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA.
Accordingly, in a further aspect, the invention provides compounds of the formula (I) and sub-groups thereof as defined herein in the form of pharmaceutical compositions.
The pharmaceutical compositions can be in any form suitable for oral, parenteral, topical, intranasal, ophthalmic, otic, rectal, intra-vaginal, or transdermal administration. Where the compositions are intended for parenteral administration, they can be formulated for intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery. The delivery can be by bolus injection, short term infusion or longer term infusion and can be via passive delivery or through the utilisation of a suitable infusion pump.
Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, co-solvents, organic solvent mixtures, cyclodextrin complexation agents, emulsifying agents (for forming and stabilizing emulsion formulations), liposome components for forming liposomes, gellable polymers for forming polymeric gels, lyophilisation protectants and combinations of agents for, inter alia, stabilising the active ingredient in a soluble form and rendering the formulation isotonic with the blood of the intended recipient. Pharmaceutical formulations for parenteral administration may also take the form of aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents (Strickly, R. G. (2004) Solubilizing Excipients in oral and injectable formulations, Pharmaceutical Research, Vol 21(2p 201-230).
Liposomes are closed spherical vesicles composed of outer lipid bilayer membranes and an inner aqueous core and with an overall diameter of <100 μm. Depending on the level of hydrophobicity, moderately hydrophobic drugs can be solubilized by liposomes if the drug becomes encapsulated or intercalated within the liposome. Hydrophobic drugs can also be solubilized by liposomes if the drug molecule becomes an integral part of the lipid bilayer membrane, and in this case, the hydrophobic drug is dissolved in the lipid portion of the lipid bilayer.
The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
The pharmaceutical formulation can be prepared by lyophilising a compound of formula (I), or sub-groups thereof. Lyophilisation refers to the procedure of freeze-drying a composition. Freeze-drying and lyophilisation are therefore used herein as synonyms.
Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
Pharmaceutical compositions of the present invention for parenteral injection can also comprise pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
The compositions of the present invention may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminium monostearate and gelatin.
In one preferred embodiment of the invention, the pharmaceutical composition is in a form suitable for i.v. administration, for example by injection or infusion. For intravenous administration, the solution can be dosed as is, or can be injected into an infusion bag (containing a pharmaceutically acceptable excipient, such as 0.9% saline or 5% dextrose), before administration.
In another preferred embodiment, the pharmaceutical composition is in a form suitable for sub-cutaneous (s.c.) administration.
Pharmaceutical dosage forms suitable for oral administration include tablets, capsules, caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions, sublingual tablets, wafers or patches and buccal patches.
Thus, tablet compositions can contain a unit dosage of active compound together with an inert diluent or carrier such as a sugar or sugar alcohol, eg; lactose, sucrose, sorbitol or mannitol; and/or a non-sugar derived diluent such as sodium carbonate, calcium phosphate, calcium carbonate, or a cellulose or derivative thereof such as methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and starches such as corn starch. Tablets may also contain such standard ingredients as binding and granulating agents such as polyvinylpyrrolidone, disintegrants (e.g. swellable crosslinked polymers such as crosslinked carboxymethylcellulose), lubricating agents (e.g. stearates), preservatives (e.g. parabens), antioxidants (e.g. BHT), buffering agents (for example phosphate or citrate buffers), and effervescent agents such as citrate/bicarbonate mixtures. Such excipients are well known and do not need to be discussed in detail here.
Capsule formulations may be of the hard gelatin or soft gelatin variety and can contain the active component in solid, semi-solid, or liquid form. Gelatin capsules can be formed from animal gelatin or synthetic or plant derived equivalents thereof.
The solid dosage forms (eg; tablets, capsules etc.) can be coated or un-coated, but typically have a coating, for example a protective film coating (e.g. a wax or varnish) or a release controlling coating. The coating (e.g. a EudragitTM type polymer) can be designed to release the active component at a desired location within the gastro-intestinal tract. Thus, the coating can be selected so as to degrade under certain pH conditions within the gastrointestinal tract, thereby selectively release the compound in the stomach or in the ileum or duodenum.
Instead of, or in addition to, a coating, the drug can be presented in a solid matrix comprising a release controlling agent, for example a release delaying agent which may be adapted to selectively release the compound under conditions of varying acidity or alkalinity in the gastrointestinal tract. Alternatively, the matrix material or release retarding coating can take the form of an erodible polymer (e.g. a maleic anhydride polymer) which is substantially continuously eroded as the dosage form passes through the gastrointestinal tract. As a further alternative, the active compound can be formulated in a delivery system that provides osmotic control of the release of the compound. Osmotic release and other delayed release or sustained release formulations may be prepared in accordance with methods well known to those skilled in the art.
The pharmaceutical compositions comprise from approximately 1% to approximately 95%, preferably from approximately 20% to approximately 90%, active ingredient. Pharmaceutical compositions according to the invention may be, for example, in unit dose form, such as in the form of ampoules, vials, suppositories, dragées, tablets or capsules.
Pharmaceutical compositions for oral administration can be obtained by combining the active ingredient with solid carriers, if desired granulating a resulting mixture, and processing the mixture, if desired or necessary, after the addition of appropriate excipients, into tablets, dragee cores or capsules. It is also possible for them to be incorporated into plastics carriers that allow the active ingredients to diffuse or be released in measured amounts.
The compounds of the invention can also be formulated as solid dispersions. Solid dispersions are homogeneous extremely fine disperse phases of two or more solids.
Solid solutions (molecularly disperse systems), one type of solid dispersion, are well known for use in pharmaceutical technology (see (Chiou and Riegelman (1971), J. Pharm. Sci., 60, 1281-1300) and are useful in increasing dissolution rates and increasing the bioavailability of poorly water-soluble drugs.
This invention also provides solid dosage forms comprising the solid solution described above. Solid dosage forms include tablets, capsules and chewable tablets. Known excipients can be blended with the solid solution to provide the desired dosage form. For example, a capsule can contain the solid solution blended with (a) a disintegrant and a lubricant, or (b) a disintegrant, a lubricant and a surfactant. A tablet can contain the solid solution blended with at least one disintegrant, a lubricant, a surfactant, and a glidant. The chewable tablet can contain the solid solution blended with a bulking agent, a lubricant, and if desired an additional sweetening agent (such as an artificial sweetener), and suitable flavours.
The pharmaceutical formulations may be presented to a patient in “patient packs” containing an entire course of treatment in a single package, usually a blister pack. Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in patient prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions.
Compositions for topical use include ointments, creams, sprays, patches, gels, liquid drops and inserts (for example intraocular inserts). Such compositions can be formulated in accordance with known methods.
Examples of formulations for rectal or intra-vaginal administration include pessaries and suppositories which may be, for example, formed from a shaped moldable or waxy material containing the active compound.
Compositions for administration by inhalation may take the form of inhalable powder compositions or liquid or powder sprays, and can be administrated in standard form using powder inhaler devices or aerosol dispensing devices. Such devices are well known. For administration by inhalation, the powdered formulations typically comprise the active compound together with an inert solid powdered diluent such as lactose.
