KR20090089849A - Macrocyclic quinazoline derivatives as vegfr3 inhibitors - Google Patents

Abstract

The present invention relates to the use of some of the macrocyclic quinazoline derivatives described in PCT publication WO2004/105765 as inhibitors of VEGFR3 mediated biological activities, especially those activities which are mediated by VEGFR3 ligands VEGF-C and/or VEGF-D. ® KIPO & WIPO 2009

Description

Macrocyclic quinazoline derivatives as VEGFR3 inhibitors {MACROCYCLIC QUINAZOLINE DERIVATIVES AS VEGFR3 INHIBITORS}

The present invention discloses that some of the macrocyclic quinazoline derivatives described in PCT Application Publication No. WO2004 / 105765 are useful as inhibitors of VEGFR3 mediated biological activity, in particular activity mediated by VEGFR3 ligand, VEGF-C and / or VEGF-D. The results of the study.

Cancer is the leading cause of death worldwide in the early 21st century, and is an important focus for ongoing research and development.

In recent years, promising methods for therapeutic intervention in cancer have been concentrated on antiangiogenic therapy. Methods for such interventions in cancer progression utilize the concept that inhibiting or otherwise limiting the blood supply to the tumor will reduce tumors of oxygen and nutrients, thereby stopping tumor cell growth and proliferation. This method has been found to be effective and currently has at least 20 anti-angiogenic agents, with various evaluation steps in phase 1, phase 2 or phase 3 clinical trials and multiple evaluation steps in preclinical development.

Although there are many different forms of cancer exhibiting various characteristics, one factor shared by many cancers is that they must metastasize to be lethal. Until the time of metastasis, it may be malignant, but the tumor is confined to one part of the body. Although it may be located prior to metastasis, it can cause discomfort and / or pain, or even lead to more serious problems, and cancer can be treated or even eliminated by surgical intervention. As long as the remaining cancer cells are inhibited, these isolated cancers can be controlled without serious problems. However, cancer cells will invade the body by metastasis, and surgical resection can remove the primary tumor, but the spread of cancer metastasis to xenosites is very difficult to handle.

Metastasis through lymphatic vessels to local lymph nodes is a common step in cancer progression. Metastasis is an important prognostic factor for many cancer types and forms the basis for surgical and radiotherapy of regional lymph nodes. Tumor metastasis processes include local infiltration and intercellular matrix destruction, intravasation into blood vessels, transport through lymph or other channels, survival in circulating blood, extravascular outflow at secondary sites, and proliferation at new sites. Is a multi-stage event (Idler, et al., Adv. Cancer Res. 28,149-250 (1978), Liotta, et al., Cancer Treatment Res. 40, 223-238 (1988), Nicolson, Biochim. Biophy. Acta 948 , 175-224 (1988) and Zetter, N. Eng. J. Med. 322,605-612 (1990)). Success in establishing metastatic deposition requires tumor cells that can achieve these steps sequentially.

Recently, a number of evidence lines have shown that lymphangiogenesis, lymphangiogenesis promotes lymphatic metastasis (Stacker et al., Nature Med. 7 (2), 186-191 (2001); Skobe et al. Nature Med. 7 (2), 192-8 (2001); Makinen et al., Nature Med. 7 (2), 199-205 (2001). Lymphatic vascular control can provide a new strategy for preventing lymph node metastasis in the treatment of cancer.

Recent studies show that a member of the vascular endothelial growth factor (VEGF) family, VEGF-C, promotes lymphatic endothelial cell growth and migration upon lymphatic neovascularization and its receptor, VEGFR3 (Karkkainen MJ). , et al., Semin Cell Dev Biol. 13: 9-18 (2002)). VEGF-C also promotes lymphatic-mediated metastasis, breast cancer metastasis and human melanoma metastasis through induction of tumor-associated lymphangiogenesis in a number of solid cancers such as gastric cancer, prostate cancer, human colorectal cancer, invasive cervical cancer Appeared to. In support of these, there is preclinical data demonstrating that VEGF-C overexpression in cancer cells significantly increases tumor-associated lymphangiogenesis, thereby elevating metastasis to regional lymph nodes (Stacker SA., Et al., FASEB J 16). : 922-34 (2002)). In addition, blocking of VEGF-C / D mediated signaling has been shown to inhibit tumor lymphangiogenesis and lymph node metastasis in mice (He Y., et al., J Natl Cancer Inst. 94: 819-25 (2002)) .

VEGFR3, a transmembrane tyrosine kinase receptor, is expressed extensively in endothelial cells at early development (Pajusola K., et al., Cancer Res. 53 (16): 3845 (1992)). In later development, expression of VEGFR-3 limits lymphatic development [Kaipainen, A., et al., Proc. Natl. Acad. Sci. USA, 92: 3566-3570 (1995). In adults, lymphatic endothelium and many high endothelial veins express VEGFR-3, and increased expression occurs in the metastases of metastatic lymph nodes and lymphangiomas. VEGFR-3 is also expressed in a subset of CD34 + hematopoietic cells that can mediate the myeloid hematopoietic activity of VEGF-C, as evidenced by overexpression studies. Due to the target destruction of the VEGFR-3 gene in the mouse fetus, remodeling of the primary vascular network ends in failure and dies after 9.5 days of embryonic day [Dumont et al., Science, 282: 946-949 (1998) ]. This study suggests an important role for VEGFR-3 in the development of fetal vasculature and lymphangiogenesis.

From the foregoing, inhibitors of VEGFR3 have great potential as therapeutic agents, and new types of new agents represent a continuing need in the art to inhibit the growth and / or spread of various tumorous or cell proliferative diseases. It will be apparent that inhibition of VEGFR3 activity, including inhibition of ligand binding to VEGFR3, is useful for the treatment of mammalian subjects diagnosed with a disease characterized by the proliferation of endothelial cells expressing VEGFR-3. For example, many tumors are characterized by vascular or lymphatic neovascularization, which includes endothelial cells expressing VEGFR-3. In many cancers, the cancer cells themselves express VEGFR3 and represent target cells. In another cancer overlapping set, the cancer cells express a VEGFR-3 ligand selected from VEGF-C and VEGF-D. Ligands are believed to be involved in recruiting endothelial cells and promoting their growth, thereby promoting the nutritional status and / or proliferation of cancer.

(Summary of invention)

The present invention is directed, in part, to methods of treating or preventing VEGFR3 mediated biological activity using certain compounds described in WO2004 / 105765, the entire contents of which are incorporated herein by reference.

In a related embodiment, the present invention comprises administering to a mammalian subject suspected of having cancer, the compound of the invention in an amount effective to inhibit metastatic spread of the cancer. Methods of inhibiting diffusion; And administering to a mammalian subject diagnosed with cancer, a composition comprising a compound of the invention in an amount effective to reduce cancer's proliferation or neoplastic spread. do. It will be appreciated that a reduction in cancer proliferation or spread (which can prolong life and sustain quality of life) indicates successful treatment. In a preferred embodiment, cancer proliferation is completely stopped. In a more preferred embodiment, the cancer is reduced or completely eradicated. Preferred therapeutic subjects are human subjects, but other animals, in particular murine, rats, dogs, cattle, pigs, primates, and other model systems for the treatment of cancer are contemplated.

In some cancers expressing VEGFR-3, direct inhibition of cancer proliferation or cancer death may be a mechanism. Treatment of all cancers expressing VEGFR-3, and all cancers characterized by angiogenesis or lymphangiogenesis in and on the proliferating tumors, is contemplated. For example, treatment consists of brain, lungs, liver, spleen, kidneys, lymph nodes, small intestine, blood cells, pancreas, colon, stomach, breast, endometrium, prostate, testes, ovaries, skin, head and neck, esophagus, bone marrow and blood. Consider cancer of the tissue, organ, or cell selected from the group consisting of: In a particular embodiment of the invention, the treatment contemplates cancer of a tissue, organ or cell selected from the group consisting of breast, lung, prostate and colon.

In one variation of the method of treatment described above, the compound is administered with a second cancer therapeutic agent. The second cancer therapeutic agent may be a chemotherapeutic agent, a radiopharmaceutical, a radiation, a nucleic acid encoding a cancer therapeutic agent, an antibody, a protein, and / or other antilymphogenic or antiangiogenic agent. The second cancer therapeutic agent may be administered before, after, or simultaneously with the compound of the present invention.

In one variant, the treated subject is diagnosed with a tumor that can be treated surgically, and the administering step is performed before, during or after excision of the tumor from the subject. Compound treatment in combination with tumor resection is intended to reduce or eliminate tumor repopulation from cancer cells that fail to ablate.

In a more genetic sense, the present invention provides a method for treating a condition characterized by VEGFR-3 binding to a natural ligand that binds to VEGFR-3, comprising administering a compound of the invention to a subject in need thereof. Provide a method.

