WO2008063853A2 - Cancer treatment method - Google Patents

Abstract

The present invention relates to a method of treating cancer in a mammal and to pharmaceutical combinations useful in such treatment. In particular, the method relates to a cancer treatment method that includes administering an erb family inhibitor and a PI3K and/or Akt inhibitor to a mammal suffering from a cancer.

Description

CANCER TREATMENT METHOD

BACKGROUND OF THE INVENTION

The present invention relates to a method of treating cancer in a mammal and to pharmaceutical combinations useful in such treatment. In particular, the method relates to a cancer treatment method that includes administering a dual erbB-2/EGFR inhibitor with an Akt inhibitor to a mammal suffering from a cancer.

Effective chemotherapy for cancer treatment is a continuing goal in the oncology field. Generally, cancer results from the deregulation of the normal processes that control cell division, differentiation and apoptotic cell death. Apoptosis (programmed cell death) plays essential roles in embryonic development and pathogenesis of various diseases, such as degenerative neuronal diseases, cardiovascular diseases and cancer. One of the most commonly studied pathways, which involves kinase regulation of apoptosis, is cellular signaling from growth factor receptors at the cell surface to the nucleus (Crews and Erikson, Cell, 74:215-17, 1993). In particular, cellular signalling from the growth factor receptors of the erbB family.

There is significant interaction among the ErbB family that regulates the cellular effects mediated by these receptors. Six different ligands that bind to EGFR include EGF, transforming growth factor, amphiregulin, heparin binding EGF, betacellulin and epiregulin (Alroy & Yarden, FEBS Letters, 410:83-86, 1997; Burden & Yarden, Neuron, 18: 847-855, 1997; Klapper et al., ProcNatlAcadSci, 4994-5000, 1999). Heregulins, another class of ligands, bind directly to HER3 and/or HER4 (Holmes et al., Science, 256:1205, 1992; Klapper et al., 1997, Oncogene, 14:2099-2109; Peles et al., Cell, 69:205, 1992). Binding of specific ligands induces homo- or heterodimerization of the receptors within members of the erbB family (Carraway & Cantley, Cell, 78:5-8, 1994; Lemmon & Schlessinger, TrendsBiochemSci, 19:459-463, 1994). In contrast with the other ErbB receptor members, a soluble ligand has not yet been identified for HER2, which seems to be transactivated following heterodimerization. The heterodimerization of the erbB-2 receptor with the EGFR, HER3, and HER4 is preferred to homodimerization (Klapper et al., 1999; Klapper et al., 1997). Receptor dimerization results in binding of ATP to the receptor's catalytic site, activation of the receptor's tyrosine kinase, and autophosphorylation on C-terminal tyrosine residues. The phosphorylated tyrosine residues then serve as docking sites for proteins such as Grb2, She, and phospholipase C, that, in turn, activate downstream signaling pathways, including the Ras/MEK/Erk and the PI3K/Akt pathways, which regulate transcription factors and other proteins involved in biological responses such as proliferation, cell motility, angiogenesis, cell survival, and differentiation (Alroy & Yarden, 1997; Burgering & Coffer, Nature, 376:599-602, 1995; Chan et al., AnnRevBiochem, 68:965-1014,1999; Lewis et al., AdvCanRes, 74:49-139,1998; Liu et al., Genes and Dev, 13:786-791 , 1999; Muthuswamy et al., Mol&CellBio, 19,10:6845-6857,1999; Riese & Stern, Bioessays, 20:41-48, 1998).

ErbB-mediated activation of Akt requires the activation of PI3K (Knuefermann et al., 22(21 ): 3205-12, 2003). This can occur via dimerization of ErbB2 or EGFR with HER3, which is able to couple to PI3K directly (Fedi et al., MoI Cell Biol., 14(1 ): 492-500, 1994), or by interaction of the receptor with the intracellular adaptor Gab1 (Rodrigues et al., MoI Cell Biol., 20(4): 1148-1459, 2000). Upon activation, PI3K converts phosphatidylinositol-4,5 bisphosphate (PIP2) to phosphatidylinositol-3,4,5 trisphosphate (Pl P3); this lipid recruits the pleckstrin-homology (PH) domain of Akt to the plasma membrane where its kinase domain is activated (Chan et al., Ann Rev Biochem., 68:965-1014, 1999). Akt, or protein kinase B, is a well-characterized serine/threonine kinase that promotes cellular survival and has three isoforms, Akt1 , Akt2, and Akt3. Activation of all three isoforms is similar in that phosphorylation of two sites, one in the activation domain and one in the COOH-terminal hydrophobic motif, are necessary for full activity. For Akt1 , phosphorylation of T308 in the activation domain by phosphoinositide-dependent kinase 1 is dependent on the products of PI3-K. Cellular levels of PIP₂ and PIP₃ are controlled by the tumor suppressor, dual-phosphatase PTEN, that dephosphorylates PIP₂ and PIP₃ at the 3' position.

Once activated, Akt can suppress apoptosis by interacting with and phosphorylating several key downstream effectors. For example, Akt phosphorylates the proapoptotic Bcl-2 partner Bad, that binds to and blocks the activity of Bcl-x, a cell survival factor (del Peso et al., Science, 278(5338):687-689,1997); inactivates the initiation caspase-9 (Cardone et al., Science, 282(5392):1318-1321 ,1998); represses the forkhead transcription factor FKHRL-1 (Brunet et al., Cell, Vol. 96, 857-868, 1999), a regulator of the expression of the apoptosis-inducing Fas ligand; and phosphorylates IKB, promoting degradation of IKB and thereby increasing the activity of N FKB, a well- known cell survival factor (Ozes et al., Nature, 401 :86-90, 1999; Romashkova & Makarov, Nature, 401 :86-90,1999). In addition to these molecules that are known to be involved in apoptosis, an increasing number of substrates involved in cell cycle regulation, protein synthesis, and glycogen metabolism are also phosphorylated by Akt (see the recent review by (Nicholson & Anderson, Cell Signal., 14(5):381-395, 2002)).

The MAP kinases ERK1 and ERK2 represent a central group of signaling kinases that are activated in response to ErbB signaling (for review see (Chang & Karin, Nature, 410, 37-40, 2001 )). The best understood mechanism for activation of ERK is via growth factor receptor or tyrosine kinase activation of Ras. ERK has been implicated in the phosphorylation of a number of transcription factors that are important for expression of genes essential for cell proliferation (Chang & Karin, 2001). The mechanism by which ERK protects cells from apoptosis is complex, and Ras, a potent ERK activator, may also promote apoptosis (Kauffmann-Zeh et al., Nature, 385(6616):544-548, 1997). In cerebellar granular cells, ERK activation by survival factors prevents apoptosis through RSK, which inactivates the pro-apoptotic protein Bad (Bonni et al., Science, 286(5443): 1358-1362, 1999). ERK may also induce growth factors that promote cell survival.

