Patent Application
20080004285
The present invention relates to a class of novel pyrido(3,2-d)pyrimidine derivatives and a method for their preparation, as well as to pharmaceutical compositions comprising one or more of said pyrido(3,2-d)pyrimidine derivatives and one or more pharmaceutically acceptable excipients. The present invention further relates to the use of said novel pyrido(3,2-d)pyrimidine derivatives as biologically active ingredients, more specifically as medicaments for the treatment of disorders and pathologic conditions such as, but not limited to, immune and auto-immune disorders, organ and cells transplant rejections, cell proliferative disorders, cardiovascular disorders, disorders of the central nervous system and viral diseases.
A huge number of pyrido(3,2-d)pyrimidine derivatives is already known in the art. For instance pyrido(3,2-d)pyrimidine derivatives with various substituents on positions 2, 4 and 6 (using the standard atom numbering for the pyrido(3,2-d)pyrimidine moiety) are known with biological activities such as competitive inhibition of pteroylglutamic acid, inhibition of thrombocyte aggregation and adhesiveness, antineoplastic activity, inhibition of dihydrofolate reductase and thymidylate synthase, e.g. from U.S. Pat. No. 2,924,599, U.S. Pat. No. 3,939,268, U.S. Pat. No. 4,460,591, U.S. Pat. No. 5,167,963 and U.S. Pat. No. 5,508,281.
Pyrido(3,2-d)pyrimidine derivatives with various substituents on positions 2, 4, 6 and 7 (using the standard atom numbering for the pyrido(3,2-d)pyrimidine moiety) are also known e.g. from U.S. Pat. No. 5,521,190, U.S. patent application publication No. 2002/0049207, U.S. patent application publication No. 2003/0186987, U.S. patent application publication No. 2003/0199526, U.S. patent application publication No. 2004/0039000, U.S. patent application publication No. 2004/0106616, U.S. Pat. No. 6,713,484, U.S. Pat. No. 6,730,682 and U.S. Pat. No. 6,723,726. Some of them show activities as antiviral agents, anti-cancer agents, EGF inhibitors, inhibitors of GSK-3 protein kinases and the like.
U.S. Pat. No. 5,654,307 discloses pyrido(3,2-d)pyrimidine derivatives which are substituted on position 4 with monoarylamino or monobenzylamino, and on positions 6 and 7 with substituents each independently selected from the group consisting of lower alkyl, amino, lower alkoxy, mono- or dialkylamino, halogen and hydroxy. WO 01/083456 discloses pyrido(3,2-d)pyrimidine derivatives which are substituted on position 4 with morpholinyl and on position 2 with hydroxyphenyl or morpholinoethoxyphenyl, having PI3K and cancer inhibiting activity. U.S. Pat. No. 6,476,031 generically discloses substituted quinazoline derivatives, including (in reaction scheme 5) a series of pyrido(3,2-d)pyrimidine derivatives which are substituted on position 4 with hydroxy, chloro or an aryl, heteroaryl (including pyridyl, pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl, benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, imidazolyl), cycloaliphatic or cycloheteroaliphatic group being optionally spaced from the pyrido(3,2-d)pyrimidine ring by a linker such as NH. WO 02/22602 and WO 02/22607 disclose pyrazole and triazole compounds, including 2-(1-trifluoromethylphenyl)-4-fluorobenzopyrazolyl-pyrido(3,2-d)pyrimidine and 2-(1-trifluoromethylphenyl)-4-methyltriazolyl-pyrido(3,2-d)pyrimidine being useful as protein kinase inhibitors. WO 03/062209 discloses pyrido(3,2-d)pyrimidine derivatives which are substituted on position 7 with aryl or heteroaryl and on position 4 with monoarylamino or monoheteroarylamino and which may further be substituted on positions 2 and/or 6, being useful as capsaicin receptor modulators. However none of these documents teaches or suggests pyrido(3,2-d)pyrimidine derivatives having the substitution pattern disclosed by the present invention.
However there is a continuous need in the art for specific and highly therapeutically active compounds, such as, but not limited to, drugs for treating immune and autoimmune disorders, organ and cells transplant rejections, cell proliferative disorders, cardiovascular disorders, disorders of the central nervous system, allergic conditions and viral diseases. In particular, there is a need in the art to provide immunosuppressive compounds, antineoplastic drugs and anti-viral drugs which are active in a minor dose in order to replace existing drugs having significant side effects and to decrease treatment costs.
Currently used immunosuppressive drugs include antiproliferative agents, such as methotrexate (a 2,4-diaminopyrido(3,2-d)pyrimidine derivative disclosed by U.S. Pat. No. 2,512,572), azathioprine, and cyclophosphamide. Since these drugs affect mitosis and cell division, they have severe toxic effects on normal cells with high turn-over rate such as bone marrow cells and the gastrointestinal tract lining. Accordingly, marrow depression and liver damage are common side effects of these antiproliferative drugs.
Anti-inflammatory compounds used to induce immunosuppression include adrenocortical steroids such as dexamethasone and prednisolone. The common side effects observed with the use of these compounds are frequent infections, abnormal metabolism, hypertension, and diabetes.
Other immunosuppressive compounds currently used to inhibit lymphocyte activation and subsequent proliferation include cyclosporine, tacrolimus and rapamycin. Cyclosporine and its relatives are among the most commonly used immunosuppressant drugs. Cyclosporine is typically used for preventing or treating organ rejection in kidney, liver, heart, pancreas, bone marrow, and heart-lung transplants, as well as for the treatment of autoimmune and inflammatory diseases such as Crohn's disease, aplastic anemia, multiple-sclerosis, myasthenia gravis, uveitis, biliary cirrhosis, etc. However, cyclosporines suffer from a small therapeutic dose window and severe toxic effects including nephrotoxicity, hepatotoxicity, hypertension, hirsutism, cancer, and neurotoxicity.
Additionally, monoclonal antibodies with immunosuppressant properties, such as OKT3, have been used to prevent and/or treat graft rejection. Introduction of such monoclonal antibodies into a patient, as with many biological materials, induces several side-effects, such as dyspnea. Within the context of many life-threatening diseases, organ transplantation is considered a standard treatment and, in many cases, the only alternative to death. The immune response to foreign cell surface antigens on the graft, encoded by the major histo-compatibility complex (hereinafter referred as MHC) and present on all cells, generally precludes successful transplantation of tissues and organs unless the transplant tissues come from a compatible donor and the normal immune response is suppressed. Other than identical twins, the best compatibility and thus, long term rates of engraftment, are achieved using MHC identical sibling donors or MHC identical unrelated cadaver donors. However, such ideal matches are difficult to achieve. Further, with the increasing need of donor organs an increasing shortage of transplanted organs currently exists. Accordingly, xenotransplantation has emerged as an area of intensive study, but faces many hurdles with regard to rejection within the recipient organism.
The host response to an organ allograft involves a complex series of cellular interactions among T and B lymphocytes as well as macrophages or dendritic cells that recognize and are activated by foreign antigen. Co-stimulatory factors, primarily cytokines, and specific cell-cell interactions, provided by activated accessory cells such as macrophages or dendritic cells are essential for T-cell proliferation. These macrophages and dendritic cells either directly adhere to T-cells through specific adhesion proteins or secrete cytokines that stimulate T-cells, such as IL-12 and IL-15. Accessory cell-derived co-stimulatory signals stimulate activation of interleukin-2 (IL-2) gene transcription and expression of high affinity IL-2 receptors in T-cells. IL-2 is secreted by T lymphocytes upon antigen stimulation and is required for normal immune responsiveness. IL-2 stimulates lymphoid cells to proliferate and differentiate by binding to IL-2 specific cell surface receptors (IL-2R). IL-2 also initiates helper T-cell activation of cytotoxic T-cells and stimulates secretion of interferon-γ which in turn activates cytodestructive properties of macrophages. Furthermore, IFN-γ and IL-4 are also important activators of MHC class II expression in the transplanted organ, thereby further expanding the rejection cascade by enhancing the immunogenicity of the grafted organ. The current model of a T-cell mediated response suggests that T-cells are primed in the T-cell zone of secondary lymphoid organs, primarily by dendritic cells. The initial interaction requires cell to cell contact between antigen-loaded MHC molecules on antigen-presenting cells (hereinafter referred as APC) and the T-cell receptor/CD3 complex on T-cells. Engagement of the TCR/CD3 complex induces CD154 expression predominantly on CD4 T-cells that in turn activate the APC through CD40 engagement, leading to improved antigen presentation. This is caused partly by upregulation of CD80 and CD86 expression on the APC, both of which are ligands for the important CD28 co-stimulatory molecule on T-cells. However, engagement of CD40 also leads to prolonged surface expression of MHC-antigen complexes, expression of ligands for 4-1BB and OX-40 (potent co-stimulatory molecules expressed on activated T-cells). Furthermore, CD40 engagement leads to secretion of various cytokines (e.g., IL-12, IL-15, TNF-α, IL-1, IL-6, and IL-8) and chemokines, all of which have important effects on both APC and T-cell activation and maturation. Similar mechanisms are involved in the development of auto-immune disease, such as type I diabetes. In humans and non-obese diabetic mice, insulin-dependent diabetes mellitus results from a spontaneous T-cell dependent auto-immune destruction of insulin-producing pancreatic .beta. cells that intensifies with age. The process is preceded by infiltration of the islets with mononuclear cells (insulitis), primarily composed of T lymphocytes. A delicate balance between auto-aggressive T-cells and suppressor-type immune phenomena determines whether expression of auto-immunity is limited to insulitis or not. Therapeutic strategies that target T-cells have been successful in preventing further progress of the auto-immune disease. These include neonatal thymectomy, administration of cyclosporine, and infusion of anti-pan T-cell, anti-CD4, or anti-CD25 (IL-2R) monoclonal antibodies. The aim of all rejection prevention and auto-immunity reversal strategies is to suppress the patient's immune reactivity to the antigenic tissue or agent, with a minimum of morbidity and mortality. Accordingly, a number of drugs are currently being used or investigated for their immunosuppressive properties. As discussed above, the most commonly used immunosuppressant is cyclosporine, which however has numerous side effects. Accordingly, in view of the relatively few choices for agents effective at immunosuppression with low toxicity profiles and manageable side effects, there exists a need in the art for identification of alternative immunosuppressive agents and for agents acting as complement to calcineurin inhibition.
The metastasis of cancer cells represents the primary source of clinical morbidity and mortality in the large majority of solid tumors. Metastasis of cancer cells may result from the entry of tumor cells into either lymphatic or blood vessels. Invasion of lymphatic vessels results in metastasis to regional draining lymph nodes. From the lymph nodes, melanoma cells for example tend to metastasize to the lung, liver, and brain. For several solid tumors, including melanoma, the absence or the presence of lymph nodes metastasis is the best predictor of patient survival. Presently, to our knowledge, no treatment is capable of preventing or significantly reducing metastasis. Hence, there is a need in the art for compounds having such anti-metastasis effect for a suitable treatment of cancer patients.
Septic shock is a major cause of death in intensive care units (about 150,000 estimated deaths annually in the United States of America, despite treatment with intravenous antibiotics and supportive care) for which very little effective treatment is available at present. Patients with severe sepsis often experience failures of various systems in the body, including the circulatory system, as well as kidney failure, bleeding and clotting. Lipopolysaccharide (hereinafter referred as LPS) is the primary mediator of Gramm-negative sepsis, the most common form of sepsis, by inducing the production of a whole array of macrophage-derived cytokines (such as TNF-α; interleukins such as IL-1, IL-6, IL-12; interferon-gamma (hereinafter referred IFN-γ), etc.). These cytokines may induce other cells (e.g. T cells, NK cells) to make cytokines as well (e.g. IFN-γ). In addition, other macrophage products (e.g. nitric oxide, hereinafter referred as NO) may also play a role in the pathogenesis of toxic shock. These substances (e.g. NO) may be induced directly due to microbial interactions or indirectly through the action of proinflammatory cytokines. LPS binds to a serum protein known as LPB and the LPS-LPB complex thus formed is recognized by the CD14 toll-like receptor 4 (hereinafter referred as Tlr 4) complex on mononuclear phagocytes. Tlr4 is a signal transducing unit, the activation of which results in the release of mediators such as TNF-α, IL-1α, IL-1β and IL-6. These cytokines are important for the pathogenesis of shock. Their administration produces the clinical symptoms of septic shock and their blockade partially protects against LPS-induced lethal shock.
Current therapeutic strategies for the treatment of septic shock are directed against LPS (e.g. antibodies against LPS or LBP-34-23) or against the cytokines induced by LPS (e.g. TNF antibodies) or against the receptor for LPS (e.a. CD14). Unfortunately the initial clinical data of these approaches are very disappointing and illustrate the redundancy of receptors and mediators involved in the pathogenesis of toxic shock. For instance flagellin seems to be another toxin that plays a role in Gramm-negative Salmonella shock syndrome and that cannot be prevented or treated by therapeutic strategies directed specifically at LPS.
Clinical trials in humans with TNF-α blocking antibodies (such as the IL-1 receptor antagonist or PAF receptor antagonists) have been unsuccessful yet, as have been approaches to down regulate inflammation (e.g. using prednisolone) or to block endotoxins. These products must be administered very early after the onset of the disease, which is in most cases not possible.
The only drug currently approved by health authorities for the treatment of adult patients with the most serious forms of sepsis, including septic shock, is a genetically engineered version of a naturally occurring human protein, Activated Protein C, known as Xigris® or drotecogin-alpha which shows only moderate efficacy. Furthermore, because Activated Protein C interferes with blood clotting, the most serious side effect associated with Xigris® is bleeding, including bleeding that causes stroke. Thus Xigris® is contra-indicated for patients who have active internal bleeding, or who are more likely to bleed because of certain medical conditions including recent strokes, recent head or spinal surgery or severe head trauma. Because treatment with Xigris® comes with potentially serious risks, the benefits and risks of treatment with Xigris® must be carefully weighed for each individual patient.
Therefore there is a strong need in the art for new medications, either alone or in combination with the currently suggested treatments, for treating the most serious forms of life-threatening illnesses caused by severe infection, such as septic shock.
TNF-α is generally considered to be the key mediator in the mammalian response to bacterial infection. It is a strong pro-inflammatory agent that will affect the function of almost any organ system, either directly or by inducing the formation of other cytokines like IL-1 or prostaglandines. TNF-α is also a potent anti-tumor agent. If administered in small quantities to humans, it causes fever, headache, anorexia, myalgia, hypotension, capillary leak syndrome, increased rates of lipolysis and skeletal muscle protein degradation (including cachexia). Its use in cancer treatment is therefore very much limited by its severe side effects.
TNF-α, a pleiotropic cytokine produced mainly by activated macro-phages, exerts an in vitro cytotoxic action against transformed cells and in vivo anti-tumor activities in animal models. However, despite the fact that TNF-α is used in cancer patients especially to treat melanoma and sarcoma, the major problem hampering its use is toxicity. Indeed, TNF-α induces shock-like symptoms such as bowel swelling and damage, liver cell necrosis, enhanced release of inflammatory cytokines such as IL-1 or IL-6, and hypo-tension probably due to the release of inducers of vessels dilatation such nitric oxide and other proinflammatory cytokines. Cardiovascular toxicity is usually dose-limiting. Hypotension can be severe with systolic blood pressure below 60 mm Hg. Respiratory compromise is common after treatment with TNF-α and may require mechanical ventilation. Upper as well as lower digestive tract symptoms are also common in this type of treatment. Nausea and vomiting can be distressing and in some cases dose-limiting. Watery diarrhea is frequently observed. Neurological sequelae of treatment with TNF-α can also occur.
Hence, compounds that inhibit the toxic effects of TNF-α but that do not inhibit TNF-α anti-tumor effect are highly desirable for the treatment of cancer patients. Presently, several clinical trials involving TNF-α are being developed for the cancer of organs such as liver, lung, kidney and pancreas, which are based on a procedure including the steps of organ isolation, injection of TNF-α into the isolated organ, and reperfusion of the treated organ. However, even for isolated organ perfusion, some TNF-α usually escapes to the general blood circulation and leads to the mortality of about 10% of the patients thus treated. Many patients treated by this procedure also require intensive care unit rescue to cope with the toxic side-effects of such TNF-α treatment.
Combined treatment of TNF-α with alkylating drugs in an isolated organ perfusion model has received considerable attention. TNF-α is currently successfully used in isolated limb perfusion of human cancer patients and, in combination with melphalan and interferon-gamma, against melanoma, sarcomas and carcinomas.
The gastrointestinal mucosa is very sensitive to chemotherapeutic drugs. Mucositis caused by chemotherapy usually begins rapidly after initiation of the treatment with inflammation and ulceration of the gastrointestinal tract and leading to diarrhea. Severe, potentially life-threatening, diarrhea may require interruption of the chemotherapeutic treatment and subsequent dose reduction of the therapeutic agent. The oral cavity is often the place of severe side effects from cancer therapy that adversely affects the quality of life of the patient and its ability to tolerate the therapy. These side effects can be caused by radiotherapy as well as chemotherapy. A relationship between both serum and mucosal levels of TNF-α and IL-1 correlates with nonhematologic toxicities, including mucositis.
Radiation injuries occurring e.g. after a single high-dose irradiation include apoptosis as well as radiation necrosis. Even normal tissues protected by shielding during irradiation may be considerably damaged. It was found in experimental animal models that the radiation injuries after a single high-dose irradiation typically used for the treatment of various malignant tumors consist of radiation necrosis and apoptosis, which were correlated with the expression of TNF-α and TGF-β1.
Irradiation may induce graft-versus-host disease (hereinafter referred as GVHD) in cancer patients. This disease may occur especially in patients receiving allogeneic bone marrow transplantation as a treatment for cancers such as leukemia or lymphoma and can lead to the death of about 25% of the relevant patients. Before bone marrow transplantation, leukaemia patients for example receive either total body or total lymphoid irradiation to suppress their immune system. However, such irradiation induces not only necrosis but also the release of proinflammatory cytokines mainly TNF-α, IL-1 and IL-6 which in turn induce direct host tissues inflammation and activation of donor cells against host antigens leading to GVHD.
Cisplatin is an effective chemotherapeutic agent used in the treatment of a wide variety of both pediatric and adult malignancies, including testicular, germ cell, head and neck (cervical), bladder and lung cancer. Dose-dependent and cumulative nephrotoxicity is the major side effect of cisplatin, sometimes requiring a reduction in dose or discontinuation of the treatment. Other side effects of cisplatin include kidney damage, loss of fertility, harmful effect on a developing baby, temporary drop in bone marrow function causing drop in white blood cell count, anaemia, drop in platelets causing bleeding, loss of appetite, numbness or tingling in limbs, loss of taste, allergic reactions, and hearing disorders (difficulty in hearing some high-pitched sounds, experiencing ringing in the ears). Blurred vision may also be a side effect with high doses of cisplatin. It was shown that TNF-α is a key element in a network of proinflammatory chemokines and cytokines activated in the kidney by cisplatin. Blockade of TNF-α action would prevent the activation of this cytokine network and would provide protection against cisplatin nephrotoxicity. Hence, compounds that inhibit the toxic effects of cisplatin but that do not inhibit cisplatin anti-tumor effects are highly desirable for the treatment of cancer patients.
A surplus of TNF-α also causes a dramatic change of endothelial cells. In particular, TNF-α is an important mediator of skeletal muscle degeneration associated with cachexia, a debilitating syndrome characterized by extreme weight loss and whole-body wasting. Cachexia is usually a secondary condition whereby there is excessive tissue catabolism in combination with deficient anabolism. It is frequently seen in patients afflicted with chronic diseases such as cancer, cardiopulmonary diseases, aging, malabsortive disorders, excessive physical stress, eating disorders and acquired immuno-deficiency syndrome (AIDS). Some authors consider that the elevated TNF-α values found in at least 50% of cancer patients in the active stage of the disease can result in cachexia. TNF-α levels in clinically healthy adults, as well as in adult cancer patients, are well documented, for instance by Nenova et al. in Archives of Hellenic Medicine (2000) 17:619-621. Serum TNF-α concentrations in healthy children as well as in children with malignancies are documented for instance by Saarinen et al. in Cancer Research (1990) 50:592-595. A very significant proportion of cancer mortalities result from cachexia rather than from tumor burden. Chronic wasting disease (cachexia) may result when excessive cellular damage results in the release of substances (TNF-α, collagenase, hyaluronidase) that further catabolize the so-called healthy tissue resulting in an inability to assimilate nutrients required for anabolic restructuring of associated tissue.
Infants infected with human immunodeficiency virus type 1 (HIV-1) show growth retardation and severe weight loss that can lead to death. The overproduction of certain cytokines has been implicated as a possible cause for this. For instance, according to Rautonen et al. in AIDS (1991) 5:1319-1325, serum IL-6 concentrations are elevated and associated with elevated TNF-α concentrations in children with HIV infection. Swapan et al. in Journal of Virology (2002) 76:11710-11714 have shown that reduction of TNF-α levels by either anti-TNF-α antibodies or human chorionic gonadotropin inhibits the expression of HIV-1 proteins and prevents cachexia and death.
Very few drugs have been suggest at present for the treatment of cachexia. Some high-dose progestins like megestrol acetate, an agent used for the treatment of metastatic breast cancer, and medroxyprogesterone acetate were shown in randomized clinical trials to provide a statistically significant advantage as regards improved appetite and body weight gain. Hence, compounds that stimulate appetite and body weight gain without inhibiting the anti-tumor effect or anti-viral effect of co-administered drugs are highly desirable for the treatment of cachexia. More specifically, there is a need in the art for treating cachexia by the administration of compounds that reduce TNF-α levels in the serum of humans.
TNF-α is also suspected to play a role, through a possible dual action in the hematopoietic environment, in the development of hematologic malignancies such as idiopathic myelodysplastic syndromes occurring most often in elderly people but also occasionally in children, these syndromes being currently regarded as the early phase of acute leukemia.
Phosphodiesterases are a family of enzymes that hydrolyse cyclic nucleotide intracellular second messengers to their non-cyclic form. Cyclic 3′,5′-adenosine monophosphate (cAMP) modulates a variety of cellular and physiologic functions in mammals, such as, cell division, endocrine function, and the immune response. The level of cAMP is controlled by a class of enzymes called phosphodiesterases, which enzymatically deactivate cAMP. There are eleven types of phosphodiesterases which are categorized according to their function and the type of cell from which they are isolated. For instance, high-affinity phosphodiesterase (PDE-3) is isolated from human platelet cells and modulates platelet aggregation. Another type of phosphodiesterase (PDE-4) is found in various tissues but is the predominant form in human leukocytes; this enzyme modulates leukocyte activation and function associated with the immune response and inflammation. Both of these phosphodiesterases implement their control by modulating the cellular level of cAMP in their respective cells. Thus, inhibition of phosphodiesterases provides a method of modulating any cellular and bodily function that is controlled by cAMP. Compounds that are non-specific phosphodiesterase inhibitors, i.e. that inhibit all or multiple types of phosphodiesterases, are known. However, since cAMP is involved in so many functions throughout the body, a non-specific phosphodiesterase inhibitor has the potential to alter all functions modulated by cAMP, thus non-specific phospho-diesterase inhibitors are of limited value because of their numerous side-effects. Phosphodiesterase-4 (hereinafter referred as PDE-4) are cAMP-specific and are the major cAMP metabolising enzymes found in inflammatory and immune cells. Thus, molecules inhibiting PDE-4 lead to an elevation of cAMP levels within inflammatory and immune cells, thus having a potential immunomodulating effect on the activation of such cells which can lead to a decreased secretion of inflammatory and immunologically important molecules such as cytokines. TNF-α is an example of such an important inflammatory cytokine. Inhibition of PDE-4 using small molecules may be expected to inhibit the production of this cytokine by inflammatory cells such as monocytes and macrophages. Preparation of Human Lymphocyte Phospho-diesterase-4, as well as Human cAMP Phosphodiesterase assays have been described for instance in U.S. Pat. No. 5,264,437. Such a biological activity is important from a therapeutic point of view since excessive inflammatory cytokine production has been associated with a number of inflammatory and immunological diseases including for example, rheumatoid arthritis, rheumatoid spondylitis asthma, Crohn's disease, inflammatory bowel disease, osteoarthritis, reperfusion injury, sepsis and septic shock, chronic obstructive pulmonary disease, graft versus host reactions and allograft rejections.
The World Health Organization estimates that world-wide 170 million people (3% of the world's population) are chronically infected with HCV. These chronic carriers are at risk of developing cirrhosis and/or liver cancer. In studies with a 10 to 20 year follow-up, cirrhosis developed in 20-30% of the patients, 1-5% of whom may develop liver cancer during the next then years. The only treatment option available today is the use of interferon a-2 (or its pegylated from) either alone or combined with ribavirin. However, sustained response to such treatment is only observed in about 40% of the patients, and treatment is associated with serious adverse effects. There is thus an urgent need in the art for potent and selective inhibitors of HCV replication in order to treat patients infected with HCV. However, investigation of specific inhibitors of HCV replication has been hampered by the fact that it is highly difficult to efficiently propagate HCV in cell culture. Since HCV and pestiviruses belong to the same virus family and share many similarities (such as, but not limited to, organisation of the genome, analogous gene products and replication cycle), pestiviruses may be adopted as a model virus and surrogate for HCV. For example the Bovine Viral Diarrhea Virus (BVDV) is closely related to hepatitis C virus (HCV) and may be used as a surrogate virus in drug development for HCV infection.
There is a strong need in the art to improve, or to provide alternatives to, the existing prophylactic or therapeutic solutions to all the aforesaid diseases. In particular there is still a need in the art for providing alternative synthetic molecules having significant TNF-α activity and/or PDE-4 activity and/or HCV replication inhibiting activity. Meeting these various needs in the art constitutes the main goal of the present invention.
The present invention is based on the unexpected finding that certain combinations of substituents on positions 2, 4, 6 and/or 7 (using the standard atom numbering for the pyrido(3,2-d)pyrimidine moiety) which are not suggested by the prior art are however able to meet one or more of the needs recited herein above, in particular have significant TNF-α activity and/or PDE-4 activity and/or HCV replication inhibiting activity.
Based on this finding the present invention relates, in a first embodiment, to a class of pyrido(3,2-d)pyrimidine derivatives represented by the structural formula (I):
wherein:
Within the above defined class of compounds, a preferred group is one wherein R1 is not hydrogen, i.e. position 2 of the pyrido(3,2-d)pyrimidine moiety is substituted. Another preferred group of compounds is one wherein R1 is amino or N-protected amino such as, but not limited to, acetamido. Another preferred group of compounds is one wherein R1 is amino or N-protected amino, and further wherein R3 is a substituted aryl group. Another preferred group of compounds is one wherein R1 is amino or N-protected amino, wherein R3 is a substituted aryl group and further wherein R4 is hydrogen.
In a second embodiment, the present invention relates to certain groups of tri-substituted pyrido(3,2-d)pyrimidines which are useful as intermediates for making some of the pyrido(3,2-d)pyrimidine derivatives represented by the structural formula (I), in particular:
In a third embodiment, the present invention relates to the unexpected finding that at least one desirable biological property is present in the said group of novel compounds such as, but not limited to:
As a result of their one or more biological properties mentioned hereinabove, compounds represented by the structural formula (I) are highly active immunosuppressive agents, or antineoplastic agents, or anti-HCV agents which, together with one or more pharmaceutically acceptable carriers, may be formulated into pharmaceutical compositions for the prevention or treatment of pathologic conditions such as, but not limited to, immune and autoimmune disorders, organ and cells transplant rejections, cell proliferative disorders, cardiovascular disorders, disorders of the central nervous system and hepatitis C. Compounds represented by the structural formula (I) are also useful for the prevention or treatment of a TNF-α-related disorder in a mammal such as, but not limited to:
Compounds represented by the structural formula (I) are also useful for the prevention or treatment of a disorder mediated by phosphodiesterase-4 activity in a mammal such as, but not limited to, erectile dysfunction.
In a further embodiment, the present invention relates to combined preparations containing at least one compound represented by the structural formula (I) and one or more drugs such as, but not limited to, immunosuppressant and/or immunomodulator drugs, antineoplastic drugs, anti-histamines, inhibitors of agents causative of allergic conditions, phosphodiesterase-4 inhibitors, and antiviral agents. In a further embodiment, the present invention relates to the prevention or treatment of the above-cited pathologic conditions by administering to the patient in need thereof an effective amount of a compound represented by the structural formula (I), optionally in the form of a pharmaceutical composition or a combined preparation with another suitable drug.
In another embodiment, the present invention relates to various processes and methods for making the novel pyrido(3,2-d)pyrimidine derivatives defined in the structural formula (I) as well as their pharmaceutically acceptable salts, N-oxides, solvates and stereoisomers, e.g. via one or more groups of tri-substituted pyrido(3,2-d)pyrimidine intermediates such as specified herein before.
In yet another embodiment, the present invention relates to the use of monosubstituted, disubstituted and trisubstituted pyrido(3,2-d)pyrimidines, whatever their substitution pattern (i.e. with a substitution pattern broader than that of structural formula (I) hereinabove, including substitution patterns of pyrido(3,2-d)pyrimidines disclosed in the section “Background of the Invention”), as phosphodiesterase-4 inhibitors. In a specific embodiment, such use includes a method of treatment of a disease mediated by phosphodiesterase-4 activity in a patient, comprising the administration of an effective amount, preferably a phosphodiesterase-4 inhibiting amount, of a pyrido(3,2-d)pyrimidine derivative. Such a disease includes, but is not limited to, erectile dysfunction, e.g. vasculogenic impotence, in a male individual.
In another embodiment the present invention relates to pyrido(3,2-d)pyrimidine
derivatives represented by the structural formula (II) (II)
or the structural formula (III)
or the structural formula (IV)
wherein:
In yet another embodiment the present invention relates to pharmaceutical compositions comprising a pyrido(3,2-d)pyrimidine derivative represented by one of the structural formulae (II), (III) and (IV) as an active ingredient especially for the treatment of immune disorders or the prevention of a transplant rejection.
Unless otherwise stated herein, the term “tri-substituted” means that three of the carbon atoms being in positions 2, 4 and 6 or, alternatively, in positions 2, 4 and 7 of the pyrido(3,2-d)pyrimidine moiety (according to standard atom numbering for the pyrido(3,2-d)pyrimidine moiety) are substituted with an atom or group of atoms other than hydrogen. The term “tetra-substituted” means that all four carbon atoms being in positions 2, 4, 6 and 7 of the pyrido(3,2-d)pyrimidine moiety are substituted with an atom or group of atoms other than hydrogen.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “C1-7 alkyl” means straight and branched chain saturated acyclic hydrocarbon monovalent radicals having from 1 to 7 carbon atoms such as, for example, methyl, ethyl, propyl, n-butyl, 1-methylethyl(isopropyl), 2-methylpropyl(isobutyl), 1,1-dimethylethyl(ter-butyl), 2-methylbutyl, n-pentyl, dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, n-heptyl and the like. By analogy, the term “C1-12 alkyl” refers to such radicals having from 1 to 12 carbon atoms, i.e. up to and including dodecyl.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “acyl” broadly refers to a substituent derived from an acid such as an organic monocarboxylic acid, a carbonic acid, a carbamic acid (resulting into a carbamoyl substituent) or the thioacid or imidic acid (resulting into a carbamidoyl substituent) corresponding to said acids, and the term “sulfonyl” refers to a substituent derived from an organic sulfonic acid, wherein said acids comprise an aliphatic, aromatic or heterocyclic group in the molecule. A more specific kind of “acyl” group within the scope of the above definition refers to a carbonyl(oxo) group adjacent to a C1-7 alkyl, a C3-10 cycloalkyl, an aryl, an arylalkyl or a heterocyclic group, all of them being such as herein defined. Suitable examples of acyl groups are to be found below.
