JP2010507626A - Macrocyclic quinazoline derivatives as VEGFR3 inhibitors - Google Patents

Abstract

The present invention is described in PCT Publication No. WO 2004/105765 as an inhibitor of biological activities mediated by VEGFR3, particularly those mediated by VEGFR3 ligands VEGF-C and / or VEGF-D. It relates to some uses of the described macrocyclic quinazoline derivatives.
[Selection figure] None

Description

FIELD OF THE INVENTION The present invention is based on the fact that some of the macrocyclic quinazoline derivatives described in PCT Publication No. WO 2004/058405 A1 are biologically mediated by VEGFR3, in particular VEGFR3 ligand VEGF-C and / or VEGF-D. Relates to the discovery that it is useful as an inhibitor of activity mediated by.

BACKGROUND OF THE INVENTION Cancer is still the leading cause of death worldwide at the beginning of the 21st century and continues to be the main focus on ongoing research and development.

  In recent years, promising methods for therapeutic intervention of cancer have focused on anti-angiogenic treatment. This method of mediating cancer progression takes advantage of the idea that hindering or otherwise restricting blood supply to the tumor will deplete the tumor with oxygen and nutrients and prevent tumor cell growth and proliferation To do. This method has been found to be effective and currently over 20 anti-angiogenic drugs and many in preclinical development undergoing various stages of evaluation in Phase I, II or III clinical trials. There are other things.

  Although there are many forms of cancer that exhibit diverse properties, one factor shared by many cancers is that they must metastasize to be lethal. Until such time as metastasis occurs, the tumor can be malignant, but it is confined to one area of the body. If it can be located before metastasis, it can treat or even remove the cancer through surgical intervention, but this can still cause discomfort and / or pain, or even more serious problems . As long as the remaining cancer cells are suppressed, such isolated cancers can be controlled without significant problems. However, although metastases can infiltrate cancer cells into the body and surgical excision can remove the primary tumor, metastatic spread of cancer to different sites is very difficult to treat.

  Metastasis to local lymph nodes via lymphatic vessels is a common stage in cancer progression. Metastasis is an important prognostic factor in many types of cancer and forms the basis for surgical and radiation treatment of local lymph nodes. The process of tumor metastasis includes local invasion and destruction of the intercellular matrix, foreign body invasion into the blood vessels, transport of lymphatic vessels or other passages, survival in the circulation, migration from the duct at the second site and new location It is a multistage event including growth in (Non-patent document 1, Non-patent document 2, Non-patent document 3, and Non-patent document 4). Successful establishment of metastatic deposition requires that tumor cells be able to achieve these steps sequentially.

  In recent years, several lines of evidence have shown that lymphangiogenesis, which is the formation of lymphatic vessels, promotes lymphatic metastasis (Non-patent document 5; Non-patent document 6; Non-patent document 7). Inhibition of lymphangiogenesis can provide a novel strategy for preventing lymph node metastasis in cancer therapy.

Recent studies have shown that VEGF-C, a member of the vascular endothelial growth factor (VEGF) group, stimulates lymphangiogenesis and lymphatic endothelial cell growth and migration when bound to its receptor, VEGFR3. (Non-patent document 8). VEGF-C is mediated by induction of tumor-associated lymphangiogenesis in many solid cancers such as gastric cancer, prostate cancer, human colorectal cancer, invasive cervical cancer, breast cancer metastasis and human melanoma metastasis. It has also been shown to promote tube-mediated metastasis. Among these supporters are preclinical data showing that VEGF-C overexpression in cancer cells significantly increases tumor-associated lymphangiogenesis and enhances metastasis to local lymph nodes (non- Patent Document 9). Furthermore, blocking of VEGF-C / D-mediated signaling has been shown to suppress tumor lymphangiogenesis and lymph node metastasis in mice (Non-Patent Document 10).

  VEGFR3, a transmembrane tyrosine kinase receptor, is extensively expressed in endothelial cells during early embryogenesis (11). During the late stages of development, VEGFR-3 expression becomes restricted to the developing lymphatic vessels [12]. In adults, the lymphatic endothelium and some high endothelial venules express VEGFR-3, with increased expression occurring in metastatic lymph nodes and lymphoid sinuses in lymphangioma. VEGFR-3 is also expressed in a subset of CD34 + hematopoietic cells, which can mediate the bone marrow hematopoietic activity of VEGF-C as shown by overexpression studies. Targeted disruption of the VEGFR-3 gene in mouse embryos leads to failure of primary vascular network remodeling and death after embryonic day 9.5 [Non-Patent Document 13]. These studies suggest an essential role for VEGFR-3 in the development of embryonic vasculature and also during lymphangiogenesis.

  In view of the above, inhibitors of VEGFR3 have great potential as therapeutic agents, and this type of novel agent is a technique for preventing the growth and / or expansion of various tumorigenic or cell proliferative disorders It will be clear that there is a continuing need in the field. Inhibition of VEGFR3 activity, including inhibition of ligands that bind to VEGFR3, is useful in the treatment of mammalian patients diagnosed with a disease characterized by proliferation of endothelial cells that express VEGFR-3. For example, many tumors are characterized by neovascularization of blood vessels or lymphatic vessels, where neovascularization includes endothelial cells that express VEGFR-3. In some cancers, the cancer cells themselves express VEGFR3 and represent target cells. In other cancers, possibly overlapping sets of cancers, the cancer cells express a VEGFR-3 ligand selected from VEGF-C and VEGF-D. Ligand appears to be involved in endothelial cell recruitment and stimulation of their growth, thereby promoting cancer nourishment and / or expansion.

International Publication No. 2004/105765 Pamphlet

Idler, et al. Author, Adv. Cancer Res. 28, 1978, 149-250. Liotta, et al. Author, Cancer Treatment Res. 40, 1988, 223-238 Nicolson, Biochim. Biophy. Acta 948, 1988, 175-224. Zetter, N.M. Author, Eng. J. et al. Med. 322, 1990, 605-612 Stacker et al. Author, Nature Med. 7 (2), 2001, 186-191. Skove et al. Author, Nature Med. 7 (2), 2001, 192-8 Makinen et al. Author, Nature Med. 7 (2), 2001, 199-205 Karkainen MJ, et al. Author, Semin Cell Dev Biol. 13: 2002, 9-18 Stacker SA. , Et al. Written by FASEB J 16: 2002, 922-34. He Y. , Et al. Written by J Natl Cancer Inst. 94: 2002, 819-25 Pajusola K.M. , Et al. Author, Cancer Res. 53 (16): 1992, 3845 Kaipainen, A .; , Et al. Author, Proc. Natl. Acad. Sci. USA, 92: 1995, 3566-3570. Dumont et al. Written, Science, 282: 1998, 946-949.

SUMMARY OF THE INVENTION The invention is mediated in part by VEGFR3 using certain compounds described in WO 2004/105765, the entire disclosure of which is incorporated herein by reference. It is aimed at a method of treating or preventing biological activity.

  In a related aspect, the present invention relates to the treatment of cancer in a mammalian patient comprising administering to a mammalian patient suspected of having cancer the compound of the present invention in an amount effective to prevent metastatic spread of the cancer. A method of preventing metastatic spread; and administering to a mammalian patient diagnosed with cancer a composition comprising a compound of the present invention in an amount effective to reduce cancer growth or tumorigenic spread. A method for treating cancer is provided. It will be appreciated that a decrease in the rate of cancer growth or expansion (which can prolong life and quality of life) is an indicator of treatment success. In a preferred embodiment, cancer growth stops completely. In an even more preferred embodiment, the cancer contracts or is eradicated entirely. The preferred patient for treatment is a human patient, although other animal systems, particularly murine, rat, dog, cow, pig, primate and other model systems for cancer treatment are contemplated.