Several strategies including monoclonal antibodies (Mab), immunoconjugates, anti-EGF vaccine, and tyrosine kinase inhibitors have been developed to target the ErbB family receptors and block their activation in cancer cells (reviewed in (Sridhar et al., Lancet, 4,7:397-406,2003)). Because ErbB2-containing heterodimers are the most stable and preferred initiating event for signaling, interrupting both ErbB2 and EGFR simultaneously is an appealing therapeutic strategy. A series of quinazoline dual ErbB- 2/EGFR TK inhibitors that possess efficacy in pre-clinical models for cancer have been synthesized (Cockerill et al., BiorgMedChemLett, 11 :1401-1405,2001 ; Rusnak et al., CanRes, 61 :7196-7203, 2001a; Rusnak et al., MolCanTher, 1 :85-94,2001 b). GW572016 is a quinazoline, orally active, reversible dual kinase inhibitor of both EGFR and ErbB2 kinases (Rusnak et al., 2001 b). In human xenograft studies, GW572016 has shown dose-dependent kinase inhibition, and selectively inhibits tumor cells overexpressing EGFR or ErbB2 (Rusnak et al., 2001 b; Xia et al., Oncogene, 21 :6255-6263, 2002).

SUMMARY OF THE INVENTION

The present inventors have now discovered a new method of treating cancer using a novel pharmaceutical combination, which can selectively treat susceptible cancers. Specifically, the novel combination of a dual EGFR/erbB-2 inhibitor and an Akt inhibitor appears to effectively inhibit growth of such tumors.

In a first aspect of the present invention, there is provided a method of treating a susceptible cancer in a mammal, comprising: administering to said mammal therapeutically effective amounts of (i) a compound of formula (I):

or salts or solvates thereof; and (ii) a compound of formula (II):

or salts or solvates thereof.

In a second aspect of the present invention, there is provided a cancer treatment combination, comprising: therapeutically effective amounts of (i) a compound of formula (I):

or salts or solvates thereof; and (ii) a compound of formula (II):

or salts or solvates thereof.

In a third aspect of the present invention, there is provided a cancer treatment combination, comprising: therapeutically effective amounts of (i) a compound of formula (I):

or salts or solvates thereof; and (ii) a compound of formula (II):

or salts or solvates thereof for use in therapy.

In a fourth aspect of the present invention, there is provided a cancer treatment combination, comprising: therapeutically effective amounts of (i) a compound of formula (I):

or salts or solvates thereof; and (ii) a compound of formula (II):

or salts or solvates thereof for use in the preparation of a medicament useful in the treatment of cancer.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term "neoplasm" refers to an abnormal growth of cells or tissue and is understood to include benign, i.e., non-cancerous growths, and malignant, i.e., cancerous growths. The term "neoplastic" means of or related to a neoplasm.

As used herein the term "agent" is understood to mean a substance that produces a desired effect in a tissue, system, animal, mammal, human, or other subject. Accordingly, the term "antineoplastic agent" is understood to mean a substance producing an anti-neoplastic effect in a tissue, system, animal, mammal, human, or other subject. It is also to be understood that an "agent" may be a single compound or a combination or composition of two or more compounds.

As used herein, the term "effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

As used herein, the term "optionally" means that the subsequently described event(s) may or may not occur, and includes both event(s), which occur, and events that do not occur.

"EGFR", also known as "erbB-1", and "erbB-2" are protein tyrosine kinase transmembrane growth factor receptors of the erbB family. Protein tyrosine kinases catalyse the phosphorylation of specific tyrosyl residues in various proteins involved in the regulation of cell growth and differentiation (A. F. Wilks, Progress in Growth Factor Research, 1990, 2, 97-111 ; S.A. Courtneidge, Dev. Supp.l, 1993, 57-64; J.A. Cooper, Semin. Cell Biol., 1994, 5{6J, 377-387; R.F. Paulson, Semin. Immunol., 1995, 7(4], 267- 277; A.C. Chan, Curr. Opin. Immunol., 1996, 8(3], 394-401 ). The ErbB family of type I receptor tyrosine kinases includes ErbB1 (also known as the epidermal growth factor receptor (EGFR or HER1 )), erbB2 (also known as Her2), erbB3, and erbB4. These receptor tyrosine kinases are widely expressed in epithelial, mesenchymal, and neuronal tissues where they play a role in regulating cell proliferation, survival, and differentiation (Sibilia and Wagner, Science, 269: 234 (1995); Threadgill et al., Science, 269: 230 (1995)). Increased expression of wild-type erbB2 or EGFR, or expression of constitutively activated receptor mutants, transforms cells in vitro (Di Fiore et al., 1987; DiMarco et al, Oncogene, 4: 831 (1989); Hudziak et al., Proc. Natl. Acad. Sci. USA., 84:7159 (1987); Qian et al., Oncogene, 10:211 (1995)). Increased expression of erbB2 or EGFR has been correlated with a poorer clinical outcome in some breast cancers and a variety of other malignancies (Slamon et al., Science, 235: 177 (1987); Slamon et al., Science, 244:707 (1989); Bacus et al, Am. J. Clin. Path, 102:S13 (1994)).

It is understood that included within the scope of the compounds of formula (I and II) are physiologically functional derivatives of the compounds of formula (I) and (II). The term "physiologically functional derivative" refers to any pharmaceutically acceptable derivative of a compound of the present invention, for example, an ester or an amide, which upon administration to a mammal is capable of providing (directly or indirectly) a compound of the present invention or an active metabolite thereof. Such derivatives are clear to those skilled in the art, without undue experimentation, and with reference to the teaching of Burger's Medicinal Chemistry And Drug Discovery, 5^th Edition, VoI 1 : Principles and Practice, which is incorporated herein by reference to the extent that it teaches physiologically functional derivatives.

Also, it is understood that included within the scope of the compounds of formula (I and II) are solvates of the compounds of formula (I) and (II). As used herein, the term "solvate" refers to a complex of variable stoichiometry formed by a solute (in this invention, compounds of formula (I) or (II) or a salt or physiologically functional derivative thereof) and a solvent. Such solvents for the purpose of the invention may not interfere with the biological activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol and acetic acid. In one embodiment, the solvent used is a pharmaceutically acceptable solvent. Examples of suitable pharmaceutically acceptable solvents include, without limitation, water, ethanol and acetic acid. In another embodiment, the solvent used is water.

Certain of the compounds described herein may contain one or more chiral atoms, or may otherwise be capable of existing as two enantiomers. The compounds of this invention include mixtures of enantiomers as well as purified enantiomers or enantiomerically enriched mixtures. Also included within the scope of the invention are the individual isomers of the compounds represented by formula (I) or (II) as well as any wholly or partially equilibrated mixtures thereof. The present invention also covers the individual isomers of the compounds represented by the formulas above as mixtures with isomers thereof in which one or more chiral centers are inverted. Also, it is understood that any tautomers and mixtures of tautomers of the compounds of formulae (I) or (II) are included within the scope of the compounds of formula (I) and (II).

As recited above, the method of treating cancer of the present invention includes administering a therapeutically effective amount of a compound of formula (I) and a compound of formula (II).