Figure 1 shows 4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzoxadiazacyclopentadecin, 17-bromo- for VEGF-C induced VEGFR3 activity. 8,9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl- (compound 2). The top frame provides the result of VEGFR-3 immune precipitates migrated to nitrocellulose membrane stained with phosphotyrosine antibody. The lower frame provides the result of VEGFR-3 immune precipitates migrated to nitrocellulose membrane stained with VEGFR-3 antibody. Left lanes 1 and 2 provide DMSO treatment in the absence and presence of negative and positive controls, ie VEGF-C.

2 shows VEGF and VEGF-C stimulation of Erk1 / 2 phosphorylation of HMVECd cells in the presence of 5 μM Compound 2. FIG. Upper lane provides staining of Ph202 / Tyr204 mouse monoclonal antibody as primary antibody and phosphorylated Erk 1/2 (P-Erk 1/2) using goat antimouse conjugated to IRDye 800 nm as secondary antibody. . The bottom lane provides staining of the entire Erk 1/2 using Erk 1/2 specific rabbit polyclonal antibody as the primary antibody and goat antirabbit conjugated at Alexa 680 nm as the secondary antibody. The left panel provides the results for VEGF induced phosphorylation of Erk-1 / 2 in HMVECd cells. The right panel provides the results for VEGF-C induced phosphorylation of Erk 1/2 of HMVECd cells.

WO-2004 / 105765 describes the preparation, formulation and pharmaceutical properties of the class of macrocyclic quinazoline derivatives of general formula (I) as multi-target kinase inhibitors.

It is now understood that certain groups of compounds in the aforementioned classes have VEGFR3 inhibitory activity which makes them useful in the treatment or prevention of VEGFR3 mediated biological activity, particularly for the treatment or prevention of metastatic spread of cancer in mammalian subjects. Figured out.

Accordingly, in one aspect, the present invention provides a general formula for the manufacture of a medicament for the treatment or prophylaxis of VEGFR3 mediated biological activity, for example, in the treatment or prevention of metastatic spread of cancer in a mammalian subject (I Compound of

Wherein Z represents NH;

Y is -C _{3 - 9} alkyl _-, -C ₁ - ₅ alkyl, -NR ¹³ -C _{1 - 5} alkyl _-, -C ₁ - ₅ alkyl, -NR ¹⁴ -CO-C _{1 - 5} alkyl _-, -C ₁ - ₃ alkyl, -NH-CO-Het ²⁰ -, or _{^{-Het 22 -CH 2 -CO-NH-}} C 1 - represents a _3-alkyl;

X ¹ is O or -OC _{1 - 2} alkyl-represents;

X ² is a direct bond, -C ₁₂ alkyl -, O, -OC ₁₂ alkyl - represents a -, NR ¹² or NR _¹² -C ¹² alkyl;

R ¹ represents hydrogen, cyano, halo or hydroxy, preferably halo;

R ² represents hydrogen, cyano, halo, or hydroxy;

R ³ represents hydrogen;

R ⁴ is C _{1 -} represents _a-4 alkyloxy;

R ¹² is hydrogen or C _{1 -} represents a _4-alkyl;

R ¹³ is hydrogen or C _{1 -} represents a _4-alkyl;

R ¹⁴ represents hydrogen or C ₁₄ alkyl;

Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl;

Het ²² stands for pyrrolidinyl, piperazinyl or piperidinyl), and pharmaceutically acceptable acid addition salts or base addition salts and stereochemical isomers thereof.

Another aspect of the invention includes administering to a mammalian subject in need of such treatment a therapeutically effective amount of a compound of Formula I, and a pharmaceutically acceptable acid addition or base addition salt and stereochemical isomer thereof A method of treating or preventing VEGFR3 mediated biological activity in the treatment or prevention of metastatic spread of cancer in a subject:

In the above formula,

Z represents NH;

Y is -C _{3 - 9} alkyl _-, -C ₁ - ₅ alkyl, -NR ¹³ -C _{1 - 5} alkyl _-, -C ₁ - ₅ alkyl, -NR ¹⁴ -CO-C _{1 - 5} alkyl _-, -C ₁ - ₃ alkyl, -NH-CO-Het ²⁰ -, or _{^{-Het 22 -CH 2 -CO-NH-}} C 1 - represents a _3-alkyl;

X ¹ is O or -OC _{1 - 2} alkyl-represents;

X ² is a direct bond, -C ₁₂ alkyl -, O, -OC ₁₂ alkyl - represents a -, NR ¹² or NR _¹² -C ¹² alkyl;

R ¹ represents hydrogen, cyano, halo or hydroxy, preferably halo;

R ² represents hydrogen, cyano, halo, or hydroxy;

R ³ represents hydrogen;

R ⁴ is C _{1 -} represents _a-4 alkyloxy;

R ¹² is hydrogen or C _{1 -} represents a _4-alkyl;

R ¹³ is hydrogen or C _{1 -} represents a _4-alkyl;

R ¹⁴ represents hydrogen or C ₁₄ alkyl;

Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl;

Het ²² represents pyrrolidinyl, piperazinyl or piperidinyl.

In addition, VEGFR3 mediated biological activity is meant to include:

Metastatic spread of cancer in mammalian subjects. Preferred therapeutic subjects are human subjects, but other animal, especially murine, rat, dog, bovine, pig, primate and other model systems for the treatment of cancer are contemplated;

-Metastasis to lymph nodes through lymphatic vessels. Thus, there is provided the use of a compound of the present invention in the treatment or prevention of lymph node metastasis;

Tumor-associated lymphangiogenesis in, for example, gastric cancer, prostate cancer, human colon rectal cancer, invasive cervical cancer, breast cancer, Kaposi's sarcoma and melanoma. Thus, the use of the compounds of the present invention in the treatment or prevention of tumor-associated lymphangiogenesis in cancer, such as gastric cancer, prostate cancer, human colon rectal cancer, invasive cervical cancer, breast cancer, Kaposi's sarcoma and melanoma;

Recruitment and proliferation of endothelial cells expressing VEGFR3 in neovascularization of cancer cells expressing VEGFR3 ligand selected from VEGF-C or VEGF-D. Thus, there is provided the use of a compound of the invention in the treatment of a cancer characterized by angiogenesis or lymphangiogenesis in a growing tumor and its surroundings.

In another embodiment, the present invention provides the preparation of a pharmaceutical composition for the treatment of cancer of a tissue, organ or cell selected from the group consisting of breast, lung (including treatment of small cell and non-small cell lung cancer), colon and prostate. For the use of the compounds of the general formula (I) above.

The present invention also relates to a method for treating breast cancer, lung cancer, colon cancer and / or prostate cancer in a mammal comprising administering to the mammal a therapeutically effective amount of a compound of formula (I) as described above.

In another embodiment, the present invention provides the use of a compound of formula (I) as described above for the manufacture of a pharmaceutical composition for treating advanced breast cancer. As used herein, the term "progressive breast cancer" is used to refer to breast cancer in a patient who does not respond to pretreatment or who is rescued after such treatment, and who has metastatic disease at diagnosis.

The invention also relates to a method of treating advanced breast cancer in a mammal, in particular a female, comprising administering to said mammal a therapeutically effective amount of a compound of formula (I) as described above.

In particular, the present invention relates to the use of a compound of formula (I), wherein one or more of the following limitation conditions apply:

Z represents NH;

Y is -C _{3 - 9} alkyl-, -C _{1 - 5} alkyl, -NR ¹⁴ -CO-C _{1 - 5} alkyl -, or -C _{1 - 3} alkyl, -NH-CO-Het ²⁰ - represents the;

X ¹ represents O;

X ² is -C ₁₂ alkyl-, O, or NR _¹² -C ¹² alkyl-represents the;

R ¹ represents hydrogen or halo; In particular R ¹ represents hydrogen or chloro; In particular R ¹ represents hydrogen;

R ² represents hydrogen or halo; In particular R ² represents hydrogen, chloro or bromo; In particular R ² represents chloro or bromo;

R ³ represents hydrogen;

R ⁴ is C _{1 -} represents a _4-alkoxy; R ⁴ represents methoxy;

R ¹² is C _{1 -} represents a _4-alkyl; In particular R ¹² represents methyl;

R ¹⁴ is a hydrogen or C ₁₄ alkyl; In particular R ¹⁴ represents hydrogen or methyl; In particular R ¹⁴ represents hydrogen;

Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl; Het ²⁰ represents piperidinyl.

Also important among the above-mentioned uses is a general formula (I) wherein R ¹ is at position 3 ', R ² is at position 5', R ⁴ is at position 7, X ² is at position 2 'using the numbering shown in formula (I). ) Compound.

