QUINAZOLINE DITOSYLATE ANHYDRATE FORMS

FIELD OF THE INVENTION The present invention relates to anhydrate forms of quinazoline ditosylate salt compounds. In particular, the invention relates to ditosylate salts of 4-quinazolineamines in anhydrate form. These compounds are inhibitors of various protein tyrosine kinases (PTKs) of the erbB family and consequently are useful in the treatment of disorders mediated by aberrant activity of such kinases.

BACKGROUND OF THE INVENTION

PTKs catalyze 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(6), 377-387; R.F. Paulson, Semin. Immunol., 1995, 7(4), 267-277; A.C. Chan, Curr. Opin. Immunol., 1996, 8(3), 394-401). Inappropriate or uncontrolled activation of many PTKs, i.e. aberrant PTK activity, for example by over-expression or mutation, has been shown to result in uncontrolled cell growth.

Aberrant protein tyrosine kinase (PTK) activity has been implicated in a variety of disorders including psoriasis, rheumatoid arthritis, bronchitis, as well as cancer. Development of effective treatments for such disorders is a constant and ongoing enterprise in the medical field. The erbB family of PTKs, which includes c-erbB-2, EGFr, and erbB-4, is one group of PTKs that has attracted interest as a therapeutic target. Currently, of special interest, is the role of erbB family PTKs in hyperproliferative disorders, particularly human malignancies. Elevated EGFr activity has, for example, been implicated in non-small cell lung, bladder, and head and neck cancers. Furthermore, increased c-erbB-2 activity has been implicated in breast, ovarian, gastric and pancreatic cancers. Consequently, inhibition of erbB family PTKs should provide a treatment for disorders characterized by aberrant erbB family PTK activity. The biological role of erbB family PTKs and their implication in various disease states is discussed, for instance in U.S. patent 5,773,476; International Patent Application WO

99/35146; M. C. Hung et al, Seminars in Oncology, 26: 4, Suppl. 12 (August) 1999, 51-59; Ullrich et al, Cell, 61: 203-212, April 20, 1990; Modjtahedi et al, Int'l. J. of Oncology, 13: 335- 342,1998; and J. R. Woodburn, Pharmacol. Ther., 82: 2-3, 241-250, 1999.

Ditosylate salts, including anhydrate form 1 and monohydrate, of N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino}methyl)-2-furyl]-4- quinazolinamine are disclosed in International Patent Application No. PCT/US01 /20706, filed

June 28, 2001 , and published as WO 02/02552 on January 10, 2002. The ditosylate salts of International Patent Application WO 02/02552 may be prepared in crystalline form and possess good moisture sorption properties (low hygroscopicity) and good physical stability. Specific ditosylate salts disclosed were crystalline forms of a monohydrate ditosylate and an anhydrate ditosylate (Form 1).

SUMMARY OF THE INVENTION

The present inventors have now discovered additional crystalline forms of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methan esulphonyl) ethyl]amino}methyl)-2-furyl]-4-quinazolinamine.

In a first aspect of the present invention, there is provided an anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methan esulphonyl)ethyl]amino} methyl) -2-furyl]-4-quinazolinamine in crystalline form, wherein the crystalline form is selected from form 2, 3, 4, 5, 6, 7, and 8.

In a second aspect of the present invention, there is provided an anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methan esulphonyl)ethyl]amino} methyl) -2-furyl]-4-quinazolinamine in crystalline form 2.

In a third aspect of the present invention, there is provided an anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyI)oxy]phenyl}-6-[5-({[2-(methan esulphonyl)ethyI]amino} methyl) -2-furyl]-4-quinazolinamine in crystalline form 3.

In a fourth aspect of the present invention, there is provided an anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methan esulphonyl)ethyl]amino} methyl) -2-furyl]-4-quinazolinamine in crystalline form 4. In a fifth aspect of the present invention, there is provided an anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methan esulphonyl)ethyl]amino} methyl) -2-furyl]-4-quinazolinamine in crystalline form 5.

In a sixth aspect of the present invention, there is provided an anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methan esulphonyl)ethyl]amino} methyl) -2-furyl]-4-quinazolinamine in crystalline form 6.

In a seventh aspect of the present invention, there is provided an anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methan esulphonyl)ethyl]amino} methyl) -2-furyl]-4-quinazolinamine in crystalline form 7.

In a eighth aspect of the present invention, there is provided an anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methan esulphonyl)ethyl]amino} methyl) -2-furyl]-4-quinazolinamine in crystalline form 8.

In a ninth aspect of the present invention, there is provided an anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methan esulphonyl)ethyl]amino} methyl) -2-furyl]-4-quinazolinamine in crystalline form, wherein the crystalline form is selected from form 2, 3, 4, 5, and 6.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 (a) depicts an X-ray powder diffraction pattern of N-{3-Chloro-4-[(3- fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino}methyl)-2-furyl]-4- quinazolinamine ditosylate anhydrate in crystalline Form 2.

Figure 1 (b) depicts an infrared spectrum of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}- 6-[5-({[2-(methanesulphonyl)ethyl] amino}methyl)-2-furyl]-4-quinazolinamine ditosylate anhydrate in crystalline Form 2.

Figure 2(a) depicts an X-ray powder diffraction pattern of N-{3-Chloro-4-[(3- fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino}methyl)-2-furyl]-4- quinazolinamine ditosylate anhydrate in crystalline Form 3.

Figure 2(b) depicts an infrared spectrum of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-

6-[5-({[2-(methanesulphonyl)ethyl] amino}methyl)-2-furyl]-4-quinazolinamine ditosylate anhydrate in crystalline Form 3.

Figure 3(a) depicts an X-ray powder diffraction pattern of N-{3-Chloro-4-[(3- fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino}methyl)-2-furyl]-4- quinazolinamine ditosylate anhydrate in crystalline Form 4.

Figure 3(b) depicts an infrared spectrum of N-{3-Chloro-4-[(3-fluorobenzyl) oxyjphenyl}- 6-[5-({[2-(methanesulphonyl)ethyl] amino}methyl)-2-furyl]-4-quinazolinamine ditosylate anhydrate in crystalline Form 4.

Figure 4(a) depicts an X-ray powder diffraction pattern of N-{3-Chloro-4-[(3- fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl)-2-furyl]-4- quinazolinamine ditosylate anhydrate in crystalline Form 5.

Figure 4(b) depicts an infrared spectrum of N-{3-ChIoro-4-[(3-fluorobenzyl) oxy]phenyl}- 6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl)-2-furyl]-4-quinazolinamine ditosylate anhydrate in crystalline Form 5.

