HK1082211A

HK1082211A - 4-anilino quinazoline derivatives for the treatment of abnormal cell growth

Info

Publication number: HK1082211A
Application number: HK06104130.7A
Authority: HK
Inventors: Charles Kath John; Liu Zhengyu; Steflik Brown Maria; Mark Winter Steven; Jane Truesdell Susan; Anthea Szewc Ruby
Original assignee: 辉瑞产品公司
Priority date: 2002-12-18
Filing date: 2003-12-08
Publication date: 2006-06-02

Description

4-anilinoquinazoline derivatives for the treatment of abnormal cell growth

Background

The present invention relates to novel bicyclic derivatives useful for treating abnormal cell growth, such as cancer, in mammals. The invention also relates to methods of using such compounds to treat abnormal cell growth in mammals, particularly humans, and to pharmaceutical compositions containing such compounds.

It is known that cells can become cancerous by conversion of a portion of their DNA into oncogenes (i.e., genes that, when activated, lead to the formation of malignant tumor cells). Many oncogenes encode proteins of abnormal tyrosine kinases that can lead to cellular transformation. On the other hand, overexpression of the normal proto-oncogenic tyrosine kinase may also lead to proliferative diseases, sometimes resulting in a malignant phenotype.

Receptor tyrosine kinases are enzymes that span the cell membrane and possess the extracellular binding domain of growth factors such as epidermal growth factor, a transmembrane domain, and an intracellular portion that functions as a kinase to phosphorylate specific tyrosine residues on proteins and thereby affect cell proliferation. Examples of receptor tyrosine kinases include c-erbB-2(HER2), c-met, tie-2, PDGFr, FGFr, and VEGFR. Such kinases are known to be normally aberrantly expressed in common human cancers such as: breast cancer; gastrointestinal cancer such as colon, rectal or gastric cancer; leukemia; and ovarian, bronchial or pancreatic cancer. It is well known that ERBB2 (protein tyrosine kinase erb B2 precursor (also known as c-ERBB-2 protein precursor or kinase-related transforming protein ERBB2) is a proto-oncogene encoding membrane-bound receptor tyrosine kinases of the Epithelial Growth Factor Receptor (EGFR) family, it is overexpressed in several types of cancers, such as breast, ovarian, gastric, pancreatic (pancreus) and colorectal cancers ERBB2 has a potential role in tumor cell proliferation, tumor invasion and tumor metastasis and drug resistance.

Thus, receptor tyrosine kinase inhibitors are believed to be useful as selective inhibitors of mammalian cancer cell growth. For example, an oncogene inhibitor, a tyrosine kinase inhibitor, selectively attenuates the growth of athymic nude mice implanted with human breast cancer expressing epidermal growth factor receptor tyrosine kinase (EGFR), but has no effect on the growth of another cancer not expressing EGF receptor. Accordingly, the compounds of the present invention are selective inhibitors of certain receptor tyrosine kinases and are useful in the treatment of abnormal cell growth, particularly cancer, in mammals. In addition to receptor tyrosine kinases, the compounds of the invention also exhibit inhibitory activity against a variety of other non-receptor tyrosine kinases (e.g., Ick, src, abl) or serine/threonine kinases (e.g., cyclin-dependent kinases).

Various other compounds such as styrene derivatives have also been demonstrated to have tyrosine kinase inhibitory activity. Five recent european patent publications relate to certain bicyclic derivatives, in particular quinazoline derivatives, which have anti-cancer properties due to tyrosine kinase inhibitory activity, namely EP 0566226 a1 (published 1993 at 10/20), EP 0602851 a1 (published 1994 at 6/22), EP 0635507 a1 (published 1995 at 1/25), EP 0635498 a1 (published 1995 at 1/25) and EP 0520722 a1 (published 1992 at 12/30). In addition, international patent application "WO 92/20642 (published 11/26.1992) relates to certain bis-mono-and bicyclic aryl and heteroaryl compounds as tyrosine kinase inhibitors for inhibiting abnormal cell proliferation. International patent applications WO96/16960 (published 6.6.1996), WO 96/09294 (published 6.3.1996), WO97/30034 (published 21.8.1997), WO 98/02434 (published 22.1.1998), WO98/02437 (published 22.1.1998) and WO98/02438 (published 22.1.22.1998) also relate to substituted bicyclic heteroaromatic derivatives as tyrosine kinase inhibitors for the same purpose. Other patent applications relating to anticancer compounds are international patent applications WO00/44728 (published on 8/3/2000), EP1029853a1 (published on 8/23/2000) and WO01/98277 (published on 12/2001), all of which are incorporated herein by reference in their entirety.

Summary of The Invention

The invention relates to a compound of the general formula1Or a pharmaceutically acceptable salt, solvate or prodrug thereof:

wherein:

R¹selected from H and C₁-C₆Alkyl groups;

R²selected from H, C₁-C₁₀Alkyl radical, C₁-C₆Alkoxy and C₁-C₆Hydroxyalkyl groups;

R³selected from H, C₁-C₆Alkyl radical, C₁-C₆Hydroxyalkyl and C (O) OR⁴Wherein R is⁴Selected from H and C₁-C₆Alkyl groups;

R⁵selected from the group consisting of-C (O) OH and- (CR⁶R⁷)_m-NR¹R⁸Wherein m is an integer of 0 to 3; r⁶And R⁷Each independently selected from H and C₁-C₆Alkyl and wherein R⁸Is selected from C₁-C₆Alkyl and-C (O) - (CR)⁶R⁷)_m-O(C₁-C₆Alkyl) groups; and it isOf the general formula1Further optionally substituted with a hydroxyl or O-glucuronic acid substituent.

The invention also relates to the preparation of the general formula1A process for the preparation of a compound by bioconversion of a microorganism, said microbial bioconversion comprising contacting a culture of a microorganism with E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] m]-quinazolin-6-yl } -allyl) -acetamide, or a salt thereof, and isolating the compound.

The invention also relates to the preparation of the general formula1A method of preparing a compound comprising the step of preparing the compound in vivo.

The invention also relates to the preparation of the general formula1A method of making a compound comprising the step of preparing the compound by a synthetic method.

The invention also relates to a process for the preparation of E-N- (3- {4- [ 3-hydroxymethyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -2-methoxy-acetamide comprising contacting a culture of the microorganism Streptomyces albus with the mesylate salt of E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide in a nutrient medium suitable for said microorganism and isolating E-N- (3- {4- [ 3-hydroxymethyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -2-methoxy-acetamide.

The invention also relates to a process for the preparation of E-N- (3- {4- [4- (6-hydroxymethyl-pyridin-3-yloxy) -3-methyl-phenylamino ] -quinazolin-6-yl } -allyl) -2-methoxy-acetamide comprising contacting a culture of the microorganism Streptomyces rimosus with the mesylate salt of E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide in a nutrient medium suitable for said microorganism and isolating E-N- (3- {4- [4- (6-hydroxymethyl-4-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide -pyridin-3-yloxy) -3-methyl-phenylamino ] -quinazolin-6-yl } -allyl) -2-methoxy-acetamide.

The invention also relates to a method of treating abnormal cell growth (such as cancer) in a mammal comprising administering to said mammal an amount of a compound of formula (I) effective to treat abnormal cell growth1A compound is provided.

The invention also relates to a method of treating abnormal cell growth in a mammal comprising administering to said mammal an amount of a compound of formula (I) effective to treat abnormal cell growth1A compound and an anti-cancer agent selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, radiation therapy, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxic agents, anti-hormones, and anti-androgens.

The invention further relates to a pharmaceutical composition for treating abnormal cell growth in a mammal comprising an amount effective to treat abnormal cell growth of formula1And a pharmaceutically acceptable carrier.

The present invention further relates to a method for determining whether a patient has been administered E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide, comprising the step of determining whether a plasma, urine, bile or fecal sample obtained from the patient shows the presence of a compound of claim 1.

The invention also relates to a kit for treating abnormal cell growth, comprising: a) containing the general formula1A pharmaceutically composition of a compound of (a) and a pharmaceutically acceptable carrier, excipient or diluent; and b) instructions describing a method for treating abnormal cell growth using the pharmaceutical composition.

Detailed Description

wherein:

R¹selected from H and C₁-C₆Alkyl groups;

R⁵selected from the group consisting of-C (O) OH and- (CR⁶R⁷)_m-NR¹R⁸Wherein m is an integer of 0 to 3; r⁶And R⁷Each independently selected from H and C₁-C₆Alkyl, and wherein R⁸Is selected from C₁-C₆Alkyl and-C (O) - (CR)⁶R⁷)_m-O(C₁-C₆Alkyl) groups; and wherein the general formula1Further optionally substituted with a hydroxyl or O-glucuronic acid substituent.

In a preferred embodiment, of formula (VII b)1The compound of (a) is substantially pure. For example, the general formula (II a) can be obtained in substantially pure form by in vivo chemical synthesis or biotransformation as described in detail below1The compound of (1).

In the general formula1In a particular embodiment of the compounds, R¹Is H, R²Is hydroxymethyl, R³Is methyl, and R⁵is-CH₂NHC(O)CH₂OCH₃。

In another specific embodiment, R¹Is H, R²Is methyl, R³Is hydroxymethyl, and R⁵is-CH₂NHC(O)CH₂OCH₃。

In another specific embodiment, R¹Is H, R²Is methyl, R³Is methyl, and R⁵is-C (O) OH.