Acyl and sulfonyl groups originating from aliphatic or cycloaliphatic monocarboxylic acids are designated herein as aliphatic or cycloaliphatic acyl and sulfonyl groups and include, but are not limited to, the following:
Acyl and sulfonyl groups may also originate from aromatic monocarboxylic acids and include, but are not limited to, the following:
Acyl groups may also originate from an heterocyclic monocarboxylic acids and include, but are not limited to, the following:
As used herein with respect to a substituting radical, and unless otherwise stated, the term “thioacyl” refers to an acyl group as defined herein-above but wherein a sulfur atom replaces the oxygen atom of the carbonyl(oxo) moiety.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “C1-7 alkylene” means the divalent hydrocarbon radical corresponding to the above defined C1-7 alkyl, such as methylene, bis(methylene), tris(methylene), tetramethylene, hexamethylene and the like.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “C3-10 cycloalkyl” means a mono- or polycyclic saturated hydrocarbon monovalent radical having from 3 to 10 carbon atoms, such as for instance cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like, or a C7-10 polycyclic saturated hydrocarbon monovalent radical having from 7 to 10 carbon atoms such as, for instance, norbornyl, fenchyl, trimethyltricycloheptyl or adamantyl.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “C3-10 cycloalkyl-alkyl” refers to an aliphatic saturated hydrocarbon monovalent radical (preferably a C1-7 alkyl such as defined above) to which a C3-10 cycloalkyl (such as defined above) is already linked such as, but not limited to, cyclohexylmethyl, cyclopentylmethyl and the like.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “C3-10 cycloalkylene” means the divalent hydrocarbon radical corresponding to the above defined C3-10 cycloalkyl.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “aryl” designate any mono- or polycyclic aromatic monovalent hydrocarbon radical having from 6 up to 30 carbon atoms such as but not limited to phenyl, naphthyl, anthracenyl, phenantracyl, fluoranthenyl, chrysenyl, pyrenyl, biphenylyl, terphenyl, picenyl, indenyl, biphenyl, indacenyl, benzocyclobutenyl, benzocyclooctenyl and the like, including fused benzo-C4-8 cycloalkyl radicals (the latter being as defined above) such as, for instance, indanyl, tetrahydronaphtyl, fluorenyl and the like, all of the said radicals being optionally substituted with one or more substituents independently selected from the group consisting of halogen, amino, trifluoromethyl, hydroxyl, sulfhydryl and nitro, such as for instance 4-fluorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl, 4-cyanophenyl, 2,6-dichlorophenyl, 2-fluorophenyl, 3-chlorophenyl, 3,5-dichlorophenyl and the like.
As used herein, e.g. with respect to a substituting radical such as the combination of substituents in certain positions of the pyrido(3,2-d)pyrimidine ring together with the carbon atoms in the same positions of said ring, and unless otherwise stated, the term “homocyclic” means a mono- or polycyclic, saturated or mono-unsaturated or polyunsaturated hydrocarbon radical having from 4 up to 15 carbon atoms but including no heteroatom in the said ring; for instance said combination of substituents may form a C2-6 alkylene radical, such as tetramethylene, which cyclizes with the carbon atoms in certain positions of the pyrido(3,2-d)pyrimidine ring.
As used herein with respect to a substituting radical (including the combination of substituents in certain positions of the pyrido(3,2-d)pyrimidine ring together with the carbon atoms in the same positions of said ring), and unless otherwise stated, the term “heterocyclic” means a mono- or polycyclic, saturated or mono-unsaturated or polyunsaturated monovalent hydrocarbon radical having from 2 up to 15 carbon atoms and including one or more heteroatoms in one or more heterocyclic rings, each of said rings having from 3 to 10 atoms (and optionally further including one or more heteroatoms attached to one or more carbon atoms of said ring, for instance in the form of a carbonyl or thiocarbonyl or selenocarbonyl group, and/or to one or more heteroatoms of said ring, for instance in the form of a sulfone, sulfoxide, N-oxide, phosphate, phosphonate or selenium oxide group), each of said heteroatoms being independently selected from the group consisting of nitrogen, oxygen, sulfur, selenium and phosphorus, also including radicals wherein a heterocyclic ring is fused to one or more aromatic hydrocarbon rings for instance in the form of benzo-fused, dibenzo-fused and naphto-fused heterocyclic radicals; within this definition are included heterocyclic radicals such as, but not limited to, diazepinyl, oxadiazinyl, thiadiazinyl, dithiazinyl, triazolonyl, diazepinonyl, triazepinyl, triazepinonyl, tetrazepinonyl, benzoquinolinyl, benzothiazinyl, benzothiazinonyl, benzoxa-thiinyl, benzodioxinyl, benzodithiinyl, benzoxazepinyl, benzothiazepinyl, benzodiazepinyl, benzodioxepinyl, benzodithiepinyl, benzoxazocinyl, benzothiazocinyl, benzodiazocinyl, benzoxathiocinyl, benzodioxocinyl, benzotrioxepinyl, benzoxathiazepinyl, benzoxadiazepinyl, benzothia-diazepinyl, benzotriazepinyl, benzoxathiepinyl, benzotriazinonyl, benzoxazolinonyl, azetidinonyl, azaspiroundecyl, dithiaspirodecyl, selenazinyl, selenazolyl, selenophenyl, hypoxanthinyl, azahypo-xanthinyl, bipyrazinyl, bipyridinyl, oxazolidinyl, diselenopyrimidinyl, benzodioxocinyl, benzopyrenyl, benzopyranonyl, benzophenazinyl, benzoquinolizinyl, dibenzo-carbazolyl, dibenzoacridinyl, dibenzophenazinyl, dibenzothiepinyl, dibenzoxepinyl, dibenzopyranonyl, dibenzoquinoxalinyl, dibenzothiazepinyl, dibenzisoquinolinyl, tetraazaadamantyl, thiatetraazaadamantyl, oxauracil, oxazinyl, dibenzothiophenyl, dibenzofuranyl, oxazolinyl, oxazolonyl, azaindolyl, azolonyl, thiazolinyl, thiazolonyl, thiazolidinyl, thiazanyl, pyrimidonyl, thiopyrimidonyl, thiamorpholinyl, azlactonyl, naphtindazolyl, naphtindolyl, naphtothiazolyl, naphtothioxolyl, naphtoxindolyl, naphto-triazolyl, naphtopyranyl, oxabicycloheptyl, azabenzimidazolyl, azacycloheptyl, azacyclooctyl, azacyclononyl, azabicyclononyl, tetrahydrofuryl, tetrahydropyranyl, tetrahydro-pyronyl, tetrahydroquinolinyl, tetrahydrothienyl and dioxide thereof, dihydrothienyl dioxide, dioxindolyl, dioxinyl, dioxenyl, dioxazinyl, thioxanyl, thioxolyl, thiourazolyl, thiotriazolyl, thiopyranyl, thiopyronyl, coumarinyl, quinoleinyl, oxyquinoleinyl, quinuclidinyl, xanthinyl, dihydropyranyl, benzodihydrofuryl, benzothiopyronyl, benzothiopyranyl, benzoxazinyl, benzoxazolyl, benzodioxolyl, benzodioxanyl, benzothiadiazolyl, benzotriazinyl, benzothiazolyl, benzoxazolyl, phenothioxinyl, phenothiazolyl, phenothienyl(benzothiofuranyl), phenopyronyl, phenoxazolyl, pyridinyl, dihydropyridinyl, tetrahydropyridinyl, piperidinyl, morpholinyl, thiomorpholinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, tetrazinyl, triazolyl, benzotriazolyl, tetrazolyl, imidazolyl, pyrazolyl, thiazolyl, thiadiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, pyrrolyl, furyl, dihydrofuryl, furoyl, hydantoinyl, dioxolanyl, dioxolyl, dithianyl, dithienyl, dithiinyl, thienyl, indolyl, indazolyl, benzofuryl, quinolyl, quinazolinyl, quinoxalinyl, carbazolyl, phenoxazinyl, phenothiazinyl, xanthenyl, purinyl, benzothienyl, naphtothienyl, thianthrenyl, pyranyl, pyronyl, benzopyronyl, isobenzofuranyl, chromenyl, phenoxathiinyl, indolizinyl, quinolizinyl, isoquinolyl, phthalazinyl, naphthiridinyl, cinnolinyl, pteridinyl, carbolinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, imidazolinyl, imidazolidinyl, benzimidazolyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, piperazinyl, uridinyl, thymidinyl, cytidinyl, azirinyl, aziridinyl, diazirinyl, diaziridinyl, oxiranyl, oxaziridinyl, dioxiranyl, thiiranyl, azetyl, dihydroazetyl, azetidinyl, oxetyl, oxetanyl, oxetanonyl, homopiperazinyl, homopiperidinyl, thietyl, thietanyl, diazabicyclooctyl, diazetyl, diaziridinonyl, diaziridinethionyl, chromanyl, chromanonyl, thiochromanyl, thiochromanonyl, thiochromenyl, benzofuranyl, benzisothiazolyl, benzocarbazolyl, benzochromonyl, benzisoalloxazinyl, benzocoumarinyl, thiocoumarinyl, phenometoxazinyl, phenoparoxazinyl, phentriazinyl, thiodiazinyl, thiodiazolyl, indoxyl, thioindoxyl, benzodiazinyl (e.g. phtalazinyl), phtalidyl, phtalimidinyl, phtalazonyl, alloxazinyl, dibenzopyronyl (i.e. xanthonyl), xanthionyl, isatyl, isopyrazolyl, isopyrazolonyl, urazolyl, urazinyl, uretinyl, uretidinyl, succinyl, succinimido, benzylsultimyl, benzylsultamyl and the like, including all possible isomeric forms thereof, wherein each carbon atom of said heterocyclic ring may furthermore be independently substituted with a substituent selected from the group consisting of halogen, nitro, C1-7 alkyl (optionally containing one or more functions or radicals selected from the group consisting of carbonyl(oxo), alcohol(hydroxyl), ether(alkoxy), acetal, amino, imino, oximino, alkyloximino, amino-acid, cyano, carboxylic acid ester or amide, nitro, thio C1-7 alkyl, thio C3-10 cycloalkyl, C1-7 alkylamino, cycloalkylamino, alkenylamino, cycloalkenylamino, alkynylamino, arylamino, arylalkylamino, hydroxylalkylamino, mercaptoalkylamino, heterocyclic-substituted alkylamino, heterocyclic amino, heterocyclic-substituted arylamino, hydrazino, alkylhydrazino, phenylhydrazino, sulfonyl, sulfonamido and halogen), C3-7 alkenyl, C2-7 alkynyl, halo C1-7 alkyl, C3-10 cycloalkyl, aryl, arylalkyl, alkylaryl, alkylacyl, arylacyl, hydroxyl, amino, C1-7 alkylamino, cycloalkylamino, alkenylamino, cycloalkenylamino, alkynylamino, arylamino, arylalkylamino, hydroxyalkylamino, mercaptoalkylamino, heterocyclic-substituted alkylamino, heterocyclic amino, heterocyclic-substituted arylamino, hydrazino, alkylhydrazino, phenylhydrazino, sulfhydryl, C1-7 alkoxy, C3-10 cycloalkoxy, aryloxy, arylalkyloxy, oxyheterocyclic, heterocyclic-substituted alkyloxy, thio C1-7 alkyl, thio C3-10 cycloalkyl, thioaryl, thioheterocyclic, arylalkylthio, heterocyclic-substituted alkylthio, formyl, hydroxylamino, cyano, carboxylic acid or esters or thioesters or amides thereof, thiocarboxylic acid or esters or thioesters or amides thereof; depending upon the number of unsaturations in the 3 to 10 atoms ring, heterocyclic radicals may be sub-divided into heteroaromatic (or “heteroaryl”) radicals and non-aromatic heterocyclic radicals; when a heteroatom of said non-aromatic heterocyclic radical is nitrogen, the latter may be substituted with a substituent selected from the group consisting of C1-7 alkyl, C3-10 cycloalkyl, aryl, arylalkyl and alkylaryl.
As used herein with respect to a substituting radical, and unless otherwise stated, the terms “C1-7 alkoxy”, “C3-10 cycloalkoxy”, “aryloxy”, “arylalkyloxy”, “oxyheterocyclic”, “thio C1-7 alkyl”, “thio C3-10 cycloalkyl”, “arylthio”, “arylalkylthio” and “thioheterocyclic” refer to substituents wherein a carbon atom of a C1-7 alkyl, respectively a C3-10 cycloalkyl, aryl, arylalkyl or heterocyclic radical (each of them such as defined herein), is attached to an oxygen atom or a divalent sulfur atom through a single bond such as, but not limited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, thiomethyl, thioethyl, thiopropyl, thiobutyl, thiopentyl, thiocyclopropyl, thiocyclobutyl, thiocyclopentyl, thiophenyl, phenyloxy, benzyloxy, mercaptobenzyl, cresoxy, and the like.
As used herein with respect to a substituting atom, and unless otherwise stated, the term halogen means any atom selected from the group consisting of fluorine, chlorine, bromine and iodine.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “halo C1-7 alkyl” means a C1-7 alkyl radical (such as above defined) in which one or more hydrogen atoms are independently replaced by one or more halogens (preferably fluorine, chlorine or bromine), such as but not limited to difluoromethyl, trifluoromethyl, trifluoroethyl, octafluoropentyl, dodecafluoroheptyl, dichloromethyl and the like.
As used herein with respect to a substituting radical, and unless otherwise stated, the terms “C2-7 alkenyl” designate a straight and branched acyclic hydrocarbon monovalent radical having one or more ethylenic unsaturations and having from 2 to 7 carbon atoms such as, for example, vinyl, 1-propenyl, 2-propenyl(allyl), 1-butenyl, 2-butenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl, 3-hexenyl, 2-hexenyl, 2-heptenyl, 1,3-butadienyl, pentadienyl, hexadienyl, heptadienyl, heptatrienyl and the like, including all possible isomers thereof.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “C3-10 cycloalkenyl” means a monocyclic mono- or polyunsaturated hydrocarbon monovalent radical having from 3 to 8 carbon atoms, such as for instance cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, cyclohepta-dienyl, cycloheptatrienyl, cyclooctenyl, cyclooctadienyl and the like, or a C7-10 polycyclic mono- or polyunsaturated hydrocarbon mono-valent radical having from 7 to 10 carbon atoms such as dicyclopentadienyl, fenchenyl (including all isomers thereof, such as α-pinolenyl), bicyclo[2.2.1]hept-2-enyl, bicyclo[2.2.1]hepta-2,5-dienyl, cyclo-fenchenyl and the like.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “C2-7 alkynyl” defines straight and branched chain hydrocarbon radicals containing one or more triple bonds and optionally at least one double bond and having from 2 to 7 carbon atoms such as, for example, acetylenyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 2-pentynyl, 1-pentynyl, 3-methyl-2-butynyl, 3-hexynyl, 2-hexynyl, 1-penten-4-ynyl, 3-penten-1-ynyl, 1,3-hexadien-1-ynyl and the like.
As used herein with respect to a substituting radical, and unless otherwise stated, the terms “arylalkyl”, “arylalkenyl” and “heterocyclic-substituted alkyl” refer to an aliphatic saturated or ethylenically unsaturated hydrocarbon monovalent radical (preferably a C1-7 alkyl or C2-7 alkenyl radical such as defined above) onto which an aryl or heterocyclic radical (such as defined above) is already bonded via a carbon atom, and wherein the said aliphatic radical and/or the said aryl or heterocyclic radical may be optionally substituted with one or more substituents independently selected from the group consisting of halogen, amino, hydroxyl, sulfhydryl, C1-7 alkyl, C1-7 alkoxy, trifluoromethyl and nitro, such as but not limited to benzyl, 4-chlorobenzyl, 4-fluorobenzyl, 2-fluorobenzyl, 3,4-dichlorobenzyl, 2,6-dichlorobenzyl, 3-methylbenzyl, 4-methylbenzyl, 4-ter-butylbenzyl, phenylpropyl, 1-naphthylmethyl, phenylethyl, 1-amino-2-phenylethyl, 1-amino-2-[4-hydroxyphenyl]ethyl, 1-amino-2-[indol-2-yl]ethyl, styryl, pyridylmethyl (including all isomers thereof, pyridylethyl, 2-(2-pyridyl)isopropyl, oxazolylbutyl, 2-thienylmethyl, pyrrolylethyl, morpholinylethyl, imidazol-1-yl-ethyl, benzodioxolylmethyl and 2-furylmethyl.
As used herein with respect to a substituting radical, and unless otherwise stated, the terms “alkylaryl” and “alkyl-substituted heterocyclic” refer to an aryl or, respectively, heterocyclic radical (such as defined above) onto which are bonded one or more aliphatic saturated or unsaturated hydrocarbon monovalent radicals, preferably one or more C1-7 alkyl, C2-7 alkenyl or C3-10 cycloalkyl radicals as defined above such as, but not limited to, o-toluoyl, m-toluoyl, p-toluoyl, 2,3-xylyl, 2,4-xylyl, 3,4-xylyl, o-cumenyl, m-cumenyl, p-cumenyl, o-cymenyl, m-cymenyl, p-cymenyl, mesityl, ter-butylphenyl, lutidinyl (i.e. dimethylpyridyl), 2-methylaziridinyl, methylbenzimidazolyl, methylbenzofuranyl, methylbenzothiazolyl, methylbenzotriazolyl, methylbenzoxazolyl and methylbenzselenazolyl.
As used herein with respect to a substituting radical, and unless otherwise stated, the term “alkoxyaryl” refers to an aryl radical (such as defined above) onto which is (are) bonded one or more C1-7 alkoxy radicals as defined above, preferably one or more methoxy radicals, such as, but not limited to, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 3,4-dimethoxyphenyl, 2,4,6-trimethoxyphenyl, methoxynaphtyl and the like.
As used herein with respect to a substituting radical, and unless otherwise stated, the terms “alkylamino”, “cycloalkylamino”, “alkenylamino”, “cycloalkenylamino”, “arylamino”, “arylalkylamino”, “heterocyclic-substituted alkylamino”, “heterocyclic-substituted arylamino”, “heterocyclic amino”, “hydroxyalkylamino”, “mercaptoalkylamino” and “alkynylamino” mean that respectively one (thus monosubstituted amino) or even two (thus disubstituted amino) C1-7 alkyl, C3-10 cycloalkyl, C2-7 alkenyl, C3-10 cycloalkenyl, aryl, arylalkyl, heterocyclic-substituted alkyl, heterocyclic-substituted aryl, heterocyclic (provided in this case the nitrogen atom is attached to a carbon atom of the heterocyclic ring), mono- or polyhydroxy C1-7 alkyl, mono- or polymercapto C1-7 alkyl, or C2-7 alkynyl radical(s) (each of them as defined herein, respectively, and including the presence of optional substituents independently selected from the group consisting of halogen, amino, hydroxyl, sulfhydryl, C1-7 alkyl, C1-7 alkoxy, trifluoromethyl and nitro) is/are attached to a nitrogen atom through a single bond such as, but not limited to, anilino, 2-bromoanilino, 4-bromoanilino, 2-chloroanilino, 3-chloroanilino, 4-chloroanilino, 3-chloro-4-methoxyanilino, 5-chloro-2-methoxyanilino, 2,3-dimethylanilino, 2,4-dimethylanilino, 2,5-dimethylanilino, 2,6-dimethylanilino, 3,4-dimethylanilino, 2-fluoroanilino, 3-fluoroanilino, 4-fluoroanilino, 3-fluoro-2-methoxyanilino, 3-fluoro-4-methoxyanilino, 2-fluoro-4-methylanilino, 2-fluoro-5-methylanilino, 3-fluoro-2-methylanilino, 3-fluoro-4-methylanilino, 4-fluoro-2-methylanilino, 5-fluoro-2-methylanilino, 2-iodoanilino, 3-iodoanilino, 4-iodoanilino, 2-methoxy-5-methylanilino, 4-methoxy-2-methylanilino, 5-methoxy-2-methylanilino, 2-ethoxyanilino, 3-ethoxyanilino, 4-ethoxyanilino, benzylamino, 2-methoxybenzylamino, 3-methoxybenzylamino, 4-methoxybenzylamino, 2-fluorobenzylamino, 3-fluorobenzylamino, 4-fluorobenzylamino, 2-chlorobenzylamino, 3-chlorobenzylamino, 4-chlorobenzylamino, 2-aminobenzylamino, diphenylmethylamino, α-naphthylamino, methylamino, dimethylamino, ethylamino, diethylamino, isopropylamino, propenylamino, n-butylamino, ter-butylamino, dibutylamino, 1,2-diaminopropyl, 1,3-diaminopropyl, 1,4-diaminobutyl, 1,5-diaminopentyl, 1,6-diaminohexyl, morpholinomethylamino, 4-morpholinoanilino, hydroxymethylamino, p-hydroxyethylamino and ethynylamino; this definition also includes mixed disubstituted amino radicals wherein the nitrogen atom is attached to two such radicals belonging to two different sub-sets of radicals, e.g. an alkyl radical and an alkenyl radical, or to two different radicals within the same sub-set of radicals, e.g. methylethylamino; among di-substituted amino radicals, symmetrically-substituted amino radicals are more easily accessible and thus usually preferred from a standpoint of ease of preparation.
As used herein with respect to a substituting radical, and unless otherwise stated, the terms “(thio)carboxylic acid ester”, “(thio)carboxylic acid thioester” and “(thio)carboxylic acid amide” refer to radicals wherein the carboxyl or thiocarboxyl group is bonded to the hydrocarbonyl residue of an alcohol, a thiol, a polyol, a phenol, a thiophenol, a primary or secondary amine, a polyamine, an amino-alcohol or ammonia, the said hydrocarbonyl residue being selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, arylalkyl, alkylaryl, alkylamino, cycloalkylamino, alkenylamino, cycloalkenylamino, arylamino, arylalkylamino, heterocyclic-substituted alkylamino, heterocyclic amino, heterocyclic-substituted arylamino, hydroxyalkylamino, mercapto-alkylamino or alkynylamino (such as above defined, respectively).
As used herein with respect to a substituting radical, and unless otherwise stated, the term “amino-acid” refers to a radical derived from a molecule having the chemical formula H2N—CHR—COOH, wherein R is the side group of atoms characterising the amino-acid type; said molecule may be one of the 20 naturally-occurring amino-acids or any similar non naturally-occurring amino-acid.
As used herein and unless otherwise stated, the term “stereoisomer” refers to all possible different isomeric as well as conformational forms which the compounds of formula (I) may possess, in particular all possible stereochemically and conformationally isomeric forms, all diastereomers, enantiomers and/or conformers of the basic molecular structure. Some compounds of the present invention may exist in different tautomeric forms, all of the latter being included within the scope of the present invention.
As used herein and unless otherwise stated, the term “enantiomer” means each individual optically active form of a compound of the invention, having an optical purity or enantiomeric excess (as determined by methods standard in the art) of at least 80% (i.e. at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.
As used herein and unless otherwise stated, the term “solvate” includes any combination which may be formed by a pyrido(3,2-d)pyrimidine derivative of this invention with a suitable inorganic solvent (e.g. hydrates) or organic solvent, such as but not limited to alcohols, ketones, esters, ethers, nitriles and the like.
In the first embodiment of the invention, the novel pyrido(3,2-d)pyrimidine derivatives are as defined in the general formula (I), wherein each of the substituents R1, R2, R3 and/or R4 may independently correspond to any of the definitions given above, in particular with any of the individual meanings (such as illustrated above) of generic terms used for substituting radicals such as, but not limited to, “C1-7 alkyl”, “C3-10 cycloalkyl”, “C2-7 alkenyl”, “C2-7 alkynyl”, “aryl”, “homocyclic”, “heterocyclic”, “halogen”, “C3-10 cycloalkenyl”, “alkylaryl”, “arylalkyl”, “alkylamino”, “cycloalkyl-amino”, “alkenylamino”, “alkynylamino”, “arylamino”, “arylalkylamino”, “heterocyclic-substituted alkylamino”, “heterocyclic amino”, “heterocyclic-substituted arylamino”, “hydroxyalkylamino”, “mercaptoalkylamino”, “alkynylamino”, “C1-7 alkoxy”, “C3-10 cycloalkoxy”, “thio C1-7 alkyl”, “thio C3-10 cycloalkyl”, “halo C1-7 alkyl” and the like.
In the second embodiment of the invention, the novel pyrido(3,2-d)pyrimidine intermediates are as specified herein before, wherein each of the substituents R1, R2, R3 and/or R4 may independently correspond to any of the definitions given with respect to the general formula (I), in particular with any of the individual meanings (such as illustrated above) of generic terms used for substituting radicals such as, but not limited to, “C1-7 alkyl”, “C3-10 cycloalkyl”, “C2-7 alkenyl”, “C2-7 alkynyl”, “aryl”, “homocyclic”, “heterocyclic”, “halogen”, “C3-10 cycloalkenyl”, “alkylaryl”, “aryl-alkyl”, “alkylamino”, “cycloalkylamino”, “alkenylamino”, “alkynylamino”, “aryl-amino”, “arylalkylamino”, “heterocyclic-substituted alkylamino”, “heterocyclic amino”, “heterocyclic-substituted arylamino”, “hydroxyalkylamino”, “mercaptoalkylamino”, “alkynylamino”, “C1-7 alkoxy”, “C3-10 cycloalkoxy”, “thio C1-7 alkyl”, “thio C3-10 cycloalkyl”, “halo C1-7 alkyl” and the like.
In another embodiment of the present invention, the novel pyrido(3,2-d)pyrimidine derivatives are as defined in one of the structural formulae (II), (III) and (IV) wherein each of the substituents R1, R2, R2′, R3, R3′ and/or R5 may independently correspond to any of the definitions given above, in particular with any of the above illustrated individual meanings of generic terms used for substituting radicals such as but not limited to “C1-7 alkyl”, “C3-10 cycloalkyl”, “C2-7 alkenyl”, “C2-7 alkynyl”, “acyl”, “thioacyl”, “aryl”, “heterocyclic”, “halogen”, “alkylaryl”, “arylalkyl”, “alkylamino”, “cycloalkylamino”, “arylamino”, “aryl C1-4 alkylamino”, “C1-4 alkylarylamino”, “hydroxy C1-7 alkylamino”, “thioalkylamino”, “C1-7 alkoxy”, “C3-10 cycloalkoxy”, “aryloxy”, “thio C1-7 alkyl”, “thio C3-10 cycloalkyl”, “thioaryl”, “halo C1-7 alkyl” and the like.
Within the class of compounds represented by the structural formula (I), a preferred group is one wherein R2 is a piperazinyl group optionally N-substituted with a substituent R5 such as defined herein above. Said piperazinyl group may be further substituted, at one or more carbon atoms, by a number n of substituents R0 wherein n is an integer from 0 to 6 and wherein, when n is at least 2, each R0 may be defined independently from the others. The presence of one or more such substituents R0 at one or more carbon atoms is a suitable way for introducing chirality into the pyrido(2,3-d)pyrimidine derivatives represented by the structural formula (I) as well as into the corresponding intermediates. In practice, the choice of such substituents R0 may be restricted by the commercial availability of the substituted piperazine. More preferably R2 is a piperazin-1-yl group, n is 0, 1 or 2, and a representative example of the substituent R0 is methyl or phenyl such as for instance in 2-methylpiperazin-1-yl, 2-phenylpiperazin-1-yl and 2,5-dimethyl-piperazin-1-yl. Within the preferred group of compounds, a more specific embodiment of the invention is one wherein one of the two nitrogen atoms of the piperazinyl group bears a substituent R5 which has a carbonyl(oxo) or thiocarbonyl(thioxo) or sulfonyl function preferably immediately adjacent to the said nitrogen atom. In other words, this specific embodiment means that when R5 is selected from, respectively, acyl, thioacyl, amide, thioamide, sulfonyl, sulfinyl, carboxylate and thiocarboxylate, then R5 together with the nitrogen atom to which it is attached forms, respectively, an amide, thioamide, urea, thiourea, sulfonamido, sulfinamido, carbamato or thiocarbamato group.
Especially useful species of pyrido(3,2-d)pyrimidine derivatives represented by the structural formula (I) are those wherein the substituent R2 is a piperazin-1-yl group, said group being substituted in the 4 position with a substituent R5, wherein R5 is selected from the group consisting of:
Especially useful species of pyrido(3,2-d)pyrimidine derivatives represented by the structural formula (I) are those wherein the substituent R1 is a group represented by the structural formula R6—NR7R12, wherein R6 is a bond or C1-3 alkylene, wherein R7 and R12 are independently selected from the group consisting of hydrogen, C1-7 alkyl, C2-7 alkenyl, C2-7 alkynyl, aryl, arylalkyl, C3-10 cycloalkyl and heteroaryl, or wherein N, R7 and R12 together form a heterocycle. Within this sub-class of derivatives, it is preferred when R6 is a bond or methylene, and/or R7 is methyl, ethyl, propyl or cyclopropylmethyl, and/or N, R7 and R12 together form morpholinyl, 2,6-dimethylmorpholinyl, pyrrolidinyl, azepanyl, 3,3,5-trimethylazepanyl, piperidinyl, 2-methylpiperidinyl or 2-ethylpiperidinyl. Methods for introducing such substituents in position 2 of the pyrido(3,2-d)pyrimidine ring are extensively described in WO 03/062209.
The present invention further provides various processes and methods for making the novel pyrido(3,2-d)pyrimidine derivatives represented by the structural formula (I). As a general rule, the preparation of these compounds is based on the principle that, starting from a suitable pyrido(3,2-d)pyrimidine precursor (usually a 2,3,6-trisubstituted pyridine), each of the substituents R2, R3, R4 and R1 may be introduced separately without adversely influencing the presence of one or more substituents already introduced at other positions on the pyrido(3,2-d)pyrimidine moiety or the capacity to introduce further substituents later on.
Methods of manufacture have been developed by the present inventors which may be used alternatively to, or may be combined with, the methods of synthesis already known in the art of pyrido(3,2-d)pyrimidine derivatives (depending upon the targeted final compound). For instance, the synthesis of mono- and di-N-oxides of the pyrido(3,2-d)pyrimidine derivatives of this invention can easily be achieved by treating the said derivatives with an oxidizing agent such as, but not limited to, hydrogen peroxide (e.g. in the presence of acetic acid) or a peracid such as chloroperbenzoic acid. The methods for making the pyrido(3,2-d)pyrimidine derivatives of the present invention will now be explained in more details by reference to the appended FIGS. 1 to 8 wherein, unless otherwise stated hereinafter, each of the substituting groups or atoms R2, R3, R4 and R1 is as defined in the structural formula (I) of the summary of the invention and, more specifically, may correspond to any of the individual meanings disclosed above.