In one embodiment, the compound of formula (I) is

(I) or salts or solvates thereof.

In another embodiment, the compound of formula (I) is a ditosylate salt of the compound of formula (I) or anhydrate or hydrate forms thereof. The ditosylate salt of the compound of formula (I) has the chemical name N-{3-chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino}methyl)-2-furyl]-4-quinazolinamine ditosylate. In one embodiment, the compound of formula (I) is the anhydrous ditosylate salt of the compound of formula (I). In another embodiment, the compound of formula (I) is the monohydrate ditosylate salt of the compound of formula (I). In another embodiment, the compound of formula (I) is a mixture of the monohydrate and anhydrous ditosylate salt of the compound of formula (I).

The free base, HCI salts, and ditosylate salts of the compound of Formula (I) may be prepared according to the procedures of International Patent Application No.

PCT/EP99/00048, filed January 8, 1999, and published as WO 99/35146 on July 15,

1999; International Patent Application No. PCT/US01/20706, filed June 28, 2001 and published as WO 02/02552 on January 10, 2002; and according to the appropriate

Examples recited below. One such procedure for preparing the ditosylate salt of the compound of formula (I) is presented following in Scheme 1.

Scheme 1

In scheme 1 , the preparation of the ditosylate salt of the compound of formula (I) proceeds in four stages: Stage 1 : Reaction of the indicated bicyclic compound and amine to give the indicated iodoquinazoline derivative; Stage 2: preparation of the corresponding aldehyde salt; Stage 3: preparation of the quinazoline ditosylate salt; and Stage 4: monohydrate ditosylate salt preparation. In one embodiment, the compound of formula (II) is

or salts or solvates thereof.

The compound of formula (II) has the chemical name 4-(2-(4-amino-1 ,2,5- oxadiazol-3-yl)-1-ethyl-7-{[(3S)-3-piperidinylmethyl]oxy}-1 H-imidazo[4,5-c]pyridin-4-yl)-2- methyl-3-butyn-2-ol and has been found to an inhibitor of all three isoforms of Akt kinase: Akt1 , Akt2, and Akt3.

The compound of Formula (II) may be prepared according to the procedures of International Patent Application No. PCT/US2006/043513, filed November 9, 2006, and published as WO 2007/058850 on 24 May 2007 and according to the appropriate Examples recited below.

The compound of Formula (II) is prepared as shown in Scheme 2 below, or by analogous methods. All of the starting materials are commercially available or are readily made from commercially available starting materials by those of skill in the art. Scheme 2

(a) Br₂, NaOAc; (b) EtNH₂; (c) SnCI₂, HCI; (d) ethyl cyanoacetate, 190 ⁰C; (e) NaNO₂, HCI; (f) NH₂OH; (g) Et₃N, dioxane; (h) n-Bul_i, THF; (i) B(OMe)₃; C) H₂O₂, NaOH; (k) 1 ,1- dimethylethyl 3-(hydroxymethyl)-1-piperidinecarboxylate, DEAD, polymer bound PPh₃, CH₂CI₂; (I) Pd(PPh₃)₄, JPr₂NH, dioxane, 100 ⁰C; (m) TFA, CH₂CI₂.

Compounds of Formula (II) can be prepared in a manner analogous to those shown in Scheme 1. Bromination of 3-nitro-4-ethoxy pyridine (1 -Scheme 2) using bromine buffered in sodium acetate gives 3-bromo-4-(ethyloxy)-5-nitropyridine (2-Scheme 2). Other alternative methods exist and are known to those skilled in the art for carrying out this transformation. A compilation of these methods can be found in standard organic synthesis texts such as Larock, "Comprehensive Organic Transformations," VCH, N.Y.(1989). The ethoxy group is then displaced by a primary amine such as ethyl amine in a polar solvent such as ethanol to give compounds such as 3-Scheme 2. In the case liquid amines, the reaction can be carried out in the absence of solvent. The reduction of the nitro group with concomitant introduction of the chloro group is achieved using tin (II) chloride according to the method described by Kelley et al. J. Med. Chem. 1995, 38(20), 4131-34. The corresponding 5-bromo-2-chloro diaminopyridine is condensed with an appropriate acid or ester such as ethyl cyanoacetate. Continued heating affects a cyclodehydration reaction to give imidazopyridines such as 4-Scheme 2. Reaction with NaNO₂ in concentrated HCI following by reaction with hydroxylamine gives a bis- oxime that cyclodehydrates in the presence of an appropriate base such as triethylamine to give an aminofurazan such as 5-Scheme 2. The hydroxyl group is introduced by generating an aryl anion by halogen-metal exchange using a suitable base such as n- butyl lithium, reacting the anion with an appropriate boron electrophile such as trimethyl borate and oxidizing the resulting aryl boronate with an appropriate oxidizing agent such as hydrogen peroxide in aqueous base to give imidazopyridinols such as 6-Scheme 2. Other bases such as Grignard reagents can also be used to affect the halogen metal exchange. Etherification of the imidazopyridinol is carried out with an appropriate alcohol such as 1 ,1-dimethylethyl 3-(hydroxymethyl)-1-piperidinecarboxylate using the methods described by Mitsunobu, Synthesis 1981 , 1 to give ethers such as 7-Scheme 2. Alternatively, the etherification can be carried out by reacting an appropriate halide such as 1 ,1-dimethylethyl 3-(chloromethyl)-1-piperidinecarboxylate with a suitable alcohol such as 6- Scheme 1 in the presence of a suitable base such as potassium carbonate. Treatment of an appropriate aryl halide such as 7-Scheme 2 with an appropriate catalyst such as tetrakistriphenylphosphine palladium and a terminal alkyne in the presence of a suitable base such as di-isopropylamine in an appropriate solvent such as dioxane gives the corresponding aryl alkyne such as 8-Scheme 2. Removal of the protecting groups is achieved using a protic or Lewis acid such as trifluoroacetic acid in a polar solvent such as methylene chloride giving compounds of Formula (I) such as 9-Scheme 2.

Alternatively, the compound of formula (II) may be administered with an EGFR inhibitor or with both an inibitor of EGFR and erbB-2. Suitable EGFR inhibitors, include but are not limited to, gefitinib and erlotinib. Gefitinib, 4-quinazolinamine,N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-4- morpholin)propoxy]; is commercially available as tablets as IRESSA®. Gefitinib is represented by the structure of formula (II)

Gefitinib is an EGFR inhibitor that is indicated as monotherapy for the treatment of patients with locally advanced or metastatic non-small-cell lung cancer after failure of both platinum-based and docetaxel chemotherapies.

The free base, HCI salts, and diHCI salts of the compound of Formula (II) may be prepared according to the procedures of International Patent Application No. PCT/GB96/00961 , filed April 23, 1996, and published as WO 96/33980 on October 31 , 1996.

Erlotinib, N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine; is commercially available as tablets as TARCEVA™. Erlotinib is represented by the structure of formula

In one embodiment, erlotinib is in the salt form: erlotinib hydrochloride. Erlotinib is an EGFR inhibitor that is indicated for the treatment of patients with locally advanced or metastatic non-small-cell lung cancer after failure of at least one prior chemotherapy regiment.