Most preferred compounds are:

4,6-ethanediylidene-19H-pyrimido [4,5-b] [6,13,1] benzodioxaxacyclo-pentadecin, 15-chloro-8,9,10,11,12, 13-hexahydro-20-methoxy-;

12H-4,6-ethanediylidene-13,17-methanopyrimido [4,5-b] [6,1,10,16] benzonetriazacyclononadecin-12-one, 21-chloro -8,9,10,11,13,14,15,16,18,23-decahydro-25-methoxy-;

4,6-ethanediylidene-12H-pyrimido [4,5-b] [6,1,10,13] benzonetriazacyclohexadecin-12-one, 18-chloro-8,9,10 , 11,13,14,15,20-octahydro-21-methoxy-13,14-dimethyl-; And

4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzonediazacyclopentadecin, 17-bromo-8,9,10,11,12,13,14 , 19-octahydro-20-methoxy-13-methyl-; a compound selected from the group consisting of; Or a pharmaceutically acceptable acid addition salt thereof. Especially valuable compounds are preferred.

Most preferred compounds for use in accordance with the present invention are compounds selected from the group consisting of compounds having the structure: Its pharmaceutically acceptable acid addition salts are:

.

.

The following compounds; Or pharmaceutically acceptable acid addition salts thereof are particularly preferred:

.

.

As used above definition and below,

Halo is the common name for fluoro, chloro, bromo and iodo;

-C _{1 - 2} alkyl defines methyl or ethyl;

-C _{1 - 3} alkyl, for example, define a straight chain and branched chain saturated hydrocarbon radicals having 3 carbon atoms such as methyl, ethyl, propyl and;

-C _{1 - 4} alkyl, e.g., methyl, ethyl, propyl, butyl, 1-methylethyl, 2-methylpropyl, 2,2-dimethylethyl, such as one to four, with straight-chain and branched-chain carbon atoms, Define a saturated hydrocarbon radical;

-C _{1 - 5} alkyl, e.g., methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 2,2-dimethylpropyl, non-woven with a 1 to 5 carbon atoms such as 2,2-dimethylethyl Define chain and branched saturated hydrocarbon radicals;

-C _{3 - 9} alkyl, e.g., propyl, butyl, pentyl, hexyl, heptyl, octyl, defines a three to nine carbon atoms, with straight-chain and branched saturated hydrocarbon radicals, such as nonylphenyl and;

-C _{1 - 4} alkyloxy is for example methoxy, ethoxy, propyloxy, butyloxy, 1-methyl-ethyloxy, 2-methylpropyl defines a straight or branched chain saturated hydrocarbon radicals such as oxy, and;

The term "CO" refers to a carbonyl group.

Pharmaceutically acceptable acid addition salts or base addition salts as described above are meant to include the therapeutically active non-toxic acid addition salts and non-toxic excitation addition salt forms that compounds of formula (I) may form. Compounds of general formula (I) having basicity can be converted to their pharmaceutically acceptable acid addition salts by treating the base form with a suitable acid. Examples of suitable acids include inorganic acids, such as hydrochloric acid, such as hydrochloric acid or hydrobromic acid; Sulfuric acid; nitric acid; Phosphoric acid and the like; Or organic acids such as acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid (ie, butanedioic acid), maleic acid, fumaric acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, Benzenesulfonic acid, p -toluenesulfonic acid, cyclic acid, salicylic acid, p -aminosalicylic acid, pamoic acid, and the like.

Compounds of general formula (I) having acidity can be converted to their pharmaceutically acceptable base addition salts by treating the acid forms with appropriate organic or inorganic bases. Examples of suitable base salt forms include salts with organic bases such as ammonium salts, alkali metal salts and alkaline earth metal salts such as lithium, sodium, potassium, calcium salts such as benzatin, N-methyl-D-glucamine , Hydravamin salts, and salts with amino acids such as arginine, lysine and the like.

The term “acid addition salt or base addition salt” also includes hydrate and solvent addition forms that the compounds of formula (I) can form. Examples of such forms are, for example, hydroxides, alcoholates and the like.

The term "stereochemically isomer of a compound of general formula (I)" as used above is composed of the same atoms bonded by the same binding sequence that a compound of general formula (I) may have, but is a non-substituted three-dimensional structure All possible compounds having are defined. Unless otherwise specified or indicated, the chemical names of compounds include mixtures of all possible stereochemical isomers that a compound may possess. The mixture may include all diastereomers and enantiomers of the basic molecular structure of the compound. All stereochemically isomeric forms of the compounds of formula (I), either in pure or mixed form, are intended to be included within the scope of the present invention.

Some compounds of formula (I) may exist as their tautomers. Such forms are not specified in the above general formula, but are intended to be included within the scope of the present invention.

Whenever used below, the term "compound of formula (I)" or "compound of the present invention" is meant to include pharmaceutically acceptable acid addition salts or base addition salts and all stereoisomers.

The compounds of the present invention may be prepared and formulated according to methods known in the art, in particular those described in the published patent specification, which is described and incorporated herein by reference; Suitable examples of compounds of formula (I) can be found in PCT Publication WO-2004 / 105765. To prepare the above-mentioned pharmaceutical compositions, a therapeutically effective amount of a particular compound as an active ingredient, in admixture form, is sufficiently mixed with a pharmaceutically acceptable carrier, which can take various forms depending on the form of preparation required for administration. do. These pharmaceutical compositions preferably include systemic administration such as oral administration, transdermal administration or parenteral administration; Or in unit dosage forms suitable for topical administration such as via inhalants, nasal sprays, eye drops or creams, gels, shampoos and the like. For example, in preparing oral dosage forms, for example, oral liquid preparations such as suspensions (including nanosuspensions), syrups, elixirs and solutions, such as water, glycols, oils, alcohols and the like; Or in the case of powders, pills, capsules and tablets, any of the usual pharmaceutical media such as solid carriers such as starch, sugar, kaolin, lubricants, binders, disintegrating agents and the like can be used. Due to their ease of administration, tablets and capsules represent the most advantageous oral unit dosage forms, in which case solid pharmaceutical carriers are dominantly used. For parenteral compositions, the carrier may include other ingredients, for example to aid solubility, but typically at least most will comprise sterile water. For example, injections can be prepared such that the carrier comprises saline, a glucose solution, or a mixture of saline and glucose solution. Injectables containing a compound of formula (I) may be formulated in oils for sustained action. Suitable oils for this are, for example, peanut oil, sesame oil, cottonseed oil, corn oil, soybean oil, synthetic glycerol esters of long chain fatty acids and mixtures of these and other oils. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be used. In a composition suitable for transdermal administration, the carrier optionally comprises a suitable additive having certain properties and a penetration promoter and / or a suitable humectant mixed in small proportions, and the additive does not have a serious adverse effect on the skin. Such additives may be useful for facilitating administration to the skin and / or for preparing a desired composition. These compositions can be administered in a variety of ways, for example as a transdermal patch, spot-on or ointment. Suitable compositions for topical use may include all compositions commonly used for topical administration of drugs, such as creams, gels, dressings, shampoos, tinctures, pastes, ointments, plasters, powders and the like. The compositions may be used in thickening compositions that can be used, for example, by aerosols with propellants, such as nitrogen, carbon dioxide, freons, or aerosols without propellants, such as pump sprays, drops, lotions, or semisolids, such as swabs. Can be used by. In particular, semisolid compositions such as plasters, creams, gels, ointments and the like will be conveniently used.

It is particularly advantageous to formulate the pharmaceutical compositions described above in unit dosage form for ease of administration and uniformity of dosage. Unit dosage forms as used herein and in the claims are physically discrete units suited as unit doses, each unit containing a predetermined amount of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Examples of such unit dosage forms include tablets (including split or coated tablets), capsules, pills, powder packets, wafers, injectable or injectable suspensions, teaspoonfuls, tablespoonfuls, and the like. There are segregated multiples.

Preferably, a therapeutically effective amount of a pharmaceutical composition comprising a compound of the present invention is administered orally or parenterally. The therapeutically effective amount is an amount that effectively prevents or reduces the size and metastasis and / or proliferation of various neoplastic diseases or cell proliferative diseases (described above) in a patient. Based on current data, compounds of the invention as active ingredients, in particular 4,6-ethanediylidenepyrimido [4,5-6] [6,1,12] benzoxdiazacyclo-pentadecin, 17- A pharmaceutical composition comprising bromo-8,9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl- is administered in a single dose or, for example, twice daily It is contemplated that it may be administered orally in an amount of from 10 mg to several (1 to 5) grams per day, as subdivided more than once, including three or even four times. Preferred amounts range from 500 to 4,000 mg per day. Particularly preferred doses for such compounds range from 750 mg to 3,000 mg per day. It will be appreciated that the amount of a compound of the invention, also referred to herein as the active ingredient, required to achieve a therapeutic effect depends, of course, on the route of administration, the age and symptoms of the recipient, and the particular disorder or condition to be treated. Optimal dosages and dosage regimens can be readily determined by one of ordinary skill in the art, using conventional methods and considering the information described herein. Such therapies may be given continuously or intermittently, including but not limited to cycles of 3 to 4 weeks with treatment given at 1 to 21 days per cycle or other schedules that appear effective and safe. .

