WO2009079547A1 - Quinazoline anhydrate forms - Google Patents

Quinazoline anhydrate forms Download PDF

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Publication number
WO2009079547A1
WO2009079547A1 PCT/US2008/087135 US2008087135W WO2009079547A1 WO 2009079547 A1 WO2009079547 A1 WO 2009079547A1 US 2008087135 W US2008087135 W US 2008087135W WO 2009079547 A1 WO2009079547 A1 WO 2009079547A1
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WO
WIPO (PCT)
Prior art keywords
methyl
ethyl
quinazolinamine
methanesulphonyl
fluorobenzyl
Prior art date
Application number
PCT/US2008/087135
Other languages
French (fr)
Inventor
Andrew Simon Craig
David M. Crowe
Michael Millan
Original Assignee
Smithkline Beecham (Cork) Limited
Smithkline Beecham Corporation
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Application filed by Smithkline Beecham (Cork) Limited, Smithkline Beecham Corporation filed Critical Smithkline Beecham (Cork) Limited
Publication of WO2009079547A1 publication Critical patent/WO2009079547A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention relates to crystalline forms of free base quinazoline compounds.
  • the invention relates to the free base of 4-quinazolineamines in crystalline anhydrate forms.
  • 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.
  • PTKs protein tyrosine kinases
  • PTKs catalyze the phosphorylation of specific tyrosyl residues in various proteins involved in the regulation of cell growth and differentiation.
  • PTK protein tyrosine kinase
  • N- ⁇ 3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl ⁇ -6-[5-( ⁇ [2-(methanesulphonyl)ethyl] amino ⁇ methyl)-2-furyl]-4-quinazolinamine is disclosed in International Patent Application No. PCT/EP99/00048, filed January 8, 1999, and published as WO 99/35146 on July 15, 1999.
  • 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 crystalline anhydrate Form 1.
  • 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 crystalline anhydrate Form 1.
  • 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 crystalline anhydrate Form 2.
  • 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 crystalline anhydrate Form 2.
  • Figure 3 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 crystalline 1 ,4-dioxane solvate (Class 1 ).
  • Figure 4 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 crystalline pyridine solvate (Class 1 ).
  • Figure 5 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 N-methyl-2-pyrrolidinone solvate.
  • 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.
  • 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.
  • pharmaceutically acceptable salts means those salts which are non-toxic and that are suitable for manufacturing and formulation as a pharmaceutical entity.
  • 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, tetrahydrofuran, trifluoroethanol/water mixture, dioxane, pyridine, cyclohexanone, dimethylformamide, dichloromethane, chloroform, acetonitrile, and acetone. As will be apparent to those skilled in the art, said solvents may also be useful as mixtures or in mixture with water.
  • 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.
  • 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.
  • 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 N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5- ( ⁇ [2-(methanesulphonyl)ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine crystalline anhydrate in forms 1 and 2.
  • the present invention also includes N- ⁇ 3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl ⁇ -6-[5-( ⁇ [2-(methanesulphonyl)ethyl] amino ⁇ methyl)-2-furyl]-4- quinazolinamine crystalline Class 1 solvates.
  • the intermediates, free base, and hydrochloride salts 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, referred to above.
  • the ditosylate salt 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
  • N- ⁇ 3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl ⁇ -6-[5-( ⁇ [2- (methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine crystalline anhydrate is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 1 (a).
  • N- ⁇ 3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl ⁇ -6-[5-( ⁇ [2-(methanesulphonyl)ethyl]amino ⁇ methyl)-2-furyl]-4- quinazolinamine crystalline anhydrate is characterized by substantially the same infrared spectrum shown in Figure 1 (b).
  • N- ⁇ 3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl ⁇ -6-[5- ( ⁇ [2-(methanesulphonyl)ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine crystalline anhydrate is characterized by substantially the same infrared spectrum shown in Figure 2(b).
  • N- ⁇ 3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl ⁇ -6-[5-( ⁇ [2- (methanesulphonyl)ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine crystalline 1 ,4- dioxane solvate (Class 1 ) is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 3.
  • 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 is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 5.
  • each of the crystalline forms of N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2- (methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine described above may be administered as the raw chemical, it is possible to present each active ingredient in crystalline form as a pharmaceutical composition.
  • the invention further provides pharmaceutical compositions, which include a therapeutically effective amount of a crystalline form of N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2- (methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine described herein 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.
  • a process for the preparation of a pharmaceutical formulation including admixing a crystalline form of N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2-(methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine described herein with one or more pharmaceutically acceptable carriers, diluents or excipients.