In some cancers expressing VEGFR-3, direct inhibition of cancer growth or extinction of the cancer may be a mechanism. Treatment of all cancers expressing VEGFR-3 and all cancers characterized by angiogenesis or lymphangiogenesis in and around the growing tumor are included. For example, from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, blood cell, pancreas, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood Treatment of cancer of the selected tissue, organ or cell is included. Particular embodiments of the invention include the treatment of tissue, organ or cell cancers selected from the group consisting of breast, lung, prostate and colon.

  In one variation of the above treatment regimen, the compound is administered with a second cancer therapeutic agent. The second agent may be any chemotherapeutic agent, radioactive agent, radiation, nucleic acid encoding cancer therapeutic agent, antibody, protein and / or other anti-lymphangiogenic or anti-angiogenic agent. it can. The second agent can be administered before, after or simultaneously with the compound of the invention.

  In one variation, the patient to be treated is diagnosed as having an operable tumor, and the administration phase is performed before, during or after the tumor is removed from the patient. Compound treatment along with tumor resection is aimed at reducing or eliminating tumor regrowth from cancer cells that have failed to be resected.

  More generally described, the present invention relates to pathology characterized by VEGFR-3 binding to a natural ligand that binds to VEGFR-3 comprising the step of administering a compound of the invention to a person in need thereof. A method of treatment is provided.

4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzoxadiazacyclopentadecin, 17-bromo-8,9,10,11 to VEGF-C induced VEGFR3 activity , 12, 13, 14, 19-octahydro-20-methoxy-13-methyl- (compound 2). The upper frame gives the results of VEGFR-3 immunoprecipitation transferred to a nitrocellulose membrane and stained with a phospho-tyrosine antibody. The lower pane gives the results of VEGFR-3 immunoprecipitation transferred to a nitrocellulose membrane and stained with VEGFR-3 antibody. Left columns 1 and 2 give DMSO treatment in the absence and presence of negative and positive standards, ie VEGF-C. VEGF and VEGF-C stimulation of Erk1 / 2 phosphorylation in HMVECd cells in the presence of 5 uM compound 2. Upper row gives phosphorylated Erk 1/2 (P-Erk 1/2) staining with Thr202 / Tyr204 mouse monoclonal antibody as primary antibody and goat anti-mouse conjugated to IRDye 800 nm as secondary antibody. . The bottom row gives total Erk 1/2 staining using Erk 1/2 specific rabbit polyclonal antibody as primary antibody and goat anti-rabbit conjugated to Alexa 680 nm as secondary antibody. The left panel gives results for VEGF-induced phosphorylation of Erk-1 / 2 in HMVECd cells. The right panel gives results for VEGF-C-induced phosphorylation of Erk 1/2 in HMVECd cells.

Detailed Description of the Invention WO 2004/105765 describes the preparation, preparation and pharmaceutical properties of certain types of macrocyclic quinazoline derivatives of formula (I) as multi-targeted kinase inhibitors (multi-targeted kinase inhibitors). It is described.

  Now, a particular group of compounds within the aforementioned classes has VEGFR3 inhibitory activity, which is in the treatment or prevention of biological activity mediated by VEGFR3, particularly in the treatment or prevention of metastatic spread of cancer in mammalian patients. It has been found to make them useful for.

Accordingly, in one aspect, the invention is in the manufacture of a medicament for the treatment or prevention of VEGFR3-mediated biological activity, such as in the treatment or prevention of metastatic spread of cancer in a mammalian patient;
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 _1-3 alkyl- represents; X ¹ represents O or —O—C _1-2 alkyl-;
X ² represents a direct bond, —C _1-2 alkyl-, O, —O—C _1-2 alkyl-, NR ¹² or NR ¹² —C _1-2 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 represents C _1-4 alkyloxy-;
R ¹² represents hydrogen or C _1-4 alkyl-;
R ¹³ represents hydrogen or C _1-4 alkyl;
R ¹⁴ represents hydrogen or C _1-4 alkyl;
Use of a compound of formula (I) wherein Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl; and Het ²² represents pyrrolidinyl, piperazinyl or piperidinyl, pharmaceutically acceptable acid or base addition salts and stereochemical isomers thereof I will provide a.

  Yet another aspect of the invention is a compound of formula I

[Where:
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 _1-3 alkyl- represents; X ¹ represents O or —O—C _1-2 alkyl-;
X ² represents a direct bond, —C _1-2 alkyl-, O, —O—C _1-2 alkyl-, NR ¹² or NR ¹² -C _1-2 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 represents C _1-4 alkyloxy-;
R ¹² represents hydrogen or C _1-4 alkyl-;
R ¹³ represents hydrogen or C _1-4 alkyl;
R ¹⁴ represents hydrogen or C _1-4 alkyl;
Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl;
Het ²² represents pyrrolidinyl, piperazinyl or piperidinyl]
And a pharmaceutically acceptable acid or base addition salt and a stereochemical isomer thereof in a therapeutically effective amount, for example in a mammalian patient in need of treatment. It is directed to a method for the treatment or prevention of VEGFR3-mediated biological activity, such as in the treatment or prevention of metastatic spread of cancer.

In addition, biological activities mediated by VEGFR3 shall include:
-Metastatic spread of cancer in mammalian patients. The preferred patient for treatment is a human patient, although other animal systems, particularly murine, rat, dog, cow, pig, primate and other model systems for cancer treatment are contemplated.
-Metastasis to local lymph nodes via lymphatic vessels. The invention therefore (it) provides the use of a compound according to the invention in the treatment or prevention of lymph node metastasis.
-Tumor-related lymphangiogenesis in cancer, eg in stomach cancer, prostate cancer, human colorectal cancer, invasive cervical cancer, breast cancer, Kaposi's sarcoma and melanoma. The present invention therefore provides the use of a compound according to the invention in the treatment or prevention of tumor-associated lymphangiogenesis in cancer, for example in gastric cancer, prostate cancer, human colorectal cancer, invasive cervical cancer, breast cancer, Kaposi's sarcoma and melanoma. I will provide a.
-Replenishment and proliferation of endothelial cells expressing VEGFR3 in neovascularization of cancer cells expressing a VEGFR3 ligand selected from VEGF-C or VEGF-D. The invention therefore provides the use of a compound according to the invention in the treatment of cancer characterized by angiogenesis or lymphangiogenesis in and around growing tumors.

  In yet another aspect, the present invention is for the treatment of cancer of tissues, organs or cells selected from the group consisting of breast, lung (including treatment of both small cell and non-small cell lung cancer), rectum and prostate. There is provided the use of a compound of formula (I) as defined above for the preparation of a pharmaceutical composition of

  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 defined above. Related.

  In yet another aspect, the present invention provides the use of a compound of formula (I) as described above for the preparation of a pharmaceutical composition for the treatment of advanced breast cancer. The term “advanced breast cancer” is used herein to refer to breast cancer that has not responded to previous treatment or has recurred after such treatment and also breast cancer in patients who present with metastatic disease at the time of diagnosis. It is done.