Figure 5(a) depicts an X-ray powder diffraction pattern of N-{3-Chloro-4-[(3- fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl)-2-furyl]-4- quinazolinamine ditosylate anhydrate in crystalline Form 6.

Figure 5(b) depicts an infrared spectrum of N-{3-Chloro-4-[(3-fluorobenzyl) oxyjphenyl}- 6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl)-2-furyl]-4-quinazolinamine ditosylate anhydrate in crystalline Form 6.

Figure 6 depicts a simulated X-ray powder diffraction pattern of N-{3-Chloro-4-[(3- fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl)-2-furyl]-4- quinazolinamine ditosylate anhydrate in crystalline Form 7.

Figure 7 depicts a simulated X-ray powder pattern of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl)-2-furyl]-4-quinazolinamine ditosylate anhydrate in crystalline Form 8.

Figure 8 depicts a simulated X-ray powder pattern of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} crystalline N-methyl-2-pyrrolidinone solvate (Class 1).

Figure 9 depicts an X-ray powder pattern of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-

6-[5-({[2-(methanesulphonyl)ethyl] amino} crystalline N-methyl-2-pyrrolidinone solvate (Class 1).

Figure 10 depicts a simulated X-ray powder pattern of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} crystalline dioxane solvate (Class 1).

Figure 11 depicts a simulated X-ray powder pattern of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} crystalline methanol solvate (Class 1).

Figure 12 depicts a simulated X-ray powder pattern of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} crystalline acetonitrile solvate (Class 1).

Figure 13 depicts a simulated X-ray powder pattern of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} crystalline N,N-dimethylformamide solvate (Class 1).

Figure 14 depicts an X-ray powder pattern of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} crystalline nitrobenzene solvate (Class D-

DETAILED DESCRIPTION OF THE INVENTION 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 "pharmaceutically acceptable salts" means those salts which are non-toxic and that are suitable for manufacturing and formulation as a pharmaceutical entity.

As used herein the term "Class 1 solvates" means those crystalline solvates of N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino}methyl)-2- furyl]-4-quinazolinamine having similar XRPD (isostructural) and are useful intermediates for generating other N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino}methyl)-2-furyl]-4-quinazolinamine crystalline forms. Suitable solvents useful to prepare Class 1 solvates include, but are not limited to, N-methyl-2-pyrrolidinone, dioxane, methanol, acetonitrile, dimethylformamide, and nitrobenzene. As will be apparent to those skilled in the art, said solvents may also be useful as mixtures or in mixture with water.

As used herein, the term "substantially the same X-ray powder diffraction pattern" is understood to mean that those X-ray powder diffraction patterns having diffraction peaks with 2 theta values within plus or minus 0.2° of the diffraction pattern referred to herein are within the scope of the referred to diffraction pattern. In a like manner, the term "at least substantially includes peaks of Table X" (where X is one of Tables 1-15) is understood to mean that those X-ray powder diffraction patterns having diffraction peaks with 2 theta values within plus or minus 0.2° of the subject Table are within the scope of the diffraction pattern referenced to the Table X. Also, in a like manner, the term "at least substantially includes the X-ray powder

diffraction (XRPD) °2θ peaks Q1 , Q2, Q3, ..." (where Q1 , Q2, Q3, ... represent specific listed peak two theta values) is understood to mean that those X-ray powder diffraction patterns having diffraction peaks with 2 theta values within plus or minus 0.2° of the subject listed peak two theta values are within the scope of the subject listed peak 2 theta values. Also, as used herein, the term "substantially the same infrared spectrum" is understood to mean that those infrared spectrum (run according to the method described) having infrared peaks with cm ^"1 values within plus or minus 2 cm ^"1 of the spectrum referred to herein are within the scope of the referred to infrared spectrum.

The present invention includes anhydrate ditosylate salts of N-{3-Chloro-4-[(3- fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl)-2-furyl]-4- quinazolinamine in crystalline forms 2, 3, 4, 5, 6, 7, and 8. In another embodiment, the present invention includes anhydrate ditosylate salts of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6- [5-({[2-(methanesulphonyl)ethyl] amino} methyl)-2-furyl]-4-quinazolinamine in crystalline forms 2, 3, 4, 5, and 6. The compound N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-

(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4-quinazolinamine and is also known as GW572016X or lapatinib and has the structure of Formula (I). (I)

The intermediates, free base, and hydrochloride salt of the compound of Formula (I) may be prepared according to procedures similar to those of International Patent Application No. PCT/EP99/00048, filed January 8, 1999, and published as WO 99/35146 on July 15, 1999.

The ditosylate salts of the compound of Formula (I) may be prepared according to procedures similar to those of International Patent Application No. PCT/US01 /20706, filed June 28, 2001 , and published as WO 02/02552 on January 10, 2002, or according to the procedures of International Patent Application No. PCT/US06/014447, filed April 18, 2006, and published as WO 06/113649 on October 26, 2006

Specific methods for the preparation of the specific anhydrate ditosylate salts of N-{3- ChIoro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2- furyl]-4-quinazolinamine in crystalline form are provided in the Examples following.

In one embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro- 4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4- quinazolinamine is form 2.

In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2- furyI]-4-quinazolinamine is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 1 (a). In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4-quinazolinamine is characterized by substantially the same infrared spectrum shown in Figure 1 (b). In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine is characterized by an X- ray powder diffraction pattern which at least substantially includes the peaks of Table I.

Table I

Pos. [°2Th.] d-spacing [A]

6.0 14.7

9.4 9.4

10.2 8.7

11.9 7.4

12.0 7.4

14.7 6.0

15.2 5.8

16.9 5.3

17.0 5.2

17.5 5.1

17.6 5.0

18.1 4.9

18.7 4.7

19.0 4.7

19.6 4.5

21.5 4.1

22.1 4.0

22.3 4.0

23.3 3.8

24.0 3.7

24.1 3.7

25.0 3.6

^* Based on Cu Ka radiation.

In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3- Chloro-4-[(3-fluorobenzyl) oxy]pheny!}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)~2- furyl]-4-quinazolinamine is characterized by an X-ray powder diffraction pattern which at least substantially includes the X-ray powder diffraction (XRPD) °2θ peaks 6.0, 9.4, 11.9, 16.9 and 24.2.

In one embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-ChIoro- 4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4- quinazolinamine is form 3.