  • the crystalline forms of N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2- (methanesulphonyl) ethyl]amino ⁇ 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, sub-lingual, 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.
  • compositions 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.
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • 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.
  • 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.
  • 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
  • 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.
  • a binder such as syrup, starch paste, acadia mucilage or solutions of cellulosic or polymeric materials and forcing through a screen.
  • 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
  • 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.
  • 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 forms 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 forms of N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2- (methanesulphonyl) ethyl]amino ⁇ 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.
  • 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.
  • compositions 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.
  • the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3(6), 318 (1986).
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • the formulations are preferably applied as a topical ointment or cream.
  • the active ingredient may be employed with either a paraffinic or a water- miscible ointment base.
  • the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • compositions 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.
  • compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.
  • compositions adapted for rectal administration may be presented as suppositories or as enemas.
  • compositions 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.
  • Fine particle dusts or mists which may be generated by means of various types of metered, dose pressurized aerosols, nebulizers or insufflators.
  • compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
  • compositions 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.
  • 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.
  • a pharmaceutical composition including a therapeutically effective amount of a crystalline form of N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2-(methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine as described herein.
  • the pharmaceutical composition further includes one or more pharmaceutically acceptable, carriers, diluents and excipients.
  • 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 form of N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2- (methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine, as described herein, to the mammal.
  • PTK erbB family protein tyrosine kinase
  • 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 over expression or mutation of the protein kinase leading to inappropriate or uncontrolled activation.
  • 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 N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2- (methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine described herein, 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.
  • the disorder treated is characterized by at least one erbB family PTK, selected from EGFr, erbB-2 and erbB-4, exhibiting aberrant activity.
  • the disorder treated is characterized by at least two erbB family PTKs, selected from EGFr, erbB-2 and erbB-4, exhibiting aberrant activity.
  • a crystalline form of N- ⁇ 3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2-(methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine inhibits at least one erbB family PTK, selected from EGFr, erbB-2 and erbB-4.
  • a crystalline form of N- ⁇ 3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl ⁇ -6-[5-( ⁇ [2-(methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine inhibits at least two erbB family PTKs selected from EGFr, c-erb-B2 and c-erb-B4.
  • a method of inhibiting at least one of EGFr, erbB-2 and erbB-4 in a mammal including administering a therapeutically effective amount of a crystalline form of N- ⁇ 3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2-(methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine.
  • a method of inhibiting at least two of EGFr, erbB-2 and erbB-4 in a mammal including administering a therapeutically effective amount of a crystalline form of N- ⁇ 3-Chloro-4- [(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2-(methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]- 4-quinazolinamine.
  • the crystalline forms are as described herein.
  • the disorder includes, but are not limited to, cancer and psoriasis.
  • the disorder is cancer.
  • 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.
  • a method of treating a cancer in a mammal comprising: administering to said mammal a therapeutically effective amount of a crystalline form of N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2- (methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine as described herein.
  • a method of treating a cancer in a mammal comprising: administering to said mammal a therapeutically effective amount of a crystalline form of N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2-
  • the crystalline forms of N- ⁇ 3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl ⁇ -6-[5-( ⁇ [2- (methanesulphonyl) ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine described herein 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.
  • 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.
  • 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.
  • a therapeutically effective amount of crystalline forms of N- ⁇ 3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl ⁇ -6-[5-( ⁇ [2-(methanesulphonyl)ethyl]amino ⁇ 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 veterinarian.
  • L liters
  • ml. milliliters
  • ⁇ l_ 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 ⁇ /, ⁇ /-dimethylformamide
  • HOAc acetic acid
  • the X-Ray Powder Diffraction (XRPD) analysis shown in the Figures were performed on a Phillips X'pert Pro powder diffracto meter, 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 °2 ⁇ , end angle: 40.0 °2 ⁇ , step size: 0.0167 °2 ⁇ , 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.
  • 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 .
  • N- ⁇ 3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl ⁇ -6- [5-( ⁇ [2-(methanesulphonyl)ethyl]amino ⁇ methyl)-2-furyl]-4-quinazolinamine is also known as GW572016X or lapatinib.
  • Lapatinib ditosylate 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, Example 1
  • 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 was stirred with water (x 2) at 52 ⁇ 3°C.
  • the layers were separated and the upper organic layer was clarified.
  • the contents were cooled to 22 ⁇ 3°C and the bottom aqueous layer was removed.
  • the contents were then concentrated by atmospheric distillation to 9 vol.
  • the solution was cooled to 68-70° and seeded with lapatinib.