In another specific embodiment, R¹Is H, R²Is methyl, R³is-COOH, and R⁵is-CH₂NHC(O)CH₂OCH₃。

In another specific embodiment, wherein, the formula1Further containing a hydroxy substituent, R¹Is H, R²Is methyl, R³Is methyl, and R⁵is-CH₂NHC(O)CH₂OCH₃. In one embodiment, the hydroxyl moiety is a substituent within the square bracket portion of the molecule as shown below:

in another specific embodiment, wherein1Further containing a hydroxy substituent, R¹Is H, R²Is methyl, R³Is hydroxymethyl, and R⁵is-CH₂NHC(O)CH₂OCH₃. In one embodiment, the hydroxyl moiety is a substituent within the square bracket portion of the molecule as shown below:

in another specific embodiment, R¹Is H, R²Is hydroxymethyl, R³Is methyl, and R⁵is-CH₂NHC(O)CH₂OCH₃。

In another specific embodiment, of formula (III)1A compound of (a) furtherContaining an-O-glucuronic acid substituent. In one embodiment, the-O-glucuronic acid substituent is located on the quinazoline ring; in one embodiment, it is located on the "phenyl" portion of the phenylamino group; in one embodiment, it is located on the pyridine ring; and in one embodiment it is located on an acyclic chain attached to the quinazoline cyclophenyl group.

Particularly preferred compounds of the invention include those selected from the group consisting of:

2-methoxy-N- (3- {4- [ 3-hydroxymethyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide;

2-methoxy-N- (3- {4- [4- (6-hydroxymethyl-pyridin-3-yloxy) -3-methyl-phenylamino ] -quinazolin-6-yl } -allyl) -acetamide;

3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) phenylamino ] -quinazolin-6-yl } -acrylic acid;

5- (4- {6- [3- (2-methoxy-acetylamino-propenyl) -quinazolin-4-ylamino ] -2-methyl-phenoxy } -pyridine-2-carboxylic acid;

2-hydroxy-N- (3- {4- [ 3-hydroxymethyl-4- (6-methyl-pyridin-3-yloxy) phenylamino ] -quinazolin-6-yl } -allyl) -acetamide;

and pharmaceutically acceptable salts, prodrugs, hydrates and solvates of the above compounds.

General formula (VII)1The compounds of (a) may exist in the form of cis (Z) or trans (E) geometric isomers. In a preferred embodiment of the invention, of the general formula1The compound of (1) is a geometric isomer of formula E.

The compounds of the invention may be used as analytical standards for in vitro or in vivo metabolic studies or as intermediates in the chemical or biological synthesis of new chemical entities. The metabolites may be isolated as solids or as solutions. The compounds of the invention may also be used to identify patients who have been administered E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide, or a pharmaceutically acceptable salt or prodrug or salt of a prodrug thereof. To identify a patient who has been administered E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide, or a pharmaceutically acceptable salt or prodrug or salt of a prodrug thereof, serum, urine, feces, or bile samples are taken from the patient and the samples are analyzed for the presence of one or more compounds of the invention.

One method of analyzing the compounds of the present invention is to use chromatography and mass spectrometry. Other methods of analysis are well known to those skilled in the art. The presence of one or more compounds of the invention in a serum, urine, feces, or bile sample indicates that the patient has been administered E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide, or a pharmaceutically acceptable salt or prodrug or salt of a prodrug thereof.

In the treatment methods of the present invention, the compounds of the present invention may be administered directly to the patient, such as in the form of a tablet, or the compounds produced in the patient by metabolism may be administered. In addition, the route of administration and dosage of the compounds that metabolically produce the compounds of the invention can be varied, if necessary, to achieve the desired in vivo concentration and yield of the compounds of the invention.

The invention also relates to the preparation of the general formula1A method of compound comprising contacting a culture of a microorganism with E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] in a nutrient medium suitable for the microorganism]-quinazolin-6-yl } -allyl) -acetamide or a salt thereof and isolating the compound.

In one embodiment, the microorganism is an actinomycete, and in one embodiment, the microorganism is a fungus.

The present invention also relates to a process for the preparation of E-N- (3- {4- [ 3-hydroxymethyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -2-methoxy-acetamide comprising: contacting a culture of the microorganism streptomyces parvulus with the mesylate salt of E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide in a nutrient medium suitable for said microorganism and dividing E-N- (3- {4- [ 3-hydroxymethyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -2-methoxy-acetamide.

In a preferred embodiment, the nutrient medium suitable for Streptomyces albugus (Streptomyces albulus) is IOWA medium.

The invention also relates to a process for the preparation of E-N- (3- {4- [4- (6-hydroxymethyl-pyridin-3-yloxy) -3-methyl-phenylamino ] -quinazolin-6-yl } -allyl) -2-methoxy-acetamide comprising contacting a culture of the microorganism Streptomyces rimosus with the mesylate salt of E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide in a nutrient medium suitable for said microorganism and isolating E-N- (3- {4- [4- (6-hydroxymethyl- Pyridin-3-yloxy) -3-methyl-phenylamino ] -quinazolin-6-yl } -allyl) -2-methoxy-acetamide.

In a preferred embodiment, the nutrient medium suitable for S.rimosus is IOWA medium.

The invention also relates to the preparation of the general formula1A method of preparing a compound comprising the step of preparing the compound in vivo (i.e., the compound is produced in vivo).

The invention also relates to a method of treating abnormal cell growth in a mammal, including a human, comprising administering to said mammal an amount of a compound of formula (la) effective to treat abnormal cell growth as described above1A compound or a pharmaceutically acceptable salt thereof,A solvate, hydrate or prodrug. In one embodiment of the method, the abnormal cell growth is cancer, including, but not limited to, lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, carcinoma of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, tumors of the Central Nervous System (CNS), primary CNS lymphoma (lymphom), spinal axis tumors, brain stem glioma, pituitary adenoma, or a combination of one or. In another embodiment of the method, the abnormal cell growth is a benign proliferative disease including, but not limited to, psoriasis, benign prostatic hypertrophy or restenosis.

The invention also relates to a method of treating abnormal cell growth in a mammal comprising administering to said mammal an amount of a compound of formula (I) effective to treat abnormal cell growth1A compound or a pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof and an anticancer agent selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxic agents, anti-hormones and anti-androgens.

The invention also relates to a pharmaceutical composition for treating abnormal cell growth in mammals including humans, comprising an effective abnormal cell growth treating amount of the general formula1A compound or a pharmaceutically acceptable salt, solvate or prodrug thereof and a pharmaceutically acceptable carrier. In one embodiment of the composition, the abnormal cell growth is a cancer, including, but not limited to, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer,A melanoma on the skin or in the eye, a carcinoma of the uterus, an ovary cancer, a rectum cancer, a cancer of the anal region, a stomach cancer, a colon cancer, a breast cancer, a carcinoma of the fallopian tubes, a carcinoma of the endometrium, a carcinoma of the cervix, a carcinoma of the vagina, a carcinoma of the vulva, hodgkin's disease, a carcinoma of the esophagus, a carcinoma of the small intestine, a cancer of the endocrine system, a cancer of the thyroid gland, a carcinoma of the parathyroid gland, a carcinoma of the adrenal gland, a sarcoma of soft tissue, a carcinoma of the urethra, a carcinoma of the penis, a prostate cancer, a chronic or acute leukemia, a lymphocytic lymphoma, a carcinoma of the bladder, a carcinoma of the kidney or ureter, a renal cell carcinoma, a carcinoma of the renal pelvis, a tumor of the central nervous system (CNS. In another embodiment of the pharmaceutical composition, the abnormal cell growth is a benign proliferative disease including, but not limited to, psoriasis, benign prostatic hypertrophy or restenosis (restinosis).

The invention also relates to a pharmaceutical composition for treating abnormal cell growth in mammals including humans, comprising an effective abnormal cell growth treating amount of the general formula1A compound or a pharmaceutically acceptable salt, solvate or prodrug thereof in combination with a pharmaceutically acceptable carrier and an anticancer agent selected from the group consisting of mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, antibodies, cytotoxic agents, anti-hormonal agents and anti-androgens.

The invention also relates to a method of treating angiogenesis-related diseases in a mammal, including a human, comprising administering to said mammal an amount of a compound of formula (la) as described above effective in treating said diseases1A compound or a pharmaceutically acceptable salt, solvate or prodrug thereof. The diseases include: cancerous tumors, such as melanoma; eye diseases such as age-related macular degeneration, ocular pseudohistoplasmosis syndrome, and retinal neovascularization due to proliferative diabetic retinopathy; rheumatoid arthritis; bone loss diseases, such as osteoporosis, Paget's disease, body fluids of malignant tumorsHypercalcemia, hypercalcemia due to tumor metastasis to bone, and glucocorticoid therapy-induced osteoporosis; restenosis of the coronary arteries; and certain microbial infections, including those associated with microbial pathogens selected from the group consisting of adenovirus, hantavirus, borrelia burgdorferi, yersinia species, bordetella pertussis, and group a streptococcus.