The VEGFR3 inhibitor can be used in combination with one or more other cancer therapies. Such combination therapy may include chemotherapy, radiation therapy, and anti-tumor therapy, including but not limited to, targeted therapies such as antibodies and small molecules being practiced. Such therapy may be combined with systemic therapy, or topical use / administration (eg, into the spinal cavity), depending on optimal efficacy / safety requirements.

In certain preferred embodiments, with respect to different tumor types, one or more other cancer therapies suitable for use with the VEGFR3 inhibitors of the invention include, but are not limited to:

Breast: Heceptin, Docetaxel, Anthracycline, Capecitabine

Prostate: docetaxel, mitoxantrone

Colon: Oxaliplatin, 5-FU, Avastin, Irinotecan, Cetuximab

lungs; Taxotere, carboplatin, gemicitabine.

VEGFR3 inhibitors and additional anticancer agents of the invention may be administered simultaneously (eg, in separate or single compositions) or sequentially in any order. In the latter case, the two compounds will be administered within a period of time in an amount and in a manner sufficient to ensure that a beneficial or synergistic effect is achieved. It will be appreciated that the preferred method and order of administration for each component of the combination, and each dosage and mode of administration, will depend upon the particular VEGFR3 inhibitor and anticancer agent administered, their route of administration, the particular tumor to be treated and the particular host to be treated. Optimal administration methods and sequences, and dosage and dosage regimens, can be readily determined by one of ordinary skill in the art, using conventional methods and considering the information described herein.

Preparation of Compounds and Formulations:

Compound 95, 12H-4,6-ethanediylidene-13,17-methanopyrimido [4,5-b] [6,1,10,16] benzonetriazacyclononadecin-12-one, Preparation of 21-Chloro-8,9,10,11,13,14,15,16,18,23-decahydro-25-methoxy-

Step 1:

Titanium, tetrakis (2-propanolato) (0.005 mol) was converted to 3-piperidinecarboxylic acid ethyl ester (0.54 g, 0.005 mol) and 4-chloro-2-nitrobenzaldehyde (0.93) in CH ₂ C1 ₂ (40 ml). g, 0.005 mol). The mixture was stirred at rt for 1 h, and NaBH (OAc) ₃ (0.0055 mol) was added. The reaction mixture was stirred at rt for 1 h, extra NaBH (OAc) ₃ (0.00275 mol) was added, the mixture was stirred at rt for 2 h and then NaHCO ₃ (saturated in H ₂ O) was added. . The organic layer was separated, dried (K ₂ CO ₃ ), filtered and the solvent was evaporated and co-evaporated with toluene to remove residual CH ₂ C1 ₂ . 3-piperidinecarboxylic acid, 1-[(4-chloro-2-nitrophenyl) methyl]-, ethyl ester as crude residue was used as such in the next reaction step.

Step 2:

A mixture of 3-piperidinecarboxylic acid, 1-[(4-chloro-2-nitrophenyl) methyl]-, ethyl ester (1.69 g, 0.005 mol) in ethanol (150 ml) was added to a thiophene solution (1 ml). In the presence, hydrogenated using Pt / C 5% (0.5 g) as catalyst. After H ₂ (3 equiv) blowing, the catalyst was filtered off and the filtrate was evaporated. The residue was redissolved in EtOAc and the solution was filtered through Extrelute. Heptane was added but no crystal appeared. The solvent was evaporated and the residue was redissolved in CH ₃ OH and filtered through a paper filter to remove silica grease. The solvent was evaporated to give 1.35 g of 3-piperidinecarboxylic acid, 1-[(2-amino-4-chlorophenyl) methyl]-, and ethyl ester as galsec oil.

Step 3:

4-Chloro-7-methoxy-6-quinazolinol 6-acetate (0.273 g, 0.00108 mol) was added to 3-piperidinecarboxylic acid in 2-propanol (appropriate), 1-[(2-amino-4-chloro Phenyl) methyl]-, ethyl ester (0.0011 mol) and then the reaction mixture was shaken at 80 ° C. overnight. The mixture was then shaken further at 80 ° C. for 6.5 hours to evaporate the solvent. 3-piperidinecarboxylic acid, 1-[[2-[[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl]-, ethyl ester (crude product) ; Used as such in the next reaction step).

Step 4:

NH ₃ / CH ₃ OH 7 N (approx. 10 ml) was diluted with 3-piperidinecarboxylic acid, 1-[[2-[[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino]- 4-chlorophenyl] methyl]-, ethyl ester (0.00114 mol) was added, the reaction mixture was shaken at 35 ° C. for 1 hour, and then the solvent was evaporated. 3-piperidinecarboxylic acid, 1-[[4-chloro-2-[(6-hydroxy-7-methoxy-4-quinazolinyl) amino] phenyl] methyl]-, ethyl ester (in the next reaction step Used as such).

Step 5:

3-piperidinecarboxylic acid, 1-[[4-chloro-2-[(6-hydroxy-7-methoxy-4-quinazolinyl) amino] phenyl] methyl]-, ethyl ester (0.00114 mol), A mixture of N- (3-bromopropyl) carbamic acid 1,1-dimethylethyl ester (1 eq) and Cs ₂ CO ₃ (1.8582 g) was stirred overnight at room temperature, and then the reaction mixture was stirred at 50 ° C. Stirred for a minute. If necessary, additional N- (3-bromopropyl) carbamic acid 1,1-dimethylethyl ester was added and the mixture was stirred at room temperature for 4 hours and at 50 ° C for 15 minutes. The solvent was evaporated (Genevac) and the residue was dissolved in CH ₂ C1 ₂ . This solution was filtered through decalite to evaporate the filtrate. 3-piperidinecarboxylic acid, 1-[[4-chloro-2-[[6- [3-[[(1,1-dimethylethoxy) carbonyl] amino] propoxy] -7-methoxy-4 -Quinazolinyl] amino] phenyl] methyl]-and ethyl ester were obtained.

Step 6:

3-piperidinecarboxylic acid in TFA / CH ₂ C1 ₂ / TIS (90/8/2) (11.4 ml), 1-[[4-chloro-2-[[6- [3-[[(1,1 -Dimethylethoxy) carbonyl] amino] propoxy] -7-methoxy-4-quinazolinyl] amino] phenyl] methyl]-, a solution of ethyl ester (residue; about 0.00114 mol) for 7-8 hours After stirring, the solvent was evaporated and the resulting residue was dried in an oven overnight. 3-piperidinecarboxylic acid, 1-[[2-[[6- (3-aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl]-. CF ₃ COOH was obtained.

Step 7:

3-piperidinecarboxylic acid, 1-[[2-[[6- (3-aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl]-. CF ₃ A solution of COOH (0.00114 mol) and DIPEA (0.00684 mol) was added to a solution of HBTU (0.00342 mol) and HOBt (0.00228 mol) in anhydrous DMF (285 ml), then the reaction mixture was allowed to react for 1 hour. The solvent was evaporated and the dry residue was purified by reverse phase high performance liquid chromatography. The product fractions were combined, Na ₂ CO ₃ was added and the organic solvent was evaporated. CH ₂ C1 ₂ was added to the aqueous concentrate, and the obtained mixture was extracted three times with CH ₂ C1 _2, and then the organic extracts were dried and collected. 12H-4,6-ethanediylidene-13,17-methanopyrimido [4,5-b] [6,1,10,16] benzonetriazacyclononadecin-12-one, 21-chloro -8,9,10,11,13,14,15,16,18,23-decahydro-25-methoxy-0.0007 mol (62% yield) was obtained.

Compound 96, 4,6-ethanediylidene-12H-pyrimido [4,5-b] [6,1,10,13] benzoxatriazacyclohexadecin-12-one, 18-chloro-8, Preparation of 9,10,11,13,14,15,20-octahydro-21-methoxy-13,14-dimethyl-, (13S)-

Step 1:

N-methyl-L-alanine methyl ester hydrochloride (1.4 g, 0.010 mol) in CH ₂ C1 ₂ (appropriate), 4-chloro-2-nitrobenzaldehyde (0.010 mol) and titanium, tetrakis (2-propanolato) (0.010 mol) was stirred at rt for 1 h. Then NaBH (OAc) ₃ (0.022 mol) was added and the mixture was stirred at rt for 3 h. Additional NaBH (OAc) ₃ (0.011 mol) was added and the mixture was stirred at rt overnight. NaHCO ₃ (saturated) was then added until basic and the mixture was filtered through a P3 filter. The organic layer was separated and the aqueous layer was extracted three times with CH ₂ C1 ₂ . The combined organic layers were dried (K ₂ CO ₃ ) and the solvent was evaporated to dryness. The crude residue, L-alanine, N-[(4-chloro-2-nitrophenyl) methyl] -N-methyl-, methyl ester was used as such in the next reaction step.