  • the mixture was stirred at 68- 70° for at least 15 min to allow crystallisation to establish, then concentrated by atmospheric distillation to 4vol.
  • 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:
  • c-ErbB-2, c-ErbB4 or EGFr were incubated for 30 minutes, at room temperature, with 1 OmM MnCl2, 1OmM 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.
  • 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.
  • 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).
  • HB4a clones were cultured in RPMI containing 10% FBS, insulin (5 ⁇ g/ml), hydrocortisone (5 ⁇ g/ml), supplemented with the selection agent hygromycin B (50 ⁇ g/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 cells/well, HB4a c5.2 3,000 cells/well, HB4a r4.2 3,000 cells/well, HFF 2500 cells/well.
  • Cell biomass was estimated by staining cells with 100 ⁇ l per well methylene blue (Sigma M9140, 0.5% in 50:50 ethanokwater), 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 10O ⁇ l 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.
  • solubilization solution 1% N-lauroyl sarcosine, Sodium salt, Sigma L5125, in PBS
  • Table XIX illustrates the inhibitory activity of GW572016X as IC 50 values in ⁇ M against a range of tumor cell lines.
  • 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.

Abstract

Crystalline forms of 4-quinazolineamines are described as well as methods of using the same in the treatment of disorders characterized by aberrant erbB family PTK activity.

Description

QUINAZOLINE ANHYDRATE FORMS
FIELD OF THE INVENTION
The present invention relates to crystalline forms of free base quinazoline compounds. In particular, the invention relates to the free base of 4-quinazolineamines in crystalline anhydrate forms. 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, 97-1 11 ; 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.
N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino}methyl)-2-furyl]-4-quinazolinamine is disclosed in International Patent Application No. PCT/EP99/00048, filed January 8, 1999, and published as WO 99/35146 on July 15, 1999.
SUMMARY OF THE INVENTION
The present inventors have now discovered crystalline anhydrate forms of N-{3- Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino}methyl)- 2-furyl]-4-quinazolinamine. Also, discovered are a series of crystalline isostructural solvates of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino}methyl)-2-furyl]-4-quinazolinamine hereinafter referred to as Class 1 solvates. In addition, crystalline N-methyl-2-pyrrolidinone solvate of N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino}methyl)-2-furyl]-4- quinazolinamine has also been discovered.
In a first aspect of the present invention, there is provided N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl] amino}methyl)-2-furyl]-4- quinazolinamine crystalline anhydrate Form 1 or 2.
In a second aspect of the present invention, there is provided N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl] amino} methyl) -2-furyl]-4- quinazolinamine crystalline anhydrate Form 1.
In a third aspect of the present invention, there is provided N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl) -2-furyl]-4- quinazolinamine crystalline anhydrate Form 2. In a fourth aspect of the present invention, there is provided N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl) -2-furyl]-4- quinazolinamine crystalline Class 1 solvates.
In a fifth aspect of the present invention, 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.
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 crystalline anhydrate Form 1.
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 crystalline anhydrate Form 1.
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 crystalline anhydrate Form 2.
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 crystalline anhydrate Form 2.
Figure 3 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 crystalline 1 ,4-dioxane solvate (Class 1 ). Figure 4 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 crystalline pyridine solvate (Class 1 ).
Figure 5 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 N-methyl-2-pyrrolidinone solvate.
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, tetrahydrofuran, trifluoroethanol/water mixture, dioxane, pyridine, cyclohexanone, dimethylformamide, dichloromethane, chloroform, acetonitrile, and acetone. 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, 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 N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5- ({[2-(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine crystalline anhydrate in forms 1 and 2. The present invention also includes N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl)-2-furyl]-4- quinazolinamine crystalline Class 1 solvates. In addition, is N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl)-2-furyl]-4- quinazolinamine N-methyl-2-pyrrolidinone solvate.
The compound N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine and is also known as GW572016X or lapatinib and has the structure of Formula (I).
(I)
Figure imgf000007_0001
The intermediates, free base, and hydrochloride salts 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, referred to above.
The ditosylate salt 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 crystalline anhydrate, Class 1 solvate, and other solvate forms of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine are provided in the Examples following.
In one embodiment, provided is N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-
({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine crystalline anhydrate Form 1.
In another embodiment, N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine crystalline anhydrate is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 1 (a). In another embodiment, N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl]amino} methyl)-2-furyl]-4- quinazolinamine crystalline anhydrate is characterized by substantially the same infrared spectrum shown in Figure 1 (b). In another embodiment, provided is N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}- 6-[5-({[2-(methanesulphonyl)ethyl]amino} methyl)-2-furyl]-4-quinazolinamine crystalline anhydrate Form 2.