In the description of the reaction steps involved in each figure, reference is made to the use of certain catalysts and/or certain types of solvents. It should be understood that each catalyst mentioned should be used in a catalytic amount well known to the skilled person with respect to the type of reaction involved. Solvents that may be used in the following reaction steps include various kinds of organic solvents such as protic solvents, polar aprotic solvents and non-polar solvents as well as aqueous solvents which are inert under the relevant reaction conditions. More specific examples include aromatic hydrocarbons, chlorinated hydrocarbons, ethers, aliphatic hydrocarbons, alcohols, esters, ketones, amides, water or mixtures thereof, as well as supercritical solvents such as carbon dioxide (while performing the reaction under supercritical conditions). The suitable reaction temperature and pressure conditions applicable to each kind of reaction step will not be detailed herein but do not depart from the relevant conditions already known to the skilled person with respect to the type of reaction involved and the type of solvent used (in particular its boiling point).
The methods described in relation to FIGS. 1 to 8 make use of an arylboronic or heteroarylboronic acid, or e.g. a pinacol ester thereof, for introducing a substituent onto the core structure. In these methods, suitable aryl-boronic acids include, but are not limited to, the following commercially available materials wherein the aryl group is 3-acetamidophenyl, 4-acetamidophenyl, 4-acetylphenyl, 3-acetylphenyl, 2-acetylphenyl, 5-acetyl-2-chlorophenyl, 4-acetyl-3-fluorophenyl, 5-acetyl-2-fluorophenyl, 3-aminophenyl, 4-aminomethylphenyl, 3-aminophenyl, 4-benzyloxybenzene, 3-benzyloxybenzene, 4-benzyloxy-2-fluorophenyl, 4-benzyloxy-3-fluorophenyl, biphenyl-3-, 3,5-bis(trifluoromethyl)benzene, 4-bromophenyl, 3-bromophenyl, 4-bromo-2,5-dimethylphenyl, 2-bromo-5-fluorophenyl, 2-Bromo-6-fluorophenyl, 4-carboxyphenyl, 2-carboxyphenyl, 2-carboxy-5-fluorophenyl, 4-carboxy-2-chlorophenyl, 5-carboxy-2-chlorophenyl, 4-carboxy-3-chlorophenyl, 3-carboxyphenyl, 2-chloro-5-formylphenyl, 2-chloro-5-hydroxyphenyl, 3-chloro-4-fluorophenyl, 2-chloro-4-fluorophenyl, 4-chloro-2-fluorophenyl, 3-chloro-5-methoxyphenyl, 2-chloro-4-methylphenyl, 2-chloro-5-methylphenyl, 2-chloro-5-trifluoromethoxyphenyl, 3-chloro-5-trifluoromethylphenyl, 4-chloro-2-trifluoromethylphenyl, 4-chlorophenyl, 3-chlorophenyl, 2-chlorophenyl, 4-cyanophenyl, 3-cyanophenyl, 2-cyanophenyl, 3,5-dibromophenyl, 2,6-dichlorophenyl, 3,4-dichlorophenyl, 2,4-dichlorophenyl, 2,3-dichlorophenyl, 3,5-dichlorophenyl, 3,5-difluorophenyl, 3,5-difluoro-2-methoxyphenyl, 3,4-difluorophenyl, 2,6-difluorophenyl, 2,5-difluorophenyl, 2,4-difluorophenyl, 2,3-difluorophenyl, 2,3-dihydro-1,4-benzodioin-6-yl, 2,4-dimethoxybenzene, 4-(N,N-dimethylamino)phenyl, 2-(N,N-dimethylaminomethyl)phenyl, 3,5-dimethylphenyl, 3,4-dimethylphenyl, 2,6-dimethylphenyl, 2,6-dimethoxyphenyl, 2,5-dimethoxyphenyl, 2,4-dimethoxyphenyl, 4-ethoxyphenyl, 2-ethoxyphenyl, 4-ethoxycarbonylphenyl, 3-ethoxycarbonylphenyl, 4-ethylphenyl, 4-fluorophenyl, 3-fluorophenyl, 2-fluorophenyl, 3-fluoro-4-formylphenyl, 4-fluoro-2-methylphenyl, 2-fluoro-5-methylphenyl, 4-fluoro-3-formylphenyl, 2-fluoro-5-methoxyphenyl, 5-fluoro-2-methoxycarbonylphenyl, 2-formyl-5-methoxyphenyl, 5-formyl-2-methoxyphenyl, 2-formyl-5-methylphenyl, 4-formylphenyl, 3-formylphenyl, 2-formylphenyl, 3-hydroxy-4-methoxycarbonylphenyl, 4-(hydroxymethyl)phenyl, 3-(hydroxymethyl)phenyl, 4-hydroxyphenyl, 3-hydroxyphenyl, 4-iodophenyl, 3-iodophenyl, 3-isopropoxycarbonylphenyl, 4-isopropoxycarbonylphenyl, 4-methanesulfonylphenyl, 2-methoxy-5-formylphenyl, 5-methoxy-2-formylphenyl, 4-methoxy-2-formylphenyl, 4-methoxycarbonylphenyl, 3-methoxycarbonylphenyl, 4-methoxyphenyl, 3-methoxyphenyl, 2-methoxyphenyl, 3,4-methylenedioxyphenyl, 4-methylphenyl, 2-methylphenyl, 4-(methylthio)phenyl, 3-(methylthio)phenyl, 4-morpholinophenyl, 3-nitrophenyl, 4-phenoxyphenyl, 4-(tert-butoxycarbonylamino)-3-methoxyphenyl, 2-(tert-butoxycarbonyl)phenyl, 3-(tert-butoxycarbonyl)phenyl, 4-(tert-butoxycarbonyl)phenyl, 4-tert-butylphenyl, 4-(tetrahdro-2H-pyran-2-yloxy)phenyl, 4-(2-thienyl)phenyl, trans-β-styrene, 4-tolyl, 3-tolyl, 2-tolyl, 4-trifluoromethoxyphenyl, 4-(trimethylammonium)methylphenyl, 2,4,6-trimethylphenyl, 3,4,5-trifluorophenyl, 3-trifluoromethylphenyl, 4-trifluoromethoxyphenyl, 3-trifluoromethoxyphenyl, 3-trifluoromethylphenyl, 2-trifluoromethylphenyl, 3,4,5-trimethoxyphenyl, 4-vinylphenyl, 6-benzyloxy-2-naphthyl, 1-naphthalene, 2-naphthalene, or 1-biphenylenyl.
In these methods, suitable heterocyclic-boronic acids include, but are not limited to, the following commercially available materials wherein the heterocyclic group is 2-acetamidopyridin-5-yl, 2-benzothienyl, 1-benzothiophen-3-yl, 1-benzothiophen-2-yl, 2-bromo-3-chloropyridin-4-yl, 5-bromo-2,3-dihydrobenzo[b]furan-7-yl, 2-bromo-3-methylpyridin-5-yl, 2-bromopyridin-5-yl, 5-bromothien-2-yl, 2-chloro-6-isopropylpyridin-3-yl, 2-chloro-3-methylpyridin-5-yl, 5-chlorothien-2-yl, dibenzo[b,d]furan-4-yl, 2-chloro-3-fluoropyridin-4-yl, dibenzo[b,d]thien-4-yl, 3,4-dihydro-2H-1,5-benzodioxepin-7-yl, 2,5-dibromo-3-pyridinyl, 2,6-dichloro-pyridin-3-yl, 2,3-dihydro-1-benzofuran-5-yl, 2,4-dimethoxypyrimidin-5-yl, 3,5-dimethylisoxazol-4-yl, 1-[1,3]dioxolan-2-ylmethyl-4-1H-pyrazolyl, 2,4-dioxo-1,2,34-tetrahydro-5-pyrimidinyl, 2,4-di(tert-butoxy)pyrimidin-5-yl, 2-ethoxypyridin-3-yl, 2-fluoro-3-methylpyridin-5-yl, 2-fluoropyridin-3-yl, 2-fluoropyridin-5-yl, 5-formyl-2-furyl, 5-formylthiophen-2-yl, furan-3-yl, furan-2-yl, 5-indolyl, isoquinolin-4-yl, 2-methoxypyrimidin-5-yl, 5-methyl-1-benzothiophen-2-yl, 5-methylfuran-2-yl, 5-methyl-3-phenyl-4-isoxazolyl, 5-(methylsulfanyl)-2-thienyl, 3-methyl-pyridin-2-yl, (5-methyl)thien-2-yl, 5-methylpyridin-2-yl, 5-methylpyridin-3-yl, 2-methoxypyridine-3-yl, (4-methyl)thien-2-yl, 2-methoxypyridin-5-yl, 1-(phenylsulfonyl)-1H-indol-3-yl, 1-(phenylsulfonyl)-1H-indol-3-yl, 5-phenyl-2-thienyl, pyridin-4-yl, pyridin-3-yl, 5-pyrimidinyl, 4-phenoxathiinyl, 8-quinolinyl, 3-quinolinyl, 1-tert-butoxycarbonyl-2-pyrrolyl, 1-(tert-butoxycarbonyl)-5-bromo-1H-indol-2-yl, 1-(tert-butoxycarbonyl)-1H-indol-2-yl, 1-(tert-butoxycarbonyl)-5-methoxy-1H-indol-2-yl, 1-thianthrenyl-3-thienyl, or 2-thienyl.
Also, as shown in certain examples below, when position 4 of the core structure is substituted with a heteroaryl group (e.g. piperazinyl or pyrrolidinyl) itself substituted with a carbamoyl or thiocarbamoyl group, a relevant method of synthesis includes a reaction step with an isocyanate or an isothiocyanate. Aryl isocyanates suitable for use in such a synthesis include, but are not limited to, 4-fluorophenyl isocyanate, phenyl isocyanate, m-tolyl isocyanate, p-tolyl isocyanate, 4-chlorophenyl isocyanate, ethyl 4-isocyanatobenzoate, 2-fluoro-phenyl isocyanate, 3-fluorophenyl isocyanate, α,α,α-trifluoro-o-tolyl isocyanate, tolylene-2,4-diisocyanate, tolylene 2,6-diisocyanate, 4-methoxyphenyl isocyanate, 4-bromophenyl isocyanate, 2-methoxy-phenyl isocyanate, 3-Methoxyphenyl isocyanate, 2,4-dichlorophenyl isocyanate, o-tolyl isocyanate, 3,4-dichlorophenyl isocyanate, 2-nitrophenyl isocyanate, 4-nitrophenyl isocyanate, 2,4-difluorophenyl isocyanate, 2-bromophenyl isocyanate, 2,6-difluoro-phenyl isocyanate, 2-(trifluoromethoxy)phenyl isocyanate, 2-chloro-5-(trifluoro-methyl)phenyl isocyanate, 4-chloro-2-(trifluoro-methyl)phenyl isocyanate, 4-chloro-3-(trifluoromethyl)phenyl isocyanate, 2,5-difluoro-phenyl isocyanate, 4-(trifluoro-methoxy)phenyl isocyanate, 2-ethoxyphenyl isocyanate, 4-ethoxyphenyl isocyanate, 4-isopropylphenyl isocyanate, 3-acetylphenyl isocyanate, 2,6-diisopropylphenyl isocyanate, 3-bromophenyl isocyanate, 3,5-dichlorophenyl isocyanate, 4-fluoro-3-nitrophenyl isocyanate, 3,5-dimethylphenyl isocyanate, 3,5-bis(trifluoromethyl)phenyl isocyanate, 3-cyanophenyl isocyanate, 4-(methylthio)phenyl isocyanate, 2-ethylphenyl isocyanate, 2,6-dimethyl-phenyl isocyanate, α,α,α-trifluoro-p-tolyl isocyanate, 2,3-dichlorophenyl isocyanate, 4-methyl-3-nitrophenyl isocyanate, 2,4-dimethoxyphenyl isocyanate, 4-(chloro-methyl)phenyl isocyanate, 4-bromo-2-chlorophenyl isocyanate, 2-bromo-4,6-difluoro-phenyl isocyanate, 4-bromo-2-fluoro-phenyl isocyanate, 4-(dimethylamino)phenyl isocyanate, 2-fluoro-5-methylphenyl isocyanate, 4-fluoro-2-nitrophenyl isocyanate, 2-fluoro-3-(trifluoromethyl)phenyl isocyanate, 2-fluoro-5-(trifluoromethyl)phenyl isocyanate, 2-fluoro-6-(trifluoromethyl)-phenyl isocyanate, 4-fluoro-2-(trifluoromethyl)phenyl isocyanate, 4-fluoro-3-(trifluoromethyl)phenyl isocyanate, 4-(heptyloxy)phenyl isocyanate, 2-iodophenyl isocyanate, 2-naphthyl isocyanate, 2-n-propylphenyl isocyanate, 4-(trifluoromethyl-thio)phenyl isocyanate, 2,3,4-trifluorophenyl isocyanate, 2,6-dichlorophenyl isocyanate, 3-nitrophenyl isocyanate, 3-chlorophenyl isocyanate, 2-chlorophenyl isocyanate, 1-naphthyl isocyanate, 2,3-dimethylphenyl isocyanate, 3-chloro-4-fluorophenyl isocyanate, 2,5-dimethylphenyl isocyanate, 3,4-difluorophenyl isocyanate, 2,3-dihydro-1-benzofuran-5-yl isocyanate, 2,3-dihydro-1,4-benzodioxin-6-yl isocyanate, 6-fluoro-4H-1,3-benzodioxin-8-yl isocyanate, 2,1,3-benzothiadiazol-4-yl isocyanate, 3,4-dihydro-2H-1,5-benzodioxepin-7-yl isocyanate, 3-(cyclopentyloxy)-4-methoxyphenyl isocyanate, 2-(methylthio)phenyl isocyanate, 2-(tert-butyl)phenyl isocyanate, 4-(tert-butyl)phenyl isocyanate, 3-chloro-2-methylphenyl isocyanate, 4-butyl-2-methylphenyl isocyanate, 2-ethyl-6-methylphenyl isocyanate, 4-chloro-3-nitrophenyl isocyanate, 4-bromo-2-methylphenyl isocyanate, 3-(methylthio)phenyl isocyanate, 5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl isocyanate, 5-fluoro-2-methylphenyl isocyanate, 4-phenoxyphenyl isocyanate, 4-methoxy-2-methyl-phenyl isocyanate, α,α,α-trifluoro-m-tolyl isocyanate, 2,6-dibromo-4-isopropylphenyl isocyanate, 2,6-dimethoxyphenyl isocyanate, 2-(4-isocyanatophenyl)thiophene, 4-(3-isocyanatophenyl)-2-methyl-1,3-thiazole, 3-(3-isocyanatophenyl)-5-methyl-1,2,4-oxadiazole, 1-benzothiophen-5-yl isocyanate, 1-(3-isocyanatophenyl)-1H-pyrrole, 1-(4-isocyanatophenyl)-1H-pyrrole, 3,5-dimethoxyphenyl isocyanate and 2,4,6-trichlorophenyl isocyanate.
Aryl isothiocyanates suitable for use in such a synthesis include, but are not limited to, phenyl isothiocyanate, 4-fluorophenyl isothiocyanate, methyl 2-isocyanatobenzoate, 2-chlorophenyl isothiocyanate, 3-chlorophenyl isothiocyanate, o-tolyl isothiocyanate, m-tolyl isothiocyanate, p-tolyl isothiocyanate, 2-methoxyphenyl isothiocyanate, 2-bromophenyl isothiocyanate, 3-bromophenyl isothiocyanate, 2,4-dichloro-phenyl isothiocyanate, 2-fluoro phenylisothiocyanate, 4-methoxyphenyl isothiocyanate, α,α,α-trifluoro-m-tolyl isothiocyanate, 3-fluorophenyl isothiocyanate, 3,5-bis(trifluoromethyl)phenyl isothiocyanate, 1-naphthyl isothiocyanate, 4-dimethylamino-1-naphthyl isothiocyanate, 4-(methylthio)phenyl isothiocyanate, 2-methoxy-5-methylphenyl isothiocyanate, 4-cyanophenyl isothiocyanate, 3-chloro-4-fluorophenyl isothiocyanate, 4-(trifluoromethoxy)phenyl isothiocyanate, 3,5-dimethylphenyl isothiocyanate, 3,5-dimethoxyphenyl isothiocyanate, 4-chlorophenyl isothiocyanate, 3,4-dimethoxyphenyl isothiocyanate, 2,6-dimethylphenyl isothiocyanate, 3-methoxyphenyl isothiocyanate, mesityl isothiocyanate, 4-(benzyloxy)phenyl isothiocyanate, 2,4-dimethylphenyl isothiocyanate, 2-bromo-5-fluorophenyl isothiocyanate, 5-fluoro-2-methylphenyl isothiocyanate, 4-chloro-2,5-dimethoxyphenyl isothiocyanate, 2,5-dichlorophenyl isothiocyanate, 2-(tert-butyl)-4,5,6-trimethyl-3-nitrophenyl isothiocyanate, 2-isopropyl-6-methylphenyl isothiocyanate, 4-ethoxyphenyl isothiocyanate, 5-chloro-2-methylphenyl isothiocyanate, 2-ethyl-6-methylphenyl isothiocyanate and 4-(trifluoromethyl)phenyl isothiocyanate, 4-nitrophenyl isothiocyanate, 4-bromophenyl isothiocyanate, 2,3-dihydro-1,4-benzodioxin-6-yl isothiocyanate, 1,3-benzodioxol-5-yl isothiocyanate, 4-(1H-pyrazol-1-yl)phenyl isothiocyanate, 2-(trifluoromethyl)phenyl isothiocyanate, 2,3-dimethylphenyl isothiocyanate, 2-isopropyl phenyl isothiocyanate, 4-iso-propylphenyl isothiocyanate, 5-chloro-2-methoxyphenyl isothiocyanate, 2,4-dimethoxyphenyl isothiocyanate, 2,4-dichloro-6-methylphenyl isothiocyanate, 2-bromo-4-isopropylphenyl isothiocyanate, and 5-chloro-2-fluorophenyl isothiocyanate.
Alkyl isocyanates and alkyl isothiocyanates may also be useful in such a synthesis, depending upon the type of carbamoyl group to be introduced onto the heteroaryl group on position 4 of the core structure.
In another particular embodiment, the invention relates to a group of pyrido(3,2-d)pyrimidine derivatives, as well as pharmaceutical compositions comprising such pyrido(3,2-d)pyrimidine derivatives as active principle, represented by one of the above structural formulae (I), (II), (III) and (IV) and being in the form of a pharmaceutically acceptable salt. The latter include any therapeutically active non-toxic addition salt which compounds represented by one of the structural formulae (I), (II), (III) and (IV) are able to form with a salt-forming agent. Such addition salts may conveniently be obtained by treating the pyrido(3,2-d)pyrimidine derivatives of the invention with an appropriate salt-forming acid or base. For instance, pyrido(3,2-d)pyrimidine derivatives having basic properties may be converted into the corresponding therapeutically active, non-toxic acid addition salt form by treating the free base form with a suitable amount of an appropriate acid following conventional procedures. Examples of such appropriate salt-forming acids include, for instance, inorganic acids resulting in forming salts such as but not limited to hydrohalides (e.g. hydrochloride and hydrobromide), sulfate, nitrate, phosphate, diphosphate, carbonate, bicarbonate, and the like; and organic monocarboxylic or dicarboxylic acids resulting in forming salts such as, for example, acetate, propanoate, hydroxyacetate, 2-hydroxypropanoate, 2-oxopropanoate, lactate, pyruvate, oxalate, malonate, succinate, maleate, fumarate, malate, tartrate, citrate, methanesulfonate, ethanesulfonate, benzoate, 2-hydroxybenzoate, 4-amino-2-hydroxybenzoate, benzene-sulfonate, p-toluenesulfonate, salicylate, p-aminosalicylate, pamoate, bitartrate, camphorsulfonate, edetate, 1,2-ethanedisulfontate, fumarate, glucoheptonate, gluconate, glutamate, hexylresorcinate, hydroxynaphthoate, hydroxyethanesulfonate, mandelate, methylsulfate, pantothenate, stearate, as well as salts derived from ethanedioic, propanedioic, butanedioic, (Z)-2-butenedioic, (E)2-butenedioic, 2-hydroxybutanedioic, 2,3-dihydroxybutane-dioic, 2-hydroxy-1,2,3-propanetricarboxylic and cyclohexanesulfamic acids and the like.
Pyrido(3,2-d)pyrimidine derivatives represented by one of the structural formulae (I), (II), (III) and (IV) having acidic properties may be converted in a similar manner into the corresponding therapeutically active, non-toxic base addition salt form. Examples of appropriate salt-forming bases include, for instance, inorganic bases like metallic hydroxides such as but not limited to those of alkali and alkaline-earth metals like calcium, lithium, magnesium, potassium and sodium, or zinc, resulting in the corresponding metal salt; organic bases such as but not limited to ammonia, alkylamines, benzathine, hydrabamine, arginine, lysine, N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylene-diamine, N-methylglucamine, procaine and the like.
Reaction conditions for treating the pyrido(3,2-d)pyrimidine derivatives represented by one of the structural formulae (I), (II), (III) and (IV) of this invention with an appropriate salt-forming acid or base are similar to standard conditions involving the same acid or base but different organic compounds with basic or acidic properties, respectively. Preferably, in view of its use in a pharmaceutical composition or in the manufacture of a medicament for treating specific diseases, the pharmaceutically acceptable salt will be designed, i.e. the salt-forming acid or base will be selected so as to impart greater water-solubility, lower toxicity, greater stability and/or slower dissolution rate to the pyrido(3,2-d)pyrimidine derivative of this invention.
The present invention further provides the use of a pyrido(3,2-d)pyrimidine derivative represented by the structural formula (I), or a pharmaceutically acceptable salt or a solvate thereof, as a biologically-active ingredient, i.e. active principle, especially as a medicine or a diagnostic agent or for the manufacture of a medicament or a diagnostic kit. In particular the said medicament may be for the prevention or treatment of a pathologic condition selected from the group consisting of:
The present invention further provides the use of a pyrido(3,2-d)pyrimidine derivative represented by one of the structural formulae (II), (III) and (IV) or a pharmaceutically acceptable salt or a solvate thereof, as a biologically-active ingredient, i.e. active principle, especially as a medicine or for the manufacture of a medicament for treating an immune disorder or for preventing a transplant rejection.
The pathologic conditions and disorders concerned by the said use, and the corresponding methods of prevention or treatment, are detailed hereinbelow. Any of the uses mentioned with respect to the present invention may be restricted to a non-medical use (e.g. in a cosmetic composition), a non-therapeutic use, a non-diagnostic use, a non-human use (e.g. in a veterinary composition), or exclusively an in-vitro use, or a use with cells remote from an animal.
The invention further relates to a pharmaceutical composition comprising:
In another embodiment, this invention provides combinations, preferably synergistic combinations, of one or more pyrido(3,2-d)pyrimidine derivatives represented by one of the structural formulae (I), (II), (III) and (IV), with one or more biologically-active drugs being preferably selected from the group consisting of immunosuppressant and/or immunomodulator drugs, antineoplastic drugs, and antiviral agents. As is conventional in the art, the evaluation of a synergistic effect in a drug combination may be made by analyzing the quantification of the interactions between individual drugs, using the median effect principle described by Chou et al. in Adv. Enzyme Reg. (1984) 22:27. Briefly, this principle states that interactions (synergism, additivity, antagonism) between two drugs can be quantified using the combination index (hereinafter referred as CI) defined by the following equation:
wherein EDx is the dose of the first or respectively second drug used alone (1a, 2a), or in combination with the second or respectively first drug (1c, 2c), which is needed to produce a given effect. The said first and second drug have synergistic or additive or antagonistic effects depending upon CI<1, CI=1, or CI>1, respectively. As will be explained in more detail herein below, this principle may be applied to a number of desirable effects such as, but not limited to, an activity against transplant rejection, an activity against immunosuppression or immunomodulation, or an activity against cell proliferation.
For instance the present invention relates to a pharmaceutical composition or combined preparation having synergistic effects against immuno-suppression or immunomodulation and containing:
Suitable immunosuppressant drugs for inclusion in the synergistic compositions or combined preparations of this invention belong to a well known therapeutic class. They are preferably selected from the group consisting of cyclosporin A, substituted xanthines (e.g. methylxanthines such as pentoxyfylline), daltroban, sirolimus, tacrolimus, rapamycin (and derivatives thereof such as defined below), leflunomide (or its main active metabolite A771726, or analogs thereof called malononitrilamides), mycophenolic acid and salts thereof (including the sodium salt marketed under the trade name Mofetil®), adrenocortical steroids, azathioprine, brequinar, gusperimus, 6-mercaptopurine, mizoribine, chloroquine, hydroxychloroquine and monoclonal antibodies with immunosuppressive properties (e.g. etanercept, infliximab or kineret). Adrenocortical steroids within the meaning of this invention mainly include glucocorticoids such as but not limited to ciprocinonide, desoxycorticisterone, fludrocortisone, flumoxonide, hydrocortisone, naflocort, procinonide, timobesone, tipredane, dexamethasone, methylprednisolone, methotrexate, prednisone, prednisolone, triamcinolone and pharmaceutically acceptable salts thereof. Rapamycin derivatives as referred herein include O-alkylated derivatives, particularly 9-deoxorapamycins, 26-dihydrorapamycins, 40-O-substituted rapamycins and 28,40-O,O-disubstituted rapamycins (as disclosed in U.S. Pat. No. 5,665,772) such as 40-O-(2-hydroxy)ethyl rapamycin—also known as SDZ-RAD—, pegylated rapamycin (as disclosed in U.S. Pat. No. 5,780,462), ethers of 7-desmethylrapamycin (as disclosed in U.S. Pat. No. 6,440,991) and polyethylene glycol esters of SDZ-RAD (as disclosed in U.S. Pat. No. 6,331,547).
Suitable immunomodulator drugs for inclusion into the synergistic immunomodulating pharmaceutical compositions or combined preparations of this invention are preferably selected from the group consisting of acemannan, amiprilose, bucillamine, dimepranol, ditiocarb sodium, imiquimod, Inosine Pranobex, interferon-β, interferon-γ, lentinan, levamisole, lisophylline, pidotimod, romurtide, platonin, procodazole, propagermanium, thymomodulin, thymopentin and ubenimex.
Synergistic activity of the pharmaceutical compositions or combined preparations of this invention against immunosuppression or immuno-modulation may be readily determined by means of one or more lymphocyte activation tests. Usually activation is measured via lymphocyte proliferation. Inhibition of proliferation thus always means immunosuppression under the experimental conditions applied. There exist different stimuli for lymphocyte activation, in particular:
Determination of the immunosuppressing or immunomodulating activity of the pyrido(3,2-d)pyrimidine derivatives of this invention, as well as synergistic combinations comprising them, is preferably based on the determination of one or more, preferably at least three lymphocyte activation in vitro tests, more preferably including at least one of the MLR test, CD3 assay and CD28 assay referred above. Preferably the lymphocyte activation in vitro tests used include at least two assays for two different clusters of differentiation preferably belonging to the same general type of such clusters and more preferably belonging to type I transmembrane proteins. Optionally the determination of the immuno-suppressing or immunomodulating activity may be performed on the basis of other lymphocyte activation in vitro tests, for instance by performing a TNF-α assay or an IL-1 assay or an IL-6 assay or an IL-10 assay or an IL-12 assay or an assay for a cluster of differentiation belonging to a further general type of such clusters and more preferably belonging to type II transmembrane proteins such as, but not limited to, CD69, CD 71 or CD134.
The synergistic effect may be evaluated by the median effect analysis method described herein before. Such tests may for instance, according to standard practice in the art, involve the use of equipment, such as flow cytometer, being able to separate and sort a number of cell subcategories at the end of the analysis, before these purified batches can be analysed further.
Synergistic activity of the pharmaceutical compositions of this invention in the prevention or treatment of transplant rejection may be readily determined by means of one or more leukocyte activation tests performed in a Whole Blood Assay (hereinafter referred as WBA) described for instance by Lin et al. in Transplantation (1997) 63:1734-1738. WBA used herein is a lymphoproliferation assay performed in vitro using lymphocytes present in the whole blood, taken from animals that were previously given the pyrido(3,2-d)pyrimidine derivative of this invention, and optionally the other immunosuppressant drug, in vivo. Hence this assay reflects the in vivo effect of substances as assessed by an in vitro read-out assay. The synergistic effect may be evaluated by the median effect analysis method described herein before. Various organ transplantation models in animals are also available in vivo, which are strongly influenced by different immunogenicities, depending on the donor and recipient species used and depending on the nature of the transplanted organ. The survival time of transplanted organs can thus be used to measure the suppression of the immune response.
The pharmaceutical composition or combined preparation with synergistic activity against immunosuppression or immunomodulation according to this invention may contain the pyrido(3,2-d)pyrimidine derivative represented by one of the structural formulae (I), (II), (III) and (IV) over a broad content range depending on the contemplated use and the expected effect of the preparation. Typically, the pyrido(3,2-d)pyrimidine derivative content in the combined preparation is within the range of from 0.1 to 99.9% by weight, preferably from 1 to 99% by weight, more preferably from about 5 to 95% by weight.
The invention further relates to a composition or combined preparation having synergistic effects against cell proliferation and containing:
Suitable antineoplastic drugs for inclusion into the synergistic antiproliferative pharmaceutical compositions or combined preparations of this invention are preferably selected from the group consisting of alkaloids, alkylating agents (including but not limited to alkyl sulfonates, aziridines, ethylenimines, methylmelamines, nitrogen mustards and nitrosoureas), antibiotics, antimetabolites (including but not limited to folic acid analogues, purine analogs and pyrimidine analogues), enzymes, interferon and platinum complexes. More specific examples include acivicin; aclarubicin; acodazole; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene; bisnafide; bizelesin; bleomycin; brequinar; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin; decitabine; dexormaplatin; dezaguanine; diaziquone; docetaxel; doxorubicin; droloxifene; dromostanolone; duazomycin; edatrexate; eflomithine; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin; erbulozole; esorubicin; estramustine; etanidazole; ethiodized oil I131; etoposide; etoprine; fadrozole; fazarabine; fenretinide; floxuridine; fludarabine; fluorouracil; flurocitabine; fosquidone; fostriecin; gemcitabine; Gold 198; hydroxyurea; idarubicin; ifosfamide; ilmofosine; interferon α-2a; interferon α-2b; interferon α-n1; interferon α-n3; interferon β-1a; interferon γ-1b; iproplatin; irinotecan; lanreotide; letrozole; leuprolide; liarozole; lometrexol; lomustine; losoxantrone; masoprocol; maytansine; mechlorethamine; megestrol; melengestrol; melphalan; menogaril; mercaptopurine; methotrexate; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone; mycophenolic acid; nocodazole; nogala-mycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin; perfosfamide; pipobroman; piposulfan; piroxantrone; plicamycin; plomestane; porfimer; porfiromycin; prednimustine; procarbazine; puromycin; pyrazofurin; riboprine; rogletimide; safingol; semustine; simtrazene; sparfosate; sparsomycin; spirogermanium; spiromustine; spiroplatin; streptonigrin; streptozocin; strontium 89 chloride; sulofenur; talisomycin; taxane; taxoid; tecogalan; tegafur; teloxantrone; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; topotecan; toremifene; trestolone; triciribine; trimetrexate; triptorelin; tubulozole; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine; vincristine; vindesine; vinepidine; vinglycinate; vinleurosine; vinorelbine; vinrosidine; vinzolidine; vorozole; zeniplatin; zinostatin; zorubicin; and their pharmaceutically acceptable salts.