The compounds of the formula (I) will generally be presented in unit dosage form and, as such, will typically contain sufficient compound to provide a desired level of biological activity. For example, a formulation may contain from 1 nanogram to 2 grams of active ingredient, e.g. from 1 nanogram to 2 milligrams of active ingredient. Within this range, particular sub-ranges of compound are 0.1 milligrams to 2 grams of active ingredient (more usually from 10 milligrams to 1 gram, e.g. 50 milligrams to 500 milligrams), or 1 microgram to 20 milligrams (for example 1 microgram to 10 milligrams, e.g. 0.1 milligrams to 2 milligrams of active ingredient).
For oral compositions, a unit dosage form may contain from 1 milligram to 2 grams, more typically 10 milligrams to 1 gram, for example 50 milligrams to 1 gram, e.g. 100 milligrams to 1 gram, of active compound.
The active compound will be administered to a patient in need thereof (for example a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect.
The skilled person will have the expertise to select the appropriate amounts of ingredients for use in the formulations. For example tablets and capsules typically contain 0-20% disintegrants, 0-5% lubricants, 0-5% flow aids and/or 0-100% fillers/or bulking agents (depending on drug dose). They may also contain 0-10% polymer binders, 0-5% antioxidants, 0-5% Pigments. Slow release tablets would in addition contain 0-100% polymers (depending on dose). The film coats of the tablet or capsule typically contain 0-10% polymers, 0-3% pigments, and/or 0-2% plasticizers.
Parenteral formulations typically contain 0-20% buffers, 0-50% cosolvents, and/or 0-100% Water for Injection (WFI) (depending on dose and if freeze dried). Formulations for intramuscular depots may also contain 0-100% oils.
Examples of Pharmaceutical Formulations
(i) Tablet Formulation
A tablet composition containing a compound of the formula (I) is prepared by mixing 50 mg of the compound with 197 mg of lactose (BP) as diluent, and 3 mg magnesium stearate as a lubricant and compressing to form a tablet in known manner.
(ii) Capsule Formulation
A capsule formulation is prepared by mixing 100 mg of a compound of the formula (I) with 100 mg lactose and filling the resulting mixture into standard opaque hard gelatin capsules.
(iii) Injectable Formulation I
A parenteral composition for administration by injection can be prepared by dissolving a compound of the formula (I) (e.g. in a salt form) in water containing 10% propylene glycol to give a concentration of active compound of 1.5% by weight. The solution is then sterilised by filtration, filled into an ampoule and sealed.
(iv) Injectable Formulation II
A parenteral composition for injection is prepared by dissolving in water a compound of the formula (I) (e.g. in salt form) (2 mg/ml) and mannitol (50 mg/ml), sterile filtering the solution and filling into sealable 1 ml vials or ampoules.
(v) Injectable formulation III
A formulation for i.v. delivery by injection or infusion can be prepared by dissolving the compound of formula (I) (e.g. in a salt form) in water at 20 mg/ml. The vial is then sealed and sterilised by autoclaving.
(vi) Injectable formulation IV
A formulation for i.v. delivery by injection or infusion can be prepared by dissolving the compound of formula (I) (e.g. in a salt form) in water containing a buffer (e.g. 0.2 M acetate pH 4.6) at 20 mg/ml. The vial is then sealed and sterilised by autoclaving.
(vii) Subcutaneous Infection Formulation
A composition for sub-cutaneous administration is prepared by mixing a compound of the formula (I) with pharmaceutical grade corn oil to give a concentration of 5 mg/ml. The composition is sterilised and filled into a suitable container.
(viii) Lyophilised formulation
Aliquots of formulated compound of formula (I) are put into 50 ml vials and lyophilized. During lyophilisation, the compositions are frozen using a one-step freezing protocol at (−45° C.). The temperature is raised to −10° C. for annealing, then lowered to freezing at −45° C., followed by primary drying at +25° C. for approximately 3400 minutes, followed by a secondary drying with increased steps if temperature to 50° C. The pressure during primary and secondary drying is set at 80 millitor.
Methods of Treatment
It is envisaged that the compounds of the formula (I) and sub-groups thereof as defined herein will be useful in the prophylaxis or treatment of a range of disease states or conditions mediated by FGFR. Examples of such disease states and conditions are set out above.
The compounds are generally administered to a subject in need of such administration, for example a human or animal patient, preferably a human.
The compounds will typically be administered in amounts that are therapeutically or prophylactically useful and which generally are non-toxic.
However, in certain situations (for example in the case of life threatening diseases), the benefits of administering a compound of the formula (I) may outweigh the disadvantages of any toxic effects or side effects, in which case it may be considered desirable to administer compounds in amounts that are associated with a degree of toxicity.
The compounds may be administered over a prolonged term to maintain beneficial therapeutic effects or may be administered for a short period only. Alternatively they may be administered in a pulsatile or continuous manner.
A typical daily dose of the compound of formula (I) can be in the range from 100 picograms to 100 milligrams per kilogram of body weight, more typically 5 nanograms to 25 milligrams per kilogram of bodyweight, and more usually 10 nanograms to 15 milligrams per kilogram (e.g. 10 nanograms to 10 milligrams, and more typically 1 microgram per kilogram to 20 milligrams per kilogram, for example 1 microgram to 10 milligrams per kilogram) per kilogram of bodyweight although higher or lower doses may be administered where required. The compound of the formula (I) can be administered on a daily basis or on a repeat basis every 2, or 3, or 4, or 5, or 6, or 7, or 10 or 14, or 21, or 28 days for example.
The compounds of the invention may be administered orally in a range of doses, for example 1 to 1500 mg, 2 to 800 mg, or 5 to 500 mg, e.g. 2 to 200 mg or 10 to 1000 mg, particular examples of doses including 10, 20, 50 and 80 mg. The compound may be administered once or more than once each day. The compound can be administered continuously (i.e. taken every day without a break for the duration of the treatment regimen). Alternatively, the compound can be administered intermittently, i.e. taken continuously for a given period such as a week, then discontinued for a period such as a week and then taken continuously for another period such as a week and so on throughout the duration of the treatment regimen. Examples of treatment regimens involving intermittent administration include regimens wherein administration is in cycles of one week on, one week off; or two weeks on, one week off; or three weeks on, one week off; or two weeks on, two weeks off; or four weeks on two weeks off; or one week on three weeks off—for one or more cycles, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more cycles.
In one particular dosing schedule, a patient will be given an infusion of a compound of the formula (I) for periods of one hour daily for up to ten days in particular up to five days for one week, and the treatment repeated at a desired interval such as two to four weeks, in particular every three weeks.
More particularly, a patient may be given an infusion of a compound of the formula (I) for periods of one hour daily for 5 days and the treatment repeated every three weeks.
In another particular dosing schedule, a patient is given an infusion over 30 minutes to 1 hour followed by maintenance infusions of variable duration, for example 1 to 5 hours, e.g. 3 hours.
In a further particular dosing schedule, a patient is given a continuous infusion for a period of 12 hours to 5 days, an in particular a continuous infusion of 24 hours to 72 hours.
Ultimately, however, the quantity of compound administered and the type of composition used will be commensurate with the nature of the disease or physiological condition being treated and will be at the discretion of the physician.
The compounds as defined herein can be administered as the sole therapeutic agent or they can be administered in combination therapy with one of more other compounds for treatment of a particular disease state, for example a neoplastic disease such as a cancer as hereinbefore defined. Examples of other therapeutic agents or treatments that may be administered together (whether concurrently or at different time intervals) with the compounds of the formula (I) include but are not limited to:
Topoisomerase I inhibitors
Antimetabolites