Step 2:

A mixture of L-alanine, N-[(4-chloro-2-nitrophenyl) methyl] -N-methyl-, methyl ester (1.03 g, 0.0036 mol) in CH ₃ OH (50 ml) was diluted with DIPE (0.5 ml). Hydrogenated using Pt / C 5% (0.5 g) as catalyst in the presence of 4% thiophene solution in water. After H ₂ (3 equiv) blowing, the catalyst was filtered off and the filtrate was evaporated. The residue was purified on silica gel (with cartridge) (eluent: 4% Et ₃ N / CH ₂ C1 ₂ ). Desired fractions were collected. Heptane was added to the desired fractions (biphasic form). The heptane layer was separated to remove silica grease. After solvent removal and drying, 0.5674 g of L-alanine, N-[(2-amino-4-chlorophenyl) methyl] -N-methyl-, methyl ester were obtained (22%; yellow oil). The crude product was used as such in the next reaction step.

Step 3:

4-Chloro-7-methoxy-6-quinazolinol 6-acetate (0.00055 mol) was converted to L-alanine, N-[(2-amino-4-chlorophenyl) methyl]-in 2-propanol (5 ml). After addition to a solution of N-methyl-, methyl ester (0.00055 mol), the reaction mixture was shaken at 80 ° C. overnight to evaporate the solvent. L-alanine, N-[[2-[[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -N-methyl-, methyl ester (crude Product, used as such in the next reaction step).

Step 4:

NH ₃ / CH ₃ OH 7 N (10 ml) was dissolved in L-alanine, N-[[2-[[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl ] Methyl] -N-methyl-, methyl ester (0.00055 mol) was added, the reaction mixture was shaken for 1-2 hours, and then the solvent was evaporated. L-alanine, N-[[4-chloro-2-[(6-hydroxy-7-methoxy-4-quinazolinyl) amino] phenyl] methyl] -N-methyl-, methyl ester (next reaction step Is used as is).

Step 5:

L-alanine, N-[[4-chloro-2-[(6-hydroxy-7-methoxy-4-quinazolinyl) amino] phenyl] methyl] -N-methyl-, methyl ester (0.00055 mol) , A mixture of N- (3-bromopropyl) carbamic acid 1,1-dimethylethyl ester (1 eq) and Cs ₂ CO ₃ (0.8965 g) was stirred overnight at room temperature, and then the reaction mixture was stirred at 50 ° C. Stir for 30 minutes. If necessary, additional CAS N- (3-bromopropyl) carbamic acid 1,1-dimethylethyl ester was added, and the mixture was stirred at room temperature for 4 hours at 50 ° C for additional 15 minutes. The solvent was evaporated (Genevac) and the residue was dissolved in CH ₂ C1 ₂ . This solution was filtered through decalite to evaporate the filtrate. L-alanine, N-[[4-chloro-2-[[6- [3-[[(1,1-dimethylethoxy) carbonyl] amino] propoxy] -7-methoxy-4-quinazoli Nil] amino] phenyl] methyl] -N-methyl-, methyl ester was obtained.

Step 6:

To L-alanine, N-[[4-chloro-2-[[6- [3-[[(1,1-dimethyl) in TFA / CH ₂ C1 ₂ / TIS (90/8/2) (5.5 ml) Oxy) carbonyl] amino] propoxy] -7-methoxy-4-quinazolinyl] amino] phenyl] methyl] -N-methyl-, a solution of methyl ester (crude residue; 0.00055 mol) 7 to 8 After stirring for hours, the solvent was evaporated and the resulting residue was dried in an oven overnight. L-alanine, N-[[2-[[6- (3-aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -N-methyl-.CF ₃ COOH was obtained.

Step 7:

L-alanine, N-[[2-[[6- (3-aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -N-methyl-.CF _{A solution of 3} COOH (0.00055 mol) and DIPEA (0.0033 mol) was added to a solution of HBTU (0.00165 mol) and HOBt (0.0011 mol) in anhydrous DMF (137 ml) and the reaction mixture was allowed to react for 1 hour. The solvent was evaporated and the dry residue was purified by reverse phase high performance liquid chromatography. The product fractions were combined, Na ₂ CO ₃ was added and the organic solvent was evaporated. CH ₂ C1 ₂ was added to the aqueous concentrate, and the obtained mixture was extracted three times with CH ₂ C1 _2, and then the organic extracts were dried and collected. 4,6-ethanediylidene-12H-pyrimido [4,5-b] [6,1,10,13] benzoxatriazacyclohexadecin-12-one, 18-chloro-8,9,10 0.011 g of, 11,13,14,15,20-octahydro-21-methoxy-13,14-dimethyl-, (13S)-was obtained.

'CH ₃ OH' used in the examples means methanol, 'Et ₃ N' means triethylamine, 'CH ₂ C1 ₂ ' means dichloromethane, and 'HBTU' means 1- [bis (Dimethylamino) methylene] -1H-benzotriazolhexahexaphosphate (1-) 3-oxide, 'DMF' means N, N-dimethylformamide, and 'NaBH (OAc) ₃ ' Means sodium triacetoxyborohydride, 'DIPEA' means N-ethyl-N- (1-methylethyl) -2-propanamine, and 'HOBt' means 1-hydroxy-1H-benzotriazole 'TFA' means trifluoroacetic acid, 'TIS' means tris (1-methylethyl) silane, 'K ₂ CO ₃ ' means potassium carbonate, 'Cs ₂ CO ₃ 'Means cesium carbonate,' Na ₂ CO ₃ 'means disodium carbonate salt,' NaHCO ₃ 'means monosodium carbonate salt.

Preparation of Compound 22, MTKI1

Suitable preparations of the preferred compounds for use in the present invention, taken from WO-2004 / 105765, are as follows:

Example A

a) Preparation of 1-pentanol, 5-[[(4-bromo-2-nitrophenyl) methyl] amino]-(intermediate 1)

Solution of 4-bromo-2-nitro-benzaldehyde (0.013 mol), 5-amino-1-pentanol (0.013 mol) and titanium, tetrakis (2-propanolato) (0.014 mol) in EtOH (15 ml) The mixture was stirred at room temperature for 1 hour, then the reaction mixture was heated to 50 ° C. and stirred for 30 minutes. The mixture was cooled to rt and NaBH ₄ (0.013 mol) was added portionwise. The reaction mixture was stirred overnight and then poured into ice water (50 ml). The resulting mixture was stirred for 20 minutes, the precipitate formed was filtered (obtained filtrate (I)), washed with H ₂ O and stirred in DCM (to dissolve the product and remove it from the Ti-salt). The mixture was filtered, then the filtrate was dried (MgSO ₄ ), filtered and finally the solvent was evaporated. The filtrate (I) was evaporated until the EtOH was removed and the aqueous concentrate was extracted twice with DCM. The organic layer was separated, dried (MgSO ₄ ), filtered and the solvent was evaporated to yield 3.8 g (93%) of intermediate 1.

Example B:

a) Preparation of 1-pentanol, 5-[[(4-bromo-2-nitrophenyl) methyl] methylamino]-(intermediate 2)

A solution of intermediate 50 (0.0047 mol), formaldehyde (0.025 mol) and titanium, tetrakis (2-propanolato) (0.0051 mol) in EtOH (150 ml) was heated to 50 ° C. and stirred for 1 hour , NaBH ₄ (0.026 mol) was added portionwise at room temperature. The reaction mixture was stirred overnight and then quenched with water (100 ml). The resulting mixture was stirred for 1 hour; The formed precipitate was washed by filtration. The organic filtrate was concentrated, then the aqueous concentrate was extracted with DCM and dried. The solvent was evaporated and the residue was filtered through silica gel (eluent: DCM / CH ₃ OH 98/2 to 95/5). The product fractions were combined and the solvent was evaporated to yield 0.5 g of intermediate 2.

b) preparation of 1-pentanol, 5-[[(4-bromo-2-nitrophenyl) methyl] methylamino]-, acetate (ester) (intermediate 3)

A solution of intermediate 2 (0.0015 mol) and pyridine (0.015 mol) in acetic anhydride (8 ml) was stirred overnight at room temperature, then the solvent was evaporated and co-evaporated with toluene to give intermediate 3.

c) Preparation of 1-pentanol, 5-[[(2-amino-4-bromophenyl) methyl] methylamino]-, acetate (ester) (intermediate 4)

A mixture of intermediate 3 (0.0015 mol) in THF (50 ml) was hydrogenated using Pt / C 5% (0.5 g) as catalyst in the presence of thiophene solution (0.5 ml) [H179-034]. After blowing H ₂ (3 equiv), the catalyst was filtered off and the filtrate was evaporated to yield 0.5 g of intermediate 4.

d) 6-quinazolinol, 4-[[2-[[[5- (acetyloxy) pentyl] methylamino] methyl] -5-bromophenyl] amino] -7-methoxy-, acetate (ester) Preparation of (Intermediate 5)

A mixture of intermediate 4 (0.0015 mol) and 4-chloro-7-methoxy-6-quinazolinol acetate (ester) (0.0015 mol) in 2-propanol (30 ml) was heated to 80 ° C. to give the reaction mixture 1 Stir for days. The solvent was evaporated under reduced pressure and the residue was used as such in the next reaction step to give 0.83 g of intermediate 5.

e) Preparation of 6-quinazolinol, 4-[[5-bromo-2-[[(5-hydroxypentyl) methylamino] methyl] phenyl] amino] -7-methoxy- (intermediate 6)

A solution of intermediate 5 (0.0015 mol) in methanol (25 ml) was stirred at room temperature, a solution of K ₂ CO ₃ (0.003 mol) in H ₂ O (2.5 ml) was added, and then the reaction mixture was heated to 60 ° C. And stirred for 18 hours. The solvent was evaporated, H ₂ O (20 ml) was added, then the mixture was neutralized with acetic acid and the precipitate formed was filtered off. The filtrate was concentrated under reduced pressure, the concentrate was extracted with DCM, filtered and then dried (MgSO ₄ ) and the mixture was concentrated under reduced pressure to give 0.5 g (70%) of intermediate 6.