The free base and HCI salts of the compound of Formula (III) may be prepared according to the procedure of Example 20 of U.S. Patent No. 5,747,498, issued May 5,1998.

The compound of formula (I) and the compound of formula (II) may be employed in combination in accordance with the invention by administration concomitantly in (1 ) a unitary pharmaceutical composition including both compounds or (2) separate pharmaceutical compositions each including one of the compounds. Alternatively, the combination may be administered separately in a sequential manner wherein, for example, the compound of formula (I) or the compound of formula (II) is administered first and the other second. Such sequential administration may be close in time or remote in time.

Typically, the salts of the present invention are pharmaceutically acceptable salts. Salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts of the compounds of this invention. Salts of the compounds of the present invention may comprise acid addition salts derived from a nitrogen on a substituent in a compound of the present invention. Representative salts include the following salts: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, monopotassium maleate, mucate, napsylate, nitrate, N-methylglucamine, oxalate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, potassium, salicylate, sodium, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, trimethylammonium and valerate. Other salts, which are not pharmaceutically acceptable, may be useful in the preparation of compounds of this invention and these form a further aspect of the invention.

While it is possible that, for use in therapy, therapeutically effective amounts of the compound of formula (I), compound of formula (II), as well as salts, solvates and physiological functional derivatives thereof, may be administered as the raw chemical, it is possible to present the active ingredient as a pharmaceutical composition. Accordingly, the invention further provides pharmaceutical compositions, which include therapeutically effective amounts of a compound of formula (I) and/or compound of formula (II) or salts, solvates and physiological functional derivatives thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The compounds of the present invention and salts, solvates and physiological functional derivatives thereof, are as described above. The carrier(s), diluent(s) or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation, capable of pharmaceutical formulation, and not deleterious to the recipient thereof. In accordance with another aspect of the invention there is also provided a process for the preparation of a pharmaceutical formulation including admixing a compound of formula (I) and/or a compound of formula (II) or salts, solvates or physiological functional derivatives thereof, with one or more pharmaceutically acceptable carriers, diluents or excipients.

Pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. As is known to those skilled in the art, the amount of active ingredient per dose will depend on the condition being treated, the route of administration and the age, weight and condition of the patient, or the pharmaceutical formulations may be presented in unit dose forms containing a predetermined amount of active ingredient per unit dose. Preferred unit dosage formulations are those containing a daily dose or sub-dose, or an appropriate fraction thereof, of an active ingredient. Furthermore, such pharmaceutical formulations may be prepared by any of the methods well known in the pharmacy art.

The compounds of formula (I) and (II) may be administered by any appropriate route. Suitable routes include oral, rectal, nasal, topical (including buccal and sublingual), vaginal, and parenteral (including subcutaneous, intramuscular, intraveneous, intradermal, intrathecal, and epidural). It will be appreciated that the preferred route may vary with, for example, the condition of the recipient of the combination. It will also be appreciated that each of the agents administered may be administered by the same or different routes and that the compounds of formula (I) and (II) may be compounded together in a pharmaceutical composition/formulation.

The method of the present invention may also be employed with other therapeutic methods of cancer treatment. In particular, in anti-neoplastic therapy, combination therapy with other chemotherapeutic, hormonal, antibody agents as well as surgical and/or radiation treatments other than those mentioned above are envisaged. Anti-neoplastic therapies are described for instance in International Application No. PCT US 02/01130, filed January 14, 2002, which application is incorporated by reference to the extent that it discloses anti-neoplastic therapies. Combination therapies according to the present invention thus include the administration of the compound of formula (I) and the compound of formula (II) as well as optional use of other therapeutic agents including other anti-neoplastic agents. Such combination of agents may be administered together or separately and, when administered separately this may occur simultaneously or sequentially in any order, both close and remote in time. The amounts of the compounds of formula (I) and (II) and the other optional pharmaceutically active agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.

Pharmaceutical formulations adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Powders are prepared by comminuting the compound to a suitable fine size and mixing with a similarly comminuted pharmaceutical carrier such as an edible carbohydrate, as, for example, starch or mannitol. Flavoring, preservative, dispersing and coloring agent can also be present.

Capsules are made by preparing a powder mixture as described above, and filling formed gelatin sheaths. Glidants and lubricants such as colloidal silica, talc, magnesium stearate, calcium stearate or solid polyethylene glycol can be added to the powder mixture before the filling operation. A disintegrating or solubilizing agent such as agar-agar, calcium carbonate or sodium carbonate can also be added to improve the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also to granulating, the powder mixture can be run through the tablet machine and the result is imperfectly formed slugs broken into granules. The granules can be lubricated be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Tablets are formulated, for example, by preparing a powder mixture, granulating or slugging, adding a lubricant and disintegrant and pressing into tablets. A powder mixture is prepared by mixing the compound, suitably comminuted, with a diluent or base as described above, and optionally, with a binder such as carboxymethylcellulose, an aliginate, gelatin, or polyvinyl pyrrolidone, a solution retardant such as paraffin, a resorption accelerator such as a quaternary salt and/or an absorption agent such as bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting with a binder such as syrup, starch paste, acadia mucilage or solutions of cellulosic or polymeric materials and forcing through a screen. As an alternative to prevent sticking to the tablet forming dies by means of the addition of stearic acid, a stearate salt, talc or mineral oil. The lubricated mixture is then compressed into tablets. The compounds of the present invention can also be combined with free flowing inert carrier and compressed into tablets directly without going through the granulating or slugging steps. A clear or opaque protective coating consisting of a sealing coat of shellac, a coating of sugar or polymeric material and a polish coating of wax can be provided. Dyestuffs can be added to these coatings to distinguish different unit dosages.

Oral fluids such as solution, syrups and elixirs can be prepared in dosage unit form so that a given quantity contains a predetermined amount of the compound. Syrups can be prepared by dissolving the compound in a suitably flavored aqueous solution, while elixirs are prepared through the use of a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersing the compound in a non-toxic vehicle. Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers, preservatives, flavor additive such as peppermint oil or natural sweeteners or saccharin or other artificial sweeteners, and the like can also be added.

Where appropriate, dosage unit formulations for oral administration can be microencapsulated. The formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax or the like.

The agents for use according to the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

Agents for use according to the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.

Pharmaceutical formulations adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical

Research, 3(6), 318 (1986).

Pharmaceutical formulations adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

For treatments of the eye or other external tissues, for example mouth and skin, the formulations are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water- miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.

Pharmaceutical formulations adapted for topical administrations to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical formulations adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.

Pharmaceutical formulations adapted for rectal administration may be presented as suppositories or as enemas.

Pharmaceutical formulations adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical formulations adapted for administration by inhalation include fine particle dusts or mists that may be generated by means of various types of metered dose pressurised aerosols, nebulizers or insufflators.