Other suitable anti-neoplastic compounds include vitamin D3 derivatives such as, but not limited to, 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; anti-androgens such as, but not limited to, benorterone, cioteronel, cyproterone, delmadinone, oxendolone, topterone, zanoterone and their pharmaceutically acceptable salts; anti-estrogens such as, but not limited to, clometherone; delmadinone; nafoxidine; nitromifene; raloxifene; tamoxifen; toremifene; trioxifene and their pharmaceutically acceptable salts; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; β-lactam derivatives; α-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors; castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; clomifene and analogues thereof; clotrimazole; collismycin A and B; combretastatin and analogues thereof; conagenin; crambescidin 816; cryptophycin and derivatives thereof; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine; cytolytic factor; cytostatin; dacliximab; dehydrodidemnin B; deslorelin; dexifosfamide; dexrazoxane; dexverapamil; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol; dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; elemene; emitefur; epristeride; estrogen agonists and antagonists; exemestane; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fluorodaunorunicin; forfenimex; formestane; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idoxifene; idramantone; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; iobenguane; iododoxorubicin; ipomeanol; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N; leinamycin; lenograstim; lentinan; leptolstatin; leukemia inhibiting factor; leuprorelin; levamisole; liarozole; lissoclinamide; lobaplatin; lombricine; lonidamine; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitors; mifepristone; miltefosine; mirimostim; mitoguazone; mitolactol; mitonafide; mitotoxin fibroblast growth factor-saporin; mofarotene; molgramostim; human chorionic gonadotrophin monoclonal antibody; mopidamol; mycaperoxide B; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone; pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; octreotide; okicenone; onapristone; ondansetron; ondansetron; oracin; osaterone; oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; peldesine; pentosan; pentostatin; pentrozole; perflubron; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine; pirarubicin; piritrexim; placetin A and B; plasminogen activator inhibitor; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein kinase C inhibitors; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; retelliptine; rhenium 186 etidronate; rhizoxin; retinamide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; saintopin; sarcophytol A; sargramostim; sizofuran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; splenopentin; spongistatin 1; squalamine; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; suradista; suramin; swainsonine; tallimustine; tamoxifen; tauromustine; tazarotene; tecogalan; tellurapyrylium; telomerase inhibitors; temozolomide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; titanocene; topsentin; tretinoin; triacetyluridine; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; variolin B; velaresol; veramine; verdins; verteporfin; vinxaltine; vitaxin; zanoterone; zilascorb; and their pharmaceutically acceptable salts.
The compounds of this invention may also be administered in combination with anti-cancer agents which act by arresting cells in the G2-M phases due to stabilized microtubules. In addition to Taxol (paclitaxel), analogues and derivatives thereof, other examples of anti-cancer agents which act by this mechanism include without limitation the following marketed drugs and drugs in development: erbulozole, dolastatin, mivobulin isethionate, discodermolide, altorhyrtins, spongistatins, cemadotin hydrochloride, epothilones desoxyepothilone, 16-aza-epothilone, 21-aminoepothilone, 21-hydroxyepothilone, 26-fluoroepothilone, auristatin, soblidotin, cryptophycin, vitilevuamide, tubulysin, canadensol, centaureidin, oncocidin, fijianolide, laulimalide, narcosine, nascapine, hemiasterlin, vanadocene acetylacetonate, monsatrol, inanocine, eleutherobins, caribaeoside, caribaeolin, halichondrin, diazonamide, taccalonolide, diozostatin, phenylahistin, myoseverin, resverastatin phosphate sodium, and their pharmaceutically acceptable salts.
Synergistic activity of the pharmaceutical compositions or combined preparations of this invention against cell proliferation may be readily determined by means of one or more tests such as, but not limited to, the measurement of the radioactivity resulting from the incorporation of 3H-thymidine in culture of tumor cell lines. For instance, different tumor cell lines may be selected in order to evaluate the anti-tumor effects of the test compounds, such as but not limited to:
In a specific embodiment of the cell proliferation synergy determination test, tumor cell lines are harvested and a suspension of 0.27×106 cells/ml in whole medium is prepared. The suspensions (150 μl) are added to a microtiter plate in triplicate. Either complete medium (controls) or the test compounds at the test concentrations (50 μl) are added to the cell suspension in the microtiter plate. Cells are incubated at 37° C. under 5% CO2 for about 16 hours. 3H-thymidine is added, and cells are incubated for another 8 hours and then harvested, and radioactivity is measured in counts per minute (CPM) in a β-counter. The 3H-thymidine cell content, and thus the measured radioactivity, is proportional to the proliferation of the cell lines. The synergistic effect is evaluated by the median effect analysis method as disclosed herein before.
The pharmaceutical composition or combined preparation with synergistic activity against cell proliferation according to this invention may contain the pyrido(3,2-d)pyrimidine derivative represented by the structural formula (I) over a broad content range depending on the contemplated use and the expected effect of the preparation. Typically, the pyrido(3,2-d)pyrimidine derivative content of the combined preparation is within the range of from 0.1 to 99.9% by weight, preferably from 1 to 99% by weight, more preferably from about 5 to 95% by weight.
The invention further relates to a pharmaceutical composition or combined preparation having synergistic effects against a viral infection and containing:
Suitable anti-viral agents for inclusion into the synergistic antiviral compositions or combined preparations of this invention include, for instance, retroviral enzyme inhibitors belonging to categories well known in the art, such as HIV-1 IN inhibitors, nucleoside reverse transcriptase inhibitors (e.g. zidovudine, lamivudine, didanosine, stavudine, zalcitabine and the like), non-nucleoside reverse transcriptase inhibitors (e.g. nevirapine, delavirdine and the like), other reverse transcriptase inhibitors (e.g. foscamet sodium and the like), and HIV-1 protease inhibitors (e.g. saquinavir, ritonavir, indinavir, nelfinavir and the like). Other suitable antiviral agents include for instance acemannan, acyclovir, adefovir, alovudine, alvircept, amantadine, aranotin, arildone, atevirdine, pyridine, cidofovir, cipamfylline, cytarabine, desciclovir, disoxaril, edoxudine, enviradene, enviroxime, famciclovir, famotine, fiacitabine, fialuridine, floxuridine, fosarilate, fosfonet, ganciclovir, idoxuridine, kethoxal, lobucavir, memotine, methisazone, penciclovir, pirodavir, somantadine, sorivudine, tilorone, trifluridine, valaciclovir, vidarabine, viroxime, zinviroxime, moroxydine, podophyllotoxin, ribavirine, rimantadine, stallimycine, statolon, tromantadine and xenazoic acid, and their pharmaceutically acceptable salts.
Especially relevant to this aspect of the invention is the inhibition of the replication of viruses selected from the group consisting of picorna-, toga-, bunya, orthomyxo-, paramyxo-, rhabdo-, retro-, arena-, hepatitis B-, hepatitis C-, hepatitis D-, adeno-, vaccinia-, papilloma-, herpes-, corona-, varicella- and zoster-virus, in particular human immunodeficiency virus (HIV). Synergistic activity of the pharmaceutical compositions or combined preparations of this invention against viral infection may be readily determined by means of one or more tests such as, but not limited to, the isobologram method, as previously described by Elion et al. in J. Biol. Chem. (1954) 208:477-488 and by Baba et al. in Antimicrob. Agents Chemother. (1984) 25:515-517, using EC50 for calculating the fractional inhibitory concentration (hereinafter referred as FIC). When the minimum FIC index corresponding to the FIC of combined compounds (e.g., FICx+FICy) is equal to 1.0, the combination is said to be additive; when it is between 1.0 and 0.5, the combination is defined as sub-synergistic, and when it is lower than 0.5, the combination is by defined as synergistic. When the minimum FIC index is between 1.0 and 2.0, the combination is defined as subantagonistic and, when it is higher than 2.0, the combination is defined as antagonistic.
The pharmaceutical composition or combined preparation with synergistic activity against viral infection according to this invention may contain the pyrido(3,2-d)pyrimidine derivative represented by the structural formula (I) over a broad content range depending on the contemplated use and the expected effect of the preparation. Typically, the pyrido(3,2-d)pyrimidine derivative content of the combined preparation is within the range of from 0.1 to 99.9% by weight, preferably from 1 to 99% by weight, more preferably from about 5 to 95% by weight.
The invention further relates to a pharmaceutical composition or combined preparation having synergistic effects against a disease mediated by phosphodiesterase-4 activity and containing:
Suitable phosphodiesterase inhibitors may be selected from the group consisting of pyrrolidinones (such as, but not limited to, rolipram, RO20-1724 and RS 33793), quinazolinediones (such as, but not limited to, nitraquazone, CP-77059 and RS-25344), xanthine derivatives (such as, but not limited to, denbufylline, arofylline and BRL 61063), phenylethyl pyridines (such as, but not limited to, CDP 840), tetrahydropyrimidones (such as, but not limited to, atizoram), diazepine derivatives (such as, but not limited to, CI 1018), oxime carbamates (such as, but not limited to, filaminast), naphthyridinones (such as, but not limited to, RS 17597), benzofurans (such as, but not limited to, 2-butyl-7-methoxy-benzofuran-4-carboxylic acid (3-5-dichloropyridin-4-yl)-amide, 2-benzyl-7-methoxy-benzofuran-4-carboxylic acid (3-5-dichloropyridin-4-yl)-amide, 7-methoxy-2-phenethyl-benzofuran-4-carboxylic acid (3-5-dichloropyridin-4-yl)-amide, 5-(2-butyl-7-methoxy-benzofuran-4-yl)-tetrahydropyrimidin-2-one, and phenyldihydrobenzofuranes), naphthalene derivatives (such as, but not limited to, T 440), purine derivatives (such as, but not limited to, V-112294A), imidazolidinones, cyclohexane carboxylic acids (such as, but not limited to, ariflo), benzamides (such as, but not limited to, piclamilast), pyridopyridazinones, benzothiophenes (such as, but not limited to, tibenelast), etazolate, S-(+)-glaucine, substituted phenyl compounds and substituted biphenyl compounds, and pyridopyridazinones.
The pharmaceutical compositions and combined preparations according to this invention may be administered orally or in any other suitable fashion. Oral administration is preferred and the preparation may have the form of a tablet, aqueous dispersion, dispersable powder or granule, emulsion, hard or soft capsule, syrup, elixir or gel. The dosing forms may be prepared using any method known in the art for manufacturing these pharmaceutical compositions and may comprise as additives sweeteners, flavoring agents, coloring agents, preservatives and the like. Carrier materials and excipients are detailed hereinbelow and may include, inter alia, calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, binding agents and the like. The pharmaceutical composition or combined preparation of this invention may be included in a gelatin capsule mixed with any inert solid diluent or carrier material, or has the form of a soft gelatin capsule, in which the ingredient is mixed with a water or oil medium. Aqueous dispersions may comprise the biologically active composition or combined preparation in combination with a suspending agent, dispersing agent or wetting agent. Oil dispersions may comprise suspending agents such as a vegetable oil. Rectal administration is also applicable, for instance in the form of suppositories or gels. Injection (e.g. intramuscularly or intraperitoneously) is also applicable as a mode of administration, for instance in the form of injectable solutions or dispersions, depending upon the disorder to be treated and the condition of the patient.
Auto-immune disorders to be prevented or treated by the pharmaceutical compositions or combined preparations of this invention include both:
Transplant rejections to be prevented or treated by the pharmaceutical compositions or combined preparations of this invention include the rejection of transplanted or grafted organs or cells (both allografts and xenografts), such as but not limited to host versus graft reaction disease. The term “organ” as used herein means all organs or parts of organs in mammals, in particular humans, such as but not limited to kidney, lung, bone marrow, hair, cornea, eye (vitreous), heart, heart valve, liver, pancreas, blood vessel, skin, muscle, bone, intestine or stomach. The term “rejection” as used herein means all reactions of the recipient body or the transplanted organ which in the end lead to cell or tissue death in the transplanted organ or adversely affect the functional ability and viability of the transplanted organ or the recipient. In particular, this means acute and chronic rejection reactions. Also included in this invention is preventing or treating the rejection of cell transplants and xenotransplantation. The major hurdle for xenotransplantation is that even before the T lymphocytes, responsible for the rejection of allografts, are activated, the innate immune system, especially T-independent B lymphocytes and macrophages are activated. This provokes two types of severe and early acute rejection called hyper-acute rejection and vascular rejection, respectively. The present invention addresses the problem that conventional immunosuppressant drugs like cyclosporin A are ineffective in xeno-transplantation. The ability of the compounds of this invention to suppress T-independent xeno-antibody production as well as macrophage activation may be evaluated in the ability to prevent xenograft rejection in athymic, T-deficient mice receiving xenogenic hamster-heart grafts.
In a particular embodiment of the invention, the pyrido(3,2-d)pyrimidine derivatives according to one of the structural formulae (II), (III) and (IV) may be used in the treatment of auto-immune disorders, or the prevention of a transplant rejection in a patient. In particular, pyrido(3,2-d)pyrimidine derivatives according to one of the structural formulae (II), (III) and (IV) may be used in the treatment a disease selected from the group consisting of rheumatoid arthritis, Crohn's disease, ulcerative colitis, uveitis, multiple sclerosis, atopic dermatitis, psoriasis and lupus erythematosus. Cell proliferative disorders to be prevented or treated by the pharmaceutical compositions or combined preparations including a pyrido(3,2-d)pyrimidine derivative represented by the structural formula (I) of this invention include any kind of tumor progression or invasion or metastasis inhibition of a cancer, preferably one selected from the group consisting of lung cancer, leukaemia, ovarian cancer, sarcoma, Kaposi's sarcoma, meningioma, colon cancer, lymph node tumor, glioblastoma multiforme, prostate cancer or skin carcinose.
CNS disorders to be prevented or treated by the pharmaceutical compositions or combined preparations including a pyrido(3,2-d)pyrimidine derivative represented by the structural formula (I) of this invention include cognitive pathologies such as dementia, cerebral ischemia, trauma, epilepsy, schizophrenia, chronic pain, and neurologic disorders such as but not limited to depression, social phobia and obsessive compulsive disorders.
Cardiovascular disorders to be prevented or treated by the pharmaceutical compositions or combined preparations including a pyrido(3,2-d)pyrimidine derivative represented by the structural formula (I) of this invention include, but are not limited to, ischemic disorders, infarct or reperfusion damage, atherosclerosis and stroke.
TNF-α-related disorders to be prevented or treated by the pharmaceutical compositions or combined preparations including a pyrido(3,2-d)pyrimidine derivative represented by the structural formula (I) of this invention include the following:
Disorders mediated by phosphodiesterase-4 activity to be prevented or treated by the pharmaceutical compositions or combined preparations including a pyrido(3,2-d)pyrimidine derivative represented by the structural formula (I) of this invention include, but are not limited to, erectile dysfunction, sepsis and septic shock. PDE-4 is particularly abundant in inflammatory and immune cells. Through modulation of cAMP levels, PDE-4 regulates leukocyte responses including the pro-inflammatory actions of monocytes, T cells and neutrophils, airway and vascular smooth muscle constriction, and neurotransmitter signaling through adenylyl cyclase linked G-protein coupled receptors (such as that for N-methyl-D-aspartate). Inhibition of PDE-4 blocks cell traffic and cell proliferation, and attenuates the production of inflammatory mediators, cytokines and reactive oxygen species. TNF-α is an important target in rheumatoid arthritis, ankylosing spondylitis, Crohn's disease and psoriasis. However, in diseases such as severe asthma and late-stage rheumatoid arthritis, neutrophils do play a key role in the pathological inflammatory process. PDE-4 inhibitors are able to suppress multiple neutrophil responses, including the production of IL-8, leukotriene B4 and superoxide anions, as well as degranulation, chemotaxis and adhesion. In addition, the smooth muscle (e.g. bronchodilatory) relaxing effect of PDE-4 inhibitors are very beneficial for the treatment of asthma. The inhibition of TNF-α production that follows inhibition of PDE-4 B isoform is cAMP-dependent and requires protein kinase A activity for protection from LPS-induced shock. The highly specialized function of PDE-4 B in macrophages and its critical role in LPS signaling are thus well known in the art, and therefore provide basis for a therapeutic strategy using subtype-selective PDE-4 inhibitors for the treatment of sepsis and septic shock.
The term “erectile dysfunction” as used herein includes any type of erectile dysfunction, such as but not limited to vasculogenic, neurogenic, endocrinologic and psychogenic impotence (“impotence” being used herein to indicate a periodic or consistent inability to achieve or sustain an erection of sufficient rigidity for sexual intercourse); Peyronie's syndrome; priapism; premature ejaculation; and any other condition, disease or disorder, regardless of cause or origin, which interferes with at least one of the three phases of human sexual response, i.e., desire, excitement and orgasm.
The medicament of this invention may be for prophylactic use, i.e. where circumstances are such that an elevation in the TNF-α level might be expected or alternatively, may be for use in reducing the TNF-α level after it has reached an undesirably high level (as defined herein above) or as the TNF-α level is rising.
The term “pharmaceutically acceptable carrier or excipient” as used herein in relation to pharmaceutical compositions and combined preparations means any material or substance with which the active principle, i.e. a pyrido(3,2-d)pyrimidine derivative represented by one of the structural formulae (I), (II), (III) and (IV), and optionally the immunosuppressant or immunomodulator or antineoplastic drug or antiviral agent, may be formulated in order to facilitate its application or dissemination to the locus to be treated, for instance by dissolving, dispersing or diffusing the said composition, and/or to facilitate its storage, transport or handling without impairing its effectiveness. The pharmaceutically acceptable carrier may be a solid or a liquid or a gas which has been compressed to form a liquid, i.e. the compositions of this invention can suitably be used as concentrates, emulsions, solutions, granulates, dusts, sprays, aerosols, pellets or powders.
Suitable pharmaceutical carriers for use in the said pharmaceutical compositions and their formulation are well known to those skilled in the art. There is no particular restriction to their selection within the present invention although, due to the usually low or very low water-solubility of the pyrido(3,2-d)pyrimidine derivatives of this invention, special attention will be paid to the selection of suitable carrier combinations that can assist in properly formulating them in view of the expected time release profile. Suitable pharmaceutical carriers include additives such as wetting agents, dispersing agents, stickers, adhesives, emulsifying or surface-active agents, thickening agents, complexing agents, gelling agents, solvents, coatings, antibacterial and antifungal agents (for example phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or sodium chloride) and the like, provided the same are consistent with pharmaceutical practice, i.e. carriers and additives which do not create permanent damage to mammals.
The pharmaceutical compositions of the present invention may be prepared in any known manner, for instance by homogeneously mixing, dissolving, spray-drying, coating and/or grinding the active ingredients, in a one-step or a multi-steps procedure, with the selected carrier material and, where appropriate, the other additives such as surface-active agents. may also be prepared by micronisation, for instance in view to obtain them in the form of microspheres usually having a diameter of about 1 to 10 μm, namely for the manufacture of microcapsules for controlled or sustained release of the biologically active ingredient(s).
Suitable surface-active agents to be used in the pharmaceutical compositions of the present invention are non-ionic, cationic and/or anionic surfactants having good emulsifying, dispersing and/or wetting properties. Suitable anionic surfactants include both water-soluble soaps and water-soluble synthetic surface-active agents. Suitable soaps are alkaline or alkaline-earth metal salts, unsubstituted or substituted ammonium salts of higher fatty acids (C10-C22), e.g. the sodium or potassium salts of oleic or stearic acid, or of natural fatty acid mixtures obtainable form coconut oil or tallow oil. Synthetic surfactants include sodium or calcium salts of polyacrylic acids; fatty sulphonates and sulphates; sulphonated benzimidazole derivatives and alkylarylsulphonates. Fatty sulphonates or sulphates are usually in the form of alkaline or alkaline-earth metal salts, unsubstituted ammonium salts or ammonium salts substituted with an alkyl or acyl radical having from 8 to 22 carbon atoms, e.g. the sodium or calcium salt of lignosulphonic acid or dodecylsulphonic acid or a mixture of fatty alcohol sulphates obtained from natural fatty acids, alkaline or alkaline-earth metal salts of sulphuric or sulphonic acid esters (such as sodium lauryl sulphate) and sulphonic acids of fatty alcohol/ethylene oxide adducts. Suitable sulphonated benzimidazole derivatives preferably contain 8 to 22 carbon atoms. Examples of alkylarylsulphonates are the sodium, calcium or alcanolamine salts of dodecylbenzene sulphonic acid or dibutyl-naphtalenesulphonic acid or a naphtalenesulphonic acid/formaldehyde condensation product. Also suitable are the corresponding phosphates, e.g. salts of phosphoric acid ester and an adduct of p-nonylphenol with ethylene and/or propylene oxide, or phospholipids. Suitable phospholipids for this purpose are the natural (originating from animal or plant cells) or synthetic phospholipids of the cephalin or lecithin type such as e.g. phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine, lysolecithin, cardiolipin, dioctanylphosphatidylcholine, dipalmitoylphosphatidylcholine and their mixtures.
Suitable non-ionic surfactants include polyethoxylated and polypropoxylated derivatives of alkylphenols, fatty alcohols, fatty acids, aliphatic amines or amides containing at least 12 carbon atoms in the molecule, alkylarenesulphonates and dialkylsulphosuccinates, such as polyglycol ether derivatives of aliphatic and cycloaliphatic alcohols, saturated and unsaturated fatty acids and alkylphenols, said derivatives preferably containing 3 to 10 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms in the alkyl moiety of the alkylphenol. Further suitable non-ionic surfactants are water-soluble adducts of polyethylene oxide with polypropylene glycol, ethylenediamino-polypropylene glycol containing 1 to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250 ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ether groups. Such compounds usually contain from 1 to 5 ethyleneglycol units per propyleneglycol unit. Representative examples of non-ionic surfactants are nonylphenol-polyethoxyethanol, castor oil polyglycolic ethers, polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethyleneglycol and octylphenoxypolyethoxyethanol. Fatty acid esters of polyethylene sorbitan (such as polyoxyethylene sorbitan trioleate), glycerol, sorbitan, sucrose and pentaerythritol are also suitable non-ionic surfactants.
Suitable cationic surfactants include quaternary ammonium salts, preferably halides, having four hydrocarbon radicals optionally substituted with halo, phenyl, substituted phenyl or hydroxy; for instance quaternary ammonium salts containing as N-substituent at least one C8-C22 alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl, oleyl and the like) and, as further substituents, unsubstituted or halogenated lower alkyl, benzyl and/or hydroxy-C1-4 alkyl radicals.
A more detailed description of surface-active agents suitable for this purpose may be found for instance in “McCutcheon's Detergents and Emulsifiers Annual” (MC Publishing Crop., Ridgewood, N.J., 1981), “Tensid-Taschenbuch”, 2nd ed. (Hanser Verlag, Vienna, 1981) and “Encyclopaedia of Surfactants (Chemical Publishing Co., New York, 1981).
Structure-forming, thickening or gel-forming agents may be included into the pharmaceutical compositions and combined preparations of the invention. Suitable such agents are in particular highly dispersed silicic acid, such as the product commercially available under the trade name Aerosil; bentonites; tetraalkyl ammonium salts of montmorillonites (e.g., products commercially available under the trade name Bentone), wherein each of the alkyl groups may contain from 1 to 20 carbon atoms; cetostearyl alcohol and modified castor oil products (e.g. the product commercially available under the trade name Antisettle).
Gelling agents which may be included into the pharmaceutical compositions and combined preparations of the present invention include, but are not limited to, cellulose derivatives such as carboxymethylcellulose, cellulose acetate and the like; natural gums such as arabic gum, xanthum gum, tragacanth gum, guar gum and the like; gelatin; silicon dioxide; synthetic polymers such as carbomers, and mixtures thereof. Gelatin and modified celluloses represent a preferred class of gelling agents.
Other optional excipients which may be included in the pharmaceutical compositions and combined preparations of the present invention include additives such as magnesium oxide; azo dyes; organic and inorganic pigments such as titanium dioxide; UV-absorbers; stabilisers; odor masking agents; viscosity enhancers; antioxidants such as, for example, ascorbyl palmitate, sodium bisulfite, sodium metabisulfite and the like, and mixtures thereof; preservatives such as, for example, potassium sorbate, sodium benzoate, sorbic acid, propyl gallate, benzylalcohol, methyl paraben, propyl paraben and the like; sequestering agents such as ethylene-diamine tetraacetic acid; flavoring agents such as natural vanillin; buffers such as citric acid and acetic acid; extenders or bulking agents such as silicates, diatomaceous earth, magnesium oxide or aluminum oxide; densification agents such as magnesium salts; and mixtures thereof.
Additional ingredients may be included in order to control the duration of action of the biologically-active ingredient in the compositions and combined preparations of the invention. Control release compositions may thus be achieved by selecting appropriate polymer carriers such as for example polyesters, polyamino-acids, polyvinyl-pyrrolidone, ethylene-vinyl acetate copolymers, methylcellulose, carboxymethylcellulose, protamine sulfate and the like. The rate of drug release and duration of action may also be controlled by incorporating the active ingredient into particles, e.g. microcapsules, of a polymeric substance such as hydrogels, polylactic acid, hydroxymethyl-cellulose, polymethyl methacrylate and the other above-described polymers. Such methods include colloid drug delivery systems like liposomes, microspheres, microemulsions, nanoparticles, nanocapsules and so on. Depending on the route of administration, the pharmaceutical composition or combined preparation of the invention may also require protective coatings.
Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation thereof. Typical carriers for this purpose therefore include biocompatible aqueous buffers, ethanol, glycerol, propylene glycol, polyethylene glycol, complexing agents such as cyclodextrins and the like, and mixtures thereof.
Pharmaceutical forms suitable for transurethral delivery, e.g. intracavernosal injection, such as needed for the treatment of erectile dysfunction are extensively disclosed in U.S. Pat. No. 6,127,363, the content of which is incorporated by reference. Transurethral drug delivery may involve an active delivery mechanism such as iontophoresis, electroporation or phonophoresis. Devices and methods for delivering drugs in this way are well known in the art. lontophoretically assisted drug delivery is, for example, described in WO96/40054. Briefly, the active agent is driven through the urethral wall by means of an electric current passed from an external electrode to a second electrode contained within or affixed to a urethral probe.
Other modes of local drug administration can also be used. For example, the selected active agent may be administered by way of intracavernosal injection, or may be administered topically, in an ointment, gel or the like, or transdermally, including transscrotally, using a conventional transdermal drug delivery system. Intracavernosal injection can be carried out by use of a syringe or any other suitable device. An example of a hypodermic syringe useful herein is described in U.S. Pat. No. 4,127,118, injection being made on the dorsum of the penis by placement of the needle to the side of each dorsal vein and inserting it deep into the corpora.
For intracavernosal injection, the active agent to be administered is preferably incorporated into a sterile liquid preparation, typically a solution or suspension in an aqueous or oleaginous medium. This solution or suspension may be formulated according to techniques known in the art using suitable carriers, dispersants, wetting agents, diluents, suspending agents or the like. Among the acceptable vehicles and solvents that may be employed are water, isotonic saline, vegetable oil, fatty esters and polyols.
Since, in the case of combined preparations including the pyrido(3,2-d)pyrimidine derivative of this invention and an immunosuppressant or immunomodulator or antineoplastic drug or antiviral agent or phosphodiesterase-4 inhibitor, both ingredients do not necessarily bring out their synergistic therapeutic effect directly at the same time in the patient to be treated, the said combined preparation may be in the form of a medical kit or package containing the two ingredients in separate but adjacent form. In the latter context, each ingredient may therefore be formulated in a way suitable for an administration route different from that of the other ingredient, e.g. one of them may be in the form of an oral or parenteral formulation whereas the other is in the form of an ampoule for intravenous injection or an aerosol.
The present invention further relates to a method for preventing or treating a disease selected from the group consisting of CNS disorders, cell proliferative disorders, viral infections, immune and auto-immune disorders, transplant rejections, PDE-4-mediated diseases and TNF-α-related disorders in a patient, preferably a mammal, more preferably a human being. The method of this invention consists of administering to the patient in need thereof an effective amount of a pyrido(3,2-d)pyrimidine derivative represented by the structural formula (I), (II), (III) or (IV), optionally together with an effective amount of another immunosuppressant or immunomodulator or antineoplastic drug or antiviral agent or phosphodiesterase-4 inhibitor, or a pharmaceutical composition comprising the same, such as disclosed above in extensive details. The effective amount is usually in the range of about 0.01 mg to 20 mg, preferably about 0.1 mg to 5 mg, per day per kg bodyweight for humans. Depending upon the pathologic condition to be treated and the patient's condition, the said effective amount may be divided into several sub-units per day or may be administered at more than one day intervals. The patient to be treated may be any warm-blooded animal, preferably a mammal, more preferably a human being, suffering from said pathologic condition.
The preferred compounds of the present invention are non-sedating. In other words, a dose of such compounds that is twice the minimum dose sufficient to provide analgesia in an animal model for determining pain relief causes only transient (i.e. lasting for no more than half the time that pain relief lasts) or preferably no statistically significant sedation in an animal model assay of sedation (using the method described by Fitzgerald et al. in Toxicology (1988) 49:433-9). Preferably, a dose that is five times the minimum dose sufficient to provide analgesia does not produce statistically significant sedation. More preferably, a compound provided herein does not produce sedation at intravenous doses of less than 10 mg/kg per day or at oral doses of less than 30 mg/kg per day. If desired, compounds provided herein may be evaluated for toxicity (a preferred compound is non-toxic when an immunomodulating amount or a cell anti-proliferative amount is administered to a subject) and/or side effects (a preferred compound produces side effects comparable to placebo when a therapeutically effective amount of the compound is administered to a subject). Toxicity and side effects may be assessed using any standard method. In general, the term “non-toxic” as used herein shall be understood as referring to any substance that, in keeping with established criteria, is susceptible to approval by the United States Federal Drug Administration for administration to mammals, preferably humans. Toxicity may be also evaluated using assays including bacterial reverse mutation assays, such as an Ames test, as well as standard teratogenicity and tumorogenicity assays. Preferably, administration of compounds provided herein within the therapeutic dose ranges disclosed hereinabove does not result in prolongation of heart QT intervals (e.g. as determined by electrocardiography in guinea pigs, minipigs or dogs). When administered daily, such doses also do not cause liver enlargement resulting in an increase of liver to body weight ratio of more than 50% over matched controls in laboratory rodents (e.g. mice or rats). Such doses also preferably do not cause liver enlargement resulting in an increase of liver to body weight ratio of more than 10% over matched untreated controls in dogs or other non-rodent mammals. The preferred compounds of the present invention also do not promote substantial release of liver enzymes from hepatocytes in vivo, i.e. the therapeutic doses do not elevate serum levels of such enzymes by more than 50% over matched untreated controls in vivo in laboratory rodents.