Tubulin targeting agents
DNA binder and topoisomerase II inhibitors
Alkylating Agents
Monoclonal Antibodies.
Anti-Hormones
Signal Transduction Inhibitors
Proteasome Inhibitors
DNA methyl transferases
Cytokines and retinoids
Chromatin targeted therapies
Radiotherapy, and,
Other therapeutic or prophylactic agents; for example agents that reduce or alleviate some of the side effects associated with chemotherapy. Particular examples of such agents include anti-emetic agents and agents that prevent or decrease the duration of chemotherapy-associated neutropenia and prevent complications that arise from reduced levels of red blood cells or white blood cells, for example erythropoietin (EPO), granulocyte macrophage-colony stimulating factor (GM-CSF), and granulocyte-colony stimulating factor (G-CSF). Also included are agents that inhibit bone resorption such as bisphosphonate agents e.g. zoledronate, pamidronate and ibandronate, agents that suppress inflammatory responses (such as dexamethazone, prednisone, and prednisolone) and agents used to reduce blood levels of growth hormone and IGF-I in acromegaly patients such as synthetic forms of the brain hormone somatostatin, which includes octreotide acetate which is a long-acting octapeptide with pharmacologic properties mimicking those of the natural hormone somatostatin. Further included are agents such as leucovorin, which is used as an antidote to drugs that decrease levels of folic acid, or folinic acid it self and agents such as megestrol acetate which can be used for the treatment of side-effects including oedema and thromoembolic episodes.
Each of the compounds present in the combinations of the invention may be given in
individually varying dose schedules and via different routes. Where the compound of the formula (I) is administered in combination therapy with one, two, three, four or more other therapeutic agents (preferably one or two, more preferably one), the compounds can be administered simultaneously or sequentially. When administered sequentially, they can be administered at closely spaced intervals (for example over a period of 5-10 minutes) or at longer intervals (for example 1, 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).
The compounds of the invention may also be administered in conjunction with non-chemotherapeutic treatments such as radiotherapy, photodynamic therapy, gene therapy; surgery and controlled diets.
For use in combination therapy with another chemotherapeutic agent, the compound of the formula (I) and one, two, three, four or more other therapeutic agents can be, for example, formulated together in a dosage form containing two, three, four or more therapeutic agents. In an alternative, the individual therapeutic agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use.
A person skilled in the art would know through his or her common general knowledge the dosing regimes and combination therapies to use.
Methods of Diagnosis
Prior to administration of a compound of the formula (I), a patient may be screened to determine whether a disease or condition from which the patient is or may be suffering is one which would be susceptible to treatment with a compound having activity against FGFR, VEGFR and/or PDGFR.
For example, a biological sample taken from a patient may be analysed to determine whether a condition or disease, such as cancer, that the patient is or may be suffering from is one which is characterised by a genetic abnormality or abnormal protein expression which leads to up-regulation of the levels or activity of FGFR, VEGFR and/or PDGFR or to sensitisation of a pathway to normal FGFR, VEGFR and for PDGFR activity, or to upregulation of these growth factor signalling pathways such as growth factor ligand levels or growth factor ligand activity or to upregulation of a biochemical pathway downstream of FGFR, VEGFR and/or PDGFR activation.
Examples of such abnormalities that result in activation or sensitisation of the FGFR, VEGFR and/or PDGFR signal include loss of, or inhibition of apoptotic pathways, up-regulation of the receptors or ligands, or presence of mutant variants of the receptors or ligands e.g PTK variants. Tumours with mutants of FGFR1, FGFR2 or FGFR3 or FGFR4 or up-regulation, in particular over-expression of FGFR1, or gain-of-function mutants of FGFR2 or FGFR3 may be particularly sensitive to FGFR inhibitors.
For example, point mutations engendering gain-of-function in FGFR2 have been identified in a number of conditions (Lemonnier, et al., (2001), J. Bone Miner. Res., 16, 832-845). In particular activating mutations in FGFR2 have been identified in 10% of endometrial tumours (Pollock et al, Oncogene, 2007, 26, 7158-7162).
In addition, genetic aberrations of the FGFR3 receptor tyrosine kinase such as chromosomal translocations or point mutations resulting in ectopically expressed or deregulated, constitutively active, FGFR3 receptors have been identified and are linked to a subset of multiple myelomas, bladder and cervical carcinomas (Powers, C. J., et al. (2000), Endocr. Rel. Cancer, 7, 165). A particular mutation T674I of the PDGF receptor has been identified in imatinib-treated patients.
In addition, a gene amplification of 8p12-p11.2 was demonstrated in  ̃50% of lobular breast cancer (CLC) cases and this was shown to be linked with an increased expression of FGFR1. Preliminary studies with siRNA directed against FGFR1, or a small molecule inhibitor of the receptor, showed cell lines harbouring this amplification to be particularly sensitive to inhibition of this signalling pathway (Reis-Filho et al. (2006) Clin Cancer Res. 12(22): 6652-6662).
Alternatively, a biological sample taken from a patient may be analysed for loss of a negative regulator or suppressor of FGFR, VEGFR or PDGFR. In the present context, the term “loss” embraces the deletion of a gene encoding the regulator or suppressor, the truncation of the gene (for example by mutation), the truncation of the transcribed product of the gene, or the inactivation of the transcribed product (e.g. by point mutation) or sequestration by another gene product.
The term up-regulation includes elevated expression or over-expression, including gene amplification (i.e. multiple gene copies) and increased expression by a transcriptional effect, and hyperactivity and activation, including activation by mutations. Thus, the patient may be subjected to a diagnostic test to detect a marker characteristic of up-regulation of FGFR, VEGFR and for PDGFR. The term diagnosis includes screening. By marker we include genetic markers including, for example, the measurement of DNA composition to identify mutations of FGFR, VEGFR and/or PDGFR. The term marker also includes markers which are characteristic of up regulation of FGFR, VEGFR and for PDGFR, including enzyme activity, enzyme levels, enzyme state (e.g. phosphorylated or not) and mRNA levels of the aforementioned proteins.
The diagnostic tests and screens are typically conducted on a biological sample selected from tumour biopsy samples, blood samples (isolation and enrichment of shed tumour cells), stool biopsies, sputum, chromosome analysis, pleural fluid, peritoneal fluid, buccal spears, biopsy or urine.
Methods of identification and analysis of mutations and up-regulation of proteins are known to a person skilled in the art. Screening methods could include, but are not limited to, standard methods such as reverse-transcriptase polymerase chain reaction (RT-PCR) or in-situ hybridization such as fluorescence in situ hybridization (FISH).
Identification of an individual carrying a mutation in FGFR, VEGFR and for PDGFR may mean that the patient would be particularly suitable for treatment with a FGFR, VEGFR and for PDGFR inhibitor. Tumours may preferentially be screened for presence of a FGFR, VEGFR and for PDGFR variant prior to treatment. The screening process will typically involve direct sequencing, oligonucleotide microarray analysis, or a mutant specific antibody. In addition, diagnosis of tumours with such mutations could be performed using techniques known to a person skilled in the art and as described herein such as RT-PCR and FISH.
In addition, mutant forms of, for example FGFR or VEGFR2, can be identified by direct sequencing of, for example, tumour biopsies using PCR and methods to sequence PCR products directly as hereinbefore described. The skilled artisan will recognize that all such well-known techniques for detection of the over expression, activation or mutations of the aforementioned proteins could be applicable in the present case.