In another embodiment, the crystalline form of the anhydrate ditosyiate salt of N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2- furyl]-4-quinazolinamine is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 2(a). In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyI) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine is characterized by substantially the same infrared spectrum shown in Figure 2(b). In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine is characterized by an X- ray powder diffraction pattern which at least substantially includes the peaks of Table II.

Table H

Pos. [°2Th.] d-spacing [A]

6.4 13.8

7.2 12.3

9.2 9.6

10.3 8.6

11.6 7.6

13.1 6.7

13.7 6.5

14.4 6.2

14.5 6.1

17.0 5.2

17.6 5.0

18.3 4.8

18.5 4.8

19.0 4.7

19.3 4.6

21.7 4.1

22.4 4.0

22.8 3.9

23.3 3.8

24.4 3.6

24.9 3.6

26.0 3.5

28.1 3.2

29.1 3.1

29.8 3.0

* Based on Cu Ka radiation.

In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-

Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphony!) ethyljamino} methy!)-2- furyI]-4-quinazolinamine is characterized by an X-ray powder diffraction pattern which at least substantially includes the X-ray powder diffraction (XRPD) °2θ peaks 6.4, 7.2, 11.6 and 19.3. In one embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro- 4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyI]-4- quinazolinamine is form 4.

In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2- furyl]-4-quinazolinamine is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 3(a). In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furylJ-4-quinazolinamine is characterized by substantially the same infrared spectrum shown in Figure 3(b). In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyljamino} methyl)-2-furylJ-4-quinazolinamine is characterized by an X- ray powder diffraction pattern which at least substantially includes the peaks of Table III.

Table ill

Pos. [°2Th.J d-spacing [A]

4.9 18.2

5.9 14.9

6.3 14.1

6.8 13.0

7.1 12.5

8.9 9.9

10.5 8.4

11.1 8.0

11.7 7.6

12.3 7.2

13.0 6.8

13.4 6.6

13.6 6.5

14.7 6.0

15.2 5.8

16.1 5.5

17.1 5.1

17.8 5.0

18.1 4.9

19.0 4.7

20.0 4.4

20.5 4.3

21.0 4.2

22.4 4.0

22.9 3.9

23.1 3.8

24.3 3.7

24.7 3.6

25.2 3.5

28.6 3.1

* Based on Cu Ka radiation.

In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyI}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2- furyl]-4-quinazolinamine is characterized by an X-ray powder diffraction pattern which at least substantially includes the X-ray powder diffraction (XRPD) °2θ peaks 6.8, 8.9, 12.3 and 20.0.

In one embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro- 4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4- quinazolinamine is form 5.

In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2- furyl]-4-quinazolinamine is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 4(a). In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine is characterized by substantially the same infrared spectrum shown in Figure 4(b). In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyljamino} methyl)-2-furyl]-4-quinazolinamine is characterized by an X- ray powder diffraction pattern which at least substantially includes the peaks of Table IV.

Table IV

Pos. [°2Th.] d-spacing [A]

4.3 20.7

5.5 16.2

6.9 12.9

8.0 11.0

8.5 10.4

9.2 9.7

11.5 7.7

12.0 7.4

12.8 6.9

13.3 6.7

13.7 6.5

14.3 6.2

14.8 6.0

15.4 5.8

16.4 5.4

17.1 5.2

17.9 5.0

18.3 4.8

19.4 4.6

20.2 4.4

21.4 4.2

22.0 4.0

22.5 4.0

23.6 3.8

24.2 3.7

24.6 3.6

25.5 3.5

28.0 3.2

* Based on Cu Ka radiation.

In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-

Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2- furyl]-4-quinazolinamine is characterized by an X-ray powder diffraction pattern which at least substantially includes the X-ray powder diffraction (XRPD) °2θ peaks 4.3, 5.5, 8.0 and 16.4.

In one embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro- 4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesuIphonyl) ethyljamino} methyl)-2-furyl]-4- quinazolinamine is form 6.

In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2- furyl]-4-quinazolinamine is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 5(a). In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyI)

ethyljamino} methyl)-2-furyl]-4-quinazolinamine is characterized by substantially the same infrared spectrum shown in Figure 5(b). In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine is characterized by an X- ray powder diffraction pattern which at least substantially includes the peaks of Table V. Table V

Pos. [°2Th.] d-spacing [A]

4.0 21.9

5.4 16.5

7.0 12.6

8.1 10.9

8.8 10.0

10.2 8.7

11.5 7.7

12.1 7.3

13.3 6.7

14.0 6.3

14.6 6.1

15.1 5.9

16.1 5.5

17.1 5.2

17.6 5.0

18.0 4.9

19.3 4.6

20.2 4.4

21.4 4.1

22.0 4.0

22.7 3.9

24.2 3.7

26.9 3.3

27.5 3.2

28.4 3.1

30.2 3.0

30.7 2.9

32.0 2.8

32.8 2.7

33.9 2.6

36.7 2.5

* Based on Cu Ka radiation.

In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-

Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2- furyl]-4-quinazolinamine is characterized by an X-ray powder diffraction pattern which at least substantially includes the X-ray powder diffraction (XRPD) °2θ peaks 4.0, 5.4, 8.1 , 14.0 and 19.3.

In one embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro- 4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4- quinazolinamine is form 7.

In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2- furyl]-4-quinazolinamine is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 6(a).

In one embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3-Chloro- 4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4- quinazolinamine is form 8.

In another embodiment, the crystalline form of the anhydrate ditosylate salt of N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2- furyl]-4-quinazolinamine is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 7(a). In one embodiment, each of the anhydrate ditosylate salts of N-{3-Chloro-4-[(3- fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4- quinazolinamine described above are pharmaceutically acceptable salts.

In one embodiment, there is provided N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5- ({[2-(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinaz olinamine crystalline Class 1 solvates.

In one embodiment, there is provided N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5- ({[2-(methanesulphonyl)ethyl] amino} methyl) -2-furyl]-4-quinazolinamine crystalline N-methyl-2- pyrrolidinone solvate (Class 1). In another embodiment, N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-

(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazol inamine crystalline N-methyl-2- pyrrolidinone solvate (Class 1) is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 8.

In one embodiment, there is provided N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5- ({[2-(methanesulphonyl)ethyl] amino} methyl) -2-furyl]-4-quinazolinamine crystalline dioxane solvate (Class 1).

In another embodiment, N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2- (methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolina mine crystalline dioxane solvate (Class 1) is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 10.

In one embodiment, there is provided N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5~ ({[2-(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinaz olinamine crystalline methanol solvate (Class 1).