The invention also relates to methods of treating abnormal cell growth in a mammal (and to pharmaceutical compositions for treating abnormal cell growth in a mammal) comprising an amount of a compound of formula1A compound or a pharmaceutically acceptable salt, solvate or prodrug thereof and an amount of one or more drugs selected from the group consisting of anti-angiogenic agents, signal transduction inhibitors and antiproliferative agents effective in combination to treat said abnormal cell growth.

Anti-angiogenic agents such as MMP-2 (matrix metalloproteinase 2) inhibitors, MMP-9 (matrix metalloproteinase 9) inhibitors, and COX-II (cyclooxygenase II) inhibitors may be combined with the general formulae in the methods and pharmaceutical compositions described herein1The compounds are combined. Examples of useful COX-II inhibitors include CELEBREX^TM(alecoxib), valdecoxib, and rofecoxib. Examples of useful matrix metalloproteinase inhibitors are described in the following documents: WO 96/33172 (published 24/10/1996); WO96/27583 (published 3/7 1996); european patent application No. EP97304971.1 (filed on 8/7/1997); european patent application No. EP 99308617.2 (filed 10/29 in 1999); WO 98/07697 (published 26/1/1998); WO 98/03516 (published on 29/1/1998); WO 98/34918 (published on 8/13 of 1998); WO 98/34915 (published on 8/13 of 1998); WO 98/33768 (published on 8/6 in 1998); WO 98/30566 (published 16.7.1998); european patent publication EP606,046 (published on 7/13 th 1994); european patent publication EP931,788 (published on 7/28/1999); WO90/05719 (published 5/31 1990); WO99/52910 (published on 21/10/1999); WO99/52889 (published 10/21 in 1999); WO 99/29667 (published on 17.6.1999); PCT International application No. PCT/IB98/01113 (1998)Submitted on day 21/7/year); european patent application No. EP 99302232.1 (filed 3/25/1999); british patent application No. GB9912961.1 (filed on 3/6/1999); U.S. provisional application No. US60/148,464 (filed 8/12/1999); US patent US5,863,949 (granted 26/1/1999); US patent US5,861,510 (granted on 19/1/1999); and european patent publication No. EP 780,386 (published on 25/6/1997), all of which are incorporated herein by reference in their entirety. Preferred MMP-2 and MMP-9 inhibitors are those that have little or no activity for inhibiting MMP-1. More preferably those inhibitors which selectively inhibit MMP-2 and/or MMP-9 relative to other matrix metalloproteinases (i.e., MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13).

Some specific examples of MMP inhibitors for use in combination with the compounds of the present invention are AG-3340, RO32-3555, RS 13-0830, and the compounds described below:

3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (1-hydroxycarbamoyl-cyclopentyl) -amino ] -propionic acid;

3-exo-3- [4- (4-chloro-phenoxy) -benzenesulfonylamino ] -8-oxa-bicyclo [3.2.1] octane-3-carboxylic acid hydroxyamide;

(2R, 3R)1- [4- (2-chloro-4-fluoro-benzyloxy) -benzenesulfonyl ] -3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide;

4- [ [4- (4-fluoro-phenoxy) -benzenesulfonylamino ] -tetrahydro-pyran-4-carboxylic acid hydroxyamide;

3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (1-hydroxycarbamoyl-cyclobutyl) -amino ] -propionic acid;

4- [ [4- (4-chloro-phenoxy) -benzenesulfonylamino ] -tetrahydro-pyran-4-carboxylic acid hydroxyamide;

3- [ [4- (4-chloro-phenoxy) -benzenesulfonylamino ] -tetrahydro-pyran-3-carboxylic acid hydroxyamide;

(2R, 3R)1- [4- (4-fluoro-2-methyl-benzyloxy) -benzenesulfonyl ] -3-hydroxy-3-methyl-piperidine-2-carboxylic acid hydroxyamide;

3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (1-hydroxycarbamoyl-1-methyl-ethyl) -amino ] -propionic acid;

3- [ [4- (4-fluoro-phenoxy) -benzenesulfonyl ] - (4-hydroxycarbamoyl-tetrahydro-pyran-4-yl) -amino ] -propionic acid;

3-endo-3- [4- (4-fluoro-phenoxy) -benzenesulfonylamino ] -8-oxa-bicyclo [3.2.1] octane-3-carboxylic acid hydroxyamide; and

3- [4- (4-fluoro-phenoxy) -benzenesulfonylamino ] -tetrahydro-furan-3-carboxylic acid hydroxyamide;

and pharmaceutically acceptable salts, solvates and prodrugs of said compounds.

General formula (VII)1The compounds and pharmaceutically acceptable salts, solvates or prodrugs thereof may also be used in combination with the following inhibitors: signal transduction inhibitors such as agents that can inhibit EGFR (epidermal growth factor receptor) response, such as EGFR antibodies, EGF antibodies, and molecules belonging to EGFR inhibitors; VEGF (vascular endothelial growth factor) inhibitors; and erbB2 receptor inhibitors, such as organic molecules or antibodies that bind to the erbB2 receptor, e.g. HERCEPTIN^TM(Genentech，Inc.of South SanFrancisco California，USA)。

EGFR inhibitors are described, for example, in WO95/19970 (published 27.7.1995), WO98/14451 (published 9.4.1998), WO 98/02434 (published 22.1.1998), and U.S. Pat. No. 5,747,498 (issued 5.5.1999). EGFR inhibitors include, but are not limited to, monoclonal antibody C225 and anti-EGFR 22Mab (Imclone systems incorporated, New York, USA, New York), compound ZD-1839(AstraZeneca), BIBX-1382(Boehringer Ingelheim), MDX-447 (Metarex Inc. of Annandale, New Jersey, USA) and OLX-103(Merck & Co. of Whitehouse State, New Jersey, USA), VRCTC-310(Ventech Research) and EGF fusion toxin (Seragen Inc. of Hopkinson, Massachusettes).

VEGF inhibitors such as SU-5416 and SU-6668(Sugen Inc. of South san Francisco, Calf. USA) may also be reacted with the general formula1The combination of compounds (1). VEGF inhibitors are described, for example, in the following documents: WO 99/24440 (published 20/5/1999), PCT International application PCT/IB99/00797 (filed 3/5/1999), WO 95/21613 (published 17/8/1999), WO 99/61422 (published 2/12/1999), US patent US5,834,504 (granted 10/11/1998), WO 98/50356 (published 12/11/1998), US patent US5,883,113 (granted 16/1999), US patent US5,886,020 (granted 23/3/1999), US patent US5,792,783 (granted 11/8/1998), WO 99/10349 (published 4/1999), WO97/32856 (published 12/1997), WO 97/22596 (published 26/1997), WO 98/54093 (published 3/1998), WO98/02438 (published 22/1998), WO 99/16755 (published 8/1999) and WO98/02437 (published 22/1998), all of these documents are incorporated herein by reference in their entirety. Other examples of certain specific VEGF inhibitors are IM862(Cytran inc. of Kirkland, Washington, USA), anti-VEGF monoclonal antibodies (Genentech, inc. of South San Francisco, California) and angiozyme, synthetic ribozymes (from Ribozyme, Boulder) and Chiron (Emeryville, California).

ErbB2 receptor inhibitors, such as GW-282974(Glaxo Welcome pic) and monoclonal antibodies AR-209(Aronex Pharmaceuticals Inc. of The Woodlans, Texas, USA) and 2B-1(Chiron) can be combined with The general formula1The compound of (1) is administered in combination. Such erbB2 inhibitors include those described in the following references: WO 98/02434 (published 22/1/1998), WO99/35146 (published 15/7/1999), WO 99/35132 (published 15/7/1999), WO98/02437 (published 22/1/1998), WO 97/13760 (published 17/4/1997), WO95/19970 (published 27/7/1995), U.S. Pat. No. 5,587,458 (published 24/12/1996) and U.S. Pat. No. 5,877,305 (published 2/3/1999),the entire contents of each of these documents are incorporated herein by reference. ErbB2 receptor inhibitors for use in the present invention are also described in U.S. provisional application Ser. No. US60/117,341 filed on 27.1.1999 and U.S. provisional application Ser. No. US60/117,346 filed on 27.1.1999, both of which are incorporated herein by reference in their entirety.

Other antiproliferative agents that may be used in combination with the compounds of the invention include farnesyl protein transferase inhibitors and receptor tyrosine kinase PDGFr inhibitors, including: the compounds disclosed and claimed in the following U.S. patent applications: US 09/221946 (filed 12 months and 28 days 1998); US 09/454058 (filed on 12/2/1999); US 09/501163 (filed 2/9/2000); US 09/539930 (filed 3/31/2000); US 09/202796 (submitted on 22/5 in 1997); US 09/384339 (filed on 26/8/1999); and US 09/383755 (filed 8/26 in 1999); and the compounds disclosed and claimed in the following U.S. provisional patent applications: US 60/168207 (filed on 30/11 in 1999); US 60/170119 (filed 10.12.1999); US 60/177718 (filed 1/21/2000); US 60/168217 (filed on 30/11 in 1999); and US 60/200834 (filed 5/1/2000). The entire contents of each of the above-mentioned patent applications and provisional patent applications are incorporated herein by reference.