In screening by RT-PCR, the level of mRNA in the tumour is assessed by creating a cDNA copy of the mRNA followed by amplification of the cDNA by PCR. Methods of PCR amplification, the selection of primers, and conditions for amplification, are known to a person skilled in the art. Nucleic acid manipulations and PCR are carried out by standard methods, as described for example in Ausubel, F. M. et al., eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc., or Innis, M. A. et al., eds. PCR Protocols: a guide to methods and applications, 1990, Academic Press, San Diego. Reactions and manipulations involving nucleic acid techniques are also described in Sambrook et al., 2001, 3rd Ed, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press. Alternatively a commercially available kit for RT-PCR (for example Roche Molecular Biochemicals) may be used, or methodology as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659, 5,272,057, 5,882,864, and 6,218,529 and incorporated herein by reference.
An example of an in-situ hybridisation technique for assessing mRNA expression would be fluorescence in-situ hybridisation (FISH) (see Angerer (1987), Meth. Enzymol., 152, 649).
Generally, in situ hybridization comprises the following major steps: (1) fixation of tissue to be analyzed; (2) prehybridization treatment of the sample to increase accessibility of target nucleic acid, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization, and (5) detection of the hybridized nucleic acid fragments. The probes used in such applications are typically labelled, for example, with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to enable specific hybridization with the target nucleic acid(s) under stringent conditions. Standard methods for carrying out FISH are described in Ausubel, F. M. et al., eds. Current Protocols in Molecular Biology, 2004, John Wiley & Sons Inc and Fluorescence In Situ Hybridization: Technical Overview by John M. S. Bartlett in Molecular Diagnosis of Cancer, Methods and Protocols, 2nd ed.; ISBN: 1-59259-760-2; March 2004, pps. 077-088; Series: Methods in Molecular Medicine.
Methods for gene expression profiling are described by (DePrimo et al. (2003), BMC Cancer, 3:3). Briefly, the protocol is as follows: double-stranded cDNA is synthesized from total RNA Using a (dT) 24 oligomer for priming first-strand cDNA synthesis, followed by second strand cDNA synthesis with random hexamer primers. The double-stranded cDNA is used as a template for in vitro transcription of cRNA using biotinylated ribonucleotides. cRNA is chemically fragmented according to protocols described by Affymetrix (Santa Clara, Calif., USA), and then hybridized overnight on Human Genome Arrays.
Alternatively, the protein products expressed from the mRNAs may be assayed by immunohistochemistry of tumour samples, solid phase immunoassay with microtitre plates, Western blotting, 2-dimensional SDS-polyacrylamide gel electrophoresis, ELISA, flow cytometry and other methods known in the art for detection of specific proteins. Detection methods would include the use of site specific antibodies. The skilled person will recognize that all such well-known techniques for detection of upregulation of FGFR, VEGFR and/or PDGFR, or detection of FGFR, VEGFR and/or PDGFR variants or mutants could be applicable in the present case.
Abnormal levels of proteins such as FGFR or VEGFR can be measured using standard enzyme assays, for example, those assays described herein. Activation or overexpression could also be detected in a tissue sample, for example, a tumour tissue. By measuring the tyrosine kinase activity with an assay such as that from Chemicon International. The tyrosine kinase of interest would be immunoprecipitated from the sample lysate and its activity measured.
Alternative methods for the measurement of the over expression or activation of FGFR or VEGFR including the isoforms thereof, include the measurement of microvessel density. This can for example be measured using methods described by Orre and Rogers (Int J Cancer (1999), 84(2), 101-8). Assay methods also include the use of markers, for example, in the case of VEGFR these include CD31, CD34 and CD105 (Mineo et al. (2004) J Clin Pathol. 57(6), 591-7).
Therefore all of these techniques could also be used to identify tumours particularly suitable for treatment with the compounds of the invention.
The compounds of the invention are particular useful in treatment of a patient having a mutated FGFR. The G697C mutation in FGFR3 is observed in 62% of oral squamous cell carcmonas and causes constitutive activation of the kinase activity. Activating mutations of FGFR3 have also been identified in bladder carcinoma cases. These mutations were of 6 kinds with varying degrees of prevelence: R248C, S249C, G372C, S373C, Y375C, K652Q. In addition, a Gly388Arg polymorphism in FGFR4 has been found to be associated with increased incidence and aggressiveness of prostate, colon, lung and breast cancer.
Therefore in a further aspect of the invention includes use of a compound according to the invention for the manufacture of a medicament for the treatment or prophylaxis of a disease state or condition in a patient who has been screened and has been determined as suffering from, or being at risk of suffering from, a disease or condition which would be susceptible to treatment with a compound having activity against FGFR.
Particular mutations a patient is screened for include G697C, R248C, S249C, G372C, S373C, Y375C, K652Q mutations in FGFR3 and Gly388Arg polymorphism in FGFR4.
In another aspect of the inventions includes a compound of the invention for use in the prophylaxis or treatment of cancer in a patient selected from a sub-population possessing a variant of the FGFR gene (for example G697C mutation in FGFR3 and Gly388Arg polymorphism in FGFR4).
MRI determination of vessel normalization (e.g. using MRI gradient echo, spin echo, and contrast enhancement to measure blood volume, relative vessel size, and vascular permeability) in combination with circulating biomarkers (circulating progenitor cells (CPCs), CECs, SDF1, and FGF2) may also be used to identify VEGFR2-resistant tumours for treatment with a compound of the invention.
General Synthetic Routes
Analytical LC-MS System and Method Description
In the examples, the compounds prepared were characterised by liquid chromatography and mass spectroscopy using commercially available systems (Waters Platform LC-MS system, Waters Fractionlynx LC-MS system), standard operating conditions and commercially available columns (Phenomenex, Waters etc) but a person skilled in the art will appreciate that alternative systems and methods could be used. Where atoms with different isotopes are present and a single mass quoted, the mass quoted for the compound is the monoisotopic mass (i.e. 35Cl; 79Br etc.).
Mass Directed Purification LC-MS System
Preparative LC-MS (or HPLC) is a standard and effective method used for the purification of small organic molecules such as the compounds described herein. The methods for the liquid chromatography (LC) and mass spectrometry (MS) can be varied to provide better separation of the crude materials and improved detection of the samples by MS. Optimisation of the preparative gradient LC method will involve varying columns, volatile eluents and modifiers, and gradients. Methods are well known in the art for optimising preparative LC-MS methods and then using them to purify compounds. Such methods are described in Rosentreter U, Huber U.; Optimal fraction collecting in preparative LC/MS; J Comb Chem.; 2004; 6(2), 159-64 and Leister W, Strauss K, Wisnoski D, Zhao Z, Lindsley C., Development of a custom high-throughput preparative liquid chromatography/mass spectrometer platform for the preparative purification and analytical analysis of compound libraries; J Comb Chem.; 2003; 5(3); 322-9.
Two such systems for purifying compounds via preparative LC-MS are the Waters Fractionlynx system or the Agilent 1100 LC-MS preparative system although a person skilled in the art will appreciate that alternative systems and methods could be used. In particular, reverse phase methods were used for preparative HPLC for the compounds described herein, but normal phase preparative LC based methods might be used in place of the reverse phase methods. Most preparative LC-MS systems utilise reverse phase LC and volatile acidic modifiers, since the approach is very effective for the purification of small molecules and because the eluents are compatible with positive ion electrospray mass spectrometry. According to the analytical trace obtained the most appropriate preparative chromatography type is chosen. A typical routine is to run an analytical LC-MS using the type of chromatography (low or high pH) most suited for compound structure. Once the analytical trace showed good chromatography a suitable preparative method of the same type is chosen. A range of chromatographic solutions e.g. normal or reverse phase LC; acidic, basic, polar, or lipophilic buffered mobile phase; basic modifiers could be used to purify the compounds. From the information provided someone skilled in the art could purify the compounds described herein by preparative LC-MS.
All compounds were usually dissolved in 100% MeOH or 100% DMSO.
General Route A