Pharmaceutical formulations adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. 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 (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

It should be understood that in addition to the ingredients particularly mentioned above, the formulations may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

Also, contemplated in the present invention is a pharmaceutical combination including the compound of formula (I) and the compound of formula (II). In another embodiment, the pharmaceutical combination includes the compound of formula (I), the compound of formula (II), and optionally at least one additional anti-neoplastic agent. The compounds of formula (I) and (II) and the additional anti-neoplastic therapy are as described above. As indicated, therapeutically effective amounts of the compound of formula (I), the compound of formula (II), and optionally additional anti-neoplastic therapies are administered to a mammal. Typically, the therapeutically effective amount of one of the administered agents of the present invention will depend upon a number of factors including, for example, the age and weight of the mammal, the precise condition requiring treatment, the severity of the condition, the nature of the formulation, and the route of administration. Ultimately, the therapeutically effective amount will be at the discretion of the attendant physician or veterinarian.

As indicated, the method of cancer treatment of the present invention, is directed to any suceptible cancer. Typically, the cancer is any cancer which is suceptible to inhibition of EGFR, erbB-2, and Akt. Examples of cancers that are suitable for treatment by the method and treatment combination of the present invention include, but are limited to, both primary and metastatic forms of head and neck, breast, lung, colon, ovary, and prostate cancers.

The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way.

EXAMPLES

As used herein the symbols and conventions used in these processes, schemes and examples are consistent with those used in the contemporary scientific literature, for example, the Journal of the American Chemical Society or the Journal of Biological Chemistry. Standard single-letter or three-letter abbreviations are generally used to designate amino acid residues, which are assumed to be in the L-configuration unless otherwise noted. Unless otherwise noted, all starting materials were obtained from commercial suppliers and used without further purification. Specifically, the following abbreviations may be used in the examples and throughout the specification:

g (grams); mg (milligrams);

L (liters); mL (milliliters); μl_ (microliters); psi (pounds per square inch); M (molar); mM (millimolar);

N (Normal) Kg (kilogram) i. v. (intravenous); Hz (Hertz);

MHz (megahertz); mol (moles); mmol (millimoles); RT (room temperature); min (minutes); h (hours); mp (melting point); TLC (thin layer chromatography);

T_r (retention time); RP (reverse phase);

DCM (dichloromethane); DCE (dichloroethane);

DMF (Λ/,Λ/-dimethylformamide); HOAc (acetic acid);

TMSE (2-(trimethylsilyl)ethyl); TMS (trimethylsilyl);

TIPS (triisopropylsilyl); TBS (f-butyldimethylsilyl);

HPLC (high pressure liquid chromatography);

THF (tetrahydrofuran); DMSO (dimethylsulfoxide);

EtOAc (ethyl acetate); DME (1 ,2-dimethoxyethane);

EDTA ethylenediaminetetraacetic acid

FBS fetal bovine serum

IMDM Iscove's Modified Dulbecco's medium

PBS phosphate buffered saline

RPMI Roswell Park Memorial Institute

RIPA buffer _*

RT room temperature

*150 mM NaCI, 50 mM Tris-HCI, pH 7.5, 0.25% (w/v) -deoxycholate, 1 % NP-40, 5 mM sodium orthovanadate, 2 mM sodium fluoride, and a protease inhibitor cocktail.

Unless otherwise indicated, all temperatures are expressed in ⁰C (degrees Centigrade). All reactions conducted under an inert atmosphere at room temperature unless otherwise noted.

¹H NMR spectra were recorded on a Varian VXR-300, a Varian Unity-300, a Varian Unity-400 instrument, or a General Electric QE-300. Chemical shifts are expressed in parts per million (ppm, δ units). Coupling constants are in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad).

Low-resolution mass spectra (MS) were recorded on a JOEL JMS-AX505HA,

JOEL SX-102, or a SCIEX-APIiii spectrometer; high resolution MS were obtained using a JOEL SX-102A spectrometer. All mass spectra were taken under electrospray ionization (ESI), chemical ionization (Cl), electron impact (El) or by fast atom bombardment (FAB) methods. Infrared (IR) spectra were obtained on a Nicolet 510 FT- IR spectrometer using a 1-mm NaCI cell. All reactions were monitored by thin-layer chromatography on 0.25 mm E. Merck silica gel plates (60F-254), visualized with UV light, 5% ethanolic phosphomolybdic acid or p-anisaldehyde solution. Flash column chromatography was performed on silica gel (230-400 mesh, Merck). Optical rotations were obtained using a Perkin Elmer Model 241 Polarimeter. Melting points were determined using a Mel-Temp Il apparatus and are uncorrected.

Example 1

Monohydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2- (methane sulphonyl) ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (monohydrate ditosylate salt of compound of formula (I))

1 (a) Preparation of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methane sulphonyl) ethyl]amino}methyl)-2-furyl]-4-quinazolinamine (free base of compound of formula (I))

The title compound was prepared according to Procedure D of International

Applications WO 02/02552: p. 16, line 19 to p. 17, line 3 and WO 99/35146: p. 56, lines 20-32 and Example 29 p. 100, lines 18-29, from 5-(4-{3-chloro-4-(3-fluorobenzyloxy)- anilino}-6-quinazolinyl)-furan-2-carbaldehyde (0.6 equiv) and 2-methanesulphonyl- ethylamine (1 equiv). ¹H NMR 400 MHz (DMSO-d6) 9.60 (bs, 1 H); 9.32 (bs, 1 H); 8.82 (bs, 1 H); 8.34 (d, 1 H); 8.0 (s, 1 H); 7.88 (d, 1 H); 7.74 (d, 1 H); 7.45 (m, 1 H); 7.34-7.23 (m, 4H); 7.17 (m, 1 H); 6.83 (d, 1 H); 5.27 (s, 2H); 4.42 (s, 2H); 3.59 (m, 2H); 3.40 (m, 2H, obscured by waterpeak); 3.12 (s, 3H); MS m/z 581 (M+H⁺).

1(b) Preparation of monohydrate ditosylate salt of N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methane sulphonyl) ethyl]amino}methyl)-2-furyl]-4- quinazolinamine (monohydrate ditosylate salt of compound of formula (III))

Stage 1: Preparation of N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-iodo-4- quinazolinamine

4-Chloro-6-iodoquinazoline (1wt) was added to a solution of fluorobenzyloxyaniline (0.894wt, 1.03equiv) in N-methylpyrrolidinone (8.26wt, 8vol) at ca 20⁰C, and after the initial exotherm had subsided, the resulting solution was stirred at 20°-25°C for at least 30 minutes. The dark solution was treated with triethylamine (0.58vol, 1.2equiv) and the mixture was stirred for 20-30 minutes, lsopropanol (2.5vol) was added and the mixture was heated to ca 50⁰C. Water (up to 3vol) was added slowly to the vessel over 10-15 minutes, while keeping the temperature at ca 50⁰C. Once crystallisation had commenced the addition was stopped and the resulting slurry was aged for 30-45 minutes at ca 50⁰C. Any residual water (from the 3vol) was added, then further water (5vol) was added to the vessel over ca 30 minutes while maintaining the temperature at ca 50⁰C. The resulting slurry was cooled to ca 20⁰C over ca 30 minutes and aged at ca 20⁰C for at least 30 minutes. The solid was collected by filtration and washed sequentially with water (2 x 5vol), then isopropanol (5vol). The product was dried in vacuo at ca 60⁰C to give the title compound as a cream crystalline solid.