General formula (VII)1The compounds of (a) may also be used in combination with other agents useful in the treatment of abnormal cell growth or cancer, including, but not limited to: agents capable of promoting an anti-tumor immune response, such as CTLA4 (cytotoxic lymphocyte antigen 4) antibodies; and other agents capable of blocking CTLA 4; and antiproliferative agents such as other inhibitors of farnesyl protein transferase, for example, as described in the references cited in the "background" section above. Specific CTLA4 antibodies that may be used in the present invention include those described in U.S. provisional application US60/113,647 (filed 12/23 of 1998), the entire contents of which are incorporated herein by reference.

As used herein, unless otherwise indicated, "abnormal cell growth" refers to cell growth that is not associated with normal regulatory mechanisms (e.g., loss of contact inhibition). It includes abnormal growth in the following cases: (1) tumor cells (tumors) that proliferate by expressing a mutant tyrosine kinase or overexpressing a receptor tyrosine kinase; (2) benign and malignant cells of other proliferative diseases in which aberrant tyrosine kinase activation occurs; (4) any tumor that proliferates through receptor tyrosine kinase activation; (5) any tumor that proliferates by aberrant serine/threonine kinase activation; and (6) benign and malignant cells of other proliferative diseases in which aberrant serine/threonine kinase activation occurs.

As used herein, unless otherwise indicated, the term "treating" or "treatment" refers to reversing, alleviating, inhibiting the development or preventing the disease or condition to which such term is directed, or one or more symptoms of the disease or condition. As used herein, unless otherwise indicated, the term "treatment" refers to a therapeutic effect such as "treatment" described above.

The term "halogen" as used herein includes, unless otherwise indicated, fluorine, chlorine, bromine or iodine. Preferred halogen groups are fluorine and chlorine.

The term "alkyl" as used herein, unless otherwise indicated, includes monovalent hydrocarbon radicals having straight, cyclic (including mono-or poly-cyclic moieties) or branched moieties. It is understood that the alkyl group includes cyclic moieties which must contain at least three carbon atoms.

Unless otherwise indicated, the term cycloalkyl as used herein includes saturated monovalent hydrocarbon radicals containing cyclic (including monocyclic or polycyclic) moieties.

The term "alkenyl", as used herein, unless otherwise indicated, includes alkyl groups as defined above containing at least one carbon-carbon double bond.

The term "alkynyl", as used herein, unless otherwise indicated, includes alkyl groups as defined above containing at least one carbon-carbon triple bond.

The term "aryl" as used herein, unless otherwise indicated, includes organic groups derived by removing a hydrogen from an aromatic hydrocarbon, such as phenyl or naphthyl.

The term "alkoxy" as used herein, unless otherwise indicated, includes-O-alkyl, wherein alkyl is as defined above.

The term "Me" refers to methyl, "Et" refers to ethyl, and "Ac" refers to acetyl.

The term "pharmaceutically acceptable salt" as used herein, unless otherwise indicated, includes salts of acidic or basic groups which may be present in the compounds of the present invention. The compounds of the invention which are basic in nature are capable of forming a wide variety of salts with a variety of inorganic and organic acids. The acids that may be used to prepare the pharmaceutically acceptable acid addition salts of these basic compounds are non-toxic acid addition salts, i.e., the formation of acids containing salts of pharmaceutically acceptable anions such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, acid sulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, mesylate, ethylsulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate [ i.e., 1' -methylene-bis- (2-hydroxy-3-naphthoic acid) ] salts. The compounds of the invention comprising a basic moiety, such as amino, may form pharmaceutically acceptable salts with various amino acids in addition to the acids described above.

The phrase "substantially pure," as used herein, unless otherwise specified, refers to the purity of a compound in the compound, wherein the compound is at least 90%, in one embodiment at least 95%, and in one embodiment at least 99%.

The acidic compounds of the present invention are capable of forming base salts with a variety of pharmaceutically acceptable cations. Examples of such salts include the alkali or alkaline earth metal salts of the compounds of the present invention, and in particular the calcium, magnesium, sodium and potassium salts.

Certain functional groups in the compounds of the invention may replace bioisosteric groups, i.e., groups that have similar steric or electronic requirements for the parent group, but exhibit different or improved physicochemical or other properties. Suitable examples are well known to those skilled in the art and include, but are not limited to, those described in Patini et al, chem. Rev, 1996, 96, 3147-.

The compounds of the invention may have asymmetric centers and may thus exist in different enantiomeric and diastereomeric forms. The present invention relates to the use of all optical isomers and stereoisomers of the compounds of the invention and mixtures thereof, and to all pharmaceutical compositions containing them and methods of treatment in which they may be used. The compounds of formula 1 may also exist as tautomers. The present invention relates to the use of all tautomers and mixtures thereof.

The invention also includes isotopically-labeled compounds, and pharmaceutically acceptable salts, solvates and prodrugs thereof, which are equivalent to those recited in formula 1, but for the fact that 1 or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine and chlorine, respectively, such as²H，³H，¹³C，¹⁴C，¹⁵N，¹⁸O，¹⁷O，³⁵S，¹⁸F, and³⁶and (4) Cl. Compounds of the present invention, prodrugs thereof, and pharmaceutically acceptable salts thereof, containing the aforementioned isotopes and/or other isotopes of other atoms are within the scope of this invention. Certain isotopically-labelled compounds of the invention, e.g. in combination with a radioactive isotope, such as³H and¹⁴c, which can be used in drug and/or stromal tissue distribution assays. Tritium, i.e.³H and carbon-14, i.e.¹⁴The C isotope is preferred because of its ease of preparation and detection. In addition, with heavier isotopes, e.g. deuterium, i.e.²H substitution may result in some of the results due to better metabolic stabilityThe resulting therapeutic advantages, such as increased half-life in vivo or reduced dosage requirements, may be preferred in certain circumstances. Isotopically labeled compounds of formula 1 of this invention and prodrugs thereof are generally prepared by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent according to the procedures disclosed in the schemes and/or in the examples and methods of preparation described below.

The invention also includes pharmaceutical compositions containing prodrugs of the compounds of formula 1 and methods of treating bacterial infections by administering prodrugs of the compounds of the invention. The compound of formula 1 having a free amino, amido, hydroxyl or carboxyl group can be converted into a prodrug. Prodrugs include compounds wherein an amino acid residue or a polypeptide chain of 2 or more (e.g., 2, 3, or 4) amino acid residues is covalently bonded via an amide or ester bond to a free amino, hydroxyl, or carboxylic acid group of the compound of formula 1. Amino acid residues include, but are not limited to, the 20 natural amino acids commonly represented by three letter symbols, and also include 4-hydroxyproline, hydroxylysine, desmosine (desosine), isodesmosine, 3-methylhistidine, pentanine, β -alanine, γ -aminobutyric acid, citrulline homocysteine (citrulline homocysteine), homoserine, ornithine and methionine sulfone. Other prodrug types are also included. For example, the free carboxyl groups may be derivatized as amides or alkyl esters. The free hydroxyl group may be derivatized with groups including, but not limited to: hemisuccinate, phosphate, dimethylaminoacetate and phosphoryloxymethoxycarbonyl, as described in Advanced Drug Delivery Reviews, 1996, 19, 115. Carbamate prodrugs of hydroxy and amino groups are also included, such as carbonate prodrugs of hydroxy groups, sulfonate esters and sulfate ester prodrugs. Also included are derivatives of hydroxyl groups as (acyloxy) methyl ethers and (acyloxy) ethyl ethers wherein the acyl group may be an alkyl ester, optionally substituted with functional groups including, but not limited to, ether, amine, and carboxylic acids, or the acyl group is an amino acid ester as described above. Such prodrugs are described in j.med.chem.1996, 39, 10. The free amines may also be derivatized to form amides, sulfonamides, or phosphonamides. All of these prodrug moieties may incorporate groups including, but not limited to, ether, amine, and carboxylic acid functional groups.

Scheme 1

The following references provide general synthetic methods for preparing the compounds of the present invention: U.S. Pat. No. 5,747,498 (published 1998 at 5.5), U.S. Pat. application Ser. No. 08/953078 (published 1997 at 10.17), WO 98/02434 (published 1998 at 1.22), WO98/02438 (published 1998 at 1.22), WO 96/40142 (published 1996 at 12.19), WO 96/09294 (published 1996 at 6.3), WO 97/03069 (published 1997 at 30.1), WO95/19774 (published 1995 at 27) and WO97/13771 (published 1997 at 4.17). Other methods are described in WO00/44728 (published as 8.3.2000), EP1029853A1 (published as 8.23.2000) and WO01/98277 (published as 12.12.2001). The foregoing patents and patent applications are incorporated herein by reference in their entirety. Certain starting materials may be prepared according to methods well known to those skilled in the art, and certain synthetic modifications may be made according to methods well known to those skilled in the art. Standard methods for preparing 6-iodoquinazolinones are found in Stevenson, t.m., Kazmierczak, f., Leonard, n.j., j.org.chem.1986, 51, 5, p.616. Palladium-catalyzed Heck couplings are described in Heck et al, Organic Reactions, 1982, 27, 345 or Cabri et al, Acc. chem. Res.1995, 28, 2.