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

In particular, the present invention relates to the use of compounds of formula (I) to which one or more of the following restrictions 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} - shows a;
X ¹ represents O;
X ² represents -C _1-2 alkyl-, O or NR ¹² -C _1-2 alkyl-;
R ¹ represents hydrogen or halo; in particular R ¹ represents hydrogen or chloro; more particularly R ¹ represents hydrogen;
R ² represents hydrogen or halo; in particular R ² represents hydrogen, chloro or bromo; more particularly R ² represents chloro or bromo;
R ³ represents hydrogen;
R ⁴ represents C _1-4 alkoxy; in particular R ⁴ represents methoxy;
R ¹² represents C _1-4 alkyl-; in particular R ¹² represents methyl;
R ¹⁴ represents hydrogen or C _1-4 alkyl; in particular R ¹⁴ represents hydrogen or methyl; more particularly R ¹⁴ represents hydrogen;
Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl; in particular Het ²⁰ represents piperidinyl.

Using the numbering shown in formula (I) above, R ¹ is in the 3 ′ position, R ² is in the 5 ′ position, R ⁴ is in the 7 position, and X ² is in the 2 ′ position. Certain compounds of formula (I) are also of interest in the above uses.

The most preferred compounds are:
4,6-ethanediylidene-19H-pyrimido [4,5-b] [6,13,1] benzodioxaazacyclo-pentadecine, 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] benzoxatriazacyclononadecin-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] benzoxatriazacyclohexadecin-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] benzoxadiazacyclopenta Decyne, 17-bromo-8,9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl-
Or a compound selected from the group consisting of pharmaceutically acceptable acid addition salts thereof. The latter compound is particularly preferred.

  The most preferred compounds for use in accordance with the present invention are the following structures:

Or selected from the group consisting of pharmaceutically acceptable acid addition salts thereof.

  The following compounds:

Or a pharmaceutically acceptable acid addition salt thereof is particularly preferred.

As used in the definitions above and below:
-Halo is generic to fluoro, chloro, bromo and iodo;
-C _1-2 alkyl defines methyl or ethyl;
-C _1-3 alkyl, straight and branched chain saturated hydrocarbon radicals having from 1 to 3 carbon atoms, defined for example methyl, ethyl, propyl and the like;
-C _1-4 alkyl, straight and branched chain saturated hydrocarbon radicals having from 1 to 4 carbon atoms, such as methyl, ethyl, propyl, butyl, 1-methylethyl, 2-methylpropyl, 2 , 2-dimethyl ethyl and the like;
-C _1-5 alkyl is a linear and branched saturated hydrocarbon group having 1 to 5 carbon atoms such as methyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 2,2-dimethyl. Define propyl, 2,2-dimethylethyl and the like;
_-C3-9alkyl defines linear and branched saturated hydrocarbon groups having 3-9 carbon atoms, such as propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and the like;
-C _1-4 alkyloxy defines methoxy, ethoxy, propyloxy, butyloxy, 1-methylethyl-oxy, straight chain or branched saturated hydrocarbon groups such as 2-methyl-propyloxy;
-The term "CO" refers to a carbonyl group.

  The pharmaceutically acceptable acid or base addition salts referred to above include the therapeutically active non-toxic acid and non-toxic base addition salt forms that the compounds of formula (I) can form. Shall be. Treatment of the base form with a suitable acid can convert the basic compounds of formula (I) into their pharmaceutically acceptable acid addition salts. Suitable acids are, for example, inorganic acids such as hydrohalic acids such as hydrochloric acid or hydrobromic acid; sulfuric acid; nitric acid; acids such as phosphoric acid; 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, cyclamic acid, Acids such as salicylic acid, p-aminosalicylic acid and pamoic acid are included.

  Treatment of the acid form with suitable organic and inorganic bases can convert acidic compounds of formula (I) into their pharmaceutically acceptable base addition salts. Suitable base salt forms include, for example, ammonium salts, alkali and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, calcium salts, salts with organic bases such as benzathine, N-methyl-D-glucamine, Hydrabamine salts, as well as salts with amino acids such as arginine, lysine and the like.

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

  The term stereochemical isomers of the compounds of formula (I) used above are composed of the same atoms bonded by the same bond permutation, but are incompatible with the compounds of formula (I). Define all possible compounds with different possible three-dimensional structures. Unless otherwise stated or indicated, the chemical name of a compound includes a mixture of all possible stereochemical isomers that the compound may have. The mixture can contain all diastereomers and / or enantiomers of the basic molecular structure of the compound. All stereochemical isomers of the compounds of formula (I), both in pure form or in admixture with each other, are intended to be included within the scope of the present invention.

  Some of the compounds of formula (I) may also exist in their tautomeric form. Such forms although not explicitly indicated in the above formula are intended to be included within the scope of the present invention.

  Whenever used below, the term “compound of formula (I)” or “compound according to the invention” is intended to also include pharmaceutically acceptable acid or base addition salts and all stereoisomers.

  The compounds according to the present invention are prepared according to methods known in the art and in particular according to the methods described in the published patent specification, the contents of which are incorporated herein by reference and by reference. Suitable examples of compounds of formula (I) can be found in PCT Publication No. WO 2004/105765, which can be prepared and prepared into pharmaceutical compositions. For the preparation of the above pharmaceutical compositions, a therapeutically effective amount of a specific compound, which can optionally be in the form of an addition salt, is used as an active ingredient and is in close contact with a pharmaceutically acceptable carrier. Combined in the mixture, the carrier can take a wide variety of forms depending on the form of preparation desired for administration. Desirably these pharmaceutical compositions are preferably administered systemically, such as orally, transdermally or parenterally; or via inhalation, nasal spray, eye drops or creams, gels, shampoos, etc. In a unit dosage form suitable for topical administration as described above. For example, in the preparation of a composition in an oral dosage form, any of the usual pharmaceutical media such as oral liquid preparations such as suspensions (including nanosuspensions), syrups, elixirs and solutions. In the case of water, glycol, oil, alcohol, etc .; or in the case of powders, pills, capsules and tablets, solid carriers such as starch, saccharides, kaolin, lubricants, binders, disintegrants and the like can be used. Because of their ease of administration, tablets and capsules provide the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will usually comprise at least a majority of sterile water, but can contain other ingredients, for example, to aid solubility. For example, an injectable solution can be prepared in which the carrier comprises saline, glucose solution or a mixture of saline and glucose solution. Injectable solutions containing a compound of formula (I) can be prepared in oil for prolonged action. Suitable oils for this purpose 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 can also be prepared, in which case suitable liquid carriers, suspending agents and the like can be employed. In a composition suitable for transdermal administration, the carrier can optionally comprise a penetration enhancer and / or a suitable wetting agent, optionally with a suitable additive of any nature in a smaller proportion. The additives can be combined and the additive does not cause significant adverse effects on the skin. The additive can facilitate administration to the skin and / or can assist in the preparation of the desired composition. These compositions can be administered in various ways, for example as a transdermal patch, as a spot-on or as an ointment. As compositions suitable for topical application, all compositions usually used for topical administration of drugs, such as creams, gels, bandages, shampoos, tinctures, coatings, ointments, salves, A powder etc. can be mentioned. The composition may be applied with a propellant such as nitrogen, carbon dioxide, freon, or by an aerosol, drop, lotion or semi-solid such as a cotton swab without a propellant such as a pump spray. Can be based on a thickened composition. In particular, semi-solid compositions such as ointments, creams, gels, ointments and the like will be easily used.

It is especially advantageous to prepare the aforementioned pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein and in the claims herein, a dosage unit form refers to a physically separated unit suitable as a single dosage, each unit calculated to produce the desired therapeutic effect. A predetermined amount of active ingredient is contained together with the required pharmaceutical carrier. Examples of such dosage unit forms are tablets (including chopped or coated tablets), capsules, pills, powder packages, wafers, injectable solutions or suspensions, tablespoons, tablespoons, and the like Were some of them.