Example C:

a) 4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzonediazacyclo-pentadecin, 17-bromo-8,9,10,11,12, Preparation of 13,14,19-octahydro-20-methoxy-13-methyl- (compound MTKI1)

A solution of intermediate 6 (0.0011 mol) in THF (50 ml) was stirred at room temperature, tributylphosphine (0.0016 mol) was added and then 1,1 '-(azodicarbonyl) bis-piperidine (0.0016 mol) was added and the reaction mixture was stirred for 2 hours. The solvent was evaporated until one third of the initial volume. The obtained precipitate was filtered off and washed. The filtrate was evaporated and the residue was purified by reverse phase high performance liquid chromatography. Product fractions were combined and the organic solvent was evaporated. The aqueous concentrate was extracted twice with DCM and the organic layer was dried (MgSO ₄ ) and then filtered. The solvent was evaporated and the residue was dried at 50 ° C. (vacuum) to afford 0.004 g (0.8%) of compound MTKI1.

Compound 2, 4,6-ethanediylidene-19H-pyrimido [4,5-b] [6,13,1] benzodioxaxacyclo-pentadecin, 15-chloro-8,9,10,11 Preparation of, 12,13-hexahydro-20-methoxy-;

Example D:

4,6-ethanediylidene-19H-pyrimido [4,5-b] [6,13,1] benzodioxaxacyclo-pentadecin, 15-chloro-8,9,10,11,12, Preparation of 13-hexahydro-20-methoxy- (Compound 2)

A solution of intermediate 9 (0.0024 mol) and triphenylphosphine (0.0036 mol) in anhydrous THF (100 ml) was stirred at room temperature and then bis (1-methylethyl) diazene dicarboxylate in THF (10 ml) (0.0036 mol) of solution was added dropwise. The reaction mixture was stirred for 6 hours, and excess bis (1-methylethyl) diazenedicarboxylate (0.35 ml) in THF (10 ml) was added. The mixture was stirred overnight and concentrated. The residue was purified by column chromatography on silica gel (eluent: DCM / CH ₃ OH / THF 90/5/5). The product fractions were combined and further purified by reverse phase high performance liquid chromatography. Product fractions were combined and concentrated. The aqueous concentrate was filtered, the residual solid was washed, dried at 65 ° C. (vacuum) to yield 0.065 g of compound 2 (melting point: 255.5-260.2 ° C.).

Example E:

a) Preparation of hexanoic acid, 6- (2-chloro-6-nitrophenoxy)-, methyl ester (intermediate 6)

A solution of 2-chloro-6-nitro-phenol (0.046 mol) in N, N-dimethylformamide (150 ml) was heated to 50 ° C., then K ₂ CO ₃ (0.069 mol) was added to the reaction mixture. Stir for 15 minutes. 6-Bromo-, methyl ester hexanoic acid (0.069 mol) was added and the mixture was stirred overnight. The reaction mixture was filtered, the filtrate was concentrated and the residue was used as such in the next step to give 13.88 g of intermediate 6.

 b) Preparation of hexanoic acid, 6- (2-amino-6-chlorophenoxy)-, methyl ester (intermediate 7)

A mixture of intermediate 6 (0.046 mol) and ethanamine (2 g) in THF (ml) was hydrogenated using Pt / C 5% (3 g) as catalyst in the presence of DIPE (2 ml). After blowing H ₂ (3 equiv), the reaction mixture was filtered through a small plug of decalite, and the filtrate was concentrated to give intermediate 7.

c) preparation of hexanoic acid, 6- [2-[[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino] -6-chlorophenoxy]-, methyl ester (intermediate 8)

A mixture of 4-chloro-6-methylcarbonyloxy-7-methoxyquinazolin (0.022 mol) and intermediate 7 (0.022 mol) in 2-propanol (170 ml) was stirred and heated at 80 ° C. for 2 hours , Concentrated, and the residue was analyzed by chromatography on silica gel (eluent: DCM / CH ₃ OH 97/3). The product fractions were combined and the solvent was evaporated to give 5.1 g of intermediate 8 (used as such in the next reaction step).

d) Preparation of 6-quinazolinol, 4-[[3-chloro-2-[(6-hydroxyhexyl) oxy] phenyl] amino] -7-methoxy- (intermediate 9)

A mixture of LAH (0.0246 mol) in THF (40 ml) was stirred at room temperature. A solution of intermediate 8 (0.006 mol) in THF (60 ml) was added dropwise. The reaction mixture was stirred for 1 day, then excess LAH (0.0123 mol) was added portionwise. The mixture was stirred over the weekend, then H ₂ O (2 ml) was added dropwise, followed by 15% NaOH solution (2 ml) and H ₂ O (6 ml) dropwise. The mixture was stirred for 15 minutes, filtered, the filtrate was concentrated, the residue was stirred in boiling CH ₃ CN, filtered and the residual solid was dried at 60 ° C. (vacuum). The solid was redissolved in CH ₃ OH / DCM (10/90) and the mixture was neutralized with HCl (1 N). The organic layer was separated, dried (MgSO ₄ ), filtered and concentrated to give 1 g of intermediate 9.

Exemplary formulations for the most preferred compound, MYKI1, are as follows:

Example F: Formulations

The product MTKI1 can be prepared as a 10-mg / mL oral solution, pH 2. It contains an excipient, Captisol® (chemical name: sulfobutyl ether-β-cyclodextrin, SBE-β-CD), citric acid, Tween® 20, HCl, and NaOH in purified water. The formulations can be refrigerated (2-8 ° C .; 36-46 ° F.), and allowed to warm up to room temperature up to 1 hour prior to dose preparation.

The product MTKI1 is a hard gelatin capsule, 50-mg, 100-mg and 300-mg oral containing the active chemical MTKI1, lactose monohydrate (200 mesh), sodium lauryl sulfate and magnesium stearate in sizes 3, 4 and 00, respectively. It can be prepared as a rapid release capsule. Capsules may also contain any or all of the following ingredients: gelatin, red iron oxide and titanium oxide.

Experimental data:

VEGFR3 inhibition

Lymph node metastasis is a poor prognostic factor for many cancer types. Recent studies have demonstrated that lymphatic vessel formation plays an important role in tumor progression, and vascular endothelial growth factors (VEGF) C and D are specific lymphatic inducers that act through activation of the cognate receptor VEGFR3. Was identified as. 4,6-ethanediylidenepyrimido [4,5-6] [6,1,12] benzoxadiazacyclo-pentadecin, 17-bromo-8,9,10, hereinafter also referred to as compound 2 , 11,12,13,14,19-octahydro-20-methoxy-13-methyl- is the only kinase inhibition profile (pan Her and Src family) and beneficial tissue that induces strong anti-tumor activity in many experimental models It is a multiple target kinase inhibitor found to have a distribution profile.

In vitro kinase assays show that the compounds of the present invention have potent inhibitory effects on EGFR, Her2, Her4 and Src family kinases (Src, Fyn, Lck, Yes, Lyn) activity.

In vitro kinase inhibition has been identified herein in cell line assays using human vascular endothelial cells (HMVECd), compound 2 inhibits VEGFR3-dependent VEGF-C stimulation of Erk1 / 2, while affecting VEGFR1-mediated VEGF signaling Did not give. Compound 2 has also been shown to inhibit VEGF-C induced phosphorylation of VEGFR3 using cell lines engineered to overexpress human VEGFR3.

These results were confirmed in a recently developed Xenopus tadpole model in which Compound 2 was found to phenocopy the VEGF-C knockout effect.

(Ny et al., Nat. Med. 2005 Sep; 11: 998-1004).