Stage 2: Preparation of 5-(4-[3-chloro-4-(3-fluorobenzyloxy)-anilino]-6- quinazolinyl)-furan-2-carbaldehyde 4-methylbenzenesulfonate

A mixture of N-{3-chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-iodo-4-quinazolinamine (1wt), boronic acid (0.37wt , 1.35equiv), and 10% palladium on charcoal (0.028wt ,50% water wet) was slurried in IMS (15vol). The resultant suspension was stirred for 5 minutes, treated with di-isopropylethylamine (0.39vol, 1.15equiv) and then heated to ca

70⁰C for ca 3 hours when the reaction was complete (determined by HPLC analysis).

The mixture was diluted with tetrahydrofuran (THF, 15vol) and then filtered (hot - through GFA filter paper) to remove catalyst. The vessel was rinsed with IMS (2vol).

A solution of p-toluenesulfonic acid monohydrate (1.54wt, 4.1equiv) in water (3vol) was added over 5-10 minutes to the filtered solution maintained at 65°C. After crystallisation the suspension was stirred at 60°-65°C for 1 hour, cooled to ca 25°C over 1 hour and stirred at this temperature for a further 2 hours. The solid was collected by filtration, washed with IMS (3vol) then dried in vacuo at ca 50⁰C to give the tile compound as a yellow-orange crystalline solid. Stage 3: Preparation of anhydrous ditosylate salt of N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methane sulphonyl) ethyl]amino}methyl)-2-furyl]-4- quinazolinamine (anhydrous ditosylate salt of compound of formula (III))

5-(4-[3-chloro-4-(3-fluorobenzyloxy)-anilino]-6-quinazolinyl)-furan-2-carbaldehyde 4-methylbenzenesulfonate (1 wt) and 2-(methylsulfonyl) ethylamine hydrochloride (0.4 wt, 1.6equiv) were suspended in THF (10vol). Sequentially, acetic acid (0.35vol, 4equiv) and di-isopropylethylamine (1.08vol, 4equiv) were added. The resulting solution was stirred at 30°-35°C for ca 1 hour then cooled to ca 23°C. Sodium triacetoxyborohydride (0.66wt, 2equiv) was then added as a continual charge over approximately 15 minutes (some effervescence is seen at this point). The resulting mixture was stirred at ca 22°C for ca 2 hours then sampled for HPLC analysis. The reaction was quenched by addition of 5M aqueous sodium hydroxide (5vol) and stirred for ca 30 minutes (some effervescence is seen at the start of the caustic addition).

The aqueous phase was then separated, extracted with THF (2vol) and the combined THF extracts were then washed with 10%w/v aqueous sodium chloride solution (4vol). A solution of p-toluenesulfonic acid monohydrate (pTSA, 1.77wt, δequiv) in THF (7 vol)¹ was prepared and warmed to ca 55°C. The THF solution of N-{3-Chloro- 4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl] amino}methyl)- 2- furyl]-4-quinazolinamine was added to the pTSA solution over at least 30minutes, maintaining the batch temperature at ca 55°±3°C ². The resulting suspension was stirred at ca 55°C for 2 hours, cooled to 20°-25°C over ca 60 minutes and aged at this temperature for ca 30 minutes. The solid was collected by filtration, washed with THF (2 x 2vol) and dried in vacuo at ca 40⁰C to give the desired compound as a pale yellow crystalline solid.

Stage 4: Preparation of monohydrate ditosylate salt of N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methane sulphonyl) ethyl]amino}methyl)-2-furyl]-4- quinazolinamine (monohydrate ditosylate salt of compound of formula (III))

A suspension of the anhydrous ditosylate salt of N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methane sulphonyl) ethyl]amino}nnethyl)-2-furyl]-4- quinazolinamine (1 wt), in tetrahydrofuran (THF, 14 vol) and water (6 vol) was heated to ca 55°-60°C for 30 minutes to give a solution which was clarified by filtration and the lines washed into the crystallisation vessel with THF/Water (7:3, 2 vol). The resultant solution was heated to reflux and tetrahydrofuran (9 vol, 95% w/w azeotrope with water) was distilled off at atmospheric pressure. The solution was seeded with N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-

({[2-(methane sulphonyl) ethyl]amino}methyl)-2-furyl]-4-quinazolinamine ditosylate monohydrate (0.002 wt). Once the crystallisation was established water (6 vol) was added while maintaining the reaction temperature above 55°C. The mixture was cooled to 5°-15°C over ca 2 hours. The solid was collected by filtration, washed with tetrahydrofuran/water (3:7 ratio, 2 x 2 vol) and dried in vacuo at 45⁰C to give N-{3- Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methane sulphonyl) ethyl] amino}methyl)-2-furyl]-4-quinazolinamine ditosylate monohydrate as a bright yellow crystalline solid. Example 2

Preparation of 2-(4-amino- 1, 2, 5-oxadiazol-3-yl)-4-chloro-1-ethyl-1H-imidazo[4, 5- c]pyridin-7-ol - Intermediate VII

I Il III IV

Vl VII

a) 5-Bromo-2-chloro-N⁴-ethyl-pyridine-3,4-diamine (II) 3-Bromo-5-nitropyridin-4-yl)amine (I, 700 g, 2.86 mol) was dissolved in cone HCI

(7L) and heated to 85 ⁰C. Tin (II) chloride (1626 g, 8.58 mol) was added in portions. The reaction was heated at reflux for 1 h and then allowd to cool to ambient temperature overnight. The resulting yellow precipitate was collected by filtration, suspended in ice water (5L) and the mixture was adjusted to pH 12 with 12N NaOH. The resulting solution was extracted with CH₂CI₂ (2 x 4 L) and the combined organic extracts were dried over Na₂SO₄. The solvent was removed under reduced pressure to give 550 g (77% yield) of the desired compound (II). This was used in the next step without further purification. MS (ES+) m/z 250(M+H)⁺.

b) N-fi-Bromo^-chloro^-fethylaminoj-S-pyridinylJ^-cyanoacetamide (III)

To a solution of 5-bromo-2-chloro-N⁴-ethyl-pyridine-3,4-diamine (II, 550 g, 2.21 mol) in CH2CI2 (5.5 L) was added 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (634.5 g, 3.31 mol), cyanoacetic acid (282 g, 3.31 mol) and N- methylmorpholine (897 g, 8.84 mol). A significant exotherm (-20 ⁰C) was observed upon the addition of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and cyanoacetic acid. After stirring at ambient temperature for 2 h, the solvent was removed under reduced pressure. The resulting reside was extracted with warm EtOAc (40 ⁰C, 20 L) and water (40 ⁰C, 8 L). The aqueous layer was washed with addition EtOAc (10 L) and the combined organic extracts were washed with water (10 L). The organic extract was concentrated under reduced pressure to a slurry and filtered. The solid was wahed with EtOAc and dried to give 534 g (76% yield) of the desired compound (III) as a white crystalline solid. This was used in the next step without further purification. MS (ES+) m/z 317(M+H)⁺.