The starting material synthesis is not specifically described above, is commercially available, or can be prepared by methods well known to those skilled in the art.

In each reaction discussed or exemplified in the above schemes, unless otherwise indicated, pressure is not critical. Pressures from about 0.5 atmospheres to about 5 atmospheres are generally acceptable, and ambient pressure, i.e., 1 atmosphere, is preferred for convenience.

With reference to scheme 1 above, a compound of formula D, wherein R5 is as defined above, can be prepared by reacting a compound of formula D, wherein R is as defined above, with a compound of formula D, wherein R is as defined above, in an anhydrous solvent¹、R²And R³The amine coupling of formula E as defined above produces the compound of formula 1 at a temperature in the range of 50-150 ℃ for a period of 1-48 hours, with solvents selected in particular from DMF (N, N-dimethylformamide), DME (ethylene glycol dimethyl ether), DCE (ethylene dichloride) and tert-butanol and phenol, or mixtures of the above solvents. The isoaryloxyalniline of formula E can be prepared by methods known to those skilled in the art, such as reduction of the corresponding nitro intermediate. By Brown, r.k., Nelson, n.a.j.org.chem.1954, p.5149; yuste, r., Saldana, M, Walls, f., tet.lett.1982, 23, 2, p.147; or the process outlined in WO 96/09294. The halonitrobenzene precursors can be prepared by nucleophilic displacement of the halide with an appropriate alcohol to produce the appropriate heteroaryloxynitrobenzene derivatives as described in Dinsmore, c.j., et al, bioorg.med.chem.lett., 7, 10, 1997, 1345; loupy, a. et al, synth. commun., 20, 18, 1990, 2855; or Brunelle, d.j., tet.lett., 25, 32, 1984, 3383. Can be prepared by mixing¹Reductive amination of parent anilines of CH (O) to prepare R¹Is C₁-C₆Alkyl group of the formula E. In which Z may be treated with a coupling agent (a coupling partner), for example a terminal alkyne, terminal alkene, vinyl halide, vinyl stannane, vinyl borane, alkyl borane, or an alkyl or alkenyl zinc reagent¹Is a reactive group, such as bromo, iodo, -N₂or-OTf (i.e., -OSO2CF3), or reactive group precursors, e.g., NO₂、NH₂Or OH to prepare a compound of formula D. With chlorinating agents, e.g. POCl₃、SOCl₂Or ClC (O) C (O) Cl/DMF, treating the compound of formula B in a halogenated solvent at a temperature of about 60 ℃ to 150 ℃ for about 2 to 24 hours, and then treating with sodium aryloxide in a solvent such as aromatic phenol at a temperature of 25 ℃ to 90 ℃ to prepare compound C. In formulas C and D, Y is-Cl or-OAr, wherein Ar is an aromatic group, such as phenyl.

Can be generated by reacting with R⁵Standard treatment of groups converts any compound of formula 1 to another compound of formula 1. These methods are known to those skilled in the art and include a) by t.w.greene and p.g.m.wuts,protective Groups in Organic Synthesis ", second edition, John Wiley and Sons, New York, 1991, remove protecting Groups; and b) replacing the leaving group (halide, mesyl ester, tosylate, etc.) with a primary or secondary amine, thiol or alcohol to form a secondary or tertiary amine, thioether or ether, respectively.

The basic compounds of formula 1 can form a variety of different salts with various organic or inorganic acids. Although these salts must be pharmaceutically acceptable for administration to animals, it is often desirable in practice to initially isolate the compound of formula 1 from the reaction mixture in the form of a pharmaceutically unacceptable salt, and then simply convert the latter to the free base compound by treatment with a basic agent, followed by conversion of the latter free base to a pharmaceutically acceptable acid addition salt. The acid addition salts thereof are readily prepared by treating the basic compounds of the present invention with substantially equivalent amounts of the selected inorganic or organic acid in an aqueous solvent medium or in a suitable organic solvent, such as methanol or ethanol. Careful evaporation of the solvent quickly gives the desired solid salt. The desired acid addition salts may also be obtained by precipitation by addition of a suitable inorganic or organic acid to a solution of the free base in an organic solvent.

The acidic compounds of formula 1 are capable of forming base salts with a variety of pharmaceutically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts, particularly sodium and potassium salts. These salts are prepared by conventional methods. Bases which can be used as reagents for preparing pharmaceutically acceptable base salts of the present invention are bases which form non-toxic basic salts with the acidic compound of formula 1. These non-toxic base salts include those derived from pharmaceutically acceptable cations such as sodium, potassium, calcium, and magnesium. These salts are readily prepared by treating the corresponding acidic compound with an aqueous solution containing the desired pharmaceutically acceptable cation and then evaporating the resulting solution to dryness, preferably under reduced pressure. Alternatively, the base salt may be prepared by mixing a lower aliphatic alcohol solution of the acidic compound with the desired alkali metal alkoxide and then evaporating the resulting solution to dryness in the manner previously described. In both cases, it is preferred to use stoichiometric amounts of reagents to ensure reaction completion and maximum yield of the desired end product. Because a single compound of the invention may include more than 1 acidic or basic moiety, a compound of the invention may include mono-, di-, or tri-salts within a single compound.

The compounds of the present invention are potent inhibitors of erbB family oncogenes and proto-oncogene protein tyrosine kinases, particularly erbB2, and are therefore suitable for use therapeutically as antiproliferative agents (anti-cancer) in mammals, particularly humans. In particular, the compounds of the invention are useful for the prevention or treatment of various human hyperproliferative diseases, such as benign or malignant tumors of the liver, kidney, bladder, breast, stomach, ovary, colon and rectum, prostate, pancreas, lung, vulva, thyroid, liver cancer, sarcomas, glioblastomas, tumors of the head and neck, and other hyperproliferative diseases, such as benign hyperplasia of the skin (e.g., psoriasis) and hyperproliferation of the prostate (e.g., BPH). In addition, it is expected that the compounds of the present invention may have activity against leukemias and lymphoid malignancies.

The compounds of the invention may be used to treat additional diseases, including aberrantly expressed ligand/receptor interactions or activation or signaling events related to various protein tyrosine kinases. These diseases may include diseases of the neurons, glia, astrocytes (astrocytal), hypothalamus and other glands, macrophages, epithelial cells, stroma and blastocoel, including aberrant function, expression, activation or signaling of erbB tyrosine kinase. In addition, the compounds of the present invention may have therapeutic utility for inflammation, angiogenic diseases and immune disorders, including the treatment of diseases caused by recognized and unrecognized tyrosine kinases that are inhibited by the compounds of the present invention.

The compounds of the present invention may also be useful as biomarkers of E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide metabolism, and may further be used to determine the rate of its absorption and metabolic breakdown in mammals, such as humans.

The in vitro activity of the compound of formula 1 can be determined by the following method.

The c-erbB2 kinase assay is similar to the method previously described in Schrang et al, anal. biochem.211, 1993, p 233-239. Nunc MaxiSorp 96-well plates were incubated overnight at 37 ℃ and coated with 100mL of 0.25mg/mL poly (Glu, Tyr) 4: 1(PGT) (Sigma Chemical Co., St. Louis, Mo.) in PBS (phosphate buffered saline) per well. Excess PGT was aspirated and the plate was washed 3 times with wash buffer (0.1% tween 20 in PBS). The kinase reaction was carried out in 50mL of 50mM HEPES (pH7.5) containing 125mM sodium chloride, 10mM magnesium chloride, 0.1mM sodium orthovanadate, 1mM ATP, 0.48mg/mL (24 ng/well) of the intracellular domain of c-erbB2 (intracellular domain). The intracellular domain of erbB2 tyrosine kinase (amino acids 674-1255) was expressed as a GST-fusion protein in baculovir, which was purified by binding to glutathione-coated beads and elution. Compounds in DMSO (dimethyl sulfoxide) were added to form final DMSO concentrates at approximately 2.5% concentration. Phosphorylation was initiated by addition of ATP (adenosine triphosphate) and continued at room temperature for 6 min with shaking. The kinase reaction was stopped by aspirating off the reaction mixture and then washed with the wash buffer (see above). Phosphorylated PGT was determined as follows: each well was incubated with 50mL of HRP-coupled PY54(OncogeneScience Inc. Uniondale, NY) anti-phosphotyrosine antibody diluted to 0.2mg/mL in blocking buffer (3% BSA in 0.05% Tween 20 in PBS) for 25 min. The antibody was aspirated and the plates were then washed 4 times with wash buffer. 50mL of TMB microwell peroxidase substrate (Kirkegaard and Perry, Gaithersburg, Md.) was added to each well to generate a colorimetric signal, and then 50mL of 0.09M sulfuric acid was added to each well to stop the reaction. The amount of phosphotyrosine was calculated by measuring absorbance at 450 nm. The contrast signal is typically between 0.6 and 1.2 absorbance units, with essentially no background signal and no PGT substrate in the wells, proportional to the incubation time over 10 minutes. Inhibitors were determined by reduction of signal relative to dishes without inhibitor and IC was determined₅₀The value, i.e. corresponding to the concentration of compound required to achieve 50% inhibition. Compounds corresponding to formula 1 exemplified herein have IC's against erbB2 kinase₅₀The value is less than 10. mu.M.