Procedure A1—Imidazopyridine Ring Formation




To a solution of Methyl 2-aminopyridine-4-carboxylate (10.0 g, 66 mmol, 1.0 equiv) in EtOH (150 ml) was added NaHCO3 (11.1 g, 132 mmol, 2.0 equiv) followed by chloroacetaldehyde (50% by weight in water, 13.0 ml, 99 mmol, 1.5 equiv). The mixture was refluxed for 2 h. Solvents were removed under reduced pressure and the crude mixture was partitioned between water and EtOAc. The resulting precipitate was washed with Et2O and recrystallised from MeOH/Et2O to afford 8.4 g of product. 1H NMR (400 MHz, DMSO-d6): 8.66 (1H, d), 8.16 (2H, s), 7.80 (1H, s), 7.33 (1H, d), 3.90 (3H, s). MS: [M+H]+ 177.
Procedure A2—Ester Hydrolysis




To a solution of methyl imidazo[1,2-a]pyridine-7-carboxylate (3.0 g, 17.04 mmol, 1.0 equiv) in EtOH (150 ml) was added 2M aqueous KOH (85 ml, 170 mmol, 10 equiv). The solution was heated for 30 min at 60° C. After cooling to room temperature, the reaction was neutralized (HCl) and solvents were removed under reduced pressure. The residue was stirred in EtOH (2×100 ml) and filtered. The solvent was removed under reduced pressure and the resulting product was used in the next step without further purification. MS: [M+H]+ 163.
Procedure A3—General Amide Bond Formation




To a solution of imidazo[1,2-a]pyridine-7-carboxylic acid (1.0 equiv) in DMF/H2O (50:1) was added TBTU (1.5 equiv) and HOBT (1.5 equiv). The reaction was stirred at room temperature for 30 min before the amine (2.0 equiv) was added. The resulting solution was stirred at room temperature for 16 h. The reaction mixture was poured onto an SCX cartridge and washed with MeOH (2 column volumes) before the product was eluted with methanolic ammonia (2 column volumes). The solvent was removed under reduced pressure and, where necessary the product was purified by chromatography on silica (0→450% MeOH/Et2O).














MS:


Amine
Product
[M + H]+

















Methyl amine
Imidazo[1,2-a]pyridine-7-carboxylic
176



acid methylamide


Dimethyl amine
Imidazo[1,2-a]pyridine-7-carboxylic
190



acid dimethylamide


Azetidine
Azetidin-1-yl-imidazo[1,2-a]pyridine-7-
202



yl-methanone









Procedure A4—Iodination




To a solution of Imidazo[1,2-a]pyridine-7-carboxylic acid amide (1.0 equiv) in DMF (280 ml) was added N-iodosuccinimide (1.2 equiv) and the resulting mixture was stirred for 2 h at room temperature. The thin brown slurry was diluted with water, 10% w/v sodium thiosulfate and sodium carbonate (1 M) and extracted with EtOAc. The aqueous was further extracted with EtOAc. The combined organic phases were washed with brine (280 ml), dried (MgSO4) and concentrated in vacuo to give a brown residue. The residue was triturated with ether, filtered and the solid was washed with ether (2×50 ml) and dried on the filter to give the product. Where necessary the product was purified by chromatography on silica (0→50% MeOH/Et2O).












NRR′
Product
MS: [M + H]+







NHMe
3-Iodoimidazo[1,2-a]pyridine-7-carboxylic
302



acid methylamide



NMe2
3-Iodoimidazo[1,2-a]pyridine-7-carboxylic
316



acid dimethylamide



 






Azetidin-1-y1-(3-iodoimidazo[1,2- a]pyridine-7-y1)-methanone
328









Procedure A5b—Suzuki Coupling with 3-([1,3,4]Thiadiazol-2-ylamino)-phenyl boronic acid pinacol ester




To a solution of 7-amido-3-iodo-imidazo[1,2-a]pyridine (1 equiv) in DME was added 3-([1,3,4]Thiadiazol-2-ylamino)-phenyl boronic acid pinacol ester (1.2 equiv), 1 M Na2CO3 (8 equiv) [reaction degassed by bubbling N2 through] followed by tetrakis(triphenylphosphine)palladium(0) (0.05 equiv). The mixture was heated at 80° C. overnight, then diluted with water and extracted with EtOAc. The organic layer was washed with brine, dried (MgSO4) and concentrated under reduced pressure. The products were purified by trituration with Et2O or by column chromatography on silica (0→50% MeOH/Et2O).














NRR′
Product









NHMe
3-[3-([1,3,4]-Thiadiazol-2-ylamino)-phenyl]-




imidazo[1,2-a]pyridine-7-carboxylic acid




methylamide



NMe2
3-[3-([1,3,4]-Thiadiazol-2-ylamino)-phenyl]-




imidazo[1,2-a]pyridine-7-carboxylic acid




dimethylamide



 







Azetidin-1-yl-{3-[3-([1,3,4]thiadiazol-2-ylamino)- phenyl]-imidazo[1,2-a]pyridine-7-yl}-methanone










General Route B





Procedure B1—Imidazo[1,2-a]pyridine-7-carboxylic acid amide




Prepared using general route A procedure A1, substituting 2-aminoisonicotinamide for methyl 2-aminopyridine-4-carboxylate. 1H NMR (400 MHz, DMSO-d6): 8.59 (1H, d), 8.16 (1H, s), 8.13 (1H, s), 8.05 (1H, s), 7.71 (1H, s), 7.52 (1H, s), 7.32 (1H, dd). MS: [M+H]+162.
Procedure B2—3-Iodoimidazo[1,2-a]pyridine-7-carboxylic acid amide




Prepared using general route A procedure A4, substituting Imidazo[1,2-a]pyridine-7-carboxylic acid amide for methyl imidazo[1,2-a]pyridine-7-carboxylate. 1H NMR (400 MHz, DMSO-d6): 8.39 (1H, d), 8.25-8.09 (3H, m), 7.86 (1H, s), 7.56-7.45 (1H, dd). MS: [M+H]+288.
Procedure B3—Suzuki Coupling




3-Iodoimidazo[1,2-a]pyridine-7-carboxylic acid amide was coupled as described in general route A, procedure A5.














Boronate
Product













3-[3-([1,3,4]-Thiadiazol-2-ylamino)- phenyl]-imidazo[1,2-a]pyridine-7- carboxylic acid amide










Examples 1 to 4
By following the methods described above, the compounds of Examples 1 to 4 set out in the Table below were prepared.