c) (7-Bromo-4-chloro-1-ethyl-1H-imidazo[4,5-c]pyridin-2-yl)-hydroxyimino-acetonitrile (V)

A solution of N-[5-bromo-2-chloro-4-(ethylamino)-3-pyridinyl]-2-cyanoacetamide III, (458 g, 1.45 mol) in glacial acetic acid (4.6 L) was heated to 100 ⁰C. After 3 h, LC/MS analysis indicated that the conversion to (7-bromo-4-chloro-1-ethyl-1 H- imidazo[4,5-c]pyridin-2-yl)acetonitrile (IV) was completed. After allowing to cool to ambient temperature, the reaction was charged with sodium nitrate (230 g, 3.34 mol) in portions. Vigorous gas evolution and foaming was observed together with a -10 ⁰C exotherm. After stirring at ambient temperature for 16 h, the solid was collected by filtration and dried to a constant weight to give 545 g of the desired product (V) as a light yellow solid. This was used in the next step without further purification. MS (ES+) m/z

328(M+H)⁺.

d) 4-(7-Bromo-4-chloro- 1 -ethyl- 1 H-imidazo[4, 5-c]pyridin-2-yl)-1 , 2, 5-oxadiazol-3- amine (Vl)

To a mixture of (7-bromo-4-chloro-1-ethyl-1 H-imidazo[4,5-c]pyridin-2-yl)- hydroxyimino-acetonitrile (V, 545 g, 1.45 mol) in dioxane (5 L) was added triethylamine (1 L) and hydroxylamine (143 g, 55% in water). The reaction was heated at reflux for 6 h. After cooling the reaction to ambient temperature, the mixture was filtered and the filtrate concentrated under reduced pressure to give a brown solid. The solid was suspended in methanol (1 L) and the suspension was stirred at 65 ⁰C for 0.5 h. The solid was collected by filtration and dried to give 321 g (70% yield) of the desired compound (Vl). This was used in the next step without further purification. MS (ES+) m/z 343(M+H)⁺.

e) 2-(4-A mi no- 1 , 2, 5-oxadiazol-3-yl) -4-chloro- 1 -ethyl- 1 H-imidazo[4, 5-c]pyridin- 7-ol (VII) A suspension of 4-(7-bromo-4-chloro-1-ethyl-1 H-imidazo[4,5-c]pyridin-2-yl)-1 ,2,5- oxadiazol-3-amine (Vl, 50 g, 0.14 mol) in THF (1 L) was cooled in a dry-ice/acetone bath until the internal temperature was below -75 ⁰C. lsopropyl magnesium chloride (225 ml_, 2M in ether, 0.45 mol) was added slowly at a rate to keep the reaction temperature below -70 ⁰C. After an additional 10 min., trimethyl borate (54 ml_, 0.48 mol) was added and the reaction was maintained in the dry-ice acetone bath for 1 h. The bath was removed and the reaction was allowed to reach ambient temperature. After 18 h., the resultant yellow suspension was cooled to 0 ⁰C. A solution of 30% hydrogen peroxide (250 ml.) and 3N NaOH (100 ml.) was added at a rate to keep the reaction temperature below 40 ⁰C. The ice bath was then removed and the reaction was stirred vigorously at ambient temperature for 2 h. The bulk of the organic solvent was removed under reduced pressure and the aqueous layer was acidified to pH 3 with 1 N HCI. After stirring the resulting suspension for 30 min., ethyl acetat (200 ml.) was added. After stirring for another 1 h, the solid was collected by filtration. The filter cake was washed sequentially with water, ethyl acetate, toluene and ethyl acetate. The solid was dried to a constant weight to give 35.9 g (88% yield) of the desired compound (VII) as a pale yellow solid. This was used without further purification. MS (ES+) m/z 281.3 (M+H)⁺.

Example 3

Preparation of 4-(2-(4-amino-1 ,2, 5-oxadiazol-3-yl)-1-ethyl-7-{[(3S)-3- piperidinylmethyl]oxy}-1H-imidazo[4, 5-c]pyridin-4-yl)-2-methyl-3-butyn-2-ol

a) 1, 1-Dimethylethyl (3S)-3-(bromomethyl)-1-piperidinecarboxylate

To a solution of 1 ,1-dimethylethyl (3S)-3-(hydroxymethyl)-1-piperidinecarboxylate (30.0 g, 139 mmol) and carbon tetrabromide (72.0 g, 217 mmol) in methylene chloride

(150 ml.) was added dropwise a solution of triphenyl phosphine (42.4 g, 162 mmol) in methylene chloride (150 ml_). An ice-bath was used to maintain an internal temperature between 20 and 25 ⁰C during the addition. After stirring the mixture at ambient temperature for 1 h, cyclohexane (500 ml.) was added. Approximately one-half of the solvent was removed under reduced pressure. The remaining solution was cooled in an ice bath and the resulting precipitate was removed by filtration. The filtrate was concentrated under reduced pressure and the residue subjected to flash chromatography (0% to 25% ethyl acetate/hexanes, silica gel) to give 35.1 g (91 % yield) of the desired product as a solid. MS (ES+) m/z 278 (M+H)⁺.

b) 1, 1-Dimethylethyl 3-({[2-(4-amino-1 ,2,5-oxadiazol-3-yl)-4-chloro-1-ethyl-1 H- imidazo[4,5-c]pyridin-7-yi]oxy}methyl)-1-pipericlinecarboxylate

A mixture consisting of intermediate VII (25.0 g, 89.1 mmol), cesium carbonate (41.0 g, 126 mmol) and the compound of Example 3(a) (35.0 g, 126 mmol) in DMF (200 mL) was stirred at 40 ⁰C for 8 h and then at 35 ⁰C for 18 h. The mixture was poured into rapidly stirring ice water (800 ml). After 10 min., ethyl acetate (300 mL) was added and the stirring continued for an additional 20 min. The solid was collected by filtration, washed with ethyl acetate (50 ml.) and dried to give 36 g (85% yield) of the desired compound. MS (ES+) m/z 478 (M+H)⁺.

c) 1, 1-Dimethylethyl 3-({[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-4-(3-hydroxy-3-methyl- i-butyn-i-ylj-IH-imidazofaδ-cJpyridin-J-ylJoxyJmethylj-i-piperidine carboxylate