Another embodiment of this invention includes the various precursor or “pro-drug” forms of the compounds of the present invention. It may be desirable to formulate the compounds of the present invention in the form of a chemical species which itself is not significantly biologically-active, but which when delivered to the body of a human being or higher mammal will undergo a chemical reaction catalyzed by the normal function of the body, inter alia, enzymes present in the stomach or in blood serum, said chemical reaction having the effect of releasing a compound as defined herein. The term “pro-drug” thus relates to these species which are converted in vivo into the active pharmaceutical ingredient.
The pro-drugs of the present invention can have any form suitable to the formulator, for example, esters are non-limiting common pro-drug forms. In the present case, however, the pro-drug may necessarily exist in a form wherein a covalent bond is cleaved by the action of an enzyme present at the target locus. For example, a C—C covalent bond may be selectively cleaved by one or more enzymes at said target locus and, therefore, a pro-drug in a form other than an easily hydrolysable precursor, inter alia an ester, an amide, and the like, may be used.
For the purposes of the present invention the term “therapeutically suitable pro-drug” is defined herein as “a compound modified in such a way as to be transformed in vivo to the therapeutically active form, whether by way of a single or by multiple biological transformations, when in contact with the tissues of humans or mammals to which the pro-drug has been administered, and without undue toxicity, irritation, or allergic response, and achieving the intended therapeutic outcome”.
The present invention will be further described with reference to certain more specific embodiments and examples, but the present invention is not limited thereto but only by the attached claims. The following examples are given by way of illustration only.
To a solution of 6-chloro-2-cyano-3-nitro-pyridine (3.03 g, 16.5 mmol) in ethanol (166 ml) and H2O (16 ml) was added iron (165 mmol, 9.2 g) and calcium chloride (2.75 g, 24.8 mmol). The reaction mixture was refluxed for 4 hours and then cooled down to room temperature. The precipitate was filtered off over Celite and the filtrate was evaporated to dryness. The residue was redissolved in ethyl acetate and extracted with brine. The aqueous layer was extracted back with ethyl acetate. The combined organic layers were evaporated in vacuo. The residue was adsorbed on silica and purified by silica gel column chromatography, the mobile phase being a ethyl acetate/hexane mixture in a ratio of 3:7, resulting in the pure title compound (1.89 g, yield 67%) which was characterised by its mass spectrum as follows MS (m/z): 172, 174 ([M+H]+, 100).
A suspension of 6-chloro-2-carboxamido-3-amino-pyridine (1.34 mmol, 230 mg) in triethyl orthoformate (10 ml) was refluxed for 3 hours. A white suspension was formed which was cooled down to room temperature. The precipitate was filtered off and dried under vacuum resulting in the pure title compound (174 mg, yield 72%) which was characterised by its mass spectrum as follows: MS (m/z): 182, 184 ([M+H]+, 100).
To a solution of 6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (200 mg, 1.1 mmol) in 1,4-dioxane (20 ml) and water (10 ml) was added 3,4-dimethoxyphenyl boronic acid (240 mg, 1.32 mmol), potassium carbonate (380 mg, 2.75 mmol) and tetrakis(triphenylphosphine)palladium(0) (63 mg, 0.055 mmol). The reaction mixture was refluxed for 3 hours, cooled down to room temperature and the solvents were evaporated in vacuo. The residue was adsorbed on silica, purified by silica gel column chromatography (the mobile phase being a acetone/dichloromethane mixture, in a ratio gradually ranging from 30:70 to 40:60) and characterised by its mass spectrum as follows: MS (m/z): 284 ([M+H]+, 100).
To a suspension of 6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one (150 mg, 0.53 mmol) in toluene (30 ml) was added phosphorus oxychloride (148 μl, 1.59 mmol) and 2,6-lutidine (185 μl, 1.59 mmol). The reaction mixture was refluxed overnight until a black solution was obtained. After evaporation to dryness, the residue was redissolved in ethyl acetate and extracted with a saturated sodium bicarbonate solution. The combined organic layers were evaporated in vacuo. The residue was purified by silica gel column chromatography, the mobile phase being an ethyl acetate/hexane mixture, in a ratio gradually ranging from 2:8 to 3:7, resulting in the pure title compound (123 mg, yield 77%) which was characterised by its mass spectrum as follows: MS (m/z): 302, 304 ([M+H]+, 100).
To a suspension of 4-chloro-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (120 mg, 0.398 mmol) in isopropanol (15 ml) was added 1-(2-phenoxyethyl)-piperazine (0.795 mmol, 164 mg). The suspension was stirred at 80° C., after which the suspension became a clear colorless solution. The solvents were evaporated in vacuo. The residue was redissolved in ethyl acetate and extracted with a NaOH solution (1 N). The combined organic layers were evaporated in vacuo and purified by silica gel column chromatography (the mobile phase being a mixture of methanol and dichloromethane in a ratio gradually ranging from 1:99 to 2:98), resulting in the title compound (157 mg, yield 84%) which was characterised by its mass spectrum as follows: MS (m/z): 472 ([M+H]+, 100).
To a solution of 6-(3,4-dimethoxyphenyl)-3-nitropyridine-2-carbonitrile (1.42 g, mmol) in ethanol (50 ml) and water (5 ml) was added iron (1.39 g, 25 mmol) and calcium chloride (6 mmol, 666 mg). The reaction mixture was refluxed for 1 hour. An additional amount of iron (1.39 g, 25 mmol) was added and the reaction was refluxed for another 3 hours. The reaction was cooled down and filtered over a paper filter, followed by washings with boiling ethyl acetate. The filtrate was evaporated in vacuo and the residue was partitioned between ethyl acetate and water. The organic layers were evaporated to dryness and the residue was purified by silica gel column chromatography (the mobile phase being a mixture of ethyl acetate and hexane in a ratio of 1:1), resulting in the pure title compound (770 mg, yield 56%) which was characterised by its mass spectrum as follows: MS (m/z): 273 [(M+H)+, 100).
A suspension of 2-carboxamido-3-amino-6-(3,4-dimethoxyphenyl)-pyridine (770 mg, 2.8 mmol) in triethyl orthoformate (28 ml) was refluxed for 12 hours. Then, the reaction mixture was cooled down and evaporated to dryness. The residue was purified by silica gel column chromatography (the mobile phase being an ethyl acetate/hexane mixture in a ratio gradually ranging from 2:8 to 3:7), resulting in the pure title compound (530 mg, yield 67%) which was characterised by its mass spectrum as follows: MS (m/z): 284 ([M+H)+, 100].
To a suspension of 4-chloro-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (227 mg, 0.8 mmol) in isopropanol (20 ml) was added piperazine-1-carboxylic acid m-tolylamide (351 mg, 1.6 mmol). The reaction mixture was stirred for 3 hours at 80° C. Then, the reaction was cooled down and evaporated to dryness. The residue was redissolved in ethyl acetate and extracted with a saturated sodium bicarbonate solution. The combined organic layers were evaporated in vacuo. The crude residue was purified by silica gel column chromatography (the mobile phase being a mixture of methanol and dichloromethane in a ratio gradually ranging from 1:99 to 2:98), resulting in the pure title compound (217 mg, yield 56%) which was characterised by its mass spectrum as follows: MZ (m/z): 485 ([M+H)+, 100).
A suspension of 2-carboxamido-3-amino-6-(3,4-dimethoxyphenyl)-pyridine (546 mg, 2 mmol) in triethyl orthoacetate (25 ml) was refluxed for 12 hours. Then, the reaction mixture was cooled down and evaporated to dryness. The residue was purified by silica gel column chromatography (the mobile phase being an ethyl acetate/hexane mixture in a ratio gradually ranging from 2:8 to 3:7), resulting in the pure title compound (437 mg, yield 73%) which was characterised by its mass spectrum as follows: MS (m/z): 297 ([M+H]+, 100).
To a solution of 2-methyl-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one (416 mg, 1.4 mmol) in toluene (28 ml) was added 2,6-lutidine (490 μl, 4.2 mmol) and POCl3 (4.2 mmol, 385 μl). The mixture was refluxed under nitrogen atmosphere for 5 hours. The reaction mixture was cooled down, diluted with ethyl acetate (50 ml) and extracted with a saturated sodium bicarbonate solution. The combined organic layers were evaporated in vacuo and the residue was purified by silica gel column chromatography (the mobile phase being an ethyl acetate/hexane mixture in a ratio of 15:85), resulting in the pure title compound (330 mg, yield 75%) which was characterised by its mass spectrum as follows: MS (m/z): 316, 318 ([M+H]+, 100).
To a suspension of 2-methyl-4-chloro-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (330 mg, 1.04 mmol) in acetonitrile (20 ml) was added piperazine-1-carboxylic acid m-tolylamide (479 mg, 2.2 mmol). The reaction mixture was refluxed for 2 hours. The mixture was cooled down and ethyl acetate was added (100 ml). The reaction mixture was extracted with a saturated sodium bicarbonate solution. The combined organic layers were evaporated to dryness. The residue was purified by a first silica gel column chromatography (the mobile phase being a methanol/dichloromethane mixture in a ratio gradually ranging from 1:99 to 2:98) and then a second silica gel column purification was performed with a mobile phase consisting of a 95:5 ethyl acetate/hexane mixture, resulting in the pure title compound (319 mg, yield 62%) which was characterised by its mass spectrum as follows: MS (m/z): 499 ([M+H]+, 100).
To a solution of 2-carboxamido-3-amino-6-(3,4-dimethoxyphenyl)-pyridine (4.10 g, 15 mmol) in 1,4-dioxane (150 ml) was added triphosgene (2.22 g, 7.5 mmol). The solution was refluxed for 25 minutes and then evaporated to dryness. The crude compound was crystallized from acetic acid (150 ml) and washed with ethyl acetate, diethyl ether and dried under vacuum over P2O5, resulting in the pure title compound (3.60 g, yield 80%) which was characterised by its mass spectrum as follows: MS (m/z): 300 ([M+H]+, 100).
To a suspension of 6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidin-2(1H)-4(3H)-dione (2.69 g, 9 mmol) in POCl3 (60 ml) was added triethylamine (3.47 ml). The reaction mixture was refluxed under nitrogen until completion. The reaction was cooled down to room temperature and evaporated to dryness. The residue was partitioned between water and dichloromethane. The organic layer was washed with brine. The combined organic layers were evaporated and the residue was purified by silica gel column chromatography (the mobile phase being a hexane/ethyl acetate mixture in a ratio 6:4), resulting in the pure title compound (yield 83%) which was characterised by its mass spectrum as follows: MS (m/z): 336, 338 ([M+H]+, 100).
To a suspension of 2,4-dichloro-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine (672 mg, 2 mmol) in THF (10 ml) was added piperazine-1-carboxylic acid m-tolylamide (484 mg, 2.2 mmol) and triethylamine (10 mmol, 1.40 ml). The reaction mixture was stirred at room temperature for 10 minutes. The mixture was evaporated to dryness. The residue was redissolved in dichloromethane and extracted with brine. The combined organic layers were evaporated in vacuo and the crude residue was purified by silica gel column chromatography (the mobile phase being a hexane/ethyl acetate mixture in a ratio 1:1), resulting in the pure title compound (760 mg, yield 73%) which was characterised by its mass spectrum as follows: MS (m/z): 519, 521 ([M+H]+, 100).
To a suspension of 2-chloro-4-(4-[3-methylphenyl)amino]carbonyl]piperazin-1-yl)-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine (0.35 mmol, 181 mg) in dioxane (5 ml) was added dimethylamine (100 μl of a 40% solution in water). The reaction was stirred at 80° C. for 1.5 hours, after which an additional amount (100 μl) of the dimethylamine solution was added. The reaction was stirred for another 18 hours and then, cooled down, and diluted with dichloromethane (50 ml). The reaction mixture was extracted with a saturated sodium bicarbonate solution. The combined organic layers were evaporated in vacuo. The residue was purified by preparative thin layer chromatography on silica (the mobile phase being a hexane/ethyl acetate mixture in a ratio 1:9), resulting in the pure title compound (57 mg, yield 31%) which was characterised by its mass spectrum as follows: MS (m/z): 528 ([M+H]+, 100).
N-(2-hydroxyethyl)morpholine (55 μl, 0.45 mmol) was dissolved in dry tetrahydrofuran (5 ml) and sodium hydride 60% (20 mg, 0.495 mmol) was added. The solution was stirred at 60° C. under nitrogen for 20 minutes and then, 2-chloro-4-(4-[3-methylphenyl)amino]carbonyl]piperazin-1-yl)-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine (156 mg, 0.3 mmol) was added. The reaction mixture was stirred for 1 hour at 60° C. The mixture was cooled down to room temperature, diluted with brine and extracted with ethyl acetate. The combined organic layers were evaporated in vacuo and purified by preparative thin layer chromatography on silica (the mobile phase being a methanol/dichloromethane mixture in a ratio 7.5:92.5), resulting in the pure title compound (166 mg, yield 90%) which was characterised by its mass spectrum as follows: MS (m/z): 614 ([M+H]+, 100).
Sodium hydride 60% (20 mg, 0.495 mmol) was dissolved in dry tetrahydrofuran (5 ml) and 1-methyl-2-pyrrolidine-ethanol (62 μl, 0.45 mmol) was added. The mixture was refluxed under an N2-atmosphere for 15 minutes. Then, 2-chloro-4-(4-[3-methylphenyl)amino]carbonyl]piperazin-1-yl)-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine (156 mg, 0.30 mmol) was added and the reaction mixture was refluxed under nitrogen for 16 hours. The reaction mixture was diluted with distilled water and extracted three times with ethyl acetate. The combined organic extracts were washed with brine and dried over Na2SO4. Upon filtration and evaporation in vacuo, the crude product was purified by preparative thin layer chromatography on silica with a dichloromethane/methanol mixture (ratio 9:1) as the mobile phase to afford 79 mg (yield 43%) of the title compound which was characterised by its mass spectrum as follows: MS (m/z): 612 ([M+H]+, 100).
Sodium hydride 60% (25 mg, 0.62 mmol) and 2-phenoxyethanol (63 mg, 0.45 mmol) were dissolved in dry tetrahydrofuran (5 ml). The reaction mixture was refluxed under a nitrogen atmosphere for 15 minutes. Then, 2-chloro-4-(4-[3-methylphenyl)amino]carbonyl]piperazin-1-yl)-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine (156 mg, 0.30 mmol) was added and the reaction was refluxed under nitrogen for 3 hours. The reaction mixture was diluted with distilled water and extracted with dichloromethane. Combined organic extracts were dried over Na2SO4. Upon filtration and evaporation in vacuo, the crude product was purified by preparative thin layer chromatography on silica with a n-hexane/ethyl acetate mixture (ratio 1.5:1) as the mobile phase. Recrystallization from ethyl acetate afforded 124 mg (yield 67%) of the title compound which was characterised by its mass spectrum as follows: MS (m/z): 621 ([M+H]+, 100).
A suspension of 2-chloro-4-(4-[3-methylphenyl)amino]carbonyl]piperazin-1-yl)-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine (156 mg, 0.30 mmol), potassium carbonate (181 mg, 1.31 mmol) and phenylboronic acid (49 mg, 0.39 mmol) in 1,4-dioxane (4.5 ml) and water (1.5 ml) was purged with a stream of nitrogen gas for 10 minutes. Tetrakis(triphenylphosphine)palladium(0) (18 mg, 15.6 μmol) was added and the reaction mixture was refluxed under a nitrogen atmosphere for 30 minutes. Upon cooling, the mixture was diluted with ethyl acetate and washed twice with brine. The organic layer was dried over Na2SO4 and subsequently filtered and evaporated in vacuo. Recrystallization from ethyl acetate afforded 74 mg (yield 44%) of the title compound which was characterised by its mass spectrum as follows: MS (m/z): 561 ([M+H]+, 100).
2,4-diamino-6-chloropyrido[3,2-d]pyrimidine (7.5 g, 38 mmole), e.g. prepared according to Colbry et al., J. Heterocycl. Chem. (1984) 21:1521, was suspended in 6 N HCl (300 ml) and the mixture was refluxed for 5 hours. After cooling, the pH was made alkaline (pH about 9-10) by means of 10 N NaOH. The precipitate obtained was filtered, washed with H2O and dried at 100° C., resulting in the pure title compound (7.0 g, yield 95%) which was characterized by its mass spectrum as follows: MS (m/z): 197 ([M+H]+, 100).
To a degassed suspension of 2-amino-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (7.30 g, 37 mmole), 3,4-dimethoxyphenyl boronic acid (7.50 g, 40 mmole) and potassium carbonate (20.70 g, 152 mmole) in a mixture of dioxane (540 ml) and H2O (120 ml), was added a catalytic amount of tetrakis(triphenylphosphine)palladium(0) (2.16 g, 18.5 mmole). The mixture was refluxed for 24 hours and, after cooling at room temperature, was filtered. The filtrate was acidified with 5 N HCl to pH 4 and the resulting precipitate was filtered and then washed successively with H2O, ethanol and diethylether, and dried under vacuum resulting in the pure title compound (8.0 g, yield 73%) which was characterized by its mass spectrum as follows: MS (m/z): 299 ([M+H]+, 100).
2-amino-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one (2.0 g, 6.70 mmole) was suspended in acetic anhydride (180 ml) and acetic acid (20 ml) and the mixture was refluxed for 16 hours. The hot suspension was filtered and the filtrate was concentrated under reduced pressure until crystallization started. The precipitate was filtered off to give the pure title compound (1.76 g, yield 77%) which was characterized by its mass spectrum as follows: MS (m/z): 341 ([M+H]+, 100).
A suspension of 1,2,4-triazole (8.28 g, 120 mmole) and phosphorus oxychloride (3.2 ml, 36 mmol) in dry acetonitrile (150 ml) was added to a stirred suspension of 2-acetamido-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidin-4(3H)-one (4.08 g, 12 mmole) and triethylamine (5.2 ml, 36 mmole) in dry acetonitrile (150 ml). The mixture was stirred at room temperature under nitrogen for 3 days and the yellow precipitate was filtered off, then successively washed with ethanol and ether, and dried over P2O5 in a vacuum dessicator resulting in the pure title compound (4.3 g, yield 90%) which was characterized by its mass spectrum as follows: MS (m/z): 392 ([M+H]+, 100), 414 ([M+Na]+; 804 [2M+Na]+
Sodium (44 mg, 2 mmol) was suspended in a suitable alcohol (10 ml) and the solution was warmed up to 50° C. until the sodium dissolved completely. Then, 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (160 mg, 0.4 mmole) was added and the mixture was stirred at room temperature for 16 hours. The mixture was then neutralized with a solution of 1 N HCl and the volatiles were removed under reduced pressure. The crude mixture was purified by silica gel column chromatography, the mobile phase consisting of CH3OH/CH2Cl2 mixtures (in a ratio gradually ranging from 2:98 to 10:90), thus providing the desired compound with yields ranging from 40 to 60%, depending upon the alcohol used. The following compounds were made according to this procedure:
A suitable alkylamine, cycloalkylamine, arylamine, heterocyclic amine or heteroarylalkylamine (2 equivalents, 0.8 mmole) was added to a stirred suspension of 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine (160 mg, 0.4 mmole) in dioxane. The mixture was heated at 50° C. for 24 hours and the volatiles were removed under reduced pressure, yielding a crude 2-acetylamino-4-alkylamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine, 2-acetylamino-4-cycloalkylamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine, 2-acetylamino-4-heteroarylalkylamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine, 2-acetylamino-4-arylamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine or 2-acetylamino-4-hetero-cyclic amino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine as an intermediate. This crude residue was resuspended in a 0.2 N sodium ethoxide (20 ml) and the mixture was stirred at room temperature for 24 hours and neutralized with 5-6 N HCl in isopropyl alcohol, yielding the crude corresponding 2-amino-4-alkylamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine, 2-amino-4-cycloalkylamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine, 2-amino-4-heteroarylalkylamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine, 2-amino-4-arylamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine or 2-amino-4-heterocyclic amino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine as the final compound. This crude residue was purified by silica gel column chromatography, the mobile phase consisting of CH3OH/CH2Cl2 mixtures (in a ratio gradually ranging from 2:98 to 10:90) with 0.5% concentrated ammonia if needed. This procedure provided the desired final compounds with yields ranging from 40 to 80%. The following final compounds were synthesized according to this procedure (each time through the corresponding intermediate having the 2-amino group protected in the form of acetamido):
To a degassed suspension of 6-chloro-2-cyano-3-nitropyridine (5.51 g, 30 mmole), 3,4-dimethoxyphenyl boronic acid (6.55 g, 36 mmole) and potassium carbonate (16.59 g, 120 mmole) in dry toluene (300 ml), was added a catalytic amount of tetrakis(triphenylphosphine)palladium (3.47 g, 3 mmole). The mixture was refluxed for 24 hours and after cooling, the volatiles were evaporated to dryness. The crude mixture was purified by silica gel column chromatography, the mobile phase consisting of hexane/CH2Cl2 mixtures (in a ratio gradually ranging from 15:85 to 0:100). The appropriated fractions were collected, evaporated to dryness and the residue was suspended in ether. The orange precipitate was filtered off, washed with ether and dried, resulting in the pure title compound (6.79 g, yield 79%).
Iron (7.14 g, 128 mmole) was added portionwise to a stirred suspension of 6-(3,4-dimethoxyphenyl)-3-nitropyridine-2-carbonitrile (4.56 g; 16 mmole) in methanol (80 ml) and 37% HCl (25 ml). The mixture was refluxed for 5 hours and, after cooling, the pH was adjusted to 9-10 by means of concentrated ammonium hydroxide (30 ml). The mixture was filtered over Celite and washed with MeOH and EtOAc. The filtrate was evaporated to dryness and the residue was purified on silica gel column chromatography, using a mixture of CH2Cl2/EtOAc (in a ratio of 95:5) as eluent, to obtain the pure title compound (2.62 g, yield 64%) which was characterized by its mass spectrum as follows: MS (m/z): 256 ([M+H]+, 100).
A solution of sodium (423 mg, 18.4 mmole) in n-butanol (180 ml) was added to 3-amino-6-(3,4-dimethoxyphenyl)pyridine-2-carbonitrile (2.36 g; 9.20 mmole) and guanidine hydrochloride (1.76 g; 18.4 mmole). The mixture was refluxed for 4 hours and, after cooling, the solvent was evaporated under reduced pressure. The residue was purified on silica gel column chromatography, using a mixture of CH2Cl2/MeOH (in a ratio of 95:5) as eluent, resulting in the pure title compound (1.88 g; yield 69%) which was characterized by its mass spectrum as follows: MS (m/z): 298 ([M+H]+, 100).
2,4-diamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine (1.27 g, 4.27 mmole) was suspended in 6 N HCl (85 ml) and the mixture was refluxed for 8 hours. After cooling, the precipitate was filtered off, washed with H2O and dried over P2O5 and KOH, resulting in the pure title compound (1.29 g; yield 90%) which was characterized by its mass spectrum as follows: MS (m/z): 299 ([M+H]+, 100)
2-amino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidin-4(3H)-one hydrochloride (332 mg; 1 mmole) was suspended in toluene (10 ml) with a catalytic amount of p-toluenesulfonic acid and ammonium sulfate. Then, 1,1,1,3,3,3-hexamethyldisilizane (3.2 ml; 15 mmole) and morpholine (0.53 ml; 6 mmol) were added. The mixture was refluxed for 24 hours and evaporated to dryness. The residue was purified by silica gel column chromatography, using a mixture of CH2Cl2/MeOH: 96:4 as eluent, resulting in the pure title compound (120 mg; yield 32%) which was characterized by its mass spectrum as follows: MS (m/z): 368 ([M+H]+, 100).
Piperazine (258 mg; 3 mmole) was added to a stirred suspension of 2-acetamido-6-(3,4-dimethoxyphenyl)-4-(1,2,4-triazolyl)pyrido[3,2-d]pyrimidine (586 mg; 1.5 mmole) in dioxane (50 ml). The mixture was stirred at room temperature for 24 hours and the volatiles were removed under reduced pressure, yielding 2-acetamido-4-(N-piperazinyl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as a crude residue. The latter was dissolved in DMF and m-tolyl isocyanate (0.66 ml, 5 mmole) was added. After 18 hours at room temperature, the solvent was removed and the residue was suspended in a mixture of CH2Cl2 (20 ml) and sodium ethoxide 0.2 N (20 ml). The suspension was stirred during 16 hours and neutralized with 5-6 N HCl in isopropyl alcohol. The crude residue was purified by silica gel column chromatography, the mobile phase consisting of a CH3OH/CH2Cl2 mixture in a ratio gradually ranging from 2:98 to 5:95, thus resulting in the pure title compound (350 mg, yield 43%) which was characterized by its mass spectrum as follows: MS (m/z): 542 ([M+H]+, 100).
1-(4-fluorophenyl)-piperazine (90 mg, 0.5 mmole) was added to a stirred suspension of 2-acetamido-6-(3,4-dimethoxyphenyl)-4-(1,2,4-triazolyl)pyrido[3,2-d]pyrimidine (120 mg, 0.3 mmole) in dioxane (10 ml). The mixture was stirred at 60° C. for 48 hours and the volatiles were removed under reduced pressure, yielding the crude 2-acetamido-4-(4-fluorophenyl-piperazin-1-yl-)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine. The latter was dissolved in a mixture of CH2Cl2 (20 ml) and sodium ethoxide 0.2 N (20 ml). The suspension was stirred during 16 hours and neutralized with 5-6 N HCl in isopropyl alcohol. The crude residue was purified by preparative thin layer chromatography, the mobile phase consisting of a CH3OH/CH2Cl2 mixture in a ratio of 5:95, resulting in the pure title compound (40 mg, yield 29%) which was characterized by its mass spectrum as follows: MS (m/z): 461 ([M+H]+, 100).
A similar procedure as in example 43 was used but starting from 1-(4-methylphenyl)-piperazine and resulted, through the corresponding 2-acetamido intermediate, in the pure title compound (49% yield) which was characterized by its mass spectrum as follows: MS (m/z): 457 ([M+H]+, 100).
A similar procedure as in example 43 was used but starting from 1-(2-phenoxy-ethyl)-piperazine and resulted, through the corresponding 2-acetamido intermediate, in the pure title compound (56% yield) which was characterized by its mass spectrum as follows: MS (m/z): 488 ([M+H]+, 100).
A similar procedure as in example 43 was used but starting from 1-(3-chlorophenyl)-piperazine and resulted, through the corresponding 2-acetamido intermediate, in the pure title compound (42% yield) which was characterized by its mass spectrum as follows: MS (m/z): 478 ([M+H]+, 100)
A similar procedure as in example 43 was used but starting from 1-(2-pyridyl)-piperazine and resulted, through the corresponding 2-acetamido intermediate, in the pure title compound (37% yield) which was characterized by its mass spectrum as follows: MS (m/z): 444 ([M+H]+, 100).
A similar procedure as in example 43 was used but starting from 2-(piperazin-1-yl)-acetic acid N-(2-thiazolyl)-amide and resulted, through the corresponding 2-acetamido intermediate, in the pure title compound (52% yield) which was characterized by its mass spectrum as follows: MS (m/z): 507 ([M+H]+, 100).
A similar procedure as in example 43 was used but starting from N-acetylpiperazine and resulted, through the corresponding 2-acetamido intermediate, in the pure title compound (33% yield) which was characterized by its mass spectrum as follows: MS (m/z): 409 ([M+H]+, 100).
A similar procedure as in example 43 was used but starting from 1-piperonyl-piperazine and resulted, through the corresponding 2-acetamido intermediate, in the pure title compound (38% yield) which was characterized by its mass spectrum as follows: MS (m/z): 501 ([M+H]+, 100).
A similar procedure as in example 43 was used but starting from 1-(2-furoyl)-piperazine instead of 1-(4-fluorophenyl)-piperazine and resulted, through the corresponding 2-acetamido intermediate, in the pure title compound which was characterized by its mass spectrum as follows: MS (m/z): 461 ([M+H]+, 100).
A similar procedure as in example 43 was used but starting from 1-benzylpiperazine and resulted, through the corresponding 2-acetamido intermediate, in the pure title compound (39% yield) which was characterized by its mass spectrum as follows: MS (m/z): 457 ([M+H]+, 100).
Piperazine (430 mg, 5 mmole) was added to a stirred suspension of 2-acetamido-6-(3,4-dimethoxyphenyl)-4-(1,2,4-triazolyl)pyrido[3,2-d]pyrimidine (977 mg, 2.5 mmole) in dioxane (70 ml). The reaction mixture was refluxed for 16 hours. The precipitate was filtered off and washed with a small amount of dioxane. The filtrate was evaporated to dryness and the residue washed with diethyl ether. Both fractions (the precipate and the washed filtrate) were combined, resulting in the pure title compound (805 mg, yield 79%) which was characterized by its mass spectrum as follows: MS (m/z): 409 ([M+H]+, 100).
To a solution of 2-acetamido-4-(piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (200 mg, 0.5 mmole) in DMF (5 ml) was added a suitable isocyanate (0.75 mmole). The reaction mixture was stirred for 16 hours at room temperature. The solvents were evaporated in vacuo yielding a crude 2-acetamido-4-(N-carbamoyl-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as an intermediate. This crude residue was dissolved in a mixture of CH2Cl2 (10 ml) and sodium ethoxide 0.2 N (10 ml), the resulting suspension was stirred for 16 hours and neutralized with 5-6 N HCl in isopropyl alcohol, yielding a crude 2-amino-4-(N-carbamoyl-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as the final compound. This crude product was purified by preparative thin layer chromatography on silica, the mobile phase consisting of a CH3OH/CH2Cl2 mixture in a ratio of 10:90, resulting in the pure desired compounds in yields varying from 20 to 40%, depending upon the isocyanate used. The following final compounds were synthesized according to this procedure (each time through the corresponding intermediate having the 2-amino group protected in the form of acetamido):
To a solution of 2-acetamido-4-(piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (200 mg, 0.5 mmole) in dioxane (15 ml) was added p-chloro-phenoxy acetyl chloride (0.75 mmol). The reaction mixture was stirred for 16 hours at 50° C. overnight. The solvents were evaporated in vacuo yielding crude 2-acetamido-4-[(N-4-chloro-phenoxy-acetyl)-piperazin-1-yl]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as an intermediate. This crude residue was dissolved in a mixture of CH2Cl2 (10 ml) and sodium ethoxide 0.2 N (10 ml). The suspension was stirred for 16 hours and neutralized with 5-6 N HCl in isopropyl alcohol, yielding crude 2-amino-4-[(N-4-chloro-phenoxy-acetyl)-piperazin-1-yl]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as the final compound. This crude product was purified by preparative thin layer chromatography on silica, the mobile phase consisting of a CH3OH/CH2Cl2 mixture in a ratio of 10:90, resulting in the pure title compound (98 mg, yield 37%) which was characterized by its mass spectrum as follows: MS (m/z): 536 ([M+H]+, 100).
A similar procedure as described in example 59 was performed, but using phenoxy acetyl chloride instead of p-chloro-phenoxy acetyl chloride and resulted, through the corresponding 2-acetamido intermediate, in the pure title compound which was characterized by its mass spectrum as follows: MS (m/z): 501 ([M+H]+, 100).