Way:

Kinase analysis in vitro:

VEGFR3 kinase reaction was performed for 10 minutes at 30 ° C. in a 96-well microtiter plate. 25 μl reaction volume was 8 mM MOPS pH 7, 200 μM EDTA, 10 mM MgAc, 10 μM unlabeled ATP, 0.5 mCi AT33P, 500 μM JAK3-tide (Ac-GGEEEEYFELVKKKK-NH2), 25 ng VEGFR3 and 2% compound.

5 μl of 3% phosphoric acid solution was added to stop the reaction. Next, 10 μl of the reaction mixture was spotted on a filtermat P30 filter (Wallac), transferred to a sealable plastic bag containing 4 ml of scintillation liquid, and read three times with 0.75% phosphoric acid for 5 minutes before methanol reading on the scintillation counter. It was washed once with 2 minutes.

VEGFR3 Cell Assay:

Inhibition of VEGF-C induced VEGFR3 activity:

For this assay, porcine aortic endothelial (PAE) cells stably expressing human VEGFR-3 were grown to confluency in 10 cm dishes. Cells were serum starved overnight and the medium was replaced with fresh serum free medium. An amount of Compound 2 or a control vehicle was added to the cells and then incubated at 37 ° C. for 15 minutes. Thereafter, VEGF-C was added at a final concentration of 100 ng / ml, the cells were incubated at 37 ° C for 10 minutes, washed with ice-cold PBS, and then lysed. Cell lysates containing protease inhibitors were immunoprecipitated with antibodies specific for VEGFR-3, and the immunoprecipitates were separated by SDS-PAGE. Proteins were transferred to nitrocellulose membranes, first probed with phosphotyrosine antibodies and then probed with VEGFR-3 antibodies.

Inhibition of Erk activation induced by human microvascular endothelial cell-dermis (HMVEC-d):

HMVEC-d cells (Cambrex) were incubated for 4 days at 37 ° C. in 5% CO ₂ in appropriate serum-containing medium (Cambrex) in 12-well plates coated with fibronectin (BD Biocoat, Becton Dickinson). Cells were then starved for 16 hours in the same medium as before, but without serum and instead containing SITE + 3 (Invitrogen). Cells were then precultured with 0.2% (solvent) or 5 μM Compound 2 in DMSO for 60 minutes, then 10 ng / ml VEGF or 100 ng / ml VEGF-C (R & D systems) were added to the cells. Before stimulation, 10 minutes after stimulation, or 30 minutes after stimulation, the medium is removed and the cells are rinsed with ice cold PBS containing 0.1 mM sodium orthovanadate, and 0.1% SDS, and 1x phosphatase and protease inhibitors (HALT, PIERCE) containing M-PER buffer (PIERCE). Cells were peeled off the plate and incubated for 30 minutes in lysis buffer on ice and then centrifuged at 16000 × g for 10 minutes at 4 ° C. Lysates were quantified using BCA reagent according to the manufacturer's instructions (PIERCE) and 5 mg of total protein mass was loaded onto SDS-polyacrylamide gel (NUPAGE, Invitrogen). Both electrophoresis and blots for PVDF membranes were performed using reagents (manufactured by NUPAGE system) according to the manufacturer's instructions. The blot was blocked with Odyssey blocking buffer (LICOR) for 1 hour, then the primary antibody (described below) was 1: 1000 in a 1: 1 mix of Odyssey blocking buffer with PBS containing 0.1% Tween-20. Diluted at 4 ° C. and used overnight at 4 ° C .: P-Erk1 / 2 (Thr202 / Tyr204, clone E1O, mouse monoclonal # 9106, Erk1 / 2 (rabbit polyclonal # 9102) (all Cell Signaling Technologies, Inc.) Blots were washed three times for 5 minutes with PBS containing 0.1% Tween and then incubated for 1 hour with secondary antibody (diluted 1: 1000 in PBS containing 0.1% Tween-20): IRDye Goat anti-mouse conjugated to 800 nm (Rockland, Inc) or goat anti-rabbit conjugated to Alexa 680 nm (Molecular Probes, Inc.) The blot was washed three times for 5 minutes with PBS containing 0.1% Tween and PBS 1 wash with 700 nm and 800 nm, with all settings and intensity 5 appropriate for membrane scanning Scan server was using LICOR system.

Xenopus analysis of VEGFR3 in vivo:

To analyze the effect of Compound 2 on lymphatic angiogenesis, Xenopus Tadpol was used as a model (Ny et al Nature Medicine 11: 998-1004 (2005)). Compounds were administered daily with a vehicle control at stage 26 to 28, i.e., in the medium of Xenopus Tedpol, which is initiated prior to the formation of blood vessels and lymphatic vessels. The effect of the compound was monitored by "bioanalysis", ie, a live observer who was trained for 4 days examined for evidence of lymphatic defects. As an additional means of confirming the results, in a limited number of experiments, Tadpoles were fixed at stages 35-36 and Prox1 was used to assess the migration of lymphatic endothelial cells (LECs) from the posterior vein (PCV) to their dorsal endpoints. In situ hybridization was performed for, indicating an essential step in lymphangiogenesis. Under normal circumstances, LECs arise in and accumulate in the region of PCV, and during the study period, they move behind to a limited “zone” (from zone 1 to zone 2 and zone 3), and then tail And caudally rostrally to form a lymphatic relationship in the trunk. Maximum migration of the cells was measured and the number of cells in these different regions of the tail was counted to quantify the migration defects of LEC from PCV.

result:

Kinase analysis in vitro:

The table below provides the pIC50 values obtained for the compounds of the present invention using the VEGFR3 kinase assay described above.

Inhibition of VEGF-C Induced VEGFR-3 Phosphorylation in Porcine Aortic Endothelial Cells Expressing VEGFR-3:

4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] -benzoxadiazacyclo-pentadecin, 17-bromo-8 on VEGF-C induced VEGFR3 activity The effect of, 9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl- (Compound 2) is evident from dose dependent inhibition in VEGFR-3 phosphorylation (FIG. 1). Upper frame). The presence of different concentrations of VEGFR-3 is confirmed upon re-staining of the western blot with the VEGFR-3 antibody (lower frame in FIG. 1).

Erk phosphorylation assay in HMVEC-d cells:

As illustrated in FIG. 2, 5 μM of compound inhibited phosphorylation of Erk1 / 2 by VEGF-C, but did not interfere with VEGF stimulation of Erk1 / 2. These data are consistent with selective inhibition of VEGFR-3 by Compound 2.

Xenopus study results:

Using live screening to evaluate the effect of Compound 2, a concentration of Compound 2 of> 50 μM induced bleeding at Tadpole 30%. At 20 μM, 10% of tadpoles showed edema and no taedpoles were seen in tadpoles treated with vehicle alone. Compound 2 dose-dependently inhibits lymphatic endothelial cell (LEC) migration in Tadpol (Table 1). Particularly in stages 35-36, LEC migration was reduced and even at the lowest concentration (0.31 μM) of the compound tested, which was more than that observed with vehicle alone. These data support the conclusion that the compound interferes with normal lymphangiogenesis.

Table 1:

Quantification of the migration of lymphatic endothelial cells from the posterior vein to the abdomen after exposure to compound 35-36 of Tadpole (n = 10-15 fetus per condition) (detected by Prox1 in situ hybridization)

Note: The above evaluation of LEC migration of Prox1 positive cells was first performed microscopically by a trained observer in a semiquantitative manner ('% of embryos reduced migration'). Then, computer processing quantitative analysis was performed. "Relative region 2-3" is the number of LECs shifted to region 2-3 relative to the control (vehicle alone). “Relative maximum shift” is the furthest LEC shift from PCV during treatment to the maximum observed in the vehicle only control.

While the foregoing specification describes the principles of the invention in conjunction with the examples given for purposes of illustration, the practice of the invention includes all conventional alterations, modifications and / or variations within the scope of the following claims and their equivalents. You can see that.