Example







Number
Structure
Name
Method
NMR Data
MS Data







1




3-[3-([1,3,4]Thiadiazol-2-ylamino)-phenyl]- imidazo[1,2- a]pyridine-7- carboxylic acid dimethylamide Hydrochloride
General Route A
1H NMR (400 MHz, Me-d3- OD): 8.80 (1H, s), 8.63 (1H, d), 8.05 (1H, s), 7.78 (1H, s), 7.70 (1H, s), 7.55 (1H, d), 7.48 (1H, t), 7.27 (1H, d), 7.04 (1H, d), 3.13 (6H, s).
[M + H] + 365


 


2




Azetidin-1-yl-{3-[3- ([1,3,4]thiadiazol-2- ylamino)-phenyl]- imidazo[1,2- a]pyridine-7-yl}- methanone
General Route A
1H NMR (400 MHz, DMSO-d6): 10.63 (1H, s), 8.98-8.91 (1H, m), 8.64 (1H, d), 8.04-7.97 (1H, m), 7.97-7.92 (1H, m). 7.90 (1H, s), 7.71 (1H, d), 7.61-7.50 (1H, m), 7.32 (1H, d), 7.24-7.13 (1H, m), 4.47 (2H, s), 4.10 (2H, d), 3.43 (2H, s), 2.37-2.25 (2H, m).
[M + H] + 377


 


3




3-[3-([1,3,4]- Thiadiazol-2- ylamino)-phenyl]- imidazo[1,2- a]pyridine-7- carboxylic acid amide
General Route B
1H NMR (400 MHz, DMSO-d6): 8.68 (1H, s), 8.64 (1H, d), 8.26 (1H, s), 8.20 (1H, s), 7.90 (2H, s), 7.56 (2H, d), 7.51-7.38 (3H, m), 7.17 (1H, d).
[M + H] + 337


 


4




3-[3-([1,3,4]- Thiadiazol-2- ylamino)-phenyl]- imidazo[1,2- a]pyridine-7- carboxylic acid methylamide
General Route B
1H NMR (400 MHz, DMSO-d6): 8.93-8.85 (1H, m), 8.72-8.62 (1H, m), 8.20 (1H, s), 8.03-7.95 (1H, m), 7.92 (1H, s), 7.67 (1H, d), 7.57-7.51 (1H, m), 7.41 (1H, dd), 7.29 (1H, d), 3.30 (1H, s), 2.88-2.79 (3H, m).
[M + H] + 351









Biological Assays

FGFR3 and PDGFR In Vitro Kinase Inhibitory Activity Assays

Enzymes (from Upstate) were prepared at 2× final concentration in 1× kinase assay buffer (as described below). Enzymes were then incubated with test compounds, biotinylated Flt3 substrate (biotin-DNEYFYV) (Cell Signalling Technology Inc.) and ATP. The reaction was allowed to proceed for 3 hours (FGFR3) or 2.5 hrs (PDGFR-beta) at room temperature on a plate shaker at 900 rpm before being stopped with 20 μl of 35 mM EDTA, pH 8 (FGFR3) or 55 mM EDTA, pH 8 (PDGFR-beta). Twenty μl of 5× detection mix (50 mM HEPES pH 7.5, 0.1% BSA, 2 nM Eu-anti-pY (PY20) (PerkinElmer) 15 nM SA-XL665 (Cisbio) for FGFR3 and 50 mM HEPES, pH 7.5, 0.5 M KF, 0.1% BSA, 11.34 nM Eu-anti-pY (PT66) (PerkinElmer), 94 nM SA-XL665 (Cisbio) for PDGFR-beta) was then added to each well and the plate sealed and incubated at room temperature for one hour on a plate shaker at 900 rpm. The plate was then read on a Packard Fusion plate reader in TRF mode.

















1 × Assay
Flt3 substrate
ATP



Enzyme
Buffer
concentration
concentration









FGFR3
A
0.125 μM
 8 μM



PDGFR-beta
B
 0.15 μM
30 μM










Kinase Assay buffers were:
A: 50 mM HEPES pH 7.5, 6 mM MnCl2, 1 mM DTT, 0.1% TritonX-100
B: 20 mM MOPS pH 7.0, 10 mM MnCl2, 0.01% Triton X-100, 0.1 mM DTT, 0.1 mM Sodium orthovanadate
Compounds of the invention have IC50 values less than 10 μM or provide at least 50% inhibition of the FGFR3 activity at a concentration of 10 μM. Preferred compounds of the invention (for example Examples 1-4) have IC50 values of less than 1 μM in the FGFR3 assay.
VEGFR2 In vitro Kinase Inhibitory Activity Assay
Assay reactions containing VEGFR2 enzyme (purchased from Upstate), and 250 μM Poly (Glu, Tyr) 4:1 substrate (CisBio) in 50 mM HEPES, pH 7.5, 6 mM MnCl2, 1 mM DTT, 0.01% TritonX-100, 5 μM ATP (2.8 Ci/mmol) were set up in the presence of compound. Reactions were stopped after 15 minutes by adding an excess of phosphoric acid. The reaction mixture was then transferred to a Millipore MAPH filter plate where the peptide binds and the unused ATP is washed away. After washing, scintillant was added and the incorporated activity measured by scintillation counting on a Packard Topcount.
FGFR1, FGFR2, FGFR4, VEGFR1 and VEGFR3 In vitro Kinase Inhibitory Activity Assays
The inhibitory activity against FGFR1, FGFR2, FGFR4, VEGFR1 and VEGFR3 can be determined at Upstate Discovery Ltd. Enzymes were prepared at 10× final concentration in enzyme buffer (20 mM MOPS, pH 7.0, 1 mM EDTA, 0.1% B-mercaptoethanol, 0.01% Brij-35, 5% glycerol, 1 mg/ml BSA). Enzymes were then incubated in assay buffer with various substrates and 33P-ATP ( ̃500 cpm/μmol) as described in the table.
The reaction was initiated by the addition of Mg/ATP. The reaction was allowed to proceed for 40 minutes at room temperature before being stopped with 5 μl of a 3% phosphoric acid solution. Ten μl of the reaction mix was transferred to either a filtermatA or P30 filtermat and washed three times in 75 mM phosphoric acid and once in methanol before being dried for scintillation counting.
Compounds were tested at the concentrations detailed below in duplicate against all kinases and the percent activity compared to control was calculated. Where inhibition was high an IC50 was determined.
















ATP



Assay

Concentration


Enzyme
Buffer
Substrate
(μM)







FGFR1
A
250 μM KKKSPGEYVNIEFG
200 μM


FGFR2
B
 0.1 mg/ml poly(Glu, Tyr) 4:1
 90 μM


FGFR4
C
 0.1 mg/ml poly(Glu, Tyr) 4:1
155 μM


VEGFR1
A
250 μM KKKSPGEYVNIEFG
200 μM


VEGFR3
A
500 μM GGEEEEYFELVKKKK
200 μM









Enzyme buffer A: 8 mM MOPS, pH 7.0, 0.2 mM EDTA, 10 mM MgAcetate

Enzyme buffer B: 8 mM MOPS, pH 7.0, 0.2 mM EDTA, 2.5 mM MnCl2, 10 mM MgAcetate

Enzyme buffer C: 8 mM Mops, pH 7.0, 0.2 mM EDTA, 10 mM MnCl2, 10 mM MgAcetate.