To a suspension of 6-chloro-3-nitro-pyridine-2-carbonitrile (9.2 g, 50 mmole) in water (100 ml), was added 20 ml of a 25% ammonia aqueous solution. The mixture was stirred at room temperature for 20 minutes. Then, Na2S2O4 (50 g, 86%, 150 mmole) was added portionwise, and the mixture was stirred at room temperature for another 2 hours. The precipitate formed was collected by filtration, washed two times with cold water (10 ml) and then dried over P2O5, resulting in the title compound (7.0 g, yield 81%) as a yellowish solid which was characterized by its mass spectrum as follows: MS (m/z): 172.1 ([M+H]+, 100).
This compound was synthesized, by using the procedure of example 61 but from 5-chloro-3-nitro-pyridine-2-carbonitrile as a starting material, in 80% yield as a yellowish solid which was characterized by its mass spectrum as follows: MS (m/z): 172.1 ([M+H]+, 100).
A suspension of 3-amino-5-chloro-pyridine-2-carboxamide (3.43 g, 20 mmole) in triethyl orthoformate (50 ml) was refluxed for 3 hours. After cooling to room temperature, the precipitate was collected by filtration and washed with hexane. The title compound was obtained as a white solid (3.4 g, yield 94%) which was characterized by its mass spectrum as follows: MS (m/z): 182.1 ([M+H]+, 100).
To a mixture of 6-chloro-pyrido[3,2-d]pyrimidin-4(3H)one (3.0 g, 16.5 mmole) and N,N-diisopropylethylamine (9 ml, 50 mmole) in toluene (150 ml), was added POCl3 (4.7 ml, 50 mmol). The resulting reaction mixture was refluxed for 1.5 hour. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was dissolved in dichloromethane (200 ml) and washed with cold water till pH=6-7. The organic phase was dried over MgSO4, filtrated and concentrated under reduced pressure to yield crude 4,6-dichloro-pyrido[3,2-d]pyrimidine which was not purified but used as such for further reactions.
To a solution of piperazine (7.0 g) in 1,4-dioxane (100 ml) was added a solution of crude 4,6-dichloro-pyrido[3,2-d]pyrimidine in 1,4-dioxane (50 ml). The resulting mixture was stirred at room temperature for 1 hour. After concentration under reduced pressure, the residue was purified by silica gel flash chromatography, the mobile phase being a methanol/dichloromethane mixture (in a ratio gradually ranging from 1:10 to 1:5), resulting in the pure title compound as a yellowish solid (3.1 g, yield 76%) which was characterized by its mass spectrum as follows: MS (m/z): 250.1 ([M+H]+, 100).
This compound was synthesized from 7-chloro-pyrido[3,2-d]pyrimidin-4(3H)one using the procedure mentioned in example 64.
The title compound was synthesized in 72% yield from 4,7-dichloro-pyrido[3,2-d]pyrimidine by the procedure of example 65 and was characterized by its mass spectrum as follows: MS (m/z): 250.1 ([M+H]+, 100).
The title compound was synthesized in 71% yield from 4,6-dichloro-pyrido[3,2-d]pyrimidine and morpholine by the procedure of example 65, and was characterized by its mass spectrum as follows: MS (m/z): 251.1 ([M+H]+, 100).
To a solution of 4-(piperazin-1-yl)-7-chloro-pyrido[3,2-d]pyrimidine (1.0 g, 4 mmole) in dichloromethane (40 ml), was added 3-chlorophenyl isocyanate (615 mg, 4 mmole). The reaction mixture was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure, resulting in the pure title compound (1.6 g, yield 99%) as a white solid which was characterized by its mass spectrum as follows: MS (m/z): 403.1 ([M+H]+, 100).
This compound was synthesized from 4-(piperazin-1-yl)-6-chloro-pyrido[3,2-d]pyrimidine (2.5 g, 10 mmole) and 3-chlorophenyl isocyanate (1.54 g, 10 mmole) using the procedure of example 69, resulting in the pure title compound (4.0 g, 99%) as a white solid which was characterized by its mass spectrum as follows: MS (m/z): 403.1 ([M+H]+, 100).
To a solution of 4-[(N-3-chlorophenylcarbamoyl)-piperazin-1-yl]-7-chloro-pyrido[3,2-d]pyrimidine (0.5 mmole) in dioxane (20 ml) and water (5 ml) was added an appropriate arylboronic acid (0.5 mmole), K2CO3 (1.5 mmole), and tetrakis (triphenylphosphine)palladium(0) (0.025 mmole). The mixture was heated at 95° C. until the starting materials disappeared on thin layer chromatography. The reaction mixture was diluted with CH2Cl2 (50 ml) and washed with a 0.5 M Na2CO3 solution (10 ml), and the organic phase was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, the mobile phase being an acetone/dichloromethane mixture (in a ratio gradually ranging from 1:3 to 1:2), resulting in the pure following compounds:
The procedure of examples 71 to 78 was repeated, using 4-[(N-3-chlorophenylcarbamoyl)-piperazin-1-yl]-6-chloro-pyrido[3,2-d]pyrimidine as the starting material, for preparing the following pure compounds:
Adding triphosgene (3.05 g, 10.14 mmole) to a solution of 6-chloro-2-carboxamido-3-amino-pyridine (3.48 g, 20.28 mmole) in dry dioxane (125 ml) under a N2 atmosphere resulted in the immediate formation of a precipitate. The dark orange reaction mixture was stirred under reflux under a N2 atmosphere for 30 minutes. Upon cooling, the solvent was removed under reduced pressure and the residue was purified by silica gel flash chromatography, the mobile phase being a CH3OH/CH2Cl2 mixture (in a ratio gradually ranging from 5:95 to 15:95), resulting in the pure title compound as a white powder (2.96 g, yield 74%) which was characterized by its mass spectrum as follows: MS (m/z): 198 ([M+H]+, 100).
A suspension of 6-chloro-pyrido[3,2-d]pyrimidin-2(1H)-4(3H)-dione (300 mg, 1.52 mmole), K2CO3 (840 mg, 6 mmole) and 3,4-dimethoxyphenylboronic acid (360 mg, 1.98 mmole) in 1,4-dioxane (22.5 ml) and water (8 ml) was purged with a nitrogen stream for 15 minutes. Tetrakis(triphenylphosphine)palladium(0) (90 mg, 76 mmole) was added and the mixture was heated to reflux for 24 hours. Upon cooling, the reaction mixture was filtered. The solid residue was recrystallized from hot acetic acid, then washed successively with acetic acid, ethyl acetate and diethyl ether, and finally dried, resulting in the pure title compound (297 mg, yield 65%) which was characterized by its mass spectrum as follows: MS (m/z): 300 ([M+H]+, 100).
6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidin-2(1H)-4(3H)-dione (2.39 g, 7.97 mmole) was suspended in POCl3 (54 ml) and triethylamine (3.1 ml, 21.8 mmole) was added. The dark brown mixture was stirred at reflux for 2.5 hours and allowed to cool down to room temperature. Most of POCl3 was removed under reduced pressure and the rest was poured into ice/water and extracted with dichloromethane. The crude residue was purified by silica gel flash chromatography, the mobile phase being a n-hexane/EtOAc mixture, in a ratio gradually ranging from 1.5:1 to 1:1, to afford the pure title compound (1.69 g, yield 63%) which was characterized by its mass spectrum as follows: MS (m/z): 336 [(M+H)+, 100].
2-chloro-4-[(N-3-methyl-phenylcarbamoyl)-piperazin-1-yl]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (156 mg, 0.3 mmole) was suspended in 1,4-dioxane (10 ml) and morpholine (0.6 mmole) was added. The reaction mixture was heated at reflux for 4 hours, allowed to cool down to room temperature and partitioned between dichloromethane and a saturated aqueous sodium bicarbonate solution. The solid residue from the organic phase was purified by preparative thin layer chromatography on silica using a mixture of ethyl acetate and n-hexane (in a ratio of 1:4) as the mobile phase, to afford the pure title compound (21 mg, yield 12%) which was characterized by its mass spectrum as follows: MS (m/z): 570 ([M+H]+, 100).
28 mg (0.7 mmole) of 60% by weight NaH in mineral oil was suspended in dry tetrahydrofuran (5 ml) under a N2 atmosphere, followed by the addition of n-butanol (0.6 mmole). Then, 2-chloro-[(N-3-methyl-phenylcarbamoyl)-piperazin-1-yl]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (149 mg, 0.29 mmole) was added. The mixture was heated at reflux under N2 for 2.5 hours and then diluted with water. The crude product was extracted four times from the reaction mixture with ethyl acetate. The organic extracts were combined, dried over MgSO4 and evaporated to dryness under reduced pressure. Preparative thin layer chromatography on silica using a n-hexane/ethyl acetate 1:4 mixture as eluent afforded the pure title compound (148 mg, yield 93%) which was characterized by its mass spectrum as follows: MS (m/z): 557 ([M+H]+, 100).
24 mg (0.6 mmole) of 60% by weight NaH in mineral oil was suspended in dry tetrahydrofuran (3 ml) under a N2 atmosphere followed by the addition of methanol (0.4 mmole). The mixture was stirred at room temperature for 15 minutes, and 2-chloro-4-[(N-3-methyl-phenylcarbamoyl)-piperazin-1-yl]-6-(3,4-dimethoxy phenyl)-pyrido[3,2-d]pyrimidine (104 mg, 0.2 mmole) was added. The solution was heated at reflux under N2 for 1 hour and diluted with water. The crude product was extracted from the reaction mixture with ethyl acetate and the organic layer was washed with brine, dried over MgSO4 and evaporated to dryness under reduced pressure. Preparative thin layer chromatography on silica, using a n-hexane/ethyl acetate mixture in a ratio of 1:5 as eluent, afforded the pure title compound (52 mg, yield 51%) which was characterized by its mass spectrum as follows: MS (m/z): 515 ([M+H]+, 100).
A white suspension of 2-chloro-4-[(N-3-methyl-phenylcarbamoyl)-piperazin-1-yl]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (104 mg, 0.2 mmole), K2CO3 (64 mg, 0.46 mmole), and p-toluidine (46 mg, 0.43 mmole) in a mixture of 1,4-dioxane/t-BuOH 5:1 (2 ml) was stirred at room temperature under nitrogen for 5 minutes. Thereafter, tetrakis(triphenylphosphine)palladium(0) (26 mg, 23 μmole) was added and the reaction mixture was heated at reflux under a N2 atmosphere for 48 hours. Upon cooling, the mixture was diluted with water and extracted three times with ethyl acetate (brine added). The combined organic extracts were dried over Na2SO4, filtered and evaporated under reduced pressure. The crude residue was purified by column chromatography on silica using an ethyl acetate/n-hexane mixture as the mobile phase (in a ratio gradually ranging from 1:1 to 3:1), resulting in the pure title compound (30 mg, yield 25%) which was characterized by its mass spectrum as follows: MS (m/z): 590 ([M+H]+, 100).
A suspension of 2-chloro-4-[(N-3-methyl-phenylcarbamoyl)-piperazin-1-yl]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (106 mg, 0.20 mmole), K2CO3 (62 mg, 0.45 mmole) and 3-chloro-4-fluoroaniline (60 mg 0.40 mmole) in a 1,4-dioxane/t-BuOH 5:1 mixture (2 ml) was purged with nitrogen for 15 minutes. Thereafter, tetrakis(triphenylphosphine)palladium(0) (28 mg, 24 μmol) was added and the reaction mixture was heated at reflux under a N2 atmosphere for 20 hours. Upon cooling, the mixture was partitioned between ethyl acetate and brine. The organic phase was evaporated under reduced pressure and the crude residue was purified by flash chromatography on silica, using an ethyl acetate/n-hexane mixture as the mobile phase (in a ratio gradually ranging from 1:1 to 4:1), thus affording the pure title compound (60 mg, yield 47%) which was characterized by its mass spectrum as follows: MS (m/z): 628 ([M+H]+, 100).
A suspension of 2,4-diamino-6-chloropyrido[3,2-d]pyrimidine (378 mg, 1.93 mmole), K2CO3 (1075 mg, 7.78 mmole) and 2-methoxy-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenol (599 mg, 2.32 mmole) in 1,4-dioxane (29 ml) and water (6 ml) was purged with a nitrogen stream for 30 minutes. Then, tetrakis(triphenylphosphine)palladium(0) (240 mg, 0.21 mmole) was added and purging with N2 was continued for 15 minutes. The reaction mixture was then heated at reflux under a N2 atmosphere for 2 hours. Upon cooling, the mixture was partitioned between CH2Cl2 and brine and the organic phase was dried over Na2SO4, filtered and evaporated under reduced pressure. Purification of the residue by silica gel flash chromatography with 10% methanol and 1% Et3N in CH2Cl2 as mobile phase, afforded the pure title compound (375 mg, yield 69%) which was characterized by its mass spectrum as follows: MS (m/z): 284 ([M+H]+, 100).
A suspension of 2,4-diamino-6-chloropyrido[3,2-d]pyrimidine (464 mg, 2.37 mmole), K2CO3 (1332 mg, 9.64 mmole), 3-chloro-4-methoxyphenyl boronic acid (907 mg, 4.86 mmole) in 1,4-dioxane (35.5 ml) and water (7 ml) was purged with a stream of nitrogen for 15 minutes. Then, tetrakis(triphenylphosphine)palladium(0) (278 mg, 0.24 mmole) was added and the reaction mixture was heated at reflux under a N2 atmosphere for 4 hours. Upon cooling, the mixture was partitioned between CH2Cl2 and a saturated aqueous sodium bicarbonate solution. The organic phase was dried over Na2SO4, filtered and evaporated under reduced pressure. The crude residue was purified by silica gel flash chromatography, using methanol and 1% Et3N in CH2Cl2 as eluent, gradually increasing the methanol concentrations from 5% to 10%, to afford the pure title compound (277 mg, yield 39%) which was characterized by its mass spectrum as follows: MS (m/z): 302 ([M+H]+, 100).
A suspension of 2,4-diamino-6-(4-hydroxy-3-methoxy)-pyrido[3,2-d]pyrimidine (268 mg, 0.95 mmole) in 6 M aqueous HCl (7.6 ml) was refluxed for 26 hours. The cooled reaction mixture was stored at 4° C. for 16 hours. The yellow precipitate obtained was filtered off, washed with water until neutral pH value of the filtrate and dried to afford 243 mg (yield 90%) of the pure title compound which was characterized by its mass spectrum as follows: MS (m/z): 285 ([M+H]+, 100)
A suspension of 2-amino-6-(4-hydroxy-3-methoxyphenyl)-pyrido[3,2-d]pyrimidin-4(3H)one (66 mg, 0.23 mmole), p-toluenesulphonic acid monohydrate (10 mg, 53 μmole), (NH4)2SO4 (11 mg, 83 μmole), 1,1,1,3,3,3-hexamethyldisilazane (1.15 mmole) and morpholine (1.83 mmole) in toluene (2 ml) was refluxed for 33 hours. The reaction mixture was allowed to cool down and partitioned between ethyl acetate and brine/saturated NaHCO3 aqueous solution. The aqueous layer was extracted two times with ethyl acetate. The combined organic layers were dried over MgSO4, filtered and evaporated under reduced pressure. The crude residue was purified by preparative thin layer chromatography on silica with 5% MeOH and 1% Et3N in CH2Cl2 as mobile phase to afford the pure title compound (68 mg, yield 84%) which was characterized by its mass spectrum as follows: MS (m/z): 354 ([M+H]+, 100).
A yellow suspension of 2-amino-4-(N-morpholino)-6-(4-hydroxy-3-methoxyphenyl)-pyrido[3,2-d]pyrimidine (32 mg, 90 μmole), anhydrous potassium carbonate (30 mg, 0.22 mmole) and iodoethane (0.36 mmole) in acetone (2 ml) was refluxed under a nitrogen atmosphere. After 24 hours, second aliquots of K2CO3 and iodoethane were added and the reaction was continued for another 24 hours. Upon cooling, the reaction mixture was partitioned between EtOAc and a 5% aqueous sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over MgSO4, filtered and evaporated under reduced pressure. Preparative thin layer chromatography of the crude residue on silica, using 5% methanol, 1% Et3N in CH2Cl2 as mobile phase, afforded the pure title compound (26 mg, yield 76%) which was characterized by its mass spectrum as follows: MS (m/z): 382 ([M+H]+, 100).
A dark orange solution of 2-amino-4-(N-morpholino)-6-(4-hydroxy-3-methoxyphenyl)-pyrido[3,2-d]pyrimidine (68 mg, 0.19 mmole), anhydrous potassium carbonate (53 mg, 0.38 mmole) and cyclopentyl iodide (0.75 mmole) in dimethylformamide (4 ml) was stirred at 60° C. After 24 hours, a second aliquot of cyclopentyl iodide was added and the reaction was continued for another 24 hours. Upon cooling, the reaction mixture was partitioned between ethyl acetate and brine/5% NaHCO3 aqueous solution. The aqueous layer was extracted two times with ethyl acetate. The combined organic layers were dried over MgSO4, filtered and evaporated under reduced pressure. Preparative thin layer chromatography of the crude residue on silica using 5% methanol in CH2Cl2 as mobile phase, afforded the pure title compound (6 mg, yield 7%) which was characterized by its mass spectrum as follows: MS (m/z): 422 ([M+H]+, 100).
To a yellow solution of 2-amino-4-(N-morpholino)-6-(3-methoxy-4-hydroxyphenyl)-pyrido[3,2-d]pyrimidine (107 mg, 0.30 mmole) in dry dimethylformamide (10 ml), was added 60% by weight NaH in mineral oil (0.93 mmole), resulting in an orange suspension. Then, 2-iodopropane (6.02 mmole) was added and the reaction mixture was stirred at room temperature for 40 minutes. The reaction mixture was partitioned between ethyl acetate and brine. The organic phase is dried over MgSO4, filtered and evaporated under reduced pressure. Preparative thin layer chromatography of the crude residue on silica, using 5% methanol, 1% Et3N in CH2Cl2 as mobile phase, afforded the title compound (83 mg, 70%) which was characterized by its mass spectrum as follows: MS (m/z): 396 ([M+H]+, 100).
A suspension of 2-amino-6-(4-hydroxy-3-methoxyphenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one (227 mg, 0.80 mmole), p-toluenesulphonic acid monohydrate (88 μmole), (NH4)2SO4 (0.12 mmole), 1,1,1,3,3,3-hexamethyldisilazane (3.98 mmole) and piperazine (11.72 mmole) in toluene (3 ml) was refluxed for 24 hours. Upon cooling, the reaction mixture was partitioned between ethyl acetate and 5% NaHCO3 aqueous solution/brine. The aqueous layer was extracted 3 times with ethyl acetate. The combined organic layers were dried over MgSO4, filtered and evaporated under reduced pressure. The crude residue was purified by preparative thin layer chromatography on silica using 15% methanol, 1% Et3N in CH2Cl2 as mobile phase, affording the title compound (74 mg, yield 62%) which was characterized by its mass spectrum as follows: MS (m/z): 353 ([M+H]+, 100).
A solution of 4-fluorophenyl isocyanate (0.39 mmole) in dimethylformamide (0.5 ml) was added to a yellow suspension of 2-amino-4-(N-piperazin-1-yl)-6-(4-hydroxy-3-methoxy)-pyrido[3,2-d]pyrimidine (0.31 mmole) in dimethylformamide (2 ml). The mixture was stirred at room temperature for 1 hour. The solvent was evaporated in vacuo. Preparative thin layer chromatography of the crude residue on silica using 5% methanol, 1% Et3N in CH2Cl2 as mobile phase, afforded the pure title compound (100 mg, yield 66%) which was characterized by its mass spectrum as follows: MS (m/z): 490 ([M+H]+, 100).
A suspension of 2-amino-4-[(N-4-fluoro-phenyl-carbamoyl)-piperazin-1-yl]-6-(4-hydroxy-3-methoxyphenyl)-pyrido[3,2-d]pyrimidine (0.13 mmole), anhydrous potassium carbonate (0.80 mmole) and iodoethane (1.23 mmole) in acetone (5 ml) was refluxed for 24 hours. Upon cooling, the reaction mixture was partitioned between ethyl acetate and brine. The aqueous layer was extracted with ethyl acetate. The combined organic layers were dried over MgSO4, filtered and evaporated under reduced pressure. Preparative thin layer chromatography of the residue on silica using 5% methanol in CH2Cl2 as mobile phase, afforded the pure title compound (15 mg, yield 22%) which was characterized by its mass spectrum as follows: MS (m/z): 518 ([M+H]+, 100).
A suspension of 2-amino-4-[(N-4-fluoro-phenyl-carbamoyl)-piperazin-1-yl]-6-(4-hydroxy-3-methoxy-phenyl)-pyrido[3,2-d]pyrimidine (96 μmole), anhydrous potassium carbonate (0.22 mmole) and 2-iodopropane (0.96 mmole) in acetone (7 ml) was refluxed under a nitrogen atmosphere for 20 hours. Then, another aliquot of 2-iodopropane was added and the reaction was continued for another 24 hours. Upon cooling, the reaction mixture was partitioned between ethyl acetate and brine and the aqueous layer was extracted several times with ethyl acetate. The combined organic layers were dried over MgSO4, filtered and evaporated under reduced pressure. Purification of the crude residue by silica gel flash chromatography, using 10% methanol in CH2Cl2 as mobile phase, afforded the pure title compound (20 mg, yield 39%) which was characterized by its mass spectrum as follows: MS (m/z): 532 ([M+H]+, 100).
m-toluoyl isocyanate (0.55 mmole) was added to a suspension of 2-amino-4-(N-piperazin-1-yl)-6-(4-hydroxy-3-methoxyphenyl)-pyrido[3,2-d]pyrimidine (0.55 mmole) in dimethylformamide (7 ml). The mixture was stirred at room temperature for 20 minutes, and then partitioned between ethyl acetate and a 5% NaHCO3 aqueous solution. The aqueous layer was extracted two times with ethyl acetate. The combined organic layers were dried over MgSO4, filtered and evaporated under reduced pressure. Purification of the crude residue by preparative thin layer chromatography on silica using 5% methanol, 1% Et3N in CH2Cl2 as eluent, afforded the pure title compound (123 mg, yield 46%) which was characterized by its mass spectrum as follows: MS (m/z): 486 ([M+H]+, 100).
To a suspension of 4-chloro-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (0.597 mmole) in isopropanol (20 ml) was added 1-(4-methyl)phenyl-piperazine (1.2 mmole). The reaction mixture was heated at 80° C. for 2 hours, after which the suspension became a yellow solution. The solvent was evaporated in vacuo. The residue was redissolved in ethyl acetate and extracted with a NaOH solution (1 N). The combined organic layers were evaporated in vacuo and purified by silica gel column chromatography (the mobile phase being a mixture of methanol and dichloromethane in a ratio gradually ranging from 1:99 to 2:98), resulting in the title compound (191 mg, yield 73%) which was characterized by its mass spectrum as follows: MS (m/z): 442 ([M+H]+, 100).
The procedure of example 105 was performed, but using 1-(4-fluoro)phenylpiperazine as the starting material, thus resulting in the pure title compound which was characterized by its mass spectrum as follows: MS (m/z): 446 ([M+H]+, 100).
To a suspension of 4-chloro-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (1.47 mmole) in isopropanol (50 ml) was added piperazine (1.2 mmole). The reaction mixture was heated at 80° C. for 2 hours. Volatiles were evaporated in vacuo. The crude residue was purified by silica gel flash chromatography, the mobile phase being a methanol/dichloromethane mixture with an 0.5% aqueous NH3 solution (in a ratio gradually ranging from 2:98 to 3:97), resulting in the pure title compound (351 mg, yield 68%) which was characterized by its mass spectrum as follows: MS (m/z): 352 ([M+H]+, 100).
To a solution of 4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]-pyrimidine (0.26 mmole) in dimethylformamide (20 ml) was added an appropriate isocyanate (0.39 mmole). The reaction mixture was stirred at room temperature for 2 hours. The solvents was evaporated in vacuo and the crude residue was purified by silica gel flash chromatography, the mobile phase being a mixture of methanol and dichloromethane in a ratio gradually ranging from 2:98 to 3:97, affording the pure title compounds in yields from 65 to 80% depending upon the relevant isocyanate. The following individual compounds were made according to this procedure:
To a solution of 4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]-pyrimidine (0.18 mmole) in dimethylformamide (20 ml) was added triethylamine (0.26 mmole) and p-chloro-phenoxy acetyl chloride (0.23 mmole). The reaction mixture was stirred at room temperature for 3 hours, then quenched with water. The aqueous phase was extracted with dichloromethane. The combined organic layers were evaporated in vacuo. The residue was purified by silica gel flash chromatography, the mobile phase being a methanol/dichloromethane mixture in a ratio of 2:98, affording the pure title compound (66 mg, yield 71%) which was characterized by its mass spectrum as follows: MS (m/z): 521 ([M+H]+, 100).
To a solution of 6-chloro-pyrido[3,2-d]pyrimidin-4(3H)one (1.94 mmole) in 1,4-dioxane (40 ml) and water (20 ml) was added 4-methoxy-3-methylphenyl boronic acid (2.33 mmole), potassium carbonate (4.85 mmole) and tetrakis(triphenylphosphine)palladium(0) (0.097 mmole). The reaction mixture was refluxed for two hours, cooled to room temperature and the solvents were evaporated in vacuo. The residue was adsorbed on silica and purified by silica gel column chromatography (the mobile phase being a methanol/dichloromethane mixture in a ratio of 3:97), affording the title compound as a pure white powder (398 mg, yield 77%) which was characterized by its mass spectrum as follows: MS (m/z): 268 ([M+H]+, 100).
To a suspension of 6-(3-methyl-4-methoxyphenyl)-pyrido[3,2-d]pyrimidin-4(3H)one (1.41 mmole) in toluene (80 ml) was added phosphorus oxychloride (4.23 mmole) and 2,6-lutidine (4.23 mmole). The reaction mixture was refluxed for 16 hours until a black solution was obtained. After evaporation to dryness, the residue was redissolved in ethyl acetate and extracted with a saturated sodium bicarbonate solution. The combined organic layers were evaporated in vacuo. The residue was purified by silica gel column chromatography (the mobile phase being a ethylacetate/hexane mixture in a ratio gradually ranging from 2:8 to 3:7), resulting in the pure title compound (300 mg, yield 74%) which was characterized by its mass spectrum as follows: MS (m/z): 287 ([M+H]+, 100).
To a suspension of 4-chloro-6-(3-methyl-4-methoxyphenyl)-pyrido[3,2-d]pyrimidine (0.99 mmole) in isopropanol (40 ml) was added piperazine (1.99 mmole). The reaction mixture was heated at 80° C. for 2 hours. The solvents were evaporated in vacuo. The crude residue was purified by silica gel flash chromatography (the mobile phase being a mixture of methanol and dichloromethane with an 0.5% aqueous NH3 solution (in a ratio gradually ranging from 2:98 to 3:97), resulting in the pure title compound (259 mg, yield 78%) which was characterized by its mass spectrum as follows: MS (m/z): 336 ([M+H]+, 100).
To a solution of 4-(N-piperazin-1-yl)-6-(3-methyl-4-methoxyphenyl)-pyrido[3,2-d]-pyrimidine (0.25 mmole) in DMF (30 ml) was added 3-chlorophenyl isocyanate (0.38 mmole). The reaction mixture was stirred at room temperature for 2 hours. The solvents were evaporated in vacuo and the crude residue was purified by silica gel flash chromatography, the mobile phase being a mixture of methanol and dichloromethane in a ratio gradually ranging from 2:98 to 3:97, affording the pure title compound (81 mg, yield 66%) which was characterized by its mass spectrum as follows: MS (m/z): 490 ([M+H]+, 100).
The procedure of example 117 was followed, but using 4-chlorophenyl isocyanate as the starting material. The pure title compound was isolated in a yield of 81% and was characterized by its mass spectrum as follows: MS (m/z): 490 ([M+H]+, 100).
To a solution of 4-[(N-3-chloro-phenylcarbamoyl)-piperazin-1-yl]-6-chloro-pyrido[3,2-d]pyrimidine (0.51 mmole) in 1,4-dioxane (15 ml) and water (5 ml) was added 2-methoxy-4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-phenol (0.51 mmole), potassium carbonate (1.53 mmole) and tetrakis(triphenylphosphine)palladium(0) (0.02 mmole). The reaction mixture was refluxed for two hours, cooled down to room temperature and the solvents were evaporated in vacuo. The residue was purified by silica gel column chromatography (the mobile phase being an acetone/dichloromethane mixture in a ratio of 20:80), affording the title compound as a pure white powder (135 mg, yield 54%) which was characterized by its mass spectrum as follows: MS (m/z): 492 ([M+H]+, 100).
To a solution of 4-[(N-4-chloro-phenylcarbamoyl)-piperazin-1-yl]-6-(3-methoxy-4-hydroxyphenyl-pyrido[3,2-d]pyrimidine (0.19 mmole) in dry dimethylformamide (15 ml) was added potassium carbonate (0.19 mmole). This mixture was stirred at room temperature for 30 minutes under nitrogen and then, ethyl iodide (0.19 mmole) was added. The reaction mixture was stirred at room temperature for 16 hours. The solvent was evaporated in vacuo and the residue was purified by silica gel flash chromatography (the mobile phase being a methanol/dichloromethane mixture in a ratio of 2:98), affording the pure title compound as a white powder (67 mg, yield 68%) which was characterized by its mass spectrum as follows: MS (m/z): 520 ([M+H]+, 100).
The procedure of example 120 was followed, but using 2-iodopropane as the starting material. The pure title compound was isolated and characterized by its mass spectrum as follows: MS (m/z): 533 ([M+H]+, 100).
A suspension of 3-chlorophenylacetic acid (2 mmole) in thionyl chloride (10 ml) was refluxed for 1 hour. The excess thionyl chloride was removed under reduced pressure to yield crude 3-chloro phenyl acetic acid chloride. This crude residue was redissolved in dichloromethane (10 ml) and this solution was added to a solution of 4-(piperazin-1-yl)-6-chloro-pyrido[3,2-d]pyrimidine (2 mmole) in dichloromethane (10 ml). The resulting mixture was stirred at room temperature for 1 hour. The solvents were removed by evaporation in vacuo. The crude residue was purified by silica gel column chromatography, the mobile phase being a MeOH/dichloromethane mixture in a ratio of 1:40, affording the pure title compound (yield 60%) as a yellowish solid which was characterized by its mass spectrum as follows: MS (m/z): 403.1 ([M+H]+, 100).
The reaction of 4-morpholino-6-chloro-pyrido[3,2-d]pyrimidine and 3,4-dichlorophenylboronic acid afforded the pure title compound (yield 97%) as a yellowish solid which was characterized by its mass spectrum as follows: MS (m/z): 361.2 ([M+H]+, 100).
The reaction of 4-morpholino-6-chloro-pyrido[3,2-d]pyrimidine and 4-chlorophenylboronic acid afforded the pure title compound (yield 92%) as a white solid solid which was characterized by its mass spectrum as follows: MS (m/z): 341.2 ([M+H]+, 100).
The reaction of 4-[(N-3-chlorophenylacetyl)piperazin-1-yl]-6-chloro-pyrido[3,2-d]pyrimidine and 3,4-dichlorophenyl boronic acid afforded the pure title compound (yield 86%) as a yellowish solid which was characterized by its mass spectrum as follows: MS (m/z): 512.2 ([M+H]+, 100).