  Preferably, a therapeutically effective amount of a pharmaceutical composition comprising a compound according to the invention is administered orally or parenterally. The therapeutically effective amount is an amount that effectively prevents or reduces the size and size of metastatic and / or cell proliferative disorders (see above) that are frequently used in a patient. Based on current data, the compounds of the invention and in particular 4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzoxiadiacyclocyclopentadecine, 17-bromo-8,9 , 10, 11, 12, 13, 14, 19-octahydro-20-methoxy-13-methyl- as an active ingredient, 10 mg to several (1-5) grams per day It is contemplated that the dose can be administered orally as a single dose or subdivided into more than one dose including, for example, 2, 3 or 4 times daily. A preferred amount is in the range of 500 to 4,000 mg per day. Particularly preferred dosages for such compounds are in the range of 750 mg to 3,000 mg per day. The amount of a compound according to the invention, also referred to herein as an active ingredient, necessary to achieve a therapeutic effect, of course, along with the route of administration, the age and condition of the recipient, and the particular disorder or disease being treated is of course It will be appreciated that it will change. Optimum dosages and dosage regimens can be readily determined by those skilled in the art using conventional methods and viewing the information provided herein. This treatment is intermittent or includes, for example, but not limited to, a 3-4 week cycle giving 1-21 days of treatment per cycle or other schedules shown to be effective and safe Can be given.

  The VEGFR3 inhibitors described above can be used in combination with one or more other cancer treatments. Such combinations can include any established anti-tumor therapy, including but not limited to chemotherapy, radiation and target-based therapy, such as antibodies and small molecules. These therapies can be combined in systemic therapy or in local instillation / administration (eg, in the subdural space) depending on optimal efficacy / safety requirements.

In certain preferred embodiments, for various tumor types, one or more other cancer treatments suitable for combination with a VEGFR3 inhibitor of the present invention;
Breast cancer: Herceptin, docetaxel, anthracycline, capecitabine
Prostate cancer: docetaxel, mitoxantrone
Colon cancer: oxaliplatin, 5-FU, avastin, irinotecan, cetuximab
Lung cancer; taxotere, carboplatin, gemicitabine
Is included, but is not limited to these.

The VEGFR3 inhibitor of the invention and the additional anti-cancer agent can 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 and in such an amount and manner that is sufficient to ensure that an advantageous or synergistic effect is achieved. The preferred method and sequence of administration and the respective dosage and dosing regimen for each component of the combination is the specific VEGFR3 inhibitor and further anti-cancer agent administered, their route of administration, the specific tumor being treated It will be appreciated that it will depend on the particular host being treated as well. Optimal administration methods and sequences and dosages and dosage regimens can be readily determined by one skilled in the art using conventional methods and viewing the information provided herein.

Compound preparation and preparation:
Compound 95, 12H-4,6-ethanediylidene-13,17-methanopyrimido [4,5-b] [6,1,10,16] benzoxatriazacyclononadecin-12-one, 21-chloro-8, 9,10,11,13,14,15,16,18,23- decahydro-25-methoxy - production of
Stage 1.
Titanium, tetrakis (2-propanolate) (0.005 mol) was added to 3-piperidinecarboxylic acid ethyl ester (0.54 g, 0.005 mol) and 4-chloro-2-nitro in CH ₂ Cl ₂ (40 ml). To a solution of benzaldehyde (0.93 g, 0.005 mol) was added. The mixture was stirred at room temperature for 1 hour and NaBH (OAc) ₃ (0.0055 mol) was added. The reaction mixture was stirred at room temperature for 1 hour, an additional portion of NaBH (OAc) ₃ (0.00275 mol) was added, the mixture was stirred at room temperature for 2 hours, and then NaHCO ₃ (saturated in H ₂ O) was added. . The organic layer was separated, dried (K ₂ CO ₃ ), filtered, the solvent was evaporated and co-evaporated with toluene to remove residual CH ₂ Cl ₂ . The 3-piperidinecarboxylic acid, 1-[(4-chloro-2-nitrophenyl) methyl]-, ethyl ester as crude residue was used as such in the next reaction step.

Stage 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 of Pt / C 5% (0.5 g) as a catalyst. After uptake of H ₂ (3 eq), the catalyst was filtered and the filtrate was evaporated. The residue was dissolved in EtOAc and the solution was filtered over Extreme. Heptane was added but no crystals appeared. The solvent was evaporated and the residue was redissolved in CH ₃ OH and filtered on filter paper to remove silica grease. The solvent was evaporated to give 1.35 g of 3-piperidinecarboxylic acid, 1-[(2-amino-4-chlorophenyl) methyl]-, ethyl ester as a brown oil.

Stage 3 .
To a solution of 3-piperidinecarboxylic acid, 1-[(2-amino-4-chlorophenyl) methyl]-, ethyl ester (0.0011 mol) in 2-propanol (sufficient amount) was added 4-chloro-7-methoxy. -6-Quinazolinol 6-acetate (0.273 g, 0.00108 mol) was added and then the reaction mixture was shaken at 80 ° C. overnight. The mixture was then shaken at 80 ° C. for a further 6.5 hours and the solvent was evaporated. Yield: 3-piperidinecarboxylic acid, 1-[[2-[[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl]-, ethyl ester (crude; Used in the reaction stage).

Step 4 .
NH ₃ / CH ₃ OH 7N (about 10 ml) was added to 3-piperidinecarboxylic acid, 1-[[2-[[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -, Ethyl ester (0.00114 mol) was added and the reaction mixture was shaken for 1 hour at 35 ° C and then the solvent was evaporated. Yield: 3-piperidinecarboxylic acid, 1-[[4-chloro-2-[(6-hydroxy-7-methoxy-4-quinazolinyl) amino] phenyl] methyl]-, ethyl ester (used directly in the next reaction step) ).

Step 5.
3-piperidinecarboxylic acid, 1-[[4-chloro-2-[(6-hydroxy-7-methoxy-4-quinazolinyl) amino] phenyl] methyl]-, ethyl ester (0.00114 mol), N- ( mixture of 3-bromo-propyl) carbamic acid, 1,1-dimethylethyl ester (1 eq.) and _Cs 2 _CO 3 _(1.8582g) was stirred at room temperature overnight, then the reaction mixture was stirred for 30 minutes at 50 ° C.. If necessary, further 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 an additional 15 minutes. The solvent was evaporated (Genevac) and the residue was dissolved in CH ₂ Cl ₂ . The solution was filtered over dicalite and the filtrate was evaporated. Harvest: 3-piperidinecarboxylic acid, 1-[[4-chloro-2-[[6- [3-[[(1,1-dimethylethoxy) carbonyl] amino] propoxy] -7-methoxy-4-quinazolinyl] Amino] phenyl] methyl]-, ethyl ester.

Step 6.
3-piperidinecarboxylic acid, 1-[[4-chloro-2-[[6- [3-[[(1,1,4 ml) in TFA / CH ₂ Cl ₂ / TIS (90/8/2) (11.4 ml). 1-dimethylethoxy) carbonyl] amino] propoxy] -7-methoxy-4-quinazolinyl] amino] phenyl] methyl]-, ethyl ester (residue; approximately 0.00114 mol) is stirred for 7-8 hours, The solvent was then evaporated and the resulting residue was dried in an oven overnight. Yield: 3-piperidinecarboxylic acid, 1-[[2-[[6- (3-aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl]-. CF ₃ COOH.