In another embodiment, N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-
(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine crystalline anhydrate is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 2(a). In another embodiment, N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5- ({[2-(methanesulphonyl)ethyl]amino} methyl)-2-furyl]-4-quinazolinamine crystalline anhydrate is characterized by substantially the same infrared spectrum shown in Figure 2(b).
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 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 1 ,4-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-quinazolinamine crystalline 1 ,4- dioxane solvate (Class 1 ) is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 3.
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 pyridine solvate (Class 1 ).
In another embodiment, N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-
(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine crystalline pyridine solvate (Class 1 ) is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 4. 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.
In another embodiment, 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 is characterized by substantially the same X-ray powder diffraction pattern shown in Figure 5.
While it is possible that, for use in therapy, therapeutically effective amounts of each of the crystalline forms of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine described above 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 form of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine described herein 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 form of N- {3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine described herein with one or more pharmaceutically acceptable carriers, diluents or excipients.
The crystalline forms of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} 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, sub-lingual, 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 forms 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 forms of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} 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 form of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine as described herein. In another embodiment, the pharmaceutical composition further includes one or more pharmaceutically acceptable, carriers, diluents and excipients.
The crystalline forms 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 form of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine, as described herein, to the mammal.
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 over expression 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 N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine described herein, 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 N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine inhibits at least one erbB family PTK, selected from EGFr, erbB-2 and erbB-4. In another embodiment of the treatment method, a crystalline form of N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine inhibits at least two erbB family PTKs selected from EGFr, c-erb-B2 and c-erb-B4. 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 N-{3- Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine. 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 N-{3-Chloro-4- [(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]- 4-quinazolinamine. The crystalline forms are as described herein.
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 N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine as described herein.
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 N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2-
(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4-quinazolinamine as described herein.
In one embodiment, there is provided a crystalline form of N-{3-Chloro-4-[(3- fluorobenzyl) oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl]amino} methyl)-2-furyl]-4- quinazolinamine, as described above, for use in therapy.
The crystalline forms of N-{3-Chloro-4-[(3-fluorobenzyl) oxy]phenyl}-6-[5-({[2- (methanesulphonyl) ethyl]amino}methyl)-2-furyl]-4-quinazolinamine described herein 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 N-{3-Chloro-4-
[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl]amino} methyl)-2-furyl]-4- quinazolinamine, as described herein, 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 N-{3-Chloro-4-
[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl]amino} methyl)-2-furyl]-4- quinazolinamine, as described herein, 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 N-{3-Chloro-4-[(3- fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl]amino}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 veterinarian.
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 (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); 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 0C (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 diffracto meter, 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.
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-(methanesulphonyl)ethyl]amino}methyl)-2-furyl]-4-quinazolinamine is also known as GW572016X or lapatinib.
Lapatinib ditosylate 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, Example 1
Preparation of crystalline Lapatinib anhydrate Form 1
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 was stirred with water (x 2) at 52±3°C. The layers were separated and the upper organic layer was clarified. The contents were cooled to 22±3°C and the bottom aqueous layer was removed. The contents were then concentrated by atmospheric distillation to 9 vol. The solution was cooled to 68-70° and seeded with lapatinib. 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 1 hour and aged for at least 1 hour. The product was isolated by filtration and washed with ethyl acetate. The product was dried in vacuo at 45±3°C to give GW572016X as a cream solid. Yield: 56% w/w. An X-ray powder diffraction pattern and an infrared spectrum were obtained and are depicted in Figure 1 (a) and Figure 1 (b) respectively.
Example 2
Preparation of crystalline Lapatinib anhydrate Form 2
Lapatinib (0.2 g) was heated to reflux in dichloromethane (20 ml) so that the solid dissolved. The solution was allowed to cool gradually and crystals were precipitated. The suspension was stirred at ambient temperature for 2 hours and then the product was filtered and dried in air. 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 3
Preparation of crystalline Lapatinib 1,4-dioxane solvate (Class 1)
Lapatinib (1.0 g) was heated in 1 ,4-dioxane (10 ml) until the solid dissolved. The solution was allowed to cool and crystals were precipitated. The resulting suspension was stirred at ambient temperature for 4 hours then the solid was filtered, washed with 1 ,4-dioxane (5 ml) and dried in vacuo for 18 hours. NMR indicated 7.3%w/w 1 ,4- dioxane. An X-ray powder diffraction pattern was obtained and is depicted in Figure 3.