In another embodiment, N-{3-ChIoro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2- (methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolina mine crystalline methanol solvate (Class 1 ) is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 11.

In one embodiment, there is provided N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5- ({[2-(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinaz olinamine crystalline acetonitrile solvate (Class 1).

In another embodiment, N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2- (methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolina mine crystalline acetonitrile solvate (Class 1) is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 12. In one embodiment, there is provided N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-

({[2-(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinaz olinamine crystalline N ₁ N- dimethylformamide solvate (Class 1).

In another embodiment, N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2- (methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolina mine crystalline N ₁ N- dimethylformamide solvate (Class 1 ) is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 13.

In one embodiment, there is provided N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5- ({[2-(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinaz olinamine crystalline nitrobenzene solvate (Class 1). In another embodiment, N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-

(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazol inamine crystalline nitrobenzene solvate (Class 1 ) is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 14.

While it is possible that, for use in therapy, therapeutically effective amounts of each of crystalline forms 2 to 8 of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine may be administered as the raw chemical, it is possible to present each active ingredient in crystalline form as a pharmaceutical composition. Accordingly, the invention further provides pharmaceutical compositions, which include a therapeutically effective amount of a crystalline anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-

(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4-quinazolinamine selected from form 2, 3, 4,

5, 6, 7, and 8 and one or more pharmaceutically acceptable carriers, diluents, or excipients. The carrier(s), diluent(s) or excipient(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. According to another aspect of the invention there is also provided a process for the preparation of a pharmaceutical formulation including admixing a crystalline anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine selected from form 2, 3, 4, 5, 6, 7, and 8, with one or more pharmaceutically acceptable carriers, diluents or excipients.

The crystalline anhydrate ditosylate salts of of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4-quinazolinamine described herein may be formulated for administration by any route, and the appropriate route will depend on the disease being treated as well as the subjects to be treated. Suitable pharmaceutical formulations include those for oral, rectal, nasal, topical (including buccal, sublingual, and transdermal), vaginal or parenteral (including intramuscular, sub-cutaneous, intravenous, and directly into the affected tissue) administration or in a form suitable for administration by inhalation or insufflation. The formulations may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well know in the pharmacy art.

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 agents 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 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 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 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 a 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 crystalline anhydrate ditosylate salts of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}- 6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine, described herein, 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.

The crystalline anhydrate ditosylate salts of N-{3-Chloro-4-[(3-fluorobenzyI) oxyjphenyl}- 6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4-quinazolinamine, as described herein, 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, which may be generated by means of various types of metered, dose pressurized 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.

In one embodiment, there is provided a pharmaceutical composition including a therapeutically effective amount of a crystalline anhydrate ditosylate salt of N-{3-Chloro-4-[(3- fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4- quinazolinamine selected wherein said crystalline salt is selected from forms 2, 3, 4, 5, 6, 7, and 8. In another embodiment, the pharmaceutical composition further includes one or more pharmaceutically acceptable, carriers, diluents and excipients. The crystalline forms of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine and the pharmaceutically acceptable carriers, diluents, and excipients are as described above.

Also provided in the present invention, is a method for treating a disorder in a mammal characterized by aberrant activity of at least one erbB family protein tyrosine kinase (PTK) which includes administering a therapeutically effective amount of a crystalline anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine selected wherein said crystalline salt is selected from forms 2, 3, 4, 5, 6, 7, and 8 to the mammal. The crystalline forms of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyljamino} methyl)-2-furyl]-4-quinazolinamine are as described above.

The aberrant PTK activity referred to herein is any erbB family PTK activity that deviates from the normal erbB family protein kinase activity expected in a particular mammalian subject. Aberrant erbB family PTK activity may take the form of, for instance, an abnormal increase in activity, or an aberration in the timing and or control of PTK activity. Such aberrant activity may result then, for example, from overexpression or mutation of the protein kinase leading to inappropriate or uncontrolled activation. Furthermore, it is also understood that unwanted PTK activity may reside in an abnormal source, such as a malignancy. That is, the level of PTK activity does not have to be abnormal to be considered aberrant, rather the activity derives from an abnormal source. The crystalline forms of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine, are inhibitors of one or more erbB family PTKs and as such have utility in the treatment of disorders in mammals which are characterized by aberrant PTK activity, particularly humans. In one embodiment of the present invention, the disorder treated is characterized by at least one erbB family PTK, selected from EGFr, erbB-2 and erbB-4, exhibiting aberrant activity. In another embodiment, the disorder treated is characterized by at least two erbB family PTKs, selected from EGFr, erbB-2 and erbB-4, exhibiting aberrant activity. In one embodiment of the treatment method, a crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3- fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4- quinazolinamine inhibit at least one erbB family PTK, selected from EGFr, erbB-2 and erbB-4, wherein said crystalline form is selected from form 2, 3, 4, 5, 6, 7, and 8. In another embodiment of the treatment method, a crystalline form of the anhydrate ditosylate salt of N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2- furyl]-4-quinazoIinamine inhibit at least two erbB family PTKs selected from EGFr, c-erb-B2 and c-erb-B4, wherein said crystalline form is selected from form 2, 3, 4, 5, 6, 7, and 8. In one embodiment, there is provided a method of inhibiting at least one of EGFr, erbB-2 and erbB-4 in a mammal, the method including administering a therapeutically effective amount of a crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxyjphenyl}- 6-[5-({[2-(methanesulphonyI) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine wherein said form is selected from forms 2, 3, 4, 5, 6, 7, and 8. In another embodiment, there is provided a method of inhibiting at least two of EGFr, erbB-2 and erbB-4 in a mammal, the method including administering a therapeutically effective amount of a crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine wherein said form is selected from forms 2, 3, 4, 5, 6, 7, and 8.

The disorders referred to may be any disorder which is characterized by aberrant PTK activity. As recited above such disorders include, but are not limited to, cancer and psoriasis. In one embodiment, the disorder is cancer. In another embodiment, the cancer is non-small cell lung, colo-rectal, bladder, prostate, liver, brain, head and neck, breast, renal, cervical, ovarian, gastric, esophageal, colorectal, or pancreatic cancers.

In one embodiment, there is provided a method of treating a cancer in a mammal, said cancer characterized by expression of at least one of EGFR, erbB-2 or erbB-4, comprising: administering to said mammal a therapeutically effective amount of a crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine wherein said form is selected from forms 2, 3, 4, 5, 6, 7, and 8.