Step 7.
3-piperidinecarboxylic acid, 1-[[2-[[6- (3-aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl]-. A solution of CF ₃ COOH (0.00114 mol) and DIPEA (0.00684 mol) is added to a solution of HBTU (0.00342 mol) and HOBt (0.00228 mol) in dry DMF (285 ml) and then the reaction The 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 collected, Na ₂ CO ₃ was added and the organic solvent was evaporated. CH ₂ Cl ₂ was added to the aqueous concentrate and the resulting mixture was extracted 3 times with CH ₂ Cl ₂ and then the organic extract was dried and collected. Harvest: 0.0007 moles 12H-4,6-ethanediylidene-13,17-methanopyrimido [4,5-b] [6,1,10,16] benzoxatriazacyclononadecin-12-one, 21- Chloro-8,9,10,11,13,14,15,16,18,23-decahydro-25-methoxy- (62% yield).

Compound 96, 4,6-ethanediylidene-12H-pyrimido [4,5-b] [6,1,10,13] benzoxatriazacyclohexadecin-12-one, 18-chloro-8,9,10, Preparation of 11,13,14,15,20-octahydro-21-methoxy-13,14-dimethyl-, (13S)-
Stage 1 .
CH ₂ Cl ₂ (sufficient amount) of N- methyl -L- alanine methyl ester hydrochloride (1.4 g, 0.010 mol), 4-chloro-2-nitrobenzaldehyde (0.010 mol) and titanium, tetrakis A mixture of (2-propanolate) (0.010 mol) was stirred at room temperature for 1 hour. NaBH (OAc) ₃ (0.022 mol) was then added and the mixture was stirred at room temperature for 3 hours. More NaBH (OAc) ₃ (0.011 mol) was added and the mixture was stirred at room temperature overnight. NaHCO ₃ (saturated) was then added until basic and the mixture was filtered over a P3 filter. The organic layer was separated and the aqueous layer was extracted 3 times with CH ₂ Cl 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.

Stage 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 added to DIPE. Hydrogenated using 5% (0.5 g) Pt / C as catalyst in the presence of a 4% thiophene solution in (0.5 ml). After uptake of H ₂ (3 eq), the catalyst was filtered and the filtrate was evaporated. The residue was purified on silica gel (using cartridge) (eluent: 4%
_{Et 3 N / CH 2 Cl 2} ). The desired fraction was collected. Heptane was added to the desired fraction (two-phase form). The heptane layer was separated to remove the silica grease. After removal of the solvent and drying, 0.5674 g of L-alanine, N-[(2-amino-4-chlorophenyl) methyl] -N-methyl-, methyl ester was obtained (22%; yellow oil) . The crude product was used as such in the next reaction step.

Stage 3.
To a solution of L-alanine, N-[(2-amino-4-chlorophenyl) methyl] -N-methyl-, methyl ester (0.00055 mol) in 2-propanol (5 ml), 4-chloro-7- Methoxy-6-quinazolinol 6-acetate (0.00055 mol) was added, then the reaction mixture was shaken at 80 ° C. overnight and the solvent was evaporated. Harvest: L-alanine, N-[[2-[[6- (acetyloxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -N-methyl-, methyl ester (crude, intact Used in the next reaction step).

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

Step 5.
L-alanine, N-[[4-chloro-2-[(6-hydroxy-7-methoxy-4-quinazolinyl) amino] phenyl] methyl] -N-methyl-, methyl ester (0.00055 mol), N - a mixture of (3-bromopropyl) carbamic acid, 1,1-dimethylethyl ester (1 eq.) and _Cs 2 _CO 3 _(0.8965g) was stirred at room temperature overnight, then the reaction mixture was stirred for 30 minutes at 50 ° C. . If necessary, more CAS 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 an additional 15 minutes. The solvent was evaporated (Genevac) and the residue was dissolved in CH ₂ Cl ₂ . The solution was filtered over dicalite and the filtrate was evaporated. Harvest: L-alanine, N-[[4-chloro-2-[[6- [3-[[(1,1-dimethylethoxy) carbonyl] amino] propoxy] -7-methoxy-4-quinazolinyl] amino] Phenyl] methyl] -N-methyl-, methyl ester.

Step 6.
L-alanine, N-[[4-chloro-2-[[6- [3-[[(1,1- 1) in TFA / CH ₂ Cl ₂ / TIS (90/8/2) (5.5 ml). Dimethylethoxy) carbonyl] amino] propoxy] -7-methoxy-4-quinazolinyl] amino] phenyl] methyl] -N-methyl-, methyl ester (crude residue; 0.00055 mol) was stirred for 7-8 hours. The solvent was then evaporated and the resulting residue was dried in an oven overnight. Harvest: L-alanine, N-[[2-[[6- (3-aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -N-methyl-. CF ₃ COOH.

Step 7.
L-alanine, N-[[2-[[6- (3-aminopropoxy) -7-methoxy-4-quinazolinyl] amino] -4-chlorophenyl] methyl] -N-methyl-. A solution of CF ₃ COOH (0.00055 mol) and DIPEA (0.0033 mol) is added to a solution of HBTU (0.00165 mol) and HOBt (0.0011 mol) in dry DMF (137 ml) and then the reaction. The 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 collected, Na ₂ CO ₃ was added and the organic solvent was evaporated. CH ₂ Cl ₂ was added to the aqueous concentrate and the resulting mixture was extracted 3 times with CH ₂ Cl ₂ and then the organic extract was dried and collected. Yield: 0.049 g 4,6-ethanediylidene-12H-pyrimido [4,5-b] [6,1,10,13] benzoxatriazacyclohexadecin-12-one, 18-chloro-8,9 , 10, 11, 13, 14, 15, 20-octahydro-21-methoxy-13,14-dimethyl-, (13S)-.

As used in the examples, “CH ₃ OH” means methanol, “Et ₃ N” means triethylamine, “CH ₂ Cl ₂ ” means dichloromethane, and “HBTU” means 1- [bis (Dimethylamino) methylene] -1H-benzotriazolium hexafluorophosphate (1-) 3-oxide, “DMF” means N, N-dimethylformamide, “NaBH (OAc) ₃ ” is sodium Triacetoxyborohydride means “DIPEA” means N-ethyl-N- (1-methylethyl) -2-propanamine, “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 a carbonate disodium salt," NaHCO ₃ "means carbonate monosodium salt.

Preparation of Compound 22, MTKI1 A suitable preparation of the preferred compound used in the present invention, obtained from WO 2004/105765, is as follows:
Example A
a) Preparation of 1-pentanol, 5-[[(4-bromo-2-nitrophenyl) methyl] amino]-(Intermediate 1) 4-Bromo-2-nitro-benzaldehyde (0) in EtOH (15 ml) 0.013 moles), 5-amino-1-pentanol (0.013 moles) and titanium, tetrakis (2-propanolate) (0.014 moles) are stirred at room temperature for 1 hour, then the reaction mixture is stirred at 50 ° C. And stirred for 30 minutes. The mixture was cooled to room temperature and NaBH ₄ (0.013 mol) was added in portions. The reaction mixture was stirred overnight and then poured out into ice water (50 ml). The resulting mixture is stirred for 20 minutes and the resulting precipitate is filtered (giving filtrate (I)), washed with H ₂ O and stirred in DCM (the product is dissolved and removed from the Ti-salt). for). The mixture was filtered, then the filtrate was dried (MgSO ₄ ), filtered and finally the solvent was evaporated. The filtrate (I) was evaporated until 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, yielding 3.8 g (93%) of intermediate 1.