Example 4 Preparation of crystalline Lapatinib pyridine solvate (Class 1)
Lapatinib (1.0 g) was heated in pyridine (5 ml) until the solid dissolved. The solution was allowed to cool under a steady flow of argon and crystals were precipitated within 1 hour. The resulting suspension was allowed to stand in the stoppered flask overnight. More pyridine (2 ml) was added to mobilize, then the solid was filtered and dried in vacuo for 30 minutes. NMR indicated 11.3%w/w pyridine. An X-ray powder diffraction pattern was obtained and is depicted in Figure 4.
Example 5 Preparation of crystalline Lapatinib N-methyl-2-pyrrolidinone solvate
Lapatinib (0.4 g) was dissolved in N-methyl-2-pyrrolidinone (4 ml) and the solution was cooled in an ice/water bath. The solution remained clear and was transferred to a Buchi flask and concentrated on the rotary evaporator for several hours with the bath temperature set at 70-800C. The oily residue was split into two vials and dried in the vacuum oven at 700C overnight. The product initially did not crystallise but the oils were scratched and dried further. The samples solidified. IR and XRPD were different from the starting material. NMR indicated sample contained 9.3%w/w N- methyl-2-pyrrolidinone. An X-ray powder diffraction pattern was obtained and is depicted in Figure 5.
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 1 OmM MnCl2, 1OmM 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 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
Figure imgf000023_0001
Figure imgf000023_0002
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 370C in a humidified 10% CO2, 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 cells/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 370C, 10% CO2 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 ethanokwater), 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 10Oμl 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 (IC50) was interpolated using nonlinear regression (Levenberg-Marquardt) and the equation, y = Vmax *(1-(x/(K+x))) + Y2, where "K" was equal to the IC50.
Table XIX illustrates the inhibitory activity of GW572016X as IC50 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
Figure imgf000024_0001
Figure imgf000024_0002

Claims

CLAIMSWe claim:
1. N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl] amino}methyl)-2-furyl]-4-quinazolinamine crystalline anhydrate in forms 1 or 2.
2. N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl) ethyl] amino} methyl) -2-furyl]-4-quinazolinamine crystalline anhydrate form 1.
3. N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl) -2-furyl]-4-quinazolinamine crystalline anhydrate form 2.
4. N-{3-Chloro-4-[(3-fluorobenzyl)oxy]phenyl}-6-[5-({[2-(methanesulphonyl)ethyl] amino} methyl) -2-furyl]-4-quinazolinamine crystalline Class 1 solvates.
5. 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.
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WO2011130831A1 (en) * 2010-04-22 2011-10-27 Apotex Pharmachem Inc. Polymorphic forms of lapatinib ditosylate and processes for their preparation
US8252805B2 (en) 2008-05-07 2012-08-28 Teva Pharmaceutical Industries Ltd. Forms of lapatinib ditosylate and processes for preparation thereof
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WO2014170910A1 (en) 2013-04-04 2014-10-23 Natco Pharma Limited Process for the preparation of lapatinib
WO2018139626A1 (en) 2017-01-30 2018-08-02 塩野義製薬株式会社 Solid preparation comprising quinazoline derivative
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US8252805B2 (en) 2008-05-07 2012-08-28 Teva Pharmaceutical Industries Ltd. Forms of lapatinib ditosylate and processes for preparation thereof
WO2009140144A1 (en) * 2008-05-15 2009-11-19 Teva Pharmaceutical Industries Ltd. Forms of crystalline lapatinib and processes for preparation thereof
WO2011130831A1 (en) * 2010-04-22 2011-10-27 Apotex Pharmachem Inc. Polymorphic forms of lapatinib ditosylate and processes for their preparation
CN103896926A (en) * 2012-12-27 2014-07-02 上海创诺医药集团有限公司 Poly-crystal-form substance of lapatinib ditosylate solvate as well as preparation method and application thereof
WO2014170910A1 (en) 2013-04-04 2014-10-23 Natco Pharma Limited Process for the preparation of lapatinib
US10329285B2 (en) 2015-07-29 2019-06-25 Shionogi & Co., Ltd. Salts of quinazoline derivative or crystals thereof, and the process for producing thereof
US10513513B2 (en) 2015-07-29 2019-12-24 Shionogi & Co., Ltd. Salts of quinazoline derivative or crystals thereof, and the process for producing thereof
WO2018139626A1 (en) 2017-01-30 2018-08-02 塩野義製薬株式会社 Solid preparation comprising quinazoline derivative
KR20190108606A (en) 2017-01-30 2019-09-24 시오노기세이야쿠가부시키가이샤 Solid preparations containing quinazoline derivatives
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