In one embodiment, there is provided a method of treating a cancer in a mammal, said cancer characterized by expression of at least two of EGFR, erbB-2 or erbB-4, comprising: administering to said mammal a therapeutically effective amount of a crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-

(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine wherein said form is selected from forms 2, 3, 4, 5, 6, 7, and 8.

In one embodiment, the mammal is a human.

The crystalline forms of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyI]-4-quinazolinamine are as described above.

In one embodiment, there is provided a crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4-quinazolinamine wherein said form is selected from forms 2, 3, 4, 5, 6, 7, and 8 for use in therapy.

The crystalline forms of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4-quinazolinamine are as described above.

The crystalline forms of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-flUorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4-quinazolinamine are useful in therapy and in the preparation of medicaments for treating a disorder in a mammal, which is characterized by aberrant activity of at least one erbB family PTK. In one embodiment of the present invention, the medicament prepared is useful in treating a disorder characterized by at least one erbB family PTK, selected from EGFr, c-erb-B2 and c-erb-B4, exhibiting aberrant activity. In another embodiment, the medicament prepared is useful in treating a

disorder characterized by at least two erbB family PTKs, selected from EGFr, c-erb-B2 and c- erb-B4, exhibiting aberrant activity.

In one embodiment, there is provided use of a crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)-2-furyl]-4-quinazolinamine wherein said form is selected from forms 2, 3, 4, 5, 6, 7, and 8 in the preparation of a medicament useful in treating cancer, said cancer characterized by expression of at least one of EGFR, erbB-2 or erbB-4.

In one embodiment, there is provided use of a crystalline form of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine wherein said form is selected from forms 2, 3, 4, 5, 6, 7, and 8 in the preparation of a medicament useful in treating cancer, said cancer characterized by expression of at least two of EGFR, erbB-2 or erbB-4.

A therapeutically effective amount of crystalline forms of the anhydrate ditosylate salt of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyljamino} methyl)- 2-furyl]-4-quinazolinamine will depend on a number of factors including, but not limited to, the age and weight of the mammal, the precise disorder requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the attendant physician or veternarian.

The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way. The physical data given for the compounds exemplified is consistent with the assigned structure of those compounds.

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 (miliimolar);

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);

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

THF (tetrahydrofuran); DMSO (dimethylsulfoxide);

EtOAc (ethyl acetate); DME (1 ,2-dimethoxyethane); DCM (dichloromethane); DCE (dichloroethane); DMF

(λ/,λ/-dimethylformamide); HOAc (acetic acid);

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

The X-Ray Powder Diffraction (XRPD) analysis shown in the Figures were performed on a Phillips X'pert Pro powder diffractometer, Model PW3040/60, serial number DY1379 using an X'Celerator detector. The acquisition conditions were; radiation: Cu Ka, generator tension: 45 kV, generator current: 4OmA, start angle: 2.0 °2θ, end angle: 40.0 °2θ, step size: 0.0167 °2θ, time per step: 31.75 seconds. The sample was prepared using silicon wafer technique. The 20 or so most intense peaks plus low angle peaks have been included in the preceding Tables I-XVI.

X-ray powder diffraction patterns generated for Example 10 (Fig 9) and Example 15 (Fig. 14) were obtained using a Bruker GADDS diffractometer that is equipped with a Hi-Star area detector. The data collection was carried out at room temperature using monochromatic CuKa radiation in the region of 2θ between 1.5 and 41.5°. The diffraction pattern was collected in two 2θ ranges with an exposure time of 90s for each frame. No background subtraction or curve smoothing is applied to the XRPD patterns.

The Infrared (IR) analyses were performed on a Perkin Elmer infrared spectrometer, model Spectrum One, using a diamond ATR attachment. The acquisition conditions were; number of scans: 16, resolution: 2 cm ^"1 .

As previously recited, the free base N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2- (methane sulphonyl) ethyl]amino}methyl)-2-furyl]-4-quinazolinamine is also know as GW572016X or lapatinib.

Lapatinib ditosylate anhydrate (form 1) and monohydrate were prepared according to methods similar to those disclosed in International Patent Application No. PCT/US01 /20706, filed June 28, 2001 , and published as WO 02/02552 on January 10, 2002,

Single crystal of crystalline anhydrate form 1 was obtained by evaporating a drop of a solution of crystalline anhydrate form 1 in DMSO. Crystal structure data for lapatinib ditosylate anhydrate form 1 was collected on a Bruker Smart CCD 6000 X-ray diffractometer

Empirical formula C ₂₉ H ₂₈ CIFN ₄ O ₄ S ^■ 2C ₇ H ₇ O ₃ S

Formula weight 925.44 Temperature 120(2) K

Wavelength 0.71073 A

Crystal system Triclinic

Space group P -1

Unit cell dimensions a = 9.1440(3) A α= 95.9840(10)°. b = 13.2319(4) A β= 90.8060(10)°. c = 18.1463(6) A γ = 100.4050(10)°.

Volume 2146.48(12) A ³

Z 2

Density (calculated) 1.432 Mg/m ³

Lapatinib free base was prepared as follows. A suspension of lapatinib ditosylate and ethyl acetate was stirred at 22+3°C. An aqueous sodium carbonate solution (10%w/v) was added and the mixture stirred for at least 60 min. The layers were separated and the upper organic layer stirred with water (x 2) at 52+3°C. The layers were separated and the upper organic layer clarified. The contents were cooled to 22±3°C and the bottom aqueous layer is removed. The contents were then concentrated by atmospheric distillation to 9vol. The solution was cooled to 68-70° and seeded with lapatinib free base. The mixture was stirred at 68-70° for at least 15 min to allow crystallisation to establish, then concentrated by atmospheric distillation to 4vol. The mixture was cooled to 22+5° over at least 1h and aged for at least 1h. The product was isolated by filtration and washed with ethyl acetate. The product was dried in vacuo at 45±3°C to give lapatinib free base as a cream solid. Expected yield: 90% theory; 56% w/w.

Example 1

Preparation of amorphous lapatinib ditosylate.

Lapatinib ditosylate monohydrate (1.0 g) was dissolved in 2,2,2-trifluoroethanol (50 ml) at ambient temperature. The solution was concentrated using a rotary evaporator to give a solid residue which was dried in the vacuum oven at 40 ⁰ C for 3 hours. An X-ray powder diffraction was run which indicated the sample was mostly non-crystalline.