Example B
a) Preparation of 1-pentanol, 5-[[(4-bromo-2-nitrophenyl) methyl] methylamino]-(Intermediate 2) Intermediate 50 (0.0047 mol) in EtOH (150 ml), formaldehyde (0.025 mol) and titanium, by heating the solution of tetrakis (2-propanolate) (0.0051 mol) in 50 ° C., stirred for 1 hour, then divided NaBH ₄ (0.026 mol) at room temperature added. The reaction mixture was stirred overnight and then quenched with water (100 ml). The resulting mixture was stirred for 1 hour and the resulting precipitate was filtered and washed. The organic filtrate was concentrated and then the aqueous concentrate was extracted with DCM and dried. The solvent was evaporated and the residue was filtered over silica gel (eluent: DCM / CH ₃ OH 98/2 to 95/5). The product fractions were collected and the solvent was evaporated, yielding 0.5 g of intermediate 2.

b) Preparation of 1-pentanol, 5-[[(4-bromo-2-nitrophenyl) methyl] methylamino]-, acetate (ester) (Intermediate 3) Intermediate 2 in acetic anhydride (8 ml) A solution of 0.0015 mol) and pyridine (0.015 mol) was stirred at room temperature overnight, 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) Intermediate 3 (0) in THF (50 ml) .0015 mol) was hydrogenated using Pt / C 5% (0.5 g) as catalyst in the presence of thiophene solution (0.5 ml) [H179-034]. After uptake of H 2 ₍₃ equiv), the catalyst was filtered off and the filtrate was evaporated, yielding intermediate 4 0.5 g.

d) 6-quinazolinol, 4-[[2-[[[5- (acetyloxy) pentyl] methylamino] methyl] -5-bromophenyl] amino] -7-methoxy-, acetate (ester) (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) at 80 ° C. Heat and stir the reaction mixture for 1 day. The solvent was evaporated under reduced pressure and the residue was used as such in the next reaction step, yielding 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) 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 the reaction mixture was then brought to 60 ° C. Heat and stir for 18 hours. The solvent was evaporated, H ₂ O (20 ml) was added, then the mixture was neutralized with acetic acid and the resulting precipitate was filtered. The filtrate was concentrated under reduced pressure and the concentrate was extracted with DCM, filtered then dried (MgSO ₄ ) and the mixture was concentrated under reduced pressure, yielding 0.5 g (70%) of intermediate 6.

Example C
a) 4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzoxiadiazacyclo-pentadecine, 17-bromo-8,9,10,11,12,13,14 , 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 and tributylphosphine (0.0016 mol). Followed by 1,1 ′-(azodicarbonyl) bis-piperidine (0.0016 mol) and the reaction mixture was stirred for 2 hours. The solvent was evaporated to 1/3 of the initial volume. The resulting precipitate was filtered and washed. The filtrate was evaporated and the residue was purified by RP high-performance liquid chromatography. The product fractions were collected and the organic solvent was evaporated. The aqueous concentrate was extracted twice with DCM and the organic layer was dried (MgSO ₄ ) then filtered. The solvent was evaporated and the residue was dried at 50 ° C. (vacuum) to give 0.004 g (0.8%) of compound MTKI1.

Compound 2, 4,6-ethanediylidene-19H-pyrimido [4,5-b] [6,13,1] benzodioxaazacyclo-pentadecine, 15-chloro-8,9,10,11,12,13- Production of hexahydro-20-methoxy- ;
Example D
4,6-ethanediylidene-19H-pyrimido [4,5-b] [6,13,1] benzodioxaazacyclo-pentadecine, 15-chloro-8,9,10,11,12,13-hexahydro-20 Preparation of -methoxy- (compound 2) A solution of intermediate 9 (0.0024 mol) and triphenylphosphine (0.0036 mol) in THF, dry (100 ml) was stirred at room temperature and then in THF (10 ml). A solution of bis (1-methylethyl) diazene dicarboxylate (0.0036 mol) was added dropwise. The reaction mixture was stirred for 6 hours and an additional portion of bis (1-methylethyl) diazene dicarboxylate (0.35 ml) in THF (10 ml) was added. The mixture was stirred overnight and concentrated. The residue was purified by column chromatography over silica gel (eluent: DCM / CH ₃ OH / THF 90/5/5). The product fractions were collected and further purified by RP high-performance liquid chromatography. The product fractions were collected and concentrated. The aqueous concentrate was filtered and the retained solid was washed and dried at 65 ° C. (vacuum) to give 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) 2-Chloro-6-nitro-phenol (0) in N, N-dimethylformamide (150 ml) 0.046 mol) solution was heated to 50 ° C., then K ₂ CO ₃ (0.069 mol) was added and the reaction mixture was stirred for 15 min. 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, yielding 13.88 g of intermediate 6.
b) Preparation of hexanoic acid, 6- (2-amino-6-chlorophenoxy)-, methyl ester (intermediate 7) Intermediate 6 (0.046 mol) and ethanime (2 g) in THF (ml) ) Was hydrogenated using Pt / C 5% (3 g) as catalyst in the presence of DIPE (2 ml). After uptake of H ₂ (3 eq), the reaction mixture was filtered over a small plug of Dicalite 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-methoxyquinazoline (0.022 mol) and intermediate 7 (0.022 mol) in 170 ml) and heated to 80 ° C. and concentrated And the residue was chromatographed on silica gel (eluent: DCM / CH ₃ OH 97/3). The product fractions were collected and the solvent was evaporated, yielding 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) LAH (0 ml) in THF (40 ml) 0.0246 mol) 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 and then an additional portion of LAH (0.0123 mol) was added in portions. The mixture was further 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). The mixture was stirred for 15 minutes, filtered and the filtrate was concentrated. The residue was stirred in boiling CH 3 _CN, filtered and the solid retained was dried at 60 ° C. the (vacuum). The solid was re-dissolved in CH ₃ OH / DCM (10/90) and the mixture was neutralized with HCl (1N). The organic layer was separated, dried (MgSO ₄ ), filtered and concentrated, yielding 1 g of intermediate 9.

An example of a typical formulation for the most preferred compound, MYKI1, is as follows:
Example F: Dispensing:
The product MTKI1 can be prepared as a 10-mg / mL oral solution, pH 2. It excipients in purified water, Captisol ^(R) (chemical name: sulfobutylether -β- cyclodextrin, SBE-β-CD), citric acid, containing Tween ^(R) 20, HCl and NaOH. Formulations can be stored refrigerated (2-8 ° C; 36-46 ° F) and allowed to warm to room temperature for up to 1 hour prior to dosage preparation.

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

Experimental data VEGFR3 inhibition Involvement of lymph nodes is a poor prognostic factor in multiple cancer types. Recent studies have shown that lymphangiogenesis plays an important role in tumor progression, with vascular endothelial growth factor (VEGF) C and D acting on the specific lymphatics that act through activation of the cognate receptor VEGFR3. Identified as a forming factor. 4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzoxadiazacyclo-pentadecine, 17-bromo-8,9,10,11,12, also referred to as compound 2 below , 13, 14, 19-octahydro-20-methoxy-13-methyl- has unique kinase inhibition aspects (pan Her and Src groups) as well as favorable tissue distribution aspects and has potent antitumor activity in multiple experimental models Is a multi-targeted kinase inhibitor that has been shown to produce

  In vitro kinase assays showed that the compounds of the invention have potent inhibition of EGFR, Her2, Her4 and Src group kinase (Src, Fyn, Lck, Yes, Lyn) activities.