Cell-Based pERK ELISA Method

LP-1 or JIM-1 multiple myeloma cells were seeded in 96 well plates at 1×106 cells/ml in 200 μl per well in serum free media. HUVEC cells were seeded at 2.5×105 cells/ml and allowed to recover for 24 h prior to transfer to serum free media. Cells were incubated for 16 h at 37° C. prior to the addition of a test compound for 30 minutes. Test compounds were administered at a 0.1% final DMSO concentration. Following this 30 minute incubation a FGF-1/Heparin (FGF-1 at 100 ng/ml final and Heparin at 100 ug/ml) mixture or VEGF165 (100 ug/ml) was added to each of the wells for a further 5 minutes. The media was removed and 50 ul ERK ELISA lysis buffer (R and D Systems DuoSet ELISA for pERK and Total ERK #DYC-1940E, DYC-1018E) added. ELISA plates and standards were prepared according to the standard DuoSet protocols and the relative amounts of pERK to total ERK in each sample calculated according to the standard curve.
In particular, compounds of the invention were tested against the LP-1 cell line (DSMZ no.: ACC 41) derived from human multiple myeloma. Many compounds of the invention were found to have IC50 values of less than 20 μM in this assay and some compounds (for example Example 2) has IC50 values of less than 1 μM.
HUVEC Cell Based Selectivity Assays
HUVEC cells were seeded in 6 well plates at 1×106 cells/well and allowed to recover for 24 h. They were transferred to serum free media for 16 hours prior to treatment with test compound for 30 minutes in 0.1% DMSO final. Following compound incubation FGF-1 (100 ng/ml) and Heparin (100 ug/ml) or VEGF165 (100 ng/ml) were added for 5 minutes. Media was removed, cells washed with ice-cold PBS and lysed in 100 ul TG lysis buffer (20 mM Tris, 130 nM NaCl, 1% Triton-X-100, 10% Glycerol, protease and phosphatase inhibitors, pH 7.5). Samples containing equivalent amounts of protein were made up with LDS sample buffer and run on SDS PAGE followed by western blotting for a number of downstream VEGFR and FGFR pathway targets including phospho-FGFR3, phospho-VEGFR2 and phospho-ERK1/2.
In Vivo Models of Hypertension
A number of animal models exist to measure the potential hypertensive effects of small molecule inhibitors. They can be classified into two main types; indirect and direct measurements. The most common indirect method is the cuff technique. Such methods have the advantages of being non-invasive and as such can be applied to a larger group of experimental animals however the process allows only intermittent sampling of blood pressure and requires the animal to be restrained in some way. Application of restraint can stress the animal and means that changes in blood pressure attributable to a specific drug effect can be hard to pick up.
Direct methodologies include those that make use of radio telemetry technology or via indwelling catheters connected to externally mounted transducers. Such methods require a high level of technical expertise for the initial surgery involved in implantation and costs involved are high. However a key advantage is that they allow continuous monitoring of blood pressure without restraint over the time period of the experiment. These methods are reviewed in Kurz et al (2005) Hypertension. 45: 299-310.
hERG Activity
The activity of compound of formula (I) against the hERG K+ion channel can be determined using the assay described in the article by M. H. Bridgland-Taylor et al., Journal of Pharmacological and Toxicological Methods, 54 (2006), 189-199. This IonWorksTM HT hERG screening assay is performed commercially by Upstate (Millipore) using the PreciSiONTM hERG-CHO cell line.
Determination of Potency Against Cytochrome P450
The potency of the compound of formula (I) against cytochrome P450 (CYP450) enzymes 1A2, 2C9, 2C19, 3A4 and 2D6 can be determined using the Pan Vera Vivid CYP450 screening kits available from Invitrogen (Paisley, UK). The CYP450s are supplied in the form of baculosomes containing the CYP450 and NADPH reductase and the substrates used are the fluorescent Vivid substrates. The final reaction mixtures are as follows:
1A2
100 mM potassium phosphate, pH 8, 1% acetonitrile, 2 μM 1A2 Blue vivid substrate, 100 μM NADP+, 4 nM CYP450 1A2, 2.66 mM glucose-6-phosphate, 0.32 U/ml glucose-6-phosphate dehydrogenase.
2C9
50 mM potassium phosphate, pH 8, 1% acetonitrile, 2 μM Green vivid substrate, 100 μM NADP+, 8 nM CYP450 2C9, 2.66 mM glucose-6-phosphate, 0.32 U/ml glucose-6-phosphate dehydrogenase.
2C19
50 mM potassium phosphate, pH 8, 1% acetonitrile, 8 μM Blue vivid substrate, 100 μM NADP+, 4 nM CYP450 2C19, 2.66 mM glucose-6-phosphate, 0.32 U/ml glucose-6-phosphate dehydrogenase.
3A4
100 mM potassium phosphate, pH 8, 1% acetonitrile, 10 μM 3A4 Blue vivid substrate, 100 μM NADP+, 2.5 nM CYP450 3A4, 2.66 mM glucose-6-phosphate, 0.32 U/ml glucose-6-phosphate dehydrogenase.
2D6
100 mM potassium phosphate, pH 8, 1% acetonitrile, 5 μM 2D6 Blue vivid substrate, 100 μM NADP+, 16 nM CYP450 2D6, 2.66 mM glucose-6-phosphate, 0.32 U/ml glucose-6-phosphate dehydrogenase.
Fluorescence is monitored for 20 minutes at 30 second intervals on a Molecular Devices Gemini fluorescence plate reader. The excitation and emission wavelengths are 390 nm and 460 nm for 1A2, 2C19 and 3A4, 390 nm and 485 nm for 2D6 and 485 nm and 530 nm for 2C9. Initial rates are determined from progress curves.
The test compound is made up in methanol or acetonitirile and tested against the CYP450s at a concentration of 10 μM.
Ba/F3-Tel-FGFR3 & Ba/F3 (Wt) Cell Proliferation Assays
Stably transfected Ba/F3-TEL-FGFR3 cells were plated out into black 96-well tissue culture plates with clear bottoms in RPMI medium containing 10% FBS and 0.25 mg/ml G418 at a density of 5×103 cells/well (200 μl per well). The parental wild-type Ba/F3 cells (DSMZ no.: ACC 300) were plated out into black 96-well tissue culture plates with clear bottoms in RPMI medium containing 10% FBS and 2 ng/ml mouse IL-3 (R&D Sysems) at a density of 2.5×103 cells/well (200 μl per well). Plates were placed in an incubator overnight before adding the compounds the following day. Dilutions of compounds were made in DMSO starting at 10 mM and were diluted into the wells to give a final DMSO concentration of 0.1% in assay. Compounds were left on the cells for 72 hours before the plates were removed from the incubator and 20 μl of Alamar BlueTM (Biosource) was added to each well. Plates were placed in the incubator for 4-6 hours before reading plates at 535 nm (excitation)/590 nm (emission) on a Fusion plate reader (Packard).
Many compounds of the invention are expected to be more active against Ba/F3-TEL-FGFR3 cell line than the parental wild-type Ba/F3 cell line, for example over 5-fold, in particular 10 fold more active against Ba/F3-TEL-FGFR3 cell line than the parental wild-type Ba/F3 cell line.

## Claims

1. A method for the treatment of a disease state or condition selected from multiple myeloma, bladder carcinoma, hepatocellular carcinoma, oral squamous cell carcinoma, cervical carcinoma, and urothelial carcinoma, which method comprises administering to a subject in need thereof a compound of the formula (I):