Example 2

Preparation of Lapatinib ditosylate anhydrate Form 2

(a) Seed preparation: Lapatinib ditosylate anhydrate Form 1 (~ 0.15 g) (Prepared by the addition p-toluenesulfonic acid to a solution of the free base in a mixture of methanokacetonitrile, 7:3 (40 volumes) at reflux then cooled) However, any preparation of anhydrate form 1 is suitable, i.e. not restricted to using a mixture of methanokacetonitrile, was charged to a 10 ml grinding jar and methanol (0.03 ml) was added. The contents of the jar were ground for 30 minutes by an 8mm grinding ball at 30 Hz using a Retsch mixer mill. An infrared spectrum was run on the resulting solid and was found to be consistent with Anhydrate Form 2.

(b) Preparation of Anhydrate Form 2 by crystallization with seeding: Lapatinib (1.0 g) was heated to reflux in a mixture of methanol (32 ml) and dimethylformamide (8 ml) so that the solid dissolved. P-toluenesulfonic acid monohydrate (0.67 g) was added at this temperature (67°C) and the heat source was removed. When the temperature had dropped to 62°C, 2% w/w anhydrate Form 2 seed (0.032 g), prepared by methods similar to those of Example 1 , was added to the solution. Crystals began to precipitate rapidly and the suspension was allowed to cool to ambient temperature and stirred for a further 1 hour. The product was filtered and dried in the vacuum oven at 40 ⁰ C. Yield 1.15 g (72%) An infrared spectrum was run on the resulting solid and was found to be consistent with Anhydrate Form 2.

(c) Preparation of Anhydrate Form 2 by seeded recrystallisation of lapatinib ditosylate monohydrate: Lapatinib ditosylate monohydrate (10.0 g) was heated to reflux in a mixture of methanol (330 ml) and dimethylformamide (70 ml) and the solution was stirred at reflux temperature (67°C) for 5 minutes. The heat source was switched off but not removed so that the solution cooled slowly. When the temperature had dropped to 62°C, 2%w/w anhydrate Form 2 seed (0.2 g) was added to the solution. Crystals began to precipitate gradually and below 55°C the heat source was removed completely so that the suspension was allowed to cool naturally and left to stand at ambient temperature overnight. The product was easily filtered and dried in the vacuum oven at 40-45 ⁰ C for 6 hours. Yield 7.34 g (73%). An X-ray powder diffraction pattern and an infrared spectrum were obtained and are depicted in Figure 1 (a) and Figure 1 (b) respectively.

(d) Single crystal structure data

Single crystal of crystalline anhydrate form 2 was obtained by recrystallisation of the monohydrate form prepared by methods similar to those disclosed in International Patent Application No. PCT/US01 /20706, filed June 28, 2001 , and published as WO 02/02552 on January 10, 2002, from DMF containing a small amount of acetonitrile as an antisolvent.

Crystal structure data for anhydrate Form 2 was collected on a Bruker Smart CCD 6000 X-ray diffractometer

Empirical formula C ₂₉ H ₂₈ CIFN ₄ O ₄ S ^■ 2C ₇ H ₇ O ₃ S Formula weight 925.44

Temperature 223(2) K

Wavelength 0.71073 A

Crystal system Triclinic

Space group P -1 Unit cell dimensions a = 9.5074(4) A α= 101.897(2)°. b = 15.4852(6) A β= 99.888(2)°.

C = 15.5537(7) A Y = 100.911(2)°.

Volume 2146.61 (16) A ³

Z 2 Density (calculated) 1.432 Mg/m ³

Example 3

Preparation of Lapatinib ditosylate anhydrate Form 3

Lapatinib free base (1.0 g) was heated in a mixture of 1 ,4-dioxane (36 ml) and water (4 ml) until the solid dissolved. P-toluenesulfonic acid monohydrate (0.66 g) was added at this temperature and the solution was allowed to cool. Crystals were precipitated and the suspension was stirred at ambient temperature for a further 1 hour. The product was filtered and dried in the vacuum oven at 60°C for 24 hours. Yield 0.96 g (60%). An X-ray powder diffraction pattern and an infrared spectrum were obtained and are depicted in Figure 2(a) and Figure 2(b) respectively.

Example 4

Preparation of Lapatinib ditosylate anhydrate Form 4

(a) A mixture of Lapatinib free base (1.0 g) and p-toluenesulfonic acid monohydrate (0.66 g) was stirred in dimethylformamide (2 ml) at ambient temperature. Initially the solids dissolved but in less than 2 minutes crystals were rapidly precipitated and amassed so that more dimethylformamide (2 ml) had to be added to mobilise. The suspension was stirred at

ambient temperature for 30 minutes then the product was filtered, washed with dimethylformamide (2 x 2 ml) and dried in the vacuum oven at 60 ⁰ C for 2 hours. An X-ray powder diffraction pattern and an infrared spectrum were obtained and are depicted in Figure 3(a) and Figure 3(b) respectively. (b) Alternative Preparation of Lapatinib ditosylate Anhydrate Form 4: Lapatinib ditosylate anhydrate form 1 (1.0 g) was heated in dimethylformamide (4 ml) until the solid dissolved and the solution was stirred at high temperature for several minutes. The solution was allowed to cool gradually and crystals were precipitated. The thick suspension was left to stand at ambient temperature overnight then the solid was filtered, washed with dimethylformamide and dried under vacuum for 3 hours. The X-ray powder diffraction pattern and the infrared spectrum were different to that obtained for Example 4a. The NMR was consistent but contained 19%w/w dimethylformamide. The sample was dried further in the vacuum oven at 45°C for 6 hours. Another X-ray powder diffraction, an infrared spectrum, and an NMR were obtained and were consistent with Form 4.

Example 5

Preparation ofLapatinib ditosylate anhydrate Form 5

Amorphous lapatinib ditosylate (1.0 g), prepared according to methods similar to those of Example 1 , was slurried in acetonitrile (10 ml) under argon for 2 hours at ambient temperature. More acetonitrile (5 ml) was added to mobilise and the solid was filtered, washed with acetonitrile and dried under vacuum for 24 hours. An X-ray powder diffraction pattern and an infrared spectrum were obtained and are depicted in Figure 4(a) and Figure 4(b) respectively.

Example 6

Preparation of Lapatinib ditosylate anhydrate Form 6

(a) Amorphous lapatinib ditosylate (0.6 g) was slurried in acetic acid (2 ml) for 2 hours at ambient temperature. More acetic acid (2 ml) was added to mobilise and the solid was filtered, washed with acetic acid and dried in the vacuum oven at 50°C for 24 hours. An X-ray powder diffraction pattern and an infrared spectrum were obtained and are depicted in Figure 5(a) and Figure 5(b) respectively.