By measuring the test compound relative to a controlThe amount of inhibition of tumor growth determines the in vivo activity of the compound of formula 1. The inhibition of tumor growth by various compounds was determined according to the methods described in the following documents, with minor modifications: corbett T.H. et al, "Metal indication Relationships in development of Transplantable Cancers of the Colon in Rice for Chemotherapy assays, with a Note on Carcinogen Structure", Cancer Res., 35, 2434. sub.2439 (1975) and Corbett T.H. et al, "A Motor Colon-mandrel Model for Experimental Therapy", Cancer Chemotherr. Rep. (Part 2), "5, 169. sub.186 (1975). Tumors were induced by left abdominal subcutaneous injection of 1-5 million log phase cultured tumor cells (mouse FRE-ErbB2 cells or human SK-OV3 ovarian cancer cells) suspended in 0.1ml RPMI 1640 medium. After sufficient time, the tumor became accessible (volume 100-³5-6mm in diameter), experimental animals (athymic female mice) were treated with the test compound (formulated at a concentration of 10-15mg/ml in 5 Gelucire) by the intraperitoneal (ip) or oral (po) route, 1 or 2 times daily for 7-10 days. To determine the antitumor effect, Tumors were measured in millimeters in 2 diametral directions with a vernier caliper, and then measured according to Geran, R.I., et al, "Protocols for screening Chemical Agents and Natural Products agricultural Animal Tumors and Biological Systems", third edition,Cancer Chemother.Rep3, 1-104(1972), tumor volume was calculated using the following formula: tumor volume (mm)³) Long x wide ═²)/2. The results are expressed as percent inhibition as calculated by the following formula: inhibition ratio (%) (TuW)_Control-TuW_{Test of})/TuW_ControlX 100%. The flank position of tumor implantation provides reproducible dose/response effects for various chemotherapeutic agents, and the (tumor diameter) measurement method is a reliable method for determining tumor growth rate.

Administration of the compounds of the present invention (hereinafter referred to as active compounds) can be accomplished by any method for delivering the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral routes of injection (including intravenous, subcutaneous, intramuscular, intravascular injection or infusion), topical administration, and rectal administration.

The amount of active compound administered will depend on the subject being treated, the severity of the disease or condition, the frequency of administration, the distribution of the compound and the judgment of the attending physician. However, an effective amount is in the range of about 0.001 to about 100mg/kg body weight per day, preferably about 1 to about 35 mg/kg/day, administered in single or divided doses. For a 70kg human, this will be about 0.05 to 7 g/day, preferably about 0.2 to about 2.5 g/day. In some cases dosage levels below the lower limit of the aforesaid range may be sufficient, and in other cases higher dosage levels may be employed without causing any harmful side effects, except that the higher dosage is first divided into smaller doses for administration throughout the day.

The present invention also advantageously provides kits for use by consumers in the treatment of disease. The kit comprises: a) pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier, excipient or diluent; and b) instructions describing a method of using the pharmaceutical composition to treat a particular disease.

Kits for use herein include containers, such as separate bottles or separate foil press packs, containing individual unit doses. The container may be in any conventional shape or form made of pharmaceutically acceptable materials known in the art, such as a box of paper or cardboard, a glass or plastic bottle or can, or a resealable bag (e.g., to refill the tablet into a different container), or a blister pack in which the single dose is extruded according to a therapeutic procedure. The containers employed depend on the particular dosage form contained therein, e.g., conventional cardboard boxes are not typically used to store liquid suspending agents. It is possible to use multiple containers in one package to sell a single dose dosage form. For example, the tablets may be packaged in bottles and then in boxes.

An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are widely used for packaging pharmaceutical unit dose dosage forms (tablets, capsules, etc.). Blister packs are generally constructed from a sheet of relatively rigid material covered with a foil, preferably of transparent plastics material. During packaging, pockets are formed in the plastic foil. The recess may have the shape and volume of a single tablet or capsule to be packaged, or may have a volume and shape to accommodate multiple tablets and/or capsules to be packaged. The tablet or capsule is then placed in the recess and a sheet of harder material is sealed to the plastic foil on the side of the opening of the foil opposite to the direction in which the recess is formed. As a result, the tablets or capsules are individually sealed or clustered as desired in the pockets formed between the plastic foil and the sheets. Preferably the strength of the sheet is such that the pockets can be manually pressed to form openings in the sheet at the location of the pockets to allow the tablets or capsules to be extruded from the blister pack. The tablet or capsule is then removed through this opening.

It may be desirable to provide a textual memo that includes information and/or guidance to the doctor, pharmacist or patient, for example, in the form of: next to the tablet or capsule is a quantity corresponding to the quantity of the given tablet or capsule to be taken on a certain day of the application, or a card containing the same information. Another example of such a memo is a calendar printed on a card, such as "first week, monday, tuesday", etc., as described below. Other types of memos are also readily apparent. A "daily dose" may be one tablet or capsule, or multiple tablets or capsules taken on a given day.

Another specific embodiment of the kit is a dispenser designed to dispense one daily dose at a time. Preferably, the dispenser is fitted with a memory aid to further facilitate compliance with the regimen. An example of such a memory aid is a mechanical counter which indicates the number of daily doses that have been dispensed. Another example of such a memory aid is a battery-driven memory microchip connected to a liquid crystal readout device or an audible cue signal, for example, to read the date the last daily dose has been taken and/or to remind the patient when the next dose is taken.

In another example of a kit, the pharmaceutical composition may also include other compounds that may be used in combination with the compounds of the present invention, or the kit may include 2 pharmaceutical compositions: one containing the compound of the invention and the other containing other compounds that may be used in combination with the compound of the invention.

The active compounds may be used as monotherapy or may include one or more other antineoplastic agents, for example selected from: mitotic inhibitors, such as vinblastine; alkylating agents, such as cisplatin, carboplatin, and cyclophosphamide; antimetabolites, such as 5-fluorouracil, cytosine arabinoside and hydroxyurea, or, for example, one of the preferred antimetabolites disclosed in European patent application No. 239362, such as N- (5- [ N- (3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl) -N-methylamino) -2-thiazoyl) -L-glutamic acid; a growth factor inhibitor; a cell cycle inhibitor; intercalating antibiotics, e.g., doxorubicin and bleomycin; enzymes, such as interferon; and anti-hormonal agents, e.g. anti-oestrogens such as Nolvadex^TM(tamoxifen) or, for example, antiandrogens such as Casodex^TM(4 '-cyano-3- (4-fluorophenylsulfonyl) -2-hydroxy-2-methyl-3' - (trifluoromethyl) -propionylanilide). Combination therapy can be achieved by administering the individual therapeutic components simultaneously, sequentially or separately.

The pharmaceutical compositions may, for example, be in the form of tablets, capsules, pills, powders, sustained release dosage forms, solutions, suspensions suitable for oral administration, sterile solutions, suspensions or emulsions suitable for parenteral injection, ointments or creams suitable for topical administration or suppositories for rectal administration. The pharmaceutical compositions may be in unit dosage form suitable for single administration of precise dosages. Pharmaceutical compositions will include the usual pharmaceutical carriers or excipients, as well as the active ingredients of the compounds of the invention. In addition, other therapeutic agents, carriers, adjuvants and the like may also be included.

The co-administration of a compound of the invention with other compounds means that the compounds may be administered as part of a composition or unit dosage form, or in separate dosage forms, at the same time or at different times.

Exemplary parenteral dosage forms include solutions or suspensions of the active compounds in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. These dosage forms can be suitably buffered, if necessary.

Suitable pharmaceutical carriers include inert diluents or fillers, water and various organic solvents. If desired, the pharmaceutical composition may contain additional components such as flavoring agents, binders, excipients, and the like. Thus, for oral administration, tablets containing various excipients such as citric acid may also contain various disintegrants such as starch, alginic acid and certain complex silicates and binding agents such as sucrose, gelatin and acacia. Additionally, lubricants such as magnesium stearate, sodium lauryl sulfate and talc are commonly used for tableting. Solid compositions of the same type may also be used in soft and hard filled capsules. Thus, preferred starting materials include lactose and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the active compound therein may be combined with various sweetening or flavoring agents, coloring matter or dyes, and emulsifying or suspending agents and diluents such as water, ethanol, propylene glycol, glycerin or combinations thereof may also be added as desired.

Methods for preparing pharmaceutical compositions containing a specific amount of active compound are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easter, Pa., 15th Edition (1975).

The examples and methods of preparation provided below further illustrate and explain the compounds of the present invention and the methods of preparing these compounds. It should be understood that the scope of the present invention is not limited in any way to the scope of the following examples and preparation methods. In the following examples, molecules having a single chiral center, unless otherwise indicated, are present in the form of a racemic mixture. Molecules with two or more chiral centers, unless otherwise indicated, exist as diastereomeric racemic mixtures. The single enantiomers/diastereomers may be obtained by methods known to those skilled in the art.

Where HPLC is involved in the following preparative methods and examples, HPLC is carried out using a Waters Alliance HPLC system (2690+996 photodiode array). Preparative HPLC was performed with a Waters 717 autosampler, 996PDA, 600 controller. Further details of chromatographic operations are provided in the examples below.