wherein
 represents a single or double bond, such that at least one bond within the 5 membered ring system is a double bond;
Ring A may be optionally substituted by 1, 2 or 3 Ra groups;
B represents a —V-carbocyclic group or a —W-heterocyclyl group wherein said carbocyclic and heterocyclyl groups may be optionally substituted by 1, 2 or 3 Ra groups;
Re, Rf and Rw independently represent hydrogen or C1-6 alkyl;
Ra represents halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, —ORx, —O—(CH2)n—ORx, haloC1-6 alkyl, haloC1-6 alkoxy, C1-6 alkanol, ═O, ═S, nitro, Si(Rx)4, —(CH2)s—CN, —S—Rx, —SO—Rx, —SO2—Rx, —CORx, —(CRxRy)s—COORz, —(CH2)s—CONRxRy, —(CH2)s—NRxRy, —(CH2)s—NRxCORy, —(CH2)s—NRxSO2—Ry, —(CH2)s—NH—SO2—NRxRy, —OCONRxRy, —(CH2)s—NRxCO2Ry, —O—(CH2)s—CRxRy—(CH2)t—ORz or —(CH2)s—SO2NRxRy groups;
Rx, Ry and Rz independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkanol, hydroxy, C1-6 alkoxy, haloC1-6 alkyl, —CO—(CH2)n—C1-6 alkoxy, C3-8 cycloalkyl or C3-8 cycloalkenyl;
R7 and R8 independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, aryl, heterocyclyl or R7 and R8 together with the nitrogen atom to which they are attached may form a nitrogen containing heterocyclyl ring, wherein said C1-6 alkyl, aryl and heterocyclyl may be optionally substituted by 1, 2 or 3 Rb groups;
R1 and Rb independently represent an Ra group or a —Y-carbocyclic or —Z-heterocyclyl group wherein said carbocyclic and heterocyclyl groups may be optionally substituted by 1, 2 or 3 Ra groups;
V and W independently represent a bond or a —(CReRf)n— group;
Y and Z independently represent a bond, —CO—(CH2)s—, —COO—, —(CH2)n—, —NRx—(CH2)s—, —(CH2)s—NRx—, —CONRx—, —NRxCO—, —SO2NRx—, —NRxSO2, —NRxCONRy—, —NRxCSNRy—, —O—(CH2)s—, —(CH2)s—O—, S—, —SO— or —(CH2)s—SO2—;
n represents an integer from 1-4;
s and t independently represent an integer from 0-4;
q represents an integer from 0-2;
or a pharmaceutically acceptable salt or solvate thereof.

2. A method as defined in claim 1, wherein
B represents an aromatic or non-aromatic heterocyclic group;
Ra represents halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, —IRx, —O—(CH2)n—ORx, haloC1-6 alkyl, haloC1-6 alkoxy, C1-6 alkanol, ═O, ═S, nitro, —(CH2)s—CN, —S—RX, —SO—Rx, —SO2—Rx, —CORx, —(CRxRy)s—COORz, —(CH2)s—CONRxRy, —(CH2)s—NRxRy, —(CH2)s—NRxCORy, —(CH2)s—NRxSO2—Ry, —OCONRxRy, —(CH2)s—NRxCO2Ry, —O—(CH2)s—CRxRy—(CH2)t—ORz or —(CH2)s—SO2NRxRy groups; and
Y and Z independently represent a bond, —CO—, —CH2—, —(CH2)2, —(CH2)3—, —O—(CH2)s— or —NH—(CH2)n—.

3. A method as defined in claim 1 wherein B represents a —W-heterocyclyl group.

4. A method as defined in claim 3 wherein W represents a bond.

5. A method as defined in claim 3 wherein the heterocyclyl group is a 5 or 6 membered monocyclic heterocyclyl group.

6. A method as defined in claim 5 wherein the heterocyclyl group is pyridyl, pyrazinyl, triazolyl, oxadiazolyl, imidazolyl or thiadiazolyl.

7. A method as defined in claim 1 wherein q represents 0.

8. A method as defined in claim 1 wherein R7 and R8 both represent hydrogen or C1-6 alkyl; or one of R7 and R8 represents hydrogen and the other represents C1-6 alkyl (optionally substituted by an —ORx group), C3-8 cycloalkyl or heterocyclyl.

9. A method as defined in claim 1 wherein R7 and R8 together with the nitrogen atom to which they are attached form a nitrogen containing heterocyclyl ring optionally substituted by 1, 2 or 3 Rb groups.

10. A method as defined in claim 1 wherein the compound of formula (I) has the following ring system:

11. A method as defined in claim 10 wherein B represents a —W-heterocyclyl group that is a 5 or 6 membered monocyclic heterocyclyl group, and W represents a bond.

12. A method as defined in claim 1 wherein Y and Z independently represent a bond, —CO—, —CH2—, —(CH2)2— or —(CH2)3—.

13. A method as defined in claim 12 wherein Z represents —CH2—.

14. A method as defined in claim 1 wherein the disease or condition is multiple myeloma.

15. A method of inhibiting FGFR kinase, which method comprises contacting the kinase with a compound of the formula (I):




wherein
 represents a single or double bond, such that at least one bond within the 5 membered ring system is a double bond;
Ring A may be optionally substituted by 1, 2 or 3 Ra groups;
B represents a —V-carbocyclic group or a —W-heterocyclyl group wherein said carbocyclic and heterocyclyl groups may be optionally substituted by 1, 2 or 3 Ra groups;
Re, Rf and Rw independently represent hydrogen or C1-6 alkyl;
Ra represents halogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, —ORx, —O—(CH2)n—ORx, haloC1-6 alkyl, haloC1-6 alkoxy, C1-6 alkanol, ═O, ═S, nitro, Si(Rx)4, —(CH2)s—CN, —S—Rx, —SO—Rx, —SO2—Rx, —CORx, —(CRxRy)s—COORz, —(CH2)s—CONRxRy, —(CH2)s—NRxRy, —(CH2)s—NRxCORy, —(CH2)s—NRxSO2—Ry, —(CH2)s—NH—SO2—NRxRy, —OCONRxRy, —(CH2)s—NRxCO2Ry, —O—(CH2)s—CRxRy—(CH2)t—ORz or —(CH2)s—SO2NRxRy groups;
Rx, Ry and Rz independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C1-6 alkanol, hydroxy, C1-6 alkoxy, haloC1-6 alkyl, —CO—(CH2)n—C1-6 alkoxy, C3-8 cycloalkyl or C3-8 cycloalkenyl;
R7 and R8 independently represent hydrogen, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C3-8 cycloalkyl, C3-8 cycloalkenyl, aryl, heterocyclyl or R7 and R8 together with the nitrogen atom to which they are attached may form a nitrogen containing heterocyclyl ring, wherein said C1-6 alkyl, aryl and heterocyclyl may be optionally substituted by 1, 2 or 3 Rb groups;
R1 and Rb independently represent an Ra group or a —Y-carbocyclic or —Z-heterocyclyl group wherein said carbocyclic and heterocyclyl groups may be optionally substituted by 1, 2 or 3 Ra groups;
V and W independently represent a bond or a —(CReRf)n— group;
Y and Z independently represent a bond, —CO—(CH2)s—, —COO—, —(CH2)n—, —NRx—(CH2)s—, —(CH2)s—NRx—, —CONRx—, —NRxCO—, —SO2NRx—, —NRxSO2—, —NRxCONRy—, —NRxCSNRy—, —O—(CH2)s—, —(CH2)s—O—, S—, —SO— or —(CH2)s—SO2—;
n represents an integer from 1-4;
s and t independently represent an integer from 0-4;
q represents an integer from 0-2;
or a pharmaceutically acceptable salt or solvate thereof.

16. A method according to claim 15 wherein R7 and R8 both represent hydrogen or C1-6 alkyl; or one of R7 and R8 represents hydrogen and the other represents C1-6 alkyl (optionally substituted by an —ORx group), C3-8 cycloalkyl or heterocyclyl.

17. A method according to claim 15 wherein Y and Z independently represent a bond, —CO—, —CH2—, —(CH2)2— or —(CH2)3—.

18. A method as defined in claim 15 wherein the compound of formula (I) has the following ring system:

19. A method as defined in claim 18 wherein B represents a —W-heterocyclyl group that is a 5 or 6 membered monocyclic heterocyclyl group, and W represents a bond.

20. A method as defined in claim 11 wherein R7 and R8 both represent hydrogen or C1-6 alkyl; or one of R7 and R8 represents hydrogen and the other represents C1-6 alkyl (optionally substituted by an —ORx group), C3-8 cycloalkyl or heterocyclyl.