(b) Alternative preparation of Lapatinib ditosylate Anhydrate Form 6: Lapatinib free base (1.0 g) was stirred in acetic acid (10 ml) at ambient temperature so that the solid dissolved. P-toluenesulfonic acid monohydrate (0.67 g) was added and crystals were precipitated in less than 2 minutes. The resulting suspension was stirred for 2 hours at ambient temperature then the solid was filtered, washed with acetic acid and dried under vacuum for 3

hours. An X-ray powder diffraction pattern, an infrared spectrum, and NMR were obtained and the X-ray powder diffraction pattern and an infrared spectrum were different. The NMR was consistent but contained 11.4%w/w acetic acid. The sample was dried further in the vacuum oven at 60 ⁰ C for 24 hours. An X-ray powder diffraction, an infrared spectrum, and an NMR were obtained and were consistent with Form 6.

Example 7

Preparation ofLapatinib ditosylate anhydrate Form 7

A single crystal of anhydrate form 2 was mounted on a Bruker Smart CCD 6000 X-ray diffractometer equipped with an Oxford cryosystems cryostream LT device and cooled to - 103 ⁰ C. Single crystal data were collected and a simulated X-ray powder pattern is depicted in Figure 6 and single crystal structure data follows.

Single crystal structure data

Empirical formula C ₂₉ H ₂₈ CIFN ₄ O ₄ S ^■ 2C ₇ H ₇ O ₃ S

Formula weight 925.44

Temperature 170(2) K

Wavelength 0.71073 A

Crystal system Triclinic

Space group P -1

Unit cell dimensions a = 16.4662(9) A α= 112.707(2)°. b = 16.8302(9) A β= 111.695(2)°.

C = 18.3787(11) A Y = 94.075(2)°.

Volume 4227.7(4) A ³

Z 4

Density (calculated) 1.454 Mg/m ³

Example 8

Preparation ofLapatinib ditosylate anhydrate Form 8

A single crystal of anhydrate form 1 , prepared by methods similar to those disclosed in International Patent Application No. PCT/US01 /20706, filed June 28, 2001 , and published as WO 02/02552 on January 10, 2002, was mounted on a Bruker Smart CCD 6000 X-ray diffractometer equipped with an Oxford cryosystems cryostream LT device and heated to 137°C. Single crystal data were collected and a simulated X-ray powder pattern is depicted in Figure 7.

Example 9

Preparation ofLapatinib ditosylate N-methyl-2-pyrrolidinone solvate (Class 1)

A portion of Lapatinib ditosylate monohydrate was dissolved in a small amount of NMP at room temperature. N-methyl-2-pyrrolidinone solvate crystallised immediately on toluene addition as an antisolvent (N-methyl-2-pyrrolidinone:toluene ratio 1 :3). Single crystal data were collected at -153 ⁰ C using a Bruker SMART CCD 6000 X-ray diffractometer equipped with an Oxford cryosystems cryostream LT device and a simulated X-ray powder pattern is depicted in Figure 8 and single crystal structure data follows.

Example 10

Preparation of Lapatinib ditosylate N-methyl-2-pyrrolidinone solvate (Class 1)

Lapatinib ditosylate monohydrate (445.9 mg) was slurried in N-methyl-2-pyrrolidinone (1.4 ml) to give Lapatinib ditosylate N-methyl-2-pyrrolidinone solvate. An X-ray powder diffraction pattern was obtained and is depicted in Figure 9.

Example 11

Preparation of Lapatinib ditosylate dioxane solvate (Class 1)

A portion of Lapatinib ditosylate monohydrate was dissolved in a mixture of acetonitrile and dioxane 4:1 at 9O ⁰ C and cooled down slowly (5°C per hour) to room temperature. Dioxane solvate crystallised on cooling. Single crystal data were collected at -153 ⁰ C using a Bruker SMART CCD 6000 X-ray diffractometer equipped with an Oxford cryosystems cryostream LT device and a simulated X-ray powder pattern is depicted in Figure 10 and single crystal structure data follows.

Single crystal structure data

Empirical formula C ₂₉ H ₂₈ CIFN ₄ O ₄ S ^■ 2C ₇ H ₇ O ₃ S ^■ C ₄ H ₈ O ₂

Formula weight 1013.54

Temperature 120(2) K Wavelength 0.71073 A

Crystal system Triclinic

Space group P -1

Unit cell dimensions a = 9.0316(18) A α= 77.58(3)°. b = 12.968(3) A β= 79.45(3)°. c = 21.325(4) A Y = 78.86(3)°.

Volume 2367.0(8) A ³

Z 2

Density (calculated) 1.422 Mg/m ³

Example 12

Preparation of Lapatinib ditosylate methanol solvate (Class 1)

A small portion of Lapatinib ditosylate monohydrate was dissolved in boiling methanol and cooled at room temperature. Methanol solvate readily crystallised on dissolution of Lapatinib ditosylate monohydrate. Single crystal data were collected at -153 ⁰ C using a Bruker SMART CCD 6000 X-ray diffractometer equipped with an Oxford cryosystems cryostream LT device and a simulated X-ray powder pattern is depicted in Figure 11 and single crystal structure data follows.

Single crystal structure data

Empirical formula C ₂₉ H ₂₈ CIFN ₄ O ₄ S ^■ 2C ₇ H ₇ O ₃ S ^■ 1.25CH ₃ OH

Formula weight 960.48

Temperature 120(2) K

Wavelength 0.71073 A

Crystal system Triclinic

Space group P -1

Unit cell dimensions a = 9.1073(5) A α= 85.140(2)°. b = 12.6966(7) A β= 84.707(2)°. c = 20.5178(11) A Y = 79.009(2)°.

Volume 2313.6(2) A ³

Z 2

Density (calculated) 1.379 Mg/m ³

Example 13

Preparation of Lapatinib ditosylate acetonitrile solvate (Class 1)

A small portion of Lapatinib ditosylate monohydrate was dissolved in boiling acetonitrile and cooled at room temperature. Acetonitrile solvate readily crystallised on dissolution of Lapatinib ditosylate monohydrate. Single crystal data were collected at -153 ⁰ C using a Bruker SMART CCD 6000 X-ray diffractometer equipped with an Oxford cryosystems cryostream LT device and a simulated X-ray powder pattern is depicted in Figure 12.