The compounds of the invention may be prepared synthetically according to scheme 1 above or may be prepared by biotransformation as known to those skilled in the art or as described below.

Examples

Example 1

General method of biotransformation

Those skilled in the art can accomplish the bioconversion by contacting the material to be converted and other necessary reactants with various living microorganisms or enzymes derived therefrom under conditions suitable for chemical action to occur. The reaction product is then isolated and the desired material is purified for elucidation of its chemical structure and physical and biological properties. The enzyme may be present in the following manner: pure reagents, crude extracts or lysates, intact cells, solutions, suspensions, covalently bound to a support surface, or embedded in a permeable matrix (e.g., agarose or alginate particles). The substrate and other necessary reactants (e.g., water, air, cofactors) are provided to chemical specifications.

Generally, the reaction is carried out in the presence of one or more liquid phases-aqueous and/or organic phases-to promote mass transfer of reactants and products. The reaction may or may not be carried out aseptically. The conditions under which the reaction proceeds and the reaction products are separated will vary depending on the physical properties of the reaction system and the chemical properties of the reactants and products, and such variations will be understood by those skilled in the art.

The following are two examples of laboratory scale processes for carrying out aerobic bioconversion, which one skilled in the art can employ to prepare the desired compounds: a nutrient medium (e.g., IOWA medium: glucose, yeast extract, dipotassium hydrogen phosphate, sodium chloride, soybean meal, water; adjusted to neutral pH) is added to one or more culture vessels (e.g., fermentation tubes or flasks) and then steam sterilized. Each vessel is aseptically inoculated with a suspension of growths, washed cells or spores obtained from an agar culture, or with a broth obtained from a culture of a liquid nutrient medium of a bioconverted microorganism. The vessel is mounted on a shaker designed for fermentation and shaken (e.g., operated at 100-. The parent compound to be converted (i.e., the substrate) is dissolved in water or a suitable water-miscible solvent (e.g., dimethyl sulfoxide, dimethylformamide, ethanol, methanol). To each bioconversion container, the resulting solution is aseptically added to achieve the desired substrate concentration (e.g., 0.1-0.2 mg/mL). The vessel to which the reactants are added is set on a shaker and shaken as before until the substrate is converted to the product by metabolism of the microorganism (e.g., 1-10 days). The contents of the bioconversion vessel may be subjected to physical treatment (e.g., filtration or centrifugation) to separate insoluble solids and cells from the aqueous phase, or extraction under conditions of optimal pH for extraction of the compound of interest (water-immiscible organic solvents include, but are not limited to, dichloromethane or ethyl acetate). Upon isolation, the solid may be extracted with a suitable water-miscible organic solvent (e.g., methanol). The solid solvent extract and liquid phase contents obtained from the vessel are recovered, combined, and concentrated by a suitable method, for example, solid phase extraction and drying under reduced pressure. The dried crude product is redissolved in a solvent compatible with the purification process (e.g., acetonitrile, methanol, water or HPLC mobile phase). Isolation and purification of the bioconversion product is achieved by, but not limited to, Solid Phase Extraction (SPE) followed by reverse phase High Performance Liquid Chromatography (HPLC).

The biotransformation products can be detected in chromatographic separations, for example with UV-absorption and photodiode array spectrograms (photodiodaray spectral profile). The fraction of the HPLC mobile phase containing the desired product is retained and the product is extracted from the mobile phase by any suitable means, for example, vacuum drying, followed by extraction with SPE or a water-immiscible organic solvent at a pH optimal for the extraction of the desired compound. The solvent eluate from the SPE extraction was recovered, filtered to remove solids, and concentrated under reduced pressure to give the pure dry bioconversion product. The structure of the isolated product was determined by Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR).

Example 2

Preparation of E-N- (3- {4- [ 3-hydroxymethyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -2-methoxy-acetamide by microbial transformation.

50mL (50mL) of IOWA medium (20 g of anhydrous glucose, 5g of yeast extract, 5g of dipotassium phosphate, 5g of sodium chloride, 5g of soybean powder, 1L of distilled water, adjusted to pH 7.2 with 1N hydrochloric acid) was added to 29 250mL Erlenmeyer flasks with foam stoppers, and steam sterilized at 15psig and 121 ℃ for 20 minutes. 0.5mL of cryopreserved (-80 ℃) mycelia of Streptomyces albulus (ATCC12757) was aseptically inoculated into 3 flasks. The inoculated flask was mounted vertically on a rotary shaker (stroke 2 inches) and shaken at 210rpm for 2 days at 29 ℃ (inoculum stage). Then, 5mL of the inoculum-stage culture was aseptically transferred to the remaining 26 flasks (biotransformation stage). The inoculated bioconversion flasks were mounted vertically on a rotary shaker (stroke 2 inches) and shaken at 210rpm for 2 days at 29 ℃. The mesylate salt of 2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide (i.e., the substrate) was dissolved in dimethyl sulfoxide (10 mg/mL). To each of the 26 bioconversion flasks, 0.5mL of the resulting solution was aseptically added to give an initial substrate concentration of 0.1mg/mL (130 mg total in 26 flasks). The flask to which the reaction was added was re-mounted vertically on a rotary shaker and shaken at 210rpm at 29 ℃ for an additional 3 days. At the end of the 3 day bioconversion cycle, the contents of the bioconversion flasks were combined. The pH of the whole broth was adjusted to 8.5 with 1N sodium hydroxide. The resulting broth was extracted 2 times with equal volume of ethyl acetate. The organic phase is concentrated using a rotary evaporator (40 ℃ water bath) and then dried under reduced pressure (Savant Speedvac, low temperature, full vacuum setting). To the residue was added dimethyl sulfoxide (0.4mL) and loaded into a pretreated Waters C18 SepPak (5g) for solid phase extraction (cartridge pre-treatment according to manufacturer's instructions). After loading, the column was eluted with 24mL of distilled water, then with 24mL of 25% aqueous methanol, then with 24mL of 50% aqueous methanol to remove excess starting material. The desired compound was eluted with 24mL of 75% aqueous methanol. The 75% aqueous methanol eluate fraction (SavantSpeedvac, low temperature set, full vacuum set) was dried under reduced pressure overnight. Methanol was added to each tube and the combined residues (about 0.6mL) were subjected to reverse phase high performance liquid chromatography (HPLC method 1) to isolate the desired compound.

HPLC method 1

A chromatographic column: waters SymmetryPrep C185. mu.19X 300 mm.

Mobile phase: linear gradients were applied from 0-20 min.

From 90: 10 to 50: 50 in 20 minutes; adjusting to 10: 90 in 20.5 minutes; keeping for 7 minutes at a ratio of 10: 90; (aqueous buffer [5mM ammonium acetate, pH 4.5: acetonitrile)

Flow rate: 12 mL/min.

A detector: UV absorbance at a wavelength of 254 nm; 200-400nm photodiode array.

The operation time is as follows: for 27 minutes.

The retention time of the title compound was about 17.2 minutes. The HPLC fractions containing the title compound were collected. The pH of the eluate was adjusted to 8.6 with 1N NaOH and then extracted 2 times with an equal volume of dichloromethane. An aliquot of the organic phase was dried in a stream of nitrogen (40 ℃ water bath) and suspended in methanol for reverse phase high performance liquid chromatography (HPLC method 2) analysis. In this assay, the retention time of the desired compound is approximately 14.7 minutes. In the same assay, elution took about 19.3 minutes to give the parent compound.

HPLC method 2

A chromatographic column: waters Symmetry C185. mu. in volume: 2.1X 150 mm.

Mobile phase: linear gradient in 0-20 min;

Flow rate: 0.3 mL/min.

The operation time is as follows: for 30 minutes.

The remaining organic phase was concentrated using a rotary evaporator under reduced pressure (40 ℃ water bath) and then reduced to dryness under reduced pressure (SavantSpeedvac, low temperature setting, full vacuum setting). The desired compound was isolated as a yellow powder (15.6 mg).

It has maximum UV-light absorbance (lambda) at 242.6nm, 312.5nm and 347nm_max). Mass spectrum: 486.5.

¹H(CD₃OD)：δ8.78(s，1H)，8.65(d，J＝1.6Hz，1H)，8.45(d，J＝2.8Hz，1H)，8.23(dd，J＝8.7，1.6Hz，1H)，8.02(d，J＝2.8Hz，1H)，7.98(dd，J＝8.7，2.8Hz，1H)，7.85(d，J＝8.7Hz，1H)，7.81(dd，J＝8.7，2.8Hz，1H)，7.78(d，J＝8.7，1H)，7.21(d，J＝8.7Hz，1H)6.78(d，J＝15.9Hz，1H)，6.64(dt，J＝15.9，5.6Hz，1H)，4.74(s，2H)，4.14(dd，J＝5.6，1.2Hz，2H)，3.98(s，2H)，3.48(s，3H)，2.73(s，3H)。¹³C(CD₃OD)δ171.6，161.2，160.9，160.5，154.8，151.1，150.4，150.0，139.0，137.9，134.8，134.6，134.3，1329，132.7，130.8，128.9，128.2，125.9，125.7，121.2，120.0，119.9，114.3，71.7，58.8，′58.7，40.6，18.9。

Example 3

Preparation of E-N- (3- {4- [4- (6-hydroxymethyl-pyridin-3-yloxy) -3-methyl-phenylamino ] -quinazolin-6-yl } -allyl) -2-methoxy-acetamide:

the desired compound, E-N- (3- {4- [4- (6-hydroxymethyl-pyridin-3-yloxy) -3-methyl-phenylamino ] -quinazolin-6-yl } -allyl) -2-methoxy-acetamide, was prepared by the method described in example 2, with the following exceptions:

the microorganism used was mycelium of Streptomyces rimosus (ATCC 23955) instead of mycelium of Streptomyces albidus (ATCC 12757). The flask with the reactants was shaken for an additional 5 days (instead of 3 days). The title compound had a retention time of about 18.5 minutes using HPLC method 1 of example 2. After collection of the HPLC fractions (from HPLC method 1), the eluate was extracted 2 times with an equal volume of dichloromethane (without adjusting the pH of the eluate). The retention time of the desired compound, corresponding to the second high performance liquid chromatography (HPLC method 2), was about 15.3 minutes. In the same assay, the parent compound elutes at about 19.3 minutes. The desired compound was isolated as a yellow powder (10.4 mg).