To a degassed suspension of 2-amino-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (6 mmole), an appropriate aryl boronic acid (6.6 mmole) and potassium carbonate (30 mmole) in a mixture of dioxane (120 ml) and H2O (30 ml), was added a catalytic amount of tetrakis(triphenylphosphine)palladium(0) (0.9 g). The mixture was refluxed for 24 hours and after cooling to room temperature, the reaction mixture was filtered. The filtrate was acidified with 5 N HCl to pH 4 and the resulting precipitate was filtered off, washed successively with H2O, ethanol and diethylether, and further dried under vacuum to afford the desired compound in a yield between 65 and 85%, depending upon the relevant aryl boronic acid used. The following compounds were synthesized according to this procedure:
A 2-amino-6-aryl-pyrido[3,2-d]pyrimidin-4(3H)-one (2.0 g) was suspended in acetic anhydride (180 ml) and acetic acid (20 ml) and the mixture was refluxed for 16 hours. The hot suspension was filtered and the filtrate was concentrated under reduced pressure until crystallization started. The precipitate was filtered off to give the pure title compound in a yield varying from 70 to 80%, depending upon the 6-aryl substituent being present in the starting material. The following compounds were synthesized according to this procedure:
A suspension of 1,2,4-triazole (120 mmole) and phosphorus oxychloride (36 mmole) in dry acetonitrile (150 ml) was added to a stirred suspension of a 2-acetamido-6-aryl-pyrido[3,2-d]pyrimidin-4(3H)-one (12 mmole) (obtained in examples 133 to 139) and triethylamine (36 mmole) in dry acetonitrile (150 ml). The mixture was stirred at room temperature under nitrogen for 70 hours and the yellow precipitate formed was filtered off, then successively washed with ethanol and ether, and further dried over P2O5 in a vacuum dessicator to afford the pure title compounds. Yields varied between 63% and 90%, depending upon the 6-aryl substituent being present. The following compounds were synthesized according to this procedure:
To a suspension of a 2-acetamido-4-(1,2,4-triazolyl)-6-aryl-pyrido[3,2-d]pyrimidine (1.25 mmole; obtained in examples 140 to 147) in dioxane (50 ml) was added piperazine (2.5 mmole). The reaction mixture was stirred for 16 hours at 50° C. The solvent was evaporated and the crude residue was purified by preparative thin layer chromatography on silica, using a methanol/dichloromethane mixture in a ratio of 20:80 as mobile phase, affording the pure title compounds in yields varying between 30 and 40%, depending upon the 6-aryl substituent being present. The following compounds were made according to this procedure:
To a solution of a 2-acetamido-4-(piperazin-1-yl)-6-aryl-pyrido[3,2-d]pyrimidine (0.5 mmole) in dimethylformamide (5 ml) was added 3-chlorophenyl isocyanate (0.75 mmole). The reaction mixture was stirred for 16 hours at room temperature. The solvent was evaporated in vacuo, affording a crude 2-acetamido-4-[(N-3-chloro-phenyl-carbamoyl)-piperazin-1-yl]-6-aryl-pyrido[3,2-d]pyrimidine as an intermediate. This crude residue was dissolved in a mixture of CH2Cl2 (10 ml) and sodium ethoxide 0.2 N (10 ml). The suspension was stirred for 16 hours and neutralized with 5-6 N HCl in isopropyl alcohol, resulting in a crude 2-amino-4-[(N-3-chloro-phenyl-carbamoyl)-piperazin-1-yl]-6-aryl-pyrido[3,2-d]pyrimidine as the final product. This crude product was purified by preparative thin layer chromatography, the mobile phase consisting of CH3OH/CH2Cl2 mixtures in a ratio of 10:90, yielding the pure title compounds, in yields varying from 20 to 40%, depending on the 6-aryl substituent being present. The following compounds were synthesized according to this procedure (each time through the corresponding intermediate having the 2-amino group protected in the form of acetamido):
To a suspension of a 2-acetamido-6-aryl-pyrido[3,2-d]pyrimidin-4(3H)-one (1 mmole) in toluene (10 ml) was added morpholine (4 mmole), p-toluene sulfonic acid (0.1 mmole), ammonium sulfate (0.1 mmole) and 1,1,1,3,3,3-hexamethyldisilazane (8 mmole). The reaction mixture was refluxed for 48 hours until a brown solution was formed. The solvent was evaporated in vacuo and the crude resulting residue was redissolved in dichloromethane and extracted successively with a saturated sodium bicarbonate aqueous solution and water. The combined organic layers were dried over sodium sulfate and evaporated in vacuo, resulting in a crude 2-amino-4-morpholino-6-aryl-pyrido[3,2-d]pyrimidine as a final product. This crude residue was purified by preparative thin layer chromatography on silica, using a methanol/dichloromethane mixture in a ratio of 10:90 as mobile phase, affording the pure final compounds in yields between 20 and 30%, depending on the 6-aryl substituent being present. The following final compounds were synthesized according to this procedure (each time through the corresponding intermediate having the 2-amino group protected in the form of acetamido):
To a suspension of a 2-acetamido-4-(1,2,4-triazolyl)-6-aryl-pyrido[3,2-d]pyrimidine (0.5 mmole) in dioxane (5 ml) was added morpholine (1 mmole). The reaction mixture was stirred for 16 hours at 50° C. The solvent was evaporated in vacuo yielding a crude 2-acetamido-4-morpholino-6-aryl-pyrido[3,2-d]pyrimidine as an intermediate product. This crude residue was dissolved in a mixture of CH2Cl2 (10 ml) and sodium ethoxide 0.2 N (10 ml). The suspension was stirred for 16 hours and neutralized with 5-6 N HCl in isopropyl alcohol, resulting in a crude 2-amino-4-morpholino-6-aryl-pyrido[3,2-d]pyrimidine as a final product. This crude product was purified by preparative thin layer chromatography, the mobile phase consisting of a CH3OH/CH2Cl2 mixtures in a ratio of 10:90, affording the pure title compounds, in yields varying from 20 to 40% depending on the 6-aryl substituent being present. The following compounds were synthesized according to this procedure (each time through the corresponding intermediate having the 2-amino group protected in the form of acetamido):
General Procedure
To a degassed suspension of 2-amino-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (1.96 g, 10 mmol), an appropriate phenyl boronic acid (11 mmol) and potassium carbonate (6.9 g, 50 mmol) in a mixture of dioxane (180 ml) and H2O (50 ml), was added a catalytic amount of tetrakis(triphenylphosphine)palladium(0) (750 mg). The suspension was refluxed for 16 hours and finally became a solution. After cooling to room temperature, the reaction mixture was filtered. The filtrate was acidified with 5 N HCl to pH 4 and the resulting precipitate was filtered off. It was washed successively with H2O, ethanol, diethylether and dried under vacuum to yield the desired product.
The following compounds were synthesized according to this procedure:
Obtained from 3-methyl-4-fluoro-phenyl boronic acid in 70% yield.
MS (m/z): 271 ([M+H]+, 100)
Obtained from 3,4-dichlorophenyl boronic acid in 91% yield.
MS (m/z): 307, 309 ([M+H]+, 100)
Obtained from 4-fluoro-phenyl boronic acid in 78% yield.
MS (m/z): 257 ([M+H]+, 100)
Obtained from 1,4-benzodioxane-6-boronic acid in 82% yield.
MS (m/z): 297 ([M+H]+, 100)
Obtained from 3,4-methylenedioxyphenyl boronic acid in 71% yield.
MS (m/z): 283 ([M+H]+, 100)
2-Amino-6-aryl-pyrido[3,2-d]pyrimidin-4(3H)-one (10 mmol) was suspended in acetic anhydride (300 ml) and the mixture was refluxed for 2 hours till a clear solution was obtained. The solution was concentrated under reduced pressure until crystallization started. The precipitate was filtered off to give the pure title compound. The following compounds were synthesized according to this procedure:
Obtained from 2-amino-6-(3-methyl-4-fluoro-phenyl)-pyrido[3,2-d]pyrimidin-4(3)-one in 90% yield.
MS (m/z): 313 ([M+H]+, 100)
Obtained from 2-amino-6-(3,4-dichloro-phenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one in 90% yield.
MS (m/z): 349, 351 ([M+H]+, 100)
Obtained from 2-amino-6-(4-fluoro-phenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one in 78% yield.
MS (m/z): 299 ([M+H]+, 100)
Obtained from 2-amino-6-(1,4-benzodioxane)-pyrido[3,2-d]pyrimidin-4(3H)-one in 68% yield.
MS (m/z): 339 ([M+H]+, 100)
Obtained from 2-amino-6-(3,4-methylenedioxyphenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one in 74% yield.
MS (m/z): 325 ([M+H]+, 100)
To a suspension of 2-acetamido-6-(3-methyl-4-fluorophenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one (312 mg, 1 mmol) in toluene (10 ml) was added morpholine (4 mmol, 0.23 ml), p-toluene sulfonic acid (0.1 mmol, 19 mg), ammonium sulfate (13 mg, 0.1 mmol) and 1,1,1,3,3,3-hexamethyldisilazane (2 ml, 8 mmol). The reaction mixture was refluxed for 48 hours till a brown solution was formed. The solvents were evaporated in vacuo, yielding crude 2-acetamido-4-(morpholino)-6-(4-methyl-3-fluoro-phenyl)-pyrido[3,2-d]pyrimidine. The residue was redissolved in a mixture of dichloromethane and ethanol (in a ratio of 80/20, 10 ml). A sodium ethoxide solution (0.2 N solution) was added till pH 12 and the resulting mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo. The crude residue was purified by preparative TLC on silica, using a methanol/dichloromethane mixture in a ratio of 10:90 as mobile phase, yielding pure 2-amino-4-(morpholino)-6-(3-methyl-4-fluorophenyl)-pyrido[3,2-d]pyrimidine (80 mg, 25%).
MS (m/z): 340 ([M+H]+, 100)
UV (MeOH, m): 211, 278, 361
General Procedure
A suspension of 1,2,4-triazole (345 mg, 5 mmol) and phosphorus oxychloride (0.11 ml, 1.25 mmol) in dry acetonitrile (10 ml) was stirred under a nitrogen atmosphere for 15 minutes. This suspension was added to another suspension of 2-acetamido-6-aryl-pyrido[3,2-d]pyrimidin-4(3H)-one (1 mmol) and triethylamine (0.4 ml, 3 mmol) in dry acetonitrile (10 ml). The resulting mixture was stirred at 50° C. under nitrogen for 24 hours. The solvents were evaporated in vacuo. The crude residue was redissolved in dichloromethane and extracted with a diluted hydrochloric acid solution (HCl 0.01 N). The combined organic layers were evaporated yielding the title compounds, which were used for further reaction without any additional purification.
The following compounds were made according to this procedure:
Obtained from 2-acetamido-6-(3,4-dichloro-phenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one in 80% yield.
MS (m/z): 400, 402 ([M+H]+, 100)
Obtained from 2-acetamido-6-(4-fluoro-phenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one in 72% yield.
MS (m/z): 350 ([M+H]+, 100)
Obtained from 2-acetamido-6-(1,4-benzodioxane)-pyrido[3,2-d]pyrimidin-4(3H)-one in 59% yield.
MS (m/z): 390 ([M+H]+, 100)
Obtained from 2-acetamido-6-(3,4-methylenedioxyphenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one in 68% yield.
MS (m/z): 376 ([M+H]+, 100)
To a suspension of 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-dichlorophenyl)-pyrido[3,2-d]pyrimidine (400 mg, 1 mmol) in dioxane (10 ml) was added morpholine (174 mg, 2 mmol). The reaction mixture was stirred overnight at 50° C. The solvents were evaporated in vacuo yielding crude 2-acetamido-4-(morpholino)-6-(3,4-dichlorophenyl)-pyrido[3,2-d]pyrimidine. The residue was redissolved in a mixture of dichloromethane and ethanol (in a ratio of 80/20, 10 ml). A sodium ethoxide solution (0.2 N solution) was added till pH 12 and the resulting mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo. The crude residue was purified by preparative TLC on silica, using a methanol/dichloromethane mixture in a ratio of 10:90 as mobile phase, yielding the pure title compound (220 mg, 60%).
MS (m/z): 376, 378 ([M+H]+, 100)
UV (MeOH, m): 282, 365
To a suspension of 2-acetamido-4-(1,2,4-triazolyl)-6-aryl-pyrido[3,2-d]pyrimidine (1 mmol) in dioxane (20 ml) was added piperazine (172 mg, 2 mmol). The reaction mixture was stirred overnight at 50° C. The solvents were evaporated in vacuo and the crude residue was purified by preparative TLC on silica, using a methanol/dichloromethane mixture in a ratio of 10:90 as mobile phase, yielding the pure title compounds.
The following compounds were prepared according to this procedure:
Obtained from 2-acetamido-4-(1,2,4-triazolyl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine in 68% yield.
MS (m/z): 368 ([M+H]+, 100)
Obtained from 2-acetamido-4-(1,2,4-triazolyl)-6-(1,4-benzodioxane)-pyrido[3,2-d]pyrimidine
MS (m/z): 407 ([M+H]+, 100)
Obtained from 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-methylenedioxyphenyl)-pyrido[3,2-d]pyrimidine
MS (m/z): 393 ([M+H]+, 100)
Obtained from 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-dichlorophenyl)-pyrido[3,2-d]pyrimidine
MS (m/z): 416, 418 ([M+H]+, 100)
To a solution of 2-acetamido-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (367 mg, 1 mmol) in DMF (10 ml) was added 4-chloro-benzyl isocyanate (201 mg, 1.2 mmol). The solution was stirred overnight at room temperature. The solvents were evaporated in vacuo yielding crude 2-acetamido-4-[(N-4-chloro-benzylcarbamoyl)-piperazin-1-yl]-6-(4-fluoro-phenyl)-pyrido[3,2-d]pyrimidine. The residue was redissolved in a mixture of dichloromethane and ethanol (in a ratio of 80/20, 10 ml). A sodium ethoxide solution (0.2 N solution) was added till pH 12 and the resulting mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo. The crude residue was purified by preparative TLC on silica, using a methanol/dichloromethane mixture in a ratio of 10:90 as mobile phase, yielding the pure title compound (280 mg, 58%).
MS (m/z): 492, 494 ([M+H]+, 100)
UV (MeOH, m): 245, 350, 460, 560
This compound was synthesized according to the procedure of example 184, using N-acetyl-piperazine and 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-methylenedioxyphenyl)-pyrido[3,2-d]pyrimidine as starting materials.
MS (m/z): 393 ([M+H]+, 100)
This compound was prepared according to the procedure of example 184, using 4-[2-(piperazin-1-yl acetic acid N-(2-thiazolyl)-amide) and 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-methylenedioxyphenyl)-pyrido[3,2-d]pyrimidine as starting materials.
MS (m/z): 491 ([M+H]+, 100)
This compound was obtained using the procedure of example 184, using 2-furoyl-piperazine and 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-methylenedioxyphenyl)-pyrido[3,2-d]pyrimidine as starting materials.
MS (m/z): 445 ([M+H]+, 100)
To a solution of 2-acetamido-4-(N-piperazin-1-yl)-6-(3,4-methylenedioxyphenyl)-pyrido[3,2-d]pyrimidine (60 mg, 0.16 mmol) in pyridine (5 ml) was added was added 4-chloro-phenoxy acetyl chloride (80 mg, 0.4 mmol). The solution was stirred overnight at 50° C. The solvents were evaporated in vacuo, thus yielding crude 2-acetamido-4-[N-(4-chlorophenoxy-acetyl)-piperazin-1-yl]-6-(3,4-methylenedioxyphenyl)-pyrido[3,2-d]pyrimidine. The residue was redissolved in 5 ml of a dichloromethane/ethanol mixture (in a volume ratio 80/20). A sodium ethoxide solution (0.2 N solution) was added till pH 12 and the resulting mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo. The crude residue was purified by preparative TLC on silica, using a methanol/dichloromethane mixture in a volume ratio 10:90 as a mobile phase, yielding the pure title compound (48 mg, 47%).
MS (m/z): 519, 521 ([M+H]+, 100)
This compound was obtained using the procedure described for the synthesis of example 193, using 2-acetamido-4-(N-piperazin-1-yl)-6-(3,4-dichlorophenyl)-pyrido[3,2-d]pyrimidine as starting material.
MS (m/z): 542, 544 ([M+H]+, 100)
This compound was obtained using the procedure described for the synthesis of example 193, using 2-acetamido-4-(N-piperazin-1-yl)-6-(1,4-benzodioxane)-pyrido[3,2-d]pyrimidine as starting material. MS (m/z): 532, 534 ([M+H]+, 100).
To a solution of 2-acetamido-4-(N-piperazin-1-yl)-6-(3,4-methylenedioxyphenyl)-pyrido[3,2-d]pyrimidine (60 mg, 0.16 mmol) in DMF (5 ml) was added m-tolyl isocyanate (31 μl, 0.24 mmol). The solution was stirred overnight at room temperature. The solvents were evaporated in vacuo yielding crude 2-acetamido-4-[N-(3-methyl-phenyl-carbamoyl)-piperazin-1-yl]-6-(3,4-methylenedioxyphenyl)-pyrido[3,2-d]pyrimidine. The residue was redissolved in a mixture of dichloromethane and ethanol (in a ratio of 80/20, 5 ml). A sodium ethoxide solution (0.2 N solution) was added till pH 12 and the resulting mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo. The crude residue was purified by preparative TLC on silica, using a methanol/dichloromethane mixture in a ratio of 10:90 as mobile phase, yielding the pure title compound (32 mg, 43%).
MS (m/z): 484 ([M+H]+, 100).
This compound was synthesized according to the procedure of example 196, using 2-acetamido-4-(N-piperazin-1-yl)-6-(3,4-dichlorophenyl)-pyrido[3,2-d]pyrimidine as the starting material. MS (m/z): 507, 509 ([M+H]+, 100).
This compound was synthesized according to the procedure of example 196, using 2-acetamido-4-(N-piperazin-1-yl)-6-(1,4-benzodioxane)-pyrido[3,2-d]pyrimidine as a starting material. MS (m/z): 498 ([M+H]+, 100).
This compound was synthesized according to the procedure of example 184, using N-acetyl-piperazine and 2-acetamido-4-(1,2,4-triazolyl)-6-(1,4-benzodioxane)-pyrido[3,2-d]pyrimidine as starting materials. MS (m/z): 407 ([M+H]+, 100).
This compound was synthesized according to the procedure of example 184, using N-acetyl-piperazine and 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-dichlorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. MS (m/z): 416, 418 ([M+H]+, 100).
This compound was prepared according to the procedure of example 184, using 2-(piperazin-1-yl acetic acid)-N-(2-thiazolyl)-amide and 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-methylenedioxyphenyl)-pyrido[3,2-d]pyrimidine as starting materials.
MS (m/z): 505 ([M+H]+, 100)
This compound was prepared according to the procedure of example 184, using 2-(piperazin-1-yl acetic acid)-N-(2-thiazolyl)-amide and 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-dichlorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. MS (m/z): 514, 516 ([M+H]+, 100).
This compound was obtained using the procedure of example 184, using 2-furoyl-piperazine and 2-acetamido-4-(1,2,4-triazolyl)-6-(1,4-benzodioxane)-pyrido[3,2-d]pyrimidine as starting materials. MS (m/z): 459 ([M+H]+, 100).
To a solution of 2-acetamido-4-(1,2,4-triazolyl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (367 mg, 1 mmol) in dioxane (10 ml) was added 1-(4-fluorophenyl)piperazine (360 mg, 2 mmol). The solution was stirred for 16 hours at 60° C. The solvents were evaporated in vacuo, yielding crude 2-acetamido-4-[N-(4-fluoro-phenyl)-piperazin-1-yl]-6-(4-fluoro-phenyl)-pyrido[3,2-d]pyrimidine. The residue was redissolved in 10 ml of a dichloromethane/ethanol mixture (in a volume ratio 80/20). A sodium ethoxide solution (0.2 N solution) was added till pH 12 and the resulting mixture was stirred for 16 hours at room temperature. The solvents were evaporated in vacuo. The crude residue was purified by preparative TLC on silica, using a methanol/dichloromethane mixture (volume ratio 10:90) as a mobile phase, yielding the pure title compound (280 mg, 69%) which was characterised as follows:
MS (m/z): 419 ([M+H]+, 100); and
UV (MeOH, m): 250, 345, 560.
To a suspension of 2-acetamido-4-(1,2,4-triazolyl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (367 mg, 1 mmol) in dioxane (10 ml) was added 1-(2-phenoxy-ethyl)-piperazine (412 mg, 2 mmol). The solution was stirred overnight at 60° C. The solvents were evaporated in vacuo yielding crude 2-acetamido-4-[N-(phenoxy-ethyl-piperazin-1-yl)]-6-(4-fluoro-phenyl)-pyrido[3,2-d]pyrimidine. The residue was redissolved in a mixture of dichloromethane and ethanol (in a ratio of 80/20, 10 ml). A sodium ethoxide solution (0.2 N solution) was added till pH 12 and the resulting mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo. The crude residue was purified by preparative TLC on silica, using a methanol/dichloromethane mixture in a ratio of 10:90 as mobile phase, yielding the pure title compound (200 mg, 45%).
MS (m/z): 445 ([M+H]+, 100)
UV (MeOH, m): 250, 345, 495, 580
To a suspension of 2-acetamido-4-(1,2,4-triazolyl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (367 mg, 1 mmol) in dioxane (20 ml) was added aniline (186 mg, 2 mmol). The solution was stirred overnight at 60° C. The solvents were evaporated in vacuo yielding crude 2-acetamido-4-anilino-6-(4-fluoro-phenyl)-pyrido[3,2-d]pyrimidine. The residue was redissolved in a mixture of dichloromethane and ethanol (in a ratio of 80/20, 10 ml). A sodium ethoxide solution (0.2 N solution) was added till pH 12 and the resulting mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo. The crude residue was purified by preparative TLC on silica, using a methanol/dichloromethane mixture in a ratio of 10:90 as mobile phase, yielding the pure title compound (160 mg, 50%).
MS (m/z): 332 ([M+H]+, 100)
UV (MeOH, m): 250, 350, 565
To a solution of 2-acetamido-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (367 mg, 1 mmol) in pyridine (10 ml) was added 4-chloro-phenoxy acetyl chloride (410 mg, 2 mmol). The solution was stirred overnight at 50° C. The solvents were evaporated in vacuo yielding crude 2-acetamido-4-[(N-4-chloro-phenoxyacetyl)-piperazin-1-yl]-6-(4-fluoro-phenyl)-pyrido[3,2-d]pyrimidine. The residue was redissolved in a mixture of dichloromethane and ethanol (in a ratio of 80/20, 10 ml). A sodium ethoxide solution (0.2 N solution) was added till pH 12 and the resulting mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo. The crude residue was purified by preparative TLC on silica, using a methanol/dichloromethane mixture in a ratio of 10:90 as mobile phase, yielding the pure title compound (250 mg, 50%).
MS (m/z): 493, 495 ([M+H]+, 100)
UV (CH3OH, m): 245, 345, 465, 560
A suspension of 2-amino-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (1.96 g, 10 mmol) in acetic anhydride (200 ml) was refluxed for 2 hours till a clear solution was obtained. The solvents were evaporated in vacuo till crystallization started. The precipitate was filtered off and dried under vacuum yielding the pure title compound (2 g, 80%).
MS (m/z): 239, 241 ([M+H]+, 100)
To a suspension of 2-acetamido-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (2.38 g, 10 mmol) in dioxane (100 ml) was added diisopropylethylamine (5.3 ml, 30 mmol). The mixture was stirred for 10 minutes at 80° C., after which phosphorus oxychloride (1.4 ml, 15 mmol) was added. This reaction mixture was stirred for 90 minutes at 80° C. The solvents were evaporated in vacuo. The residue was redissolved in dichloromethane and extracted with water. The combined organic layers were evaporated till a volume of 50 ml. Then, morpholine (870 mg, 10 mmol) was added and the reaction was stirred overnight at room temperature. The solvents were evaporated in vacuo. The residue was redissolved in a mixture of dichloromethane and ethanol (80/20, 100 ml). A sodium ethoxide solution (0.2 N solution) was added till pH=11. The mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo. The residue was redissolved in dichloromethane and washed with water. The combined organic layers were combined and evaporated in vacuo, yielding the title compound (1 g, 40%).
MS (m/z): 266, 268 ([M+H]+, 100)
A solution of 2-amino-4-morpholino-6-chloro-pyrido[3,2-d]pyrimidine (265 mg, 1 mmol), potassium carbonate (690 mg, 5 mmol), tetrakis(triphenylphosphine)palladium(0) (100 mg) in dioxane (10 ml) and water (3 ml) was refluxed. To this refluxing solution was added dropwise (with a speed of 0.25 ml/min) a solution of 2-bromo-phenyl boronic acid (220 mg, 1.1 mmol) in dioxane (2 ml). Once the addition was complete, the reaction mixture was refluxed for another 2 hours. The reaction mixture was cooled down and the solvents were evaporated in vacuo. The residue was redissolved in dichloromethane and extracted with water. The combined organic layers were dried over Na2SO4 and the crude residue was purified by preparative TLC on silica, using a methanol/dichloromethane mixture in a ratio of 10:90 as mobile phase, yielding the pure title compound (100 mg, 30%).
MS (m/z): 386, 388 ([M+H]+, 100)
The procedure of example 120 was followed, but using cyclopropylmethyl bromide as a starting material. The pure title compound was isolated and characterized by its mass spectrum as follows: MS (m/z): 560, 562 ([M+H]+, 100).
To a solution of 4-[N-(3-chloro-phenylcarbamoyl)-piperazin-1-yl]-6-chloro-pyrido[3,2-d]pyrimidine (650 mg, 1.61 mmol) in 1,4-dioxane (40 ml) and water (13 ml) was added 2-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl acetate (470 mg, 1.61 mmol), potassium carbonate (667 mg, 4.83 mmol) and tetrakis(triphenylphosphine)palladium(0) (93 mg, 0.0805 mmol). The reaction mixture was refluxed for 3 hours, then cooled down to room temperature and the solvents were evaporated in vacuo. The residue was purified by silica gel column chromatography, the mobile phase being an acetone/dichloromethane mixture (in a ratio ranging from 20:80 to 30:70), yielding the title compound as a pure white powder (513 mg, 63%). MS (m/z): 506, 508 ([M+H]+, 100).
To a solution of 4-[N-(3-chloro-phenylcarbamoyl)-piperazin-1-yl]-6-(3-hydroxy-4-methoxy-phenyl)-pyrido[3,2-d]pyrimidine (100 mg, 0.20 mmol) in dry DMF (10 ml) was added potassium carbonate (42 mg, 0.3 mmol). This mixture was stirred at room temperature for 30 minutes under nitrogen and then, the appropriate alkyl halide (0.3 mmol) was added. After stirring for 5 hours, there was still starting material left and therefore an additional amount of the alkyl halide (0.3 mmol) and potassium carbonate (0.3 mmol) was added. The reaction mixture was further stirred at room temperature overnight. The solvents were evaporated in vacuo and purified by silica gel flash chromatography, the mobile phase being a mixture of methanol/dichloromethane (in a ratio ranging from 2:98 to 3:97), yielding the title compound as white powders, in yields varying from 60% to 70%, depending on the alkyl halide used.
The following compounds were synthesized according to this procedure:
This compound was obtained from ethyl iodide as starting material.
MS (m/z): 534, 536 ([M+H]+, 100)
This compound was obtained from isopropyl iodide as starting material. MS (m/z): 548, 550 ([M+H]+, 100).
This compound was obtained from cyclopropylmethyl bromide as starting material.
MS (m/z): 560, 562 ([M+H]+, 100)
To a suspension of 2-acetamido-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (360 mg, 1.51 mmol) in dioxane (30 ml) was added diisopropylethylamine (788 μl, 4.53 mmol) and POCl3 (422 μl, 4.53 mmol). The reaction was heated at 100° C. overnight till a black solution was obtained. The solvents were evaporated in vacuo. The crude residue was redissolved in dichloromethane and was extracted three times with ice-cold water. The combined organic layers were evaporated in vacuo and used for further reactions without any additional purification. MS (m/z): 257, 259 ([M+H]+, 100).
To a solution of 2-acetamido-4,6-dichloro-pyrido[3,2-d]pyrimidine (the crude residue obtained in the previous example 216a) in dioxane (20 ml) was added (S)-3-(Boc-amino)pyrrolidine (563 mg, 3.02 mmol). The reaction mixture was stirred at room temperature for 2 hours. The reaction was diluted with water and extracted with dichloromethane. The combined organic layers were evaporated in vacuo. The crude residue was purified by silica gel flash chromatography, the mobile phase being a MeOH/CH2Cl2 mixture in a ratio of 4:96, yielding two pure compounds, i.e.:
To a solution of 2-acetamido-4-[(S)-3-(Boc-amino)pyrrolidine]-6-chloro-pyrido[3,2-d]pyrimidine in methanol (10 ml) was added a solution of potassium carbonate (360 mg) in water (5 ml). The reaction was heated at 80° C. for 2 hours. The reaction was cooled down, diluted with water and extracted with dichloromethane. The combined organic layers were evaporated in vacuo and the crude residue was purified by flash chromatography on silica, the mobile phase being a mixture of acetone/CH2Cl2 (in a ratio of 40:60), followed by a mixture of CH3OH/CH2Cl2 in a ratio of 4:96, yielding the title compound as a pure white solid (133 mg, 71%). MS (m/z): 365, 367 ([M+H]+, 100).
To a solution of 2-amino-4-[(S)-3-(Boc-amino)pyrrolidine]-6-chloro-pyrido[3,2-d]pyrimidine (100 mg, 0.27 mmol) in 1,4-dioxane (20 ml) and water (7 ml) was added 3,4-dimethoxyphenyl boronic acid (65 mg, 0.36 mmol), potassium carbonate (114 mg, 0.82 mmol) and tetrakis(triphenylphosphine)palladium(0) (16 mg, 0.014 mmol). The reaction mixture was refluxed for three hours, cooled down to room temperature and the solvents were evaporated in vacuo. The residue was purified by silica gel column chromatography, the mobile phase being a CH3OH/dichloromethane mixture (in a ratio of 4:96), yielding the title compound as a pure white powder (79 mg, 63%).
MS (m/z): 467 ([M+H]+, 100).
A solution of 2-amino-4-[(S)-3-(Boc-amino)pyrrolidine]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (113 mg, 0.24 mmol) in dichloromethane (10 ml) and trifluoroacetic acid (4 ml) was stirred at room temperature for 30 minutes. The solvents were evaporated. The salt was redissolved in water and the solution was made alkaline (pH=9) by the addition of a 33% aqueous ammonia solution. The solvents were evaporated in vacuo and the residue was purified by silica gel flash chromatography, the mobile phase being a mixture of CH3OH/CH2Cl2 in a ratio of 4:96, containing 0.5% of an aqueous 33% ammonia solution, yielding the title compound as a pure white solid (76 mg, 87%). MS (m/z): 367 ([M+H]+, 100).