Claims (20)

VEGFR3 mediation in a mammalian subject comprising administering to the mammalian subject in need thereof a therapeutically effective amount of a compound of Formula I, and a pharmaceutically acceptable acid addition or base addition salt and stereochemical isomer thereof How to treat or prevent biological activity:

In the above formula, Z represents NH; Y is -C _{3 - 9} alkyl _-, -C ₁ - ₅ alkyl, -NR ¹³ -C _{1 - 5} alkyl _-, -C ₁ - ₅ alkyl, -NR ¹⁴ -CO-C _{1 - 5} alkyl _-, -C ₁ - ₃ alkyl, -NH-CO-Het ²⁰ -, or _{^{-Het 22 -CH 2 -CO-NH-}} C 1 - represents a _3-alkyl; X ¹ is O or -OC _{1 - 2} alkyl-represents; X ² is a direct bond, -C ₁₂ alkyl -, O, -OC ₁₂ alkyl - represents a -, NR ¹² or NR ^₁₂ -C ¹² alkyl; R ¹ represents hydrogen, cyano, halo or hydroxy; R ² represents hydrogen, cyano, halo, or hydroxy; R ³ represents hydrogen; R ⁴ is C _{1 -} represents _a-4 alkyloxy; R ¹² is hydrogen or C _{1 -} represents a _4-alkyl; R ¹³ is hydrogen or C _{1 -} represents a _4-alkyl; R ¹⁴ represents hydrogen or C ₁₄ alkyl; Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl; Het ²² represents pyrrolidinyl, piperazinyl or piperidinyl. The compound according to claim 1, wherein in the compound of general formula (I), Z represents NH; Y is -C _{3 - 9} alkyl-, -C _{1 - 5} alkyl, -NR ¹⁴ -CO-C _{1 - 5} alkyl -, or -C _{1 - 3} alkyl, -NH-CO-Het ²⁰ - represents the; X ¹ represents O; X ² is -C ₁₂ alkyl-, O, or NR ^₁₂ -C ¹² alkyl-represents the; R ¹ represents hydrogen or halo; R ² represents hydrogen or halo; R ³ represents hydrogen; R ⁴ is C _{1 -} represents a _4-alkoxy; R ¹² is C _{1 -} represents a _4-alkyl; R ¹⁴ is a hydrogen or C ₁₄ alkyl; Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl. The compound of formula (I) according to claim 1, wherein the compound of formula (I) is selected from the group consisting of 15-chloro-8,9,10,11,12,13-hexahydro-20-methoxy-; 12H-4,6-ethanediylidene-13,17-methanopyrimido [4,5-b] [6,1,10,16] benzoxatria zcyclononadecin-12-one, 21-chloro -8,9,10,11,13,14,15,16,18,23-decahydro-25-methoxy-; 4,6-ethanediylidene-12H-pyrimido [4,5-b] [6,1,10,13] benzonetriazacyclohexadecin-12-one, 18-chloro-8,9,10 , 11,13,14,15,20-octahydro-21-methoxy-13,14-dimethyl-; And 4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzonediazacyclopentadecin, 17-bromo-8,9,10,11,12,13,14 , 19-octahydro-20-methoxy-13-methyl-; Or a pharmaceutically acceptable acid addition salt thereof. The compound of claim 3, wherein the compound is 4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzoxadiazacyclo-pentadecin, 17-bromo-8,9, 10,11,12,13,14,19-octahydro-20-methoxy-13-methyl-; Or a pharmaceutically acceptable acid addition salt thereof. The method of claim 5, wherein the therapeutically effective amount of the compound is administered orally or parenterally. The method of claim 1, wherein the compound of formula (I) is administered in combination with an additional anticancer agent. 7. The method of claim 6, wherein the additional anticancer agent is heceptin, docetaxel, anthracycline and capecitabine for breast cancer; For prostate cancer, docetaxel and mitoxantrone; Oxaliplatin, 5-FU, avastin, irinotecan and cetuximab for colon cancer; And in the case of lung cancer, taxotere, carboplatin and zemisitabine. The method of claim 1, wherein the VEGFR3 mediated biological activity is selected from the group consisting of metastatic spread of cancer in a mammalian subject; Metastasis through lymphatic vessels to regional lymph nodes; Tumor associated lymphangiogenesis in cancer; Recruitment and proliferation of endothelial cells expressing VEGFR3 in neovascularization of cancer cells expressing VEGFR3 ligands; And combinations thereof. The method of claim 8, wherein the VEGFR3 ligand is in the form of VEGF-C, VEGF-D, or a combination thereof. The method of claim 1, wherein the VEGFR3 mediated biological activity is advanced breast cancer of a mammal comprising administering to the mammal a therapeutically effective amount of the compound of any one of claims 1-4. Use of a compound of formula I, and a pharmaceutically acceptable acid addition salt or base addition salt and stereochemical isomer thereof in the manufacture of a medicament for the treatment of VEGFR3 mediated biological activity:

In the above formula, Z represents NH; Y is -C _{3 - 9} alkyl _-, -C ₁ - ₅ alkyl, -NR ¹³ -C _{1 - 5} alkyl _-, -C ₁ - ₅ alkyl, -NR ¹⁴ -CO-C _{1 - 5} alkyl _-, -C ₁ - ₃ alkyl, -NH-CO-Het ²⁰ -, or _{^{-Het 22 -CH 2 -CO-NH-}} C 1 - represents a _3-alkyl; X ¹ is O or -OC _{1 - 2} alkyl-represents; X ² is a direct bond, -C ₁₂ alkyl -, O, -OC ₁₂ alkyl - represents a -, NR ¹² or NR ^₁₂ -C ¹² alkyl; R ¹ represents hydrogen, cyano, halo or hydroxy, preferably halo; R ² represents hydrogen, cyano, halo, or hydroxy; R ³ represents hydrogen; R ⁴ is C _{1 -} represents _a-4 alkyloxy; R ¹² is hydrogen or C _{1 -} represents a _4-alkyl; R ¹³ is hydrogen or C _{1 -} represents a _4-alkyl; R ¹⁴ represents hydrogen or C ₁₄ alkyl; Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl; Het ²² represents pyrrolidinyl, piperazinyl or piperidinyl. The compound of claim 10, wherein in the compound of general formula (I): Z represents NH; Y is -C _{3 - 9} alkyl-, -C _{1 - 5} alkyl, -NR ¹⁴ -CO-C _{1 - 5} alkyl -, or -C _{1 - 3} alkyl, -NH-CO-Het ²⁰ - represents the; X ¹ represents O; X ² is -C ₁₂ alkyl-, O, or NR ^₁₂ -C ¹² alkyl-represents the; R ¹ represents hydrogen or halo; In particular R ¹ represents hydrogen or chloro; In particular R ¹ represents hydrogen; R ² represents hydrogen or halo; In particular R ² represents hydrogen, chloro or bromo; In particular R ² represents chloro or bromo; R ³ represents hydrogen; R ⁴ is C _{1 - 4} denotes an alkyl-oxy; R ⁴ represents methoxy; R ¹² is C _{1 -} represents a _4-alkyl; In particular R ¹² represents methyl; R ¹⁴ is a hydrogen or C ₁₄ alkyl; In particular R ¹⁴ represents hydrogen or methyl; In particular R ¹⁴ represents hydrogen; Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl; Het ²⁰ represents piperidinyl. 12. The compound of claim 11, wherein the compound of formula (I) is 4,6-ethanediylidene-19H-pyrimido [4,5-b] [6,13,1] benzodioxaazacyclo-pentadecin, 15-chloro-8,9,10,11,12,13-hexahydro-20-methoxy-; 12H-4,6-ethanediylidene-13,17-methanopyrimido [4,5-b] [6,1,10,16] benzonetriazacyclononadecin-12-one, 21-chloro -8,9,10,11,13,14,15,16,18,23-decahydro-25-methoxy-; 4,6-ethanediylidene-12H-pyrimido [4,5-b] [6,1,10,13] benzonetriazacyclohexadecin-12-one, 18-chloro-8,9,10 , 11,13,14,15,20-octahydro-21-methoxy-13,14-dimethyl-; And 4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzonediazacyclopentadecin, 17-bromo-8,9,10,11,12,13,14 , 19-octahydro-20-methoxy-13-methyl-; Or a pharmaceutically acceptable acid addition salt thereof. 12. The compound of claim 11, wherein the compound is 4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzoxadiazacyclo-pentadecine, 17-bromo-8,9, 10,11,12,13,14,19-octahydro-20-methoxy-13-methyl-; Or a pharmaceutically acceptable acid addition salt thereof. Use according to any one of claims 11 to 14, characterized in that the medicament is for oral or parenteral administration. Use according to claim 15, characterized in that the compound of formula (I) is administered in combination with an additional anticancer agent. The method of claim 16, wherein the additional anticancer agent is heceptin, docetaxel, anthracycline, and capecitabine for breast cancer; For prostate cancer, docetaxel and mitoxantrone; Oxaliplatin, 5-FU, avastin, irinotecan and cetuximab for colon cancer; And in the case of lung cancer, it is selected from the group consisting of taxotere, carboplatin and zemisitabine. The method of claim 11, wherein the VEGFR3 mediated biological activity is Metastatic spread of cancer in mammalian subjects; Metastasis through lymphatic vessels to local lymph nodes; Tumor-associated lymphangiogenesis in cancers such as gastric cancer, prostate cancer, human colorectal cancer, invasive cervical cancer, breast cancer, Kaposi's sarcoma and melanoma; And Use of the group consisting of recruitment and proliferation of endothelial cells expressing VEGFR3 in neovascularization of cancer cells expressing VEGFR3 ligands. Use according to claim 8, characterized in that the VEGFR3 ligand is in the form of VEGF-C, VEGF-D, or a combination thereof. Use according to any of the preceding claims, wherein the VEGFR3 mediated biological activity is advanced breast cancer.

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