  Here, we confirmed in vitro kinase inhibition in a cell-based assay using human vascular endothelial cells (HMVECd), where compound 2 prevented VEGFR3-dependent VEGF-C stimulation of Erk 1/2, It did not affect VEGFR1 mediated VEGF signaling. Compound 2 was also shown herein to inhibit VEGF-C-induced phosphorylation of VEGFR3 using a cell line engineered to express human VEGFR3.

These results were further confirmed herein in a recently developed Xenopus tadpole model, where compound 2 was found to phenotype the effects of VEGF-C knockout.
(Ny et al., Nat. Med. 2005 Sep; 11: 998-1004).

Methods In vitro kinase assay The VEGFR3 kinase reaction was performed in a 96-well microtiter plate at 30 ° C for 10 minutes. The 25 μl reaction volume was 8 mM MOPS pH 7, 200 μM EDTA, 10 mM MgAc, 10 μM unlabeled ATP, 0.5 mCi AT ³³ P, 500 μM JAK3-tide (Ac-GGEEEEYFELVKKKK-NH ₂ ), 25 ng VEGFR 3 and 2 in 100% DMSO. % Compound.

  The reaction was stopped by the addition of 5 μl of 3% phosphoric acid solution. 10 μl of the reaction mixture was then spotted on a Filtermat P30 filter (Wallac), washed 3 times for 5 minutes in 0.75% phosphoric acid and once for 2 minutes in methanol, then 4 ml Was transferred to a sealable plastic bag containing a scintillation fluid and read on a scintillation counter.

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 confluence on 10 cm dishes. Overnight, the cells were serum starved and the medium was replaced with a new serum-free medium. A predetermined amount of Compound 2 or standard vehicle was added to the cells and then incubated at 37 ° C. for 15 minutes. This was followed by the addition of VEGF-C to a final concentration of 100 ng / ml and the cells were incubated at 37 ° C. for 10 minutes, after which they were washed with ice-cold PBS and then lysed. Cell lysates containing protease inhibitors were subjected to immunoprecipitation using an antibody specific for VEGFR-3, and the immunoprecipitates were separated by SDS-PAGE. The protein was transferred to a nitrocellulose membrane and then probed first with a phospho-tyrosine antibody and then with a VEGFR-3 antibody.

Human microvascular endothelial cells - the skin (HMVEC-d) of Erk activation induced by VEGF-C in inhibiting HMVEC-d cells (Cambrex), the 37C in 5% CO _2, fibronectin-coated 12- Cultures were carried out for 4 days in a suitable serum-containing medium (Cambrex) on well plates (BD Biocoat, Becton Dickinson). The cells were then starved for 16 hours in the same medium as before except without serum and containing SITE + 3 (Invitrogen) instead. The cells were then preincubated with 0.2% DMSO (solvent) or 5 uM Compound 2 for 60 minutes, then either 10 ng / ml VEGF or 100 ng / ml VEGF-C (R & D systems) were applied to the cells. added. Before or 10 and 30 minutes after stimulation, the medium is removed, cells are rinsed with ice-cold PBS containing 0.1 mM sodium orthovanadate, 0.1% SDS and 1x phosphatase and protease inhibitors ( Lysed in M-PER buffer (PIERCE) containing HALT, PIERCE). Cells were scraped from the plate, incubated in lysis buffer for 30 minutes on ice, and then centrifuged at 16000 × g for 10 minutes at 4 ° C. Lysates were quantified using BCA reagent according to manufacturer's instructions (PIERCE) and 5 mg of total protein was loaded onto an SDS-polyacrylamide gel (NUPAGE, Invitrogen). All electrophoresis and blotting onto PVDF membranes were performed using reagents from the NUPAGE system according to the manufacturer's instructions. Block blot with Odyssey Blocking Buffer (LICOR) for 1 hour, then primary antibody in a 1: 1 mixture of Odyssey Blocking Buffer and 0.1% Tween-20 (see below) Was diluted 1: 1000 and applied overnight at 4 ° C .: P-Erk1 / 2 (Thr202 / Tyr204, clone E10, mouse monoclonal # 9106, Erk1 / 2 (rabbit polyclonal # 9102) (all from Cell Signaling Technologies, Inc.) The blot was washed 3 times over 5 minutes in PBS containing 0.1% Tween and then secondary antibody (diluted 1: 1000 in PBS containing 0.1% Tween-20) 1 hour incubation with: IRDye at 800 nm Goat engaged anti - mouse (Rockland, Inc) or goat anti conjugated to Alexa 680 nm - rabbit (Molecular Probes, Inc).

  All suitable for intensity 5 and membrane scanning at 700 nm and 800 nm using the LICOR system, washing the blot 3 times in PBS containing 0.1% Tween for 3 minutes over 5 minutes and once in PBS The blots were scanned using the settings of

VEGFR3 In Vivo Xenops Assay To analyze the effect of Compound 2 on lymphangiogenesis, Xenopus tadpoles were used as a model (Ny et al, Nature Medicine 11: 2005, 998-1004). The xenopus tadpole medium was administered daily in a dose-dependent manner with vehicle standards, starting before stage 26-28, the formation of both blood vessels and lymphatic vessels. The effect of the compounds was monitored by a “trained observer” by “live analysis”, ie viable embryos were examined after 4 days for evidence of lymphatic vessel-deficiency. As an additional means of confirming the results in a limited number of experiments, tadpoles were fixed in steps 35-36, in situ hybridized with Prox1, and from the posterior main vein (PCV) of lymphatic endothelial cells (LECs) Their migration to the dorsal endpoint is assessed, which reflects the essential stage of lymphangiogenesis. Under normal circumstances, LECs originate from the PCV region and accumulate there, and during the study period they are limited to “areas” (from region 1 to region 2 and region 3) in the back direction. They then migrate further in the rostrally and caudally directions to form lymphatic structures in the tail and trunk. Defects in the migration of LECs from PCV were quantified by determining the maximum migration of cells, as well as by counting the number of cells in these various regions in the tail.

Results In vitro kinase assay The following table provides the pIC50 values obtained for the compounds of the invention using the VEGFR3 kinase assay described above.

Inhibition of VEGF-C-induced VEGFR-3 phosphorylation in VEGFR-3-expressing porcine aortic endothelial cells 4,6-ethanediylidenepyrimido [4,5-b] [6,1, to VEGF-C-induced VEGFR3 activity 12] Benzoxadiazacyclo-pentadecine, 17-bromo-8,9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl- (compound 2) has the effect of VEGFR- This is evident from the dose-dependent decrease in 3 phosphorylation (top box in FIG. 1). Re-staining Western blots with VEGFR-3 antibody confirms the presence of VEGFR-3 at various concentrations (bottom frame of figure).

Erk phosphorylation assay in HMVEC-d cells As shown in FIG. 2, Compound 2 at 5 μM prevented phosphorylation of Erk1 / 2 by VEGF-C, but did not prevent VEGF stimulation of Erk1 / 2. These data are consistent with selective inhibition of VEGFR-3 by compound 2.

Xenops Study Results Live screening was used to assess the effect of Compound 2, and a concentration of> 50 uM of Compound 2 caused bleeding in 30% of tadpoles. At 20 uM, 10% of tadpoles developed edema, which was not seen in tadpoles treated with vehicle alone. Compound 2 blocks lymphatic endothelial cell (LEC) migration in a tadpole in a dose-dependent manner (Table 1). Even at the lowest concentration of compound tested (0.31 uM), LEC migration in stages 35-36 was particularly reduced and this was more than observed with vehicle alone. These data support the conclusion that this compound prevents normal lymphangiogenesis.