A thick-walled pressure vessel was charged with the compound of Example 3(b) (36 g, 75.3 mmol), 2-methyl-3-buty-2-ol (16 ml_, 165 mmol), (Ph3P)4Pd (1 g, 0.86 mmol), Zn dust (1.0 g., 14.8 mmol), NaI (2.20 g, 14.8 mmol), DBU (16 ml_, 107 mmol), triethylamine (15 ml_, 109 mmol) and DMSO (300 ml_). The mixture was purged with argon for 10 min. The pressure vessel was then sealed and heated at 80 ⁰C for 4 h. The mixture was then poured into rapidly stirring ice water (1000 ml_). After 10 min., ethyl acetate (300 ml.) was added and stirring continued for an additional 20 min. The solid was collected by filtration, washed with ethyl acetate (50 ml.) and dried to give 35.5 g (90% yield) of the desired compound. MS (ES+) m/z 526 (M+H)⁺.

d) 4-{2-(4-Amino-1 ,2, 5-oxadiazol-3-yl)-1-ethyl-7-[(3-pipeήdinylmethyl)oxy]-1H- imidazo[4,5-c]pyήdin-4-yl}-2-methyl-3-butyn-2-ol

The compound of Example 3(c) (35.0 g, 66.6 mmol) and TFA (350 mL of a 20% solution in methylene chloride, 808 mmol) was stirred at ambient temperature for 2.5 h. The solution was poured slowly into rapidly stirring mixture of water, NaOH (36 g, 900 mmol), ethyl acetate (200 mL) and THF (1000 mL). The organic layer was separated and the aqueous layer was extracted with additional ethyl acetate/THF (1 :5 v/v, 150 mL). The combined organic extract were washed with sat. NaCI, dried over Na₂SO₄. The solvent was removed under vacuum and the resulting solid was recrystallized from hot ethanol (1200 mL) to give 26.3 g (93% yield) of the title compound as a white crystalline solid. MS (ES+) m/z 426 (M+H)⁺.

Example 4 - Methods

GW572016F or lapatinib ditosylate is

whose chemical name is N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2- (methane sulphonyl) ethyl]amino}methyl)-2-furyl]-4-quinazolinamine ditosylate monhydrate.

GSK690693C is the mono-chloride salt of

whose chemical name is 4-(2-(4-amino-1 ,2,5-oxadiazol-3-yl)-1-ethyl-7-{[(3S)-3- piperidinylmethyl]oxy}-1 H-imidazo[4,5-c]pyridin-4-yl)-2-methyl-3-butyn-2-ol.

HN5 cells are LICRON-HN5 head and neck carcinoma cells, which were a gift from the Institute of Cancer Research, Surrey, U.K.. BT474 cells are human breast adenocarcinoma cells originally obtained from the American Type Culture Collection.

Human breast (BT474) and head/neck (HN5) cells were cultured in a humidified incubator at 37⁰C in 95% air, 5% CO₂ in the following medium: BT474, Dulbecco's modified Eagle medium (DMEM) containing 10 % fetal bovine serum (FBS); HN5, RPMI 1640 containing 10 % FBS.

Synergistic interaction between compounds was analyzed by the median effect method described by Chou and Talalay (Adv. Enzyme Regul. 22: 27-55, 1984). Briefly, if the two compounds fit the mutually exclusive model of Chou, one calculates the combination index (Cl) using the formula Cl = ((DV(D_x)₁) + ((D)₂/(D_X)₂) (1 ) where (D)₁ is the concentration of drug 1 in the combination that gives "x" percent apoptosis, (D)₂ is the same for drug 2, and (D_x)₁ and (D_x)₂ are the concentrations of drug 1 or 2 that give "x" percent apoptosis when used alone. (D)₁ and (D)₂ are known from the composition of the combination and (D_x)₁ and (D_x)₂ can be calculated from the equation Dχ = D_m * [f_a/(1-f_a)]^1/m (2) where D_m is the concentration of drug giving 50% effect, f_a is the fraction affected, and m is the slope from the median effect plot of log (f_a/f_u) where f_u is the fraction unaffected versus log (D). A Cl less than 1 indicates synergy, equal to 1 indicates additivity and greater than 1 antagonism.

Example 5 Dosing with Lapatinib and GSK690693C

The combination of GSK690693C and lapatinib (GW572016F) was compared to each monotherapy using a modified version of Chou and Talalay Combination Index (Cl) analysis. Human breast (BT474) and head/neck (LICRON-HN5, HN5) cells were cultured as indicated above. Cells were assayed in a 96-well tissue culture plate (Falcon 3075) with the following plating densities: BT474 10,000 cells/well, HN5 3,000 cells/well. Approximately 24 hours after plating cells were exposed to ten, two-fold serial dilutions of GSK690693C (30 micromolar to 0.017 micromolar), lapatinib (1.875 micromolar to 0.001 micromolar) or the combination of the two agents. The final concentration of DMSO in all wells was 0.6%. Cells were incubated in the presence of compound for 3 days. Medium was then removed by aspiration. Cell biomass was estimated by staining cells with 90 microliters per well methylene blue (Sigma M9140, 0.5% in 1 :1 ethanol:water), and incubation at room temperature for at least 30 minutes. Stain was removed, and the plates rinsed by immersion in deionized water and air-dried. To release stain from the cells 100 microliters of solubilization solution was added (1 % N- lauroyl sarcosine, Sodium salt, Sigma L5125, in PBS), and plates were shaken gently for about 30 minutes. Optical density at 620 nM was measured on a microplate reader. Percent inhibition of cell growth was calculated relative to vehicle treated control wells. Concentration of compound that inhibits 50% of control cell growth (IC₅₀) was interpolated using nonlinear regression and the equation, y = Vmax*(1-(x/(K+x))) + Y2. Combination Index values are generated by inserting the interpolated values into the mutually exclusive equation derived by Chou and Talalay: Cl = D_a/IC₅₀(_a) + D_b/IC₅₀(b), where IC_50(a) is the IC₅₀ of lapatinib, IC_50(b) is the IC₅₀ for GSK690693C, D_a is the concentration of lapatinib in combination with GSK690693C that inhibits 50 % of cell growth and D_b is the concentration of GSK690693C in combination with lapatinib that inhibits 50% of cell growth. When the Cl value is equal to 1.0, the combination of the two agents is deemed additive. Values for Cl of less than 1.0 are indicative of synergistic growth inhibition by the combination.

Results

Claims

CLAIMSWe claim:

1. A method of treating a susceptible cancer in a mammal, comprising: administering to said mammal therapeutically effective amounts of (i) a compound of formula (I):

or salts or solvates thereof; and (ii) a compound of formula (II):

or salts or solvates thereof.

2. A cancer treatment combination, comprising: therapeutically effective amounts of (i) a compound of formula (I):

or salts or solvates thereof; and (ii) a compound of formula (II):

or salts or solvates thereof.

3. A cancer treatment combination, comprising: therapeutically effective amounts of (i) a compound of formula (I):

or salts or solvates thereof; and (ii) a compound of formula (II):

or salts or solvates thereof for use in therapy.

4. A cancer treatment combination, comprising: therapeutically effective amounts of (i) a compound of formula (I):

or salts or solvates thereof; and (ii) a compound of formula (II):

or salts or solvates thereof for use in the preparation of a medicament useful in the treatment of cancer.