Example 14

Preparation of Lapatinib ditosylate N,N-dimethylformamide solvate (Class 1)

A portion of Lapatinib ditosylate monohydrate was dissolved in a small amount of N ₁ N- dimethylformamide at 90°C. The solution was cooled to room temperature, seeded with methanol solvate crystals and left overnight. Single crystal data were collected at -153 ⁰ C using

a Bruker SMART CCD 6000 X-ray diffractometer equipped with an Oxford cryosystems cryostream LT device and a simulated X-ray powder pattern is depicted in Figure 13.

Example 15

Preparation of Lapatinib ditosylate nitrobenzene solvate (Class 1)

Nitrobenzene (5 ml) was added to Lapatinib ditosylate monohydrate. The slurry was kept at 100 ⁰ C for 1.5 hours in the oven without stirring, then removed from the oven and cooled to room temperature. The solid was collected by filtration, then left on the filter to dry for 4 days. An X-ray powder diffraction pattern was obtained and is depicted in Figure 14.

Single crystal structure data

Empirical formula C ₂₉ H ₂₈ CIFN ₄ O ₄ S ^■ 2C ₇ H ₇ O ₃ S ^• C ₅ H ₇ NO

Formula weight 1024.57

Temperature 120(2) K

Wavelength 0.71073 A

Crystal system Triclinic

Space group P -1

Unit cell dimensions a = 9.1718(3) A α= 80.504(2)° b = 12.8831 (5) A β= 79.901 (2)° c = 21.0578(8) A γ= 79.565(2)°

Volume 2386.24(15) A ³

Z 2

Density (calculated) 1.426 Mg/m ³

Biological Data

GW572016X has been tested for erbB family protein tyrosine kinase inhibitory activity in substrate phosphorylation assays and cell proliferation assays. See International Patent Application PCT/EP99/00048 filed January 8, 1999, and published as WO 99/35146 on July 15, 1999. The salts of the present invention may be tested for erbB family protein tyrosine kinase inhibitory activity in substrate phosphorylation assays and cell proliferation assays as follows.

Substrate Phosphorylation Assay

The substrate phosphorylation assays use baculovirus expressed, recombinant constructs of the intracellular domains of c-erbB-2 and c-erbB-4 that are constitutively active and EGFr isolated from solubilised A431 cell membranes. The method measures the ability of

the isolated enzymes to catalyse the transfer of the g-phosphate from ATP onto tyrosine residues in a biotinylated synthetic peptide (Biotin-GluGluGluGluTyrPheGluLeuVal). Substrate phosphorylation was detected following either of the following two procedures: a.) c-ErbB-2, c-ErbB4 or EGFr were incubated for 30 minutes, at room temperature, with 10mM MnCl2, 10mM ATP, 5 mM peptide, and test compound (diluted from a 5mM stock in DMSO, final DMSO concentration is 2%) in 4OmM HEPES buffer, pH 7.4. The reaction was stopped by the addition of EDTA (final concentration 0.15mM) and a sample was transferred to a streptavidin-coated 96-well plate. The plate was washed and the level of phosphotyrosine on the peptide was determined using a Europium-labelled antiphosphotyrosine antibody and quantified with a time-resolved fluorescence technique. b.) ErbB2 was incubated for 50 minutes at room temperature with 15 mM MnCI2, 2

33 mM ATP, 0.25 mCi [g- P] ATP/well, 5 mM peptide substrate, and test compound (diluted from a 1OmM stock in DMSO, final DMSO concentration is 2%) in 50 mM MOPS pH 7.2. The reaction was terminated by the addition of 200 ml of PBS containing 2.5 mg/ml streptavidin- coated SPA beads (Amersham Inc.), 50 mM ATP, 10 mM EDTA and 0.1%TX-100. The microtitre plates were sealed and SPA beads were allowed to settle for at least six hours. The SPA signal was measured using a Packard Topcount 96-well plate scintillation counter (Packard Instrument Co., Meriden, CT).

Results for GW572016X are shown in Table XVIII for EGFR, erbB2, and erbB4 tyrosine kinase inhibition. The structure of the free base (GW572016X) is given.

Table XVIII

Cellular assays: Methylene Blue Growth Inhibition Assay

Human breast (BT474), head and neck (HN5) and gastric tumor (N87) cell lines and human foreskin Fibroblasts (HFF) were cultured in low glucose DMEM (Life Technologies 12320-032) containing 10% fetal bovine serum (FBS) at 37 ⁰ C in a humidified 10% CO ₂ , 90% air incubator. The SV40 transformed human mammary epithelial cell line HB4a was transfected with either human H-ras cDNA (HB4a r4.2) or the human c-erbB2 cDNA (HB4a c5.2). The HB4a clones were cultured in RPMI containing 10% FBS, insulin (5 μg/ml), hydrocortisone (5 μg/ml), supplemented with the selection agent hygromycin B (50μg/ml). Cells were harvested using trypsin/EDTA, counted using a haemocytometer, and plated in 100 ml of the appropriate media, at the following densities, in a 96-well tissue culture plate (Falcon 3075): BT474 10,000 cells/well, HN5 3,000 cells/well, N87 10,000 ceils/well, HB4a c5.2 3,000 cells/well, HB4a r4.2 3,000 cells/well, HFF 2500 cells/well. The next day, compounds were diluted in DMEM containing 100 mg/ml gentamicin, at twice the final required concentration, from 1OmM stock solutions in DMSO. 100ml/well of these dilutions were added to the 100ml of media currently on the cell plates. Medium containing 0.6% DMSO was added to control wells. Compounds diluted in DMEM were added to all cell lines, including the HB4a r4.2 and HB4a c5.2 cell lines.

The final concentration of DMSO in all wells was 0.3%. Cells were incubated at 37 ⁰ C, 10% CO ₂ for 3 days. Medium was removed by aspiration. Cell biomass was estimated by staining cells with 100μl per well methylene blue (Sigma M9140, 0.5% in 50:50 ethanol:water), and incubation at room temperature for at least 30 minutes. Stain was removed, and the plates rinsed under a gentle stream of water, and air-dried. To release stain from the cells 100μl 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 cell growth (IC ₅₀ ) was interpolated using nonlinear regression (Levenberg-Marquardt) and the equation, y = V _max *(1- (x/(K+x))) + Y2, where "K" was equal to the IC ₅₀ .

Table XIX illustrates the inhibitory activity of GW572016X as IC ₅₀ values in μM against a range of tumor cell lines. Using HFF as a representative human normal cell line, values for cytotoxicity are supplied as IC50 values in micromolar. A measure of selectivity between normal and tumor lines is provided as well.

Table XIX