To a solution of 2-amino-4-[(S)-3-(amino)pyrrolidine]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (76 mg, 0.21 mmol) in DMF (10 ml) was added triethylamine (38 μl, 0.27 mmol) and p-chloro-phenoxy acetyl chloride (51 mg, 0.25 mmol). The reaction was stirred at 60° C. for 2 hours. The solvents were evaporated in vacuo and the crude residue was purified by silica gel flash chromatography, the mobile phase being a mixture of CH3OH/CH2Cl2 in a ratio of 4:96, yielding the pure title compound (87 mg, 78%). MS (m/z): 535, 537 ([M+H]+, 100).
To a solution of 2-amino-4-[(S)-3-(amino)pyrrolidine]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (113 mg, 0.25 mmol) in dichloromethane (10 ml) was added m-tolyl isocyanate (0.28 mmol, 35 μl). The reaction was stirred at room temperature for 2 hours. The solvents were evaporated in vacuo and the crude residue was purified by silica gel flash chromatography, the mobile phase being a mixture of CH3OH/CH2Cl2 in a ratio of 3:97, yielding the pure title compound (77 mg, 62%). MS (m/z): 500 ([M+H]+, 100).
A suspension of 2-amino-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidin-4(3H)one (100 mg, 0.34 mmol) and phosphorus pentasulfide (163 mg, 0.37 mmol) in pyridine (10 ml) was refluxed for 4 hours. The solvents were evaporated in vacuo. The residue was resuspended in a small amount of water and filtered off, yielding the title compound which was used without any further purification. MS (m/z): 315 ([M+H]+, 100).
The crude compound obtained in example 222 was dissolved in NaOH 1 N. Then, methyl iodide (18 μl, 0.29 mmol) was added and the reaction mixture was stirred at room temperature for 2 hours. Then, an additional amount of methyl iodide (9 μl) was added and the reaction was stirred for another hour at room temperature. A yellow precipitate was formed, which was filtered off. The precipitate was adsorbed on silica and purified by silica gel flash chromatography, the mobile phase being a methanol/dichloromethane mixture (in a ratio of 1:99), yielding the pure title compound (52 mg, 47%). MS (m/z): 329 ([M+H]+, 100).
To a suspension of 6-chloro-3-nitro-pyridine-2-carbonitrile (5.5 g, 30 mmol) in water (100 ml), was added acetic acid (5.4 ml, 90 mmol). The mixture was stirred at room temperature for 20 minutes. Then, Na2S2O4 (20 g, 86%, 90 mmol) was added slowly. The reaction mixture was stirred at room temperature for another 2 hours. The precipitate was filtered off and washed with cold water (2×10 ml). The precipitate was dried over P2O5 yielding the title compound as a yellowish solid (3.7 g, 80%) which was characterised as follows:
Rf=0.64 (EtOAc/CH2Cl2 1:4); and
MS (m/z): 154, 156 ([M+H]+, 100).
A mixture consisting of 3-amino-6-chloro-pyridine-2-carbonitrile (4.6 g, 30 mmol), chloroformamidine hydrochloride (6.9 g, 60 mmol) and dimethylsulfon (12 g) was heated at 165° C. for 30 minutes. After cooling to room temperature, water (500 ml) was added. The solution was neutralized with a 30% NaOH solution to pH 9-10. The precipitate was filtered off, washed with water, dried over P2O5, yielding the title compound as a yellow solid (4.0 g, 68%) which was characterised as follows:
Rf=0.40 (MeOH/CH2Cl2 1:9); and
MS (m/z): 196, 198 ([M+H]+, 100).
To a suspension of 6-chloro-3-nitro-pyridine-2-carbonitrile (4 g, 22 mmol) in water (40 ml) was added a 33% aqueous solution of ammonia in water (8.8 ml). This suspension was stirred at room temperature for 30 minutes. Then, sodium dithionite (21.8 g, 124 mmol) was added portionwise. The resulting mixture was stirred for another 2 hours at room temperature. The precipitate was filtered off and washed with a small amount of water, yielding the title compound (2.7 g, 72%). MS (m/z): 172, 174 ([M+H]+, 100)
Method A
A suspension of 2,4-diamino-6-chloro-pyrido[3,2-d]pyrimidine (3.5 g, 17 mmol) in 5 N HCl (150 ml) was refluxed for 3 hours. After cooling to room temperature, the mixture was neutralized with a 30% NaOH solution to pH 6-7. The precipitate was filtered off, washed with water, dried over P2O5, yielding the title compound as a yellow solid (3.2 g, 90%).
Method B
A mixture of 3-amino-6-chloro-pyridine-2-carboxamide (2.4 g, 14 mmol), chloroform-amidine hydrochloride (3.2 g, 28 mmol), dimethylsulfone (6 g) and sulfolane (0.8 ml) was heated at 165° C. for 30 minutes. After cooling to room temperature, water (600 ml) was added and the pH was adjusted to 7-8 with a 25% ammonia solution in water. The precipitate was filtered off, washed with water and dried over P2O5, yielding the title compound as a yellow solid (2.7 g, 98%) which was characterised as follows:
Rf=0.33 (MeOH/CH2Cl2 1:4); and
MS (m/z): 197, 199 ([M+H]+, 100).
A suspension of 2-amino-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)one (3.2 g, 16 mmol) in acetic anhydride (20 ml) was refluxed for 2 hours. After cooling to room temperature, the precipitate was filtered off, washed with diethyl ether and dried under vacuum yielding the title compound as a yellowish solid (3.2 g, 85%) which was characterised as follows:
Rf=0.75 (MeOH/CH2Cl2 1:4); and
MS (m/z): 238, 240 ([M+H]+, 100).
A mixture of 2-acetamido-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)one (2.4 g, 10 mmol), N,N-diisopropylethylamine (5.4 ml, 30 mmol) and POCl3 (2.8 ml, 30 mmol) in dioxane (100 ml), was stirred at room temperature for 2 hours. After concentration under reduced pressure, the residue was redissolved in dichloromethane (200 ml) and extracted with cold water till pH 6-7. The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure to yield crude 2-acetamido-4,6-dichloro-pyrido[3,2-d]pyrimidine. This crude residue was dissolved in 1,4-dioxane (100 ml) and morpholine (5 ml) was added. The resulting reaction mixture was stirred at 50° C. for 1 hour. After concentration under reduced pressure, the residue was purified by silica gel flash chromatography, the mobile phase being a mixture of MeOH/dichloromethane (in a ratio of 1:40), yielding the title compound as a yellowish solid (1.6 g, 68%) which was characterised as follows:
Rf=0.82 (MeOH/CH2Cl2 1:19); and
MS (m/z): 308, 310 ([M+H]+, 100).
A suspension of 2-acetamido-4-morpholino-6-chloro-pyrido[3,2-d]pyrimidine (500 mg, 1.6 mmol) and K2CO3 (660 mg, 4.8 mmol) in MeOH (30 ml) and water (10 ml) was refluxed for 2 hours. After cooling to room temperature, the mixture was extracted with dichloromethane (100 ml), washed with water and dried over MgSO4. After filtration and concentration, the residue was purified by silica gel flash chromatography, the mobile phase being a MeOH/CH2Cl2 mixture (in a ratio of 1:35) yielding the title compound as yellowish solid (425 mg, 98%) which was characterised as follows:
Rf=0.64 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O): 245, 330, and 455 nm; and
MS (m/z): 266, 268 ([M+H]+, 100)
To a solution of 2-amino-4-morpholino-6-chloro-pyrido[3,2-d]pyrimidine (53 mg, 0.2 mmol) in 1,4-dioxane (15 ml) and water (5 ml) was added an appropriate aryl or heteroaryl boronic acid (0.2 mmol), potassium carbonate (280 mg, 2 mmol) and tetrakis(triphenylphosphine)palladium(0) (30 mg, 0.026 mmol). The reaction mixture was refluxed for three hours, cooled down to room temperature and the solvents were evaporated in vacuo. The residue was purified by silica gel column chromatography, the mobile phase being a CH3OH/dichloromethane mixture, thus resulting in the pure desired compounds in the following yields:
was obtained from 3,4-dichlorophenylboronic acid as a yellowish solid (79%) and was characterised as follows:
Rf=0.55 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 283.8, 365.9; and
MS (m/z): 376, 378 ([M+H]+, 100).
Was obtained from 2-furanboronic acid as a yellow solid (79%) and was characterised as follows:
Rf=0.36 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 212.9, 290.9, 377.9; and
MS (m/z): 298 ([M+H]+, 100).
Was obtained from 3-thiopheneboronic acid as a yellowish solid (73%) and was characterised as follows:
Rf=0.50 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 215.3, 279.1, 362.5; and
MS (m/z): 314 ([M+H]+, 100).
Was obtained from 4-pyridine boronic acid as a yellowish solid (90%) and was characterised as follows:
Rf=0.63 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 214.1, 236.5, 280.3, 341, 356.6; and
MS (m/z): 309 ([M+H]+, 100)
Was obtained from 5-methyl-2-thiophene boronic acid as a yellowish solid (69%) and was characterised as follows:
Rf=0.60 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 214.1, 298.1, 380.3; and
MS (m/z): 328 ([M+H]+, 100).
Was obtained from 6-methoxy-2-pyridine boronic acid as a yellowish solid (75%) and was characterised as follows:
Rf=0.44 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 214.1, 283.8, 359.5; and
MS (m/z): 339 ([M+H]+, 100).
Was obtained from 5-indole boronic acid as a yellowish solid (90%) and was characterised as follows:
Rf=0.25 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 216.5, 314.7, 422.5, 441.9; and
MS (m/z): 347 ([M+H]+, 100).
Was obtained from 2-thiophene boronic acid as a yellowish solid (72%) and was characterised as follows:
Rf=0.70 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 214.1, 293.3, 377.9; and
MS (m/z): 314 ([M+H]+, 100).
Was obtained from 4-methyl-2-thiophene boronic acid as a yellowish solid (76%) and was characterised as follows:
Rf=0.45 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 212.9, 298.1, 380.3;
MS (m/z): 328 ([M+H]+, 100).
Was obtained from 3-pyridine boronic acid as a yellowish solid (90%) and was characterised as follows:
Rf=0.55 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 214.1, 247.1, 285, 363.5; and
MS (m/z): 309 ([M+H]+, 100).
Was obtained from 5-chloro-2-thiophene boronic acid as a yellowish solid (29%) and was characterised as follows:
Rf=0.65 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 212.9, 298.1, 380.3; and
MS (m/z): 348 ([M+H]+, 100).
Was obtained from 3-chloro-4-fluorophenyl boronic acid as a yellowish solid (75%) and was characterised as follows:
Rf=0.55 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 345, 480, 560; and
MS (m/z): 360 ([M+H]+, 100).
Was obtained from 3,4-difluorophenyl boronic acid as a yellowish solid (75%) and was characterised as follows:
Rf=0.64 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 345, 465, 560; and
MS (m/z): 344 ([M+H]+, 100).
Was obtained from 4-fluoro-3-methylphenyl boronic acid as a white solid (81%) and was characterised as follows:
Rf=0.60 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 280.3, 365.9; and
MS (m/z): 340 ([M+H]+, 100).
Was obtained from 4-fluorophenyl boronic acid as a white solid (85%) and was characterised as follows:
Rf=0.64 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 250, 470, 560; and
MS (m/z): 326 ([M+H]+, 100).
Was obtained from 3,5-dimethylisoxazole-4-boronic acid as a yellowish solid (62%) and was characterised as follows:
Rf 0.60 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 214.1, 269.6, 356.6; and
MS (m/z): 327 ([M+H]+, 100).
This compound was synthesized from homopiperazine according to the procedure of example 229, yielding the pure title compound as a yellowish solid (49%) which characterised as follows:
Rf=0.17 (MeOH/CH2Cl2 1:4); and
MS (m/z): 321, 323 ([M+H]+, 100).
To a solution of 2-acetamido-6-chloro-4-(N-homopiperazin-1-yl)-pyrido[3,2-d]pyrimidine (95 mg, 0.3 mmol) in dichloromethane (10 ml) was added m-tolylisocyanate (40 mg, 0.3 mmol). The solution was stirred at room temperature for 1 hour. The solvents were evaporated in vacuo yielding the crude title compound, which was used for further reaction without any purification.
To a solution of crude 2-acetamido-6-chloro-4-[N-(3-methylphenylcarbamoyl)-homopiperazin-1-yl]-pyrido[3,2-d]pyrimidine (130 mg, 0.3 mmol) in dioxane (15 ml) and water (5 ml) was added 3,4-dimethoxyphenyl boronic acid (55 mg, 0.3 mmol), potassium carbonate (280 mg, 2 mmol) and tetrakis(triphenylphosphine)palladium(0) (30 mg, 0.026 mmol). The reaction mixture was refluxed for 30 minutes. The solvents were evaporated in vacuo. The crude residue was purified by silica gel flash chromatography, the mobile phase being a MeOH/CH2Cl2 mixture (in a ratio of 1:40), yielding the pure title compound (126 mg, 78%). MS (m/z): 556 ([M+H]+, 100).
A solution of 2-acetamido-4-[(N-3-methylphenylcarbamoyl)-homopiperazin-1-yl]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (110 mg, 0.24 mmol) and potassium carbonate (83 mg, 0.6 mmol) in methanol (10 ml) and water (5 ml) was heated at 50° C. for 2 hours. The solvents were evaporated in vacuo and the crude residue was purified by silica gel flash chromatography, the mobile phase being a MeOH/CH2Cl2 mixture in a volume ratio of 1:30, yielding the pure title compound (96 mg, 93%) which characterised as follows:
Rf=0.55 (MeOH/CH2Cl2 1/9);
UV (MeOH/H2O, m): 245, 490, 565; and
MS (m/z): 514 ([M+H]+, 100).
This compound was prepared from (R)-3-Boc-amino-pyrrolidine according to the procedure of example 229, yielding the title compound as a yellowish solid (46%) which characterised as follows:
Rf=0.55 (MeOH/CH2Cl2 1:9); and
MS (m/z): 407, 409 ([M+H]+, 100).
This compound was synthesized from the compound of example 251. In a first step, a Suzuki coupling with 3,4-dimethoxyphenyl boronic acid (general procedure as in examples 231 to 246) was performed. In a second step, alkaline hydrolysis of the acetyl group (using the procedure for the synthesis of example 230) yielded the pure title compound (81%) which characterised as follows:
Rf=0.54 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 280, 470, 565; and
MS (m/z): 467 ([M+H]+, 100).
A suspension of 2-amino-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidin-4(3H)one (298 mg, 1.0 mmol), 1,1,1,3,3,3-hexamethyldisilazane (1 ml, 4.7 mmol), an appropriate amine (4.0 mmol), p-toluenesulfonic acid (20 mg, 0.1 mmol) and ammonium sulfate (20 mg, 0.15 mmol) in pyridine (5 ml) was refluxed for 12 to 48 hours (depending upon the amine used; the reaction mixture became clear when reaction was completed). The solvents were evaporated in vacuo and the residue was purified by silica gel flash chromatography, the mobile phase being a MeOH/dichloromethane mixture (in a volume ratio of 1:20 to 1:30, depending upon the amine used), resulting into the title compounds as yellow solids in the following yields.
Was obtained from ethylene diamine as a yellowish solid (64%) which characterised as follows:
Rf=0.25 (MeOH/CH2Cl2 1:4); and
MS (m/z): 341 ([M+H]+, 100).
Was obtained from 1,3-diaminopropane as a yellowish solid (68%) which characterised as follows:
Rf=0.28 (MeOH/CH2Cl2 1:4); and
MS (m/z): 355 ([M+H]+, 100).
was obtained from 4-amino-N-Boc-piperidine as a yellowish solid (92%) which characterised as follows:
Rf=0.58 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 250, 480, 565; and
MS (m/z): 481 ([M+H]+, 100).
was obtained from ammonium chloride as a yellowish solid (56%) which characterised as follows:
Rf=0.23 (MeOH/CH2Cl2 1:4);
UV (MeOH/H2O, m): 245, 585; and
MS (m/z): 298 ([M+H]+, 100);
was obtained from 3-amino-N-Boc-piperidine as a yellowish solid (70%) which characterised as follows:
Rf=0.60 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 250, 490, 565; and
MS (m/z): 481 ([M+H]+, 100).
was synthesized from 3-amino-1-benzyloxycarbonyl-piperidine, yielding the title compound (63%). MS (m/z): 515 ([M+H]+, 100).
To a suspension of 2-amino-4-[(R)-3-Boc-aminopyrrolidin-1-yl]-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidine (94 mg, 0.2 mmol) in dichloromethane (5 ml) was added trifluoroacetic acid (2 ml). The resulting solution was stirred at room temperature for 30 minutes. The solvents were removed under reduced pressure. The residue was extracted with chloroform and washed with a 0.2 M Na2CO3 solution. The combined organic layers were evaporated in vacuo. The crude residue was purified by silica gel flash chromatography, the mobile phase being a MeOH/CH2Cl2 mixture in a volume ratio of 2:3, yielding the pure title compound (70 mg, 96%). MS (m/z): 367 ([M+H]+, 100).
To a solution of 2-amino-4-[(R)-3-Boc-aminopyrrolidin-1-yl)]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (55 mg, 0.12 mmol) in dichloromethane (5 ml) was added trifluoroacetic acid (2 ml). The mixture was stirred at room temperature for 30 minutes. The solvents were evaporated in vacuo. To a suspension of this crude residue in dichloromethane (5 ml) was added N,N-diisopropylethylamine (0.5 ml) and m-tolyl isocyanate (16 μl). The reaction mixture was stirred at room temperature for 30 minutes. The solvents were evaporated in vacuo. The crude residue was purified by silica gel chromatography, the mobile phase being a MeOH/CH2Cl2 mixture (in a ratio of 1:20), yielding the pure title compound (50 mg, 85%) which characterised as follows:
Rf=0.42 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 240, 470, 560; and
MS (m/z): 500 ([M+H]+, 100).
To a solution of 2-amino-4-(ethylenediamine-1-N-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (70 mg, 0.2 mmol) in dichloromethane (10 ml) was added N,N-diisopropylethylamine (200 μl) and m-tolyl isocyanate (26 μl). The solution was stirred at room temperature for 1 hour. The solvents were evaporated in vacuo. The crude residue was purified by silica gel flash chromatography, the mobile phase being a MeOH/CH2Cl2 mixture, in a ratio of 1:15, yielding the pure title compound (72 mg, 76%) which characterised as follows:
Rf=0.32 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 250, 560; and
MS (m/z): 474 ([M+H]+, 100).
This compound was obtained from 2-amino-4-(3-aminopropanamine-1-N-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine and m-tolyl isocyanate (using the procedure described for the synthesis of example 261) in 82% yield and was characterised as follows:
Rf=0.38 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 250, 480, 560; and
MS (m/z): 488 ([M+H]+, 100).
This compound was obtained from 2-amino-4-(1-Boc-piperidin-4-yl-amino)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as a yellowish solid (82%) which characterised as follows:
Rf=0.40 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 250, 470, 560; and
MS (m/z): 514 ([M+H]+, 100).
This compound was synthesized from 2-amino-4-(1-Boc-piperidin-3-ylamino)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine by Boc-deprotection and coupling with m-tolyl isocyanate (using the procedure described for example 260), as a yellowish solid (88%) which was characterised as follows:
Rf=0.32 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 250, 370, 560; and
MS (m/z): 514 ([M+H]+, 100).
To a suspension of 2-amino-4-(ethylenediamine-1-N-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (50 mg, 0.15 mmol) in dichloromethane (10 ml) was added DIPEA (200 μl) and 4-chloro-phenoxy acetyl chloride (30 mg, 0.15 mmol). The mixture was stirred at room temperature for 1 hour. The solvents were evaporated in vacuo. The crude residue was purified by flash chromatography, the mobile phase being a MeOH/CH2Cl2 mixture (in a ratio of 1:20), yielding the pure title compound (40 mg, 53%) which was characterised as follows:
Rf=0.35 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 250, 480, 560; and
MS (m/z): 509, 511 ([M+H]+, 100).
This compound was synthesized from 2-amino-4-(3-aminopropanamine-1-N-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine and 4-chlorophenoxyacetyl chloride, using the procedure described for the synthesis of example 265, yielding the pure title compound (56%) which was characterised as follows:
Rf=0.36 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 250, 560; and
MS (m/z): 523, 525 ([M+H]+, 100).
This compound was synthesized from 2-amino-4-[(3-(R)-Boc-aminopyrrolidin-1-yl]-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine in two steps. The Boc group was deprotected (using the procedure described for example 259) and then, the free amino group was coupled with 4-chlorophenoxyacetyl chloride (using the procedure described for example 265), yielding the pure title compound (68%) which was characterised as follows:
Rf=0.30 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 280, 470, 560; and
MS (m/z): 535, 537 ([M+H]+, 100).
To a suspension of 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (0.5 mmol) and N,N-diisopropylethylamine (3 mmol) in 1,4-dioxane (20 ml) was added an appropriate amine (1.5 mmol). The reaction mixture was refluxed for 2 hours. The solvents were evaporated in vacuo and the residue was redissolved in methanol (20 ml). A solution of K2CO3 (3 mmol) in water (5 ml) was added and the resulting reaction mixture was refluxed for 2 hours. After cooling to room temperature, the mixture was extracted with dichloromethane (100 ml). The organic phase was washed with a 0.5 M Na2CO3 solution and concentrated under reduced pressure. The residue was purified by silica gel flash chromatography, the mobile phase being a mixture of MeOH and dichloromethane, thus resulting into the pure title compounds in the following yields.
This compound was obtained from piperidine-3-carboxylic acid isobutyl amide, as a yellowish solid (60%) which was characterised as follows:
Rf=0.25 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 245, 560; and
MS (m/z): 465 ([M+H]+, 100).
This compound was obtained from 4-(4-chlorophenyl)-4-hydroxy-piperidine, as a white solid (58%) which was characterised as follows:
Rf=0.42 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 285, 365, 560; and
MS (m/z): 492, 494 ([M+H]+, 100).
This compound was synthesized from N-(2-phenylethyl)-2-piperazin-1-yl-acetamide as a yellowish solid (54%) which was characterised as follows:
Rf=0.38 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 245, 560; and
MS (m/z): 528 ([M+H]+, 100).
This compound was obtained from 2-amino-1-(4-benzylpiperazin-1-yl)-ethanone as a yellowish solid (54%) which was characterised as follows:
Rf=0.32 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 265, 585;
MS (m/z): 514 ([M+H]+, 100)
This compound was obtained from 1-(4-acetylpiperazin-1-yl)-3-aminopropan-1-one as a yellowish solid (60%) which was characterised as follows:
Rf=0.30 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 250, 505, 580; and
MS (m/z): 480 ([M+H]+, 100)
This compound was synthesized from 2-piperazine-1-yl-1-pyrrolidin-1-yl-ethanone (yield 59%) as a yellowish solid which was characterised as follows:
Rf=0.27 (MeOH/CH2Cl2 1/9);
UV (MeOH/H2O, m): 245, 580; and
MS (m/z): 478 ([M+H]+, 100)
This compound was synthesized from 2-piperazin-1-yl-N-pyridin-2-yl-acetamide as a yellowish solid (53%) which was characterised as follows:
Rf=0.33 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 245, 365, 560; and
MS (m/z): 501 ([M+H]+, 100)
This compound was synthesized from N-[2-(1-piperazino)-acetyl]-morpholino as a yellowish solid (57%) which was characterised as follows:
Rf=0.45 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 275, 365; and
MS (m/z): 494 ([M+H]+, 100)
This compound was synthesized from 2-amino-1-(4-methylpiperazin-1-yl)-ethanone as a yellowish solid (57%) which was characterised as follows:
Rf=0.20 (MeOH/CH2Cl2 1:4);
UV (MeOH/H2O, m): 270, 355, 495;
MS (m/z): 438 ([M+H]+, 100)
To a suspension of 2-acetamido-4-(1,2,4-triazolyl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (0.5 mmol) and N,N-diisopropylethylamine (3 mmol) in 1,4-dioxane (20 ml) was added an appropriate amine (1.5 mmol). The reaction mixture was refluxed for 2 hours. The solvents were evaporated in vacuo and the residue was purified by silica gel chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 1:30) yielding the pure final compounds as follows:
This compound was synthesized from 2-piperazin-1-yl-N-pyridin-3-yl-acetamide as a yellowish solid (40%) which was characterised as follows:
Rf=0.40 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 245, 370; and
MS (m/z): 543 ([M+H]+, 100)
This compound was synthesized from N-methyl-N-phenyl-2-piperazin-1-yl-acetamide as a yellowish solid (38%) which was characterised as follows:
Rf=0.45 (MeOH/CH2Cl2 1:9);
UV (MeOH/H2O, m): 255, 360; and
MS (m/z): 556 ([M+H]+, 100)
To a suspension of 6-chloro-3-nitro-pyridine-2-carbonitrile (11.01 g, 60 mmol) in methanol (120 ml), was added Raney-Nickel (3 g, washed with methanol to remove water) and the mixture was shaken under a H2-atmosphere at room temperature for 4 hours. The catalyst was removed by filtration, washed with methanol (500 ml). Both filtrates were combined and then evaporated to dryness. The residue was dissolved in dichloromethane and the solution was filtered through a short and wide column with silica gel (100 g). The column was additionally washed with CH2Cl2/MeOH (200 ml, 4:1). The filtrate and washings were combined and evaporated to small volume. The formed precipitate was filtered off to give 3-amino-6-chloro-pyridine-2-carboxamide (8.1 g). The final filtrate was evaporated to dryness and the residue purified by column chromatography on silica gel (30 g). The compound was eluted with the following solvent systems: CH2Cl2 (200 ml), CH2Cl2/MeOH 100:1 (200 ml). The appropriate fractions were evaporated in vacuo yielding an additional 1.15 g of 3-amino-6-chloro-pyridine-2-carboxamide (total yield 9.25 g, i.e. 90%) which was characterised as follows:
M.p. 176-177° C.;
UV (MeOH): 212 (3.76), 256 (4.14), 348 (3.76); and
Elemental analysis: calculated for C6H6ClN3O (171.6): C, 42.00; H, 3.52; N, 24.49. Found: C, 42.42; H, 3.54; H, 24.11.
A mixture of 3-amino-6-chloro-pyridine-2-carboxamide (5.1 g, 30 mmol), chloroform-amidine hydrochloride (6.99 g, 60 mmol), dimethylsulfone (24 g) and sulfolane (2.4 ml) was heated at 165° C. for 30 min. To the hot mixture was added water (50 ml). After cooling to room temperature, a diluted ammonium hydroxide solution was slowly added dropwise till pH 7. The resulting precipitate was filtered off, washed with water and dried overnight at 100° C. to give the pure title compound (5.8 g, 98%). The obtained compound was used a such for further reactions without additional purification. M.p. >330° C.; elemental analysis calc. for C7H5ClN4O (196.6): C, 42.77; H, 2.56; N, 28.50. Found: C, 41.61; H, 2.74; N, 28.76.
A suspension of 2-amino-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (6.2 g, 31.54 mmol) in acetic anhydride (620 ml) was refluxed with stirring for 4 hours. The hot mixture was filtered to remove insoluble material and the filtrate was evaporated to dryness. To the residue was added methanol (50 ml). The precipitate was filtered, washed with methanol and dried yielding the title compound (5.3 g, 70%) which was characterised as follows:
M.p. 317-319° C.;
UV (MeOH): 208 (4.13), 216 (sh 4.17), 280 (4.13), 310 (sh 3.44); and
Elemental analysis: calc. for C9H7ClN4O2 (238.6): C, 45.30; H, 2.96; N, 23.48. Found: C, 45.61; H, 3.53; N, 23.28.
To a mixture of 2-acetamido-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (0.72 g, 3 mmol), triphenylphosphine (1.18 g, 4.5 mmol), and the appropriate alcohol (4.5 mmol) in dioxane (50 ml) was added diisopropyl azodicarboxylate (0.91 g, 0.87 ml, 4.5 mmol). The mixture was stirred at room temperature for 24-36 hr and then evaporated in vacuo. The residue was purified by silica gel flash chromatography. The compound was eluted with the following solvent systems: CH2Cl2 (500 ml), CH2Cl2/AcOEt 5:1 (600 ml), CH2Cl2/AcOEt 4:1 (500 ml), CH2Cl2/AcOEt 1:1 (300 ml), CH2Cl2/MeOH 100:5 (500 ml). Evaporation of the product fractions gave the desired 4-alkyloxy-2-amino-6-chloropyrido[3,2-d]pyrimidine in yields of 45-60%, depending on the alcohol used. Analytical samples were obtained by crystallization of the 2-amino-4-alkoxy-6-chloro-pyrido[3,2-d]pyrimidine from ethyl acetate, diethylether or methanol. Unreacted 2-acetamido-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (40 to 20%) was also isolated during chromatography. The following compounds were synthesized according to this general procedure:
From ethanol (210 mg, 4.5 mmol) to give the pure title compound (0.48 g, 60%) which was characterised as follows:
M.p. 233° C.;
UV (MeOH): 237 (4.58), 266 (4.15), 274 (4.14), 321 (3.73).
Calc. for C11H11ClN4O2 (266.7): C, 49.54; H, 4.16; N, 21.01. Found: C, 49.01; H, 4.30; N, 20.70.
From n-propanol (270 mg, 4.5 mmol) to give the pure title compound (0.42 g, 50%) which was characterised as follows:
M.p. 191° C.;
UV (MeOH): 237 (4.58), 266 (4.15), 274 (4.14), 321 (3.73); and
Calc. for C12H13ClN4O2 (280.7): C, 51.35; H, 4.67; N, 19.96. Found: C, 51.16; H, 4.69; N, 19.94.
From isopropanol (270 mg, 4.5 mmol) to give the pure title compound (0.479 g, 57%) which was characterised as follows:
M.p. 244° C.;
UV (MeOH): 237 (4.59), 266 (4.15), 274 (4.15), 321 (3.73);
Calc. for C12H13ClN4O2 (280.7): C, 51.35; H, 4.67; N, 19.96. Found: C, 51.30; H, 4.71; N, 20.05.
From n-butanol (270 mg, 4.5 mmol) to give the pure title compound (0.504 g, 57%) which was characterised as follows:
M.p. 158-159° C.;
UV (MeOH): 237 (4.59), 266 (4.15), 274 (4.15), 321 (3.73); and
Calc. for C13H15ClN4O2 (294.7): C, 52.98; H, 5.13; N, 19.01. Found: C, 52.11; H, 5.16; N, 18.68.
From isobutanol (333 mg, 4.5 mmol) to yield the pure title compound (0.46 g, 52%) which was characterised as follows:
M.p. 168° C.;
UV (MeOH): 237 (4.59), 266 (4.16), 274 (4.15), 321 (3.75);
Calc. for C13H15ClN4O2 (294.7): C, 52.98; H, 5.13; N, 19.01. Found: C, 52.87; H, 5.16; N, 19.07.
From sec-butanol (400 mg, 4.5 mmol) to yield the pure title compound (0.442 g, 50%) which was characterised as follows:
M.p. 143-144° C.;
UV (MeOH): 237 (4.56), 266 (4.13), 274 (4.18), 321 (3.71); and
Calc. for C13H15ClN4O2 (294.7): C, 52.98; H, 5.13; N, 19.01. Found