The compound has UV-light maximum absorption (lambda) at 242.6nm, 312.5nm and 347nm_max). Mass spectrum: 486.5.

¹H(CD₃OD)：δ8.53(s，1H)，8.40(d，J＝1.2Hz，1H)，8.22(d，J＝2.8Hz，1H)，8.03(dd，J＝8.7，2.0Hz，1H)，7.72(d，J＝2.8Hz，1H)，7.76(d，J＝8.7Hz，1H)，7.64(dd，J＝8.7，2.8Hz，1H)，7.54(d，J＝8.7Hz，1H)，7.41(dd，J＝8.7，2.8Hz，1H)，7.04(d，J＝8.7Hz，1H)，6.76(d，J＝15.9Hz，1H)，6.53(dt，J＝15.9，5.6Hz，1H)，4.70(s，2H)，4.12(m，2H)，3.99(s，2H)，3.48(s，3H)，2.29(s，3H)。¹³C(CD₃OD)δ171.6，159.3，155.1，154.3，153.7，151.2，147.1，138.0，136.7，135.3，131.9，130.7，130.1，128.5，127.0，126.0，125.4，123.1，122.2，120.4，120.3，115.5，71.7，64.2，58.6，40.6，15.4。

Example 4

Preparation of E-3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -acrylic acid

To a cooled (0 ℃) stirred suspension of NaH (4.8g, 60%, 0.12mol) in anhydrous DMF (60mL) was added dropwise a solution of PhOH (11.3g, 0.12mol) in dry DMF (50 mL). After dropwise addition, 4-chloro-6-iodoquinazoline (29g, 0.1mol) was added in portions. The cooling bath was then removed, the resulting solution was stirred at room temperature for 1.5 hours, and the reaction was quenched with water (300 mL). The product precipitated and was then extracted with EtOAc (400 mL). Washing the separated organic layer with aqueous NaOH, water, brine, and Na₂SO₄Drying and concentration gave 6-iodo-4-phenoxyquinazoline (32.9g, 94%) as a yellow-white solid. Crystallization from EtOAc gave white, soft and short needles.

With N₂The 6-iodo-4-phenoxyquinazoline (3.5g, 10mmol) prepared in the previous paragraph, methyl propionate (6g, 70mmol), Pd (OAc)₂(140mg, 0.62mmol) and Ph₃P (320mg, 1.22mmol) in Et₃The mixture in N/DMF was purged and the pressure reactor was capped tightly. The reaction was then heated and stirred at 110 ℃ on an oil bath. Thin layer chromatography showed the reaction was complete after 3 hours. The product mixture was then transferred to a round bottom flask and charged with N₂The gas stream was purified to remove methyl propionate. The residue was then dissolved in ethyl acetate, washed with water, brine, dried over sodium sulfate and concentrated to give crude E-3- (4-phenoxy-quinazolin-6-yl) -acrylic acid methyl ester as a yellow solid, which was recrystallized from ethyl acetate to give 2.3g (70%) of light yellow solid crystals in 2 batches.

A mixture of the product obtained from the previous paragraph (E-3- (4-phenoxy-quinazolin-6-yl) -acrylic acid methyl ester) (307mg, 1mmol) and the desired aniline (215mg, 1mmol) was dissolved in phenol (2g) and the resulting mixture was then heated on an oil bath at 100 ℃ to give a clear liquid. After heating for 20 hours, the brown liquid was distilled under reduced pressure to remove phenol. The residue was partitioned between dilute NaOH and dichloromethane. The separated organic phase was washed with brine, dried over sodium sulfate and concentrated to give the crude product which was purified by chromatography to give pure E-3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -acrylic acid methyl ester (480 mg).

The methyl ester (450mg, 1mmol) and LiOH. H₂O (0.63g, 15mmol) was refluxed in a methanol/water (10/1ml) mixture for 3 hours. After cooling, the reaction mixture was neutralized to pH6.0 with acetic acid (0.9g, 15mmol) in 2mL of water. A clear liquid was initially obtained and the precipitated acid product was subsequently obtained as a yellow solid which was collected by vacuum filtration and dried to give the final product E-3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino as a yellow solid]-quinazolin-6-yl } -acrylic acid (280mg, 68%).

¹H(CD₃OD)：δ2.24(s，3H)，2.48(s，3H)，6.70(d，J＝16Hz，1H)，6.98(d，1H)，7.28(m，2H)，7.6(m，1H)，7.69(m，1H)，7.76(m，1H)，7.78(d，J＝16Hz，1H)，8.1(m，2H)，8.5(s，1H)，8.6(d，1H)。m/z(ES⁺) (M +1) 413.4. HPLC Rt 4.831 min.

The compounds of the present invention may also be prepared as mixtures of metabolites of E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide, of the structure shown below.

Thus, E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide may be cultured with mouse, rat, monkey, dog, and human liver tissue preparations (slices, homogenates, hepatocytes, microsomes) or with recombinant enzymes (e.g., human CYP containing insect cell microsomes). Bile, urine and plasma samples may be collected and further processed to obtain a sample of metabolite mixture. These samples can then be separated by HPLC and analyzed by standard detection methods, such as mass spectrometry, NMR and UV.

Claims

1. A compound of formula 1 or a pharmaceutically acceptable salt, solvate or prodrug thereof:

wherein:

R¹selected from H and C₁-C₆Alkyl groups;

R⁵selected from the group consisting of-C (O) OH and- (CR⁶R⁷)_m-NR¹R⁸Wherein m is an integer of 0 to 3; r⁶And R⁷Each independently selected from H and C₁-C₆Alkyl and wherein R⁸Is selected from C₁-C₆Alkyl and-C (O) - (CR)⁶R⁷)_m-O(C₁-C₆Alkyl) groups; wherein the general formula1Further optionally substituted with a hydroxyl or O-glucuronic acid substituent.

2. The compound of claim 1, wherein R¹Is H, R²Is hydroxymethyl, R³Is methyl, and R⁵is-CH₂NHC(O)CH₂OCH₃。

3. The compound of claim 1, wherein R¹Is H, R²Is methyl, R³Is hydroxymethyl, and R⁵is-CH₂NHC(O)CH₂OCH₃。

4. The compound of claim 1, wherein R¹Is H, R²Is methyl, R³Is methyl, and R⁵is-C (O) OH.

5. The compound of claim 1, wherein R¹Is H, R²Is methyl, R³is-COOH, and R⁵is-CH₂NHC(O)CH₂OCH₃。

6. The compound of claim 1, wherein the compound of formula 1The compound further contains a hydroxy substituent, and wherein R¹Is H, R²Is methyl, R³Is methyl, and R⁵is-CH₂NHC(O)CH₂OCH₃。

7. The compound of claim 1, wherein the formula1Further containing a hydroxy substituent, and wherein R¹Is H, R²Is methyl, R³Is hydroxymethyl, and R⁵is-CH₂NHC(O)CH₂OCH₃。

8. The compound of claim 1, wherein R¹Is H, R²Is hydroxymethyl, R³Is methyl, and R⁵is-CH₂NHC(O)CH₂OH。

9. The compound of claim 1, wherein the formula1The compound of (1) further contains an-O-glucuronic acid substituent.

10. The compound of claim 1, wherein the compound is substantially pure.

11. A method of treating abnormal cell growth in a mammal comprising the step of administering to said mammal an amount of a compound of claim 1 effective to treat abnormal cell growth.

12. A pharmaceutical composition for treating abnormal cell growth in a mammal comprising an abnormal cell growth treating effective amount of a compound of claim 1 and a pharmaceutically acceptable carrier.

13. A method for determining whether a patient has been administered E-2-methoxy-N- (3- {4- [ 3-methyl-4- (6-methyl-pyridin-3-yloxy) -phenylamino ] -quinazolin-6-yl } -allyl) -acetamide, comprising the step of determining whether a plasma, urine, bile or fecal sample obtained from the patient shows the presence of a compound of claim 1.

14. A kit for treating abnormal cell growth, comprising: a) a pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrier, excipient or diluent; and b) instructions describing a method for treating abnormal cell growth using the pharmaceutical composition.