  Although the foregoing specification has described the principles of the invention and examples have been given for purposes of illustration, the practice of the invention will generally fall within the scope of the following claims and their equivalents. It will be understood to encompass all of the changes, applications and / or modifications.

Claims (20)

Formula (I)
[Where:
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 _1-3 alkyl- represents; X ¹ represents O or —O—C _1-2 alkyl-;
X ² represents a direct bond, —C _1-2 alkyl-, O, —O—C _1-2 alkyl-, NR ¹² or NR ¹² -C _1-2 alkyl-;
R ¹ represents hydrogen, cyano, halo or hydroxy;
R ² represents hydrogen, cyano, halo or hydroxy;
R ³ represents hydrogen;
R ⁴ represents C _1-4 alkyloxy-;
R ¹² represents hydrogen or C _1-4 alkyl-;
R ¹³ represents hydrogen or C _1-4 alkyl;
R ¹⁴ represents hydrogen or C _1-4 alkyl;
Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl; and Het ²² represents pyrrolidinyl, piperazinyl or piperidinyl]
VEGFR3 in a mammalian patient comprising administering to a mammalian patient in need of treatment a therapeutically effective amount of a compound of the invention, a pharmaceutically acceptable acid or base addition salt thereof and a stereochemical isomer. A method of treating or preventing a biological activity mediated by. In compounds of 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} - indicates;
X ¹ represents O;
X ² is _{-C 1-2} alkyl -, O or ^NR 12 _{-C 1-2} alkyl - indicates;
R ¹ represents hydrogen or halo;
R ² represents hydrogen or halo;
R ³ represents hydrogen;
R ⁴ represents C _1-4 alkoxy;
R ¹² represents C _1-4 alkyl-;
The process according to claim 1, wherein R ¹⁴ represents hydrogen or C _1-4 alkyl; and Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl. The compound of formula (I) is 4,6-ethanediylidene-19H-pyrimido [4,5-b] [6,13,1] benzodioxaazacyclo-pentadecine, 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] benzoxatriazacyclononadecin-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] benzoxatriazacyclohexadecin-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] benzoxiadiazacyclopentadecine, 17-bromo-8,9,10,11,12,13,14, 19-octahydro-20-methoxy-13-methyl-;
Or the method of claim 1 selected from the group consisting of pharmaceutically acceptable acid addition salts thereof. The compound is 4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzoxiadiazacyclopentadecin, 17-bromo-8,9,10,11,12,13,14 , 19-octahydro-20-methoxy-13-methyl-;
Or a pharmaceutically acceptable acid addition salt thereof.   2. The method of claim 1, wherein a 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 another anti-cancer agent.   Further anti-cancer agents are herceptin, docetaxel, anthracycline and capecitabine in breast cancer; docetaxel and mitoxantrone in prostate cancer; colon cancer Oxaliplatin, 5-FU, Avastin, irinotecan and cetuximab; and Taxotere and carboplatin and geplatin in the case of lung cancer The method according to claim 6.   Biological activity mediated by VEGFR3 is metastatic spread of cancer in mammalian patients; metastasis to local lymph nodes via lymphatic vessels; tumor-associated lymphangiogenesis in cancer; neovascularization of cancer cells expressing VEGFR3 ligand 2. The method of claim 1 selected from the group consisting of: recruitment and proliferation of endothelial cells expressing VEGFR3 in formation; and combinations thereof.   9. The method of claim 8, wherein the VEGFR3 ligand is VEGF-C, VEGF-D or a combination thereof.   The biological activity mediated by VEGFR3 is advanced breast cancer in a mammal, comprising the step of administering to the mammal a therapeutically effective amount of a compound as defined in any one of claims 1-4. The method of claim 1. Formula (I) in the manufacture of a medicament for the treatment of a biological activity mediated by VEGFR3
[Where:
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 _1-3 alkyl- represents; X ¹ represents O or —O—C _1-2 alkyl-;
X ² represents a direct bond, —C _1-2 alkyl-, O, —O—C _1-2 alkyl-, NR ¹² or NR ¹² -C _1-2 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 represents C _1-4 alkyloxy-;
R ¹² represents hydrogen or C _1-4 alkyl-;
R ¹³ represents hydrogen or C _1-4 alkyl;
R ¹⁴ represents hydrogen or C _1-4 alkyl;
Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl;
Het ²² represents pyrrolidinyl, piperazinyl or piperidinyl]
The use of pharmaceutically acceptable acid or base addition salts and stereochemical isomers thereof. In compounds of 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} - indicates;
X ¹ represents O;
X ² is _{-C 1-2} alkyl -, O or ^NR 12 _{-C 1-2} alkyl - indicates;
R ¹ represents hydrogen or halo; in particular R ¹ represents hydrogen or chloro; more particularly R ¹ represents hydrogen;
R ² represents hydrogen or halo; in particular R ² represents hydrogen, chloro or bromo; more particularly R ² represents chloro or bromo;
R ³ represents hydrogen;
R ⁴ represents C _1-4 alkyloxy; in particular R ⁴ represents methoxy;
R ¹² represents C _1-4 alkyl-; in particular R ¹² represents methyl;
R ¹⁴ represents hydrogen or C _1-4 alkyl; in particular R ¹⁴ represents hydrogen or methyl; more particularly R ¹⁴ represents hydrogen; and Het ²⁰ represents pyrrolidinyl, piperazinyl or piperidinyl; in particular Het ²⁰ represents 11. Use according to claim 10 indicating piperidinyl. A compound of formula (I);
4,6-ethanediylidene-19H-pyrimido [4,5-b] [6,13,1] benzodioxaazacyclo-pentadecine, 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] benzoxatriazacyclononadecin-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] benzoxatriazacyclohexadecin-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] benzoxadiazacyclopenta Decyne, 17-bromo-8,9,10,11,12,13,14,19-octahydro-20-methoxy-13-methyl-;
12. Use according to claim 11 selected from the group consisting of or a pharmaceutically acceptable acid addition salt thereof. The compound of formula (I) is 4,6-ethanediylidenepyrimido [4,5-b] [6,1,12] benzoxiadiazacyclopentadecin, 17-bromo-8,9,10,11, 12, 13, 14, 19-octahydro-20-methoxy-13-methyl-;
Or use according to claim 11 which is a pharmaceutically acceptable acid addition salt thereof.   15. Use according to any one of claims 11 to 14, wherein the medicament is for oral or parenteral administration.   The use according to claim 15, wherein the compound of formula (I) is administered in combination with another anti-cancer agent.   Further anti-cancer agents are herceptin, docetaxel, anthracycline and capecitabine for breast cancer; docetaxel and mitoxantrone for prostate cancer; oxaliplatin, 5-FU, Avastin, irinotecan and cetuximab for colon cancer And use according to claim 16, selected from the group consisting of taxotere, carboplatin and gemicitabine in the case of lung cancer. A biological activity mediated by VEGFR3;
-Metastatic spread of cancer in mammalian patients;
-Metastasis to local lymph nodes via lymphatic vessels;
-Tumor-related lymphangiogenesis in cancers such as gastric cancer, prostate cancer, human colorectal cancer, invasive cervical cancer, breast cancer, Kaposi's sarcoma and melanoma; and-in neovascularization of cancer cells expressing VEGFR3 ligand Recruitment and proliferation of endothelial cells expressing VEGFR3;
Use according to any of claims 11 to 14, selected from the group consisting of:   9. Use according to claim 8, wherein the VEGF3 ligand is VEGF-C, VEGF-D or a combination thereof.   The use according to any one of claims 1 to 4, which is a breast cancer having an advanced biological activity mediated by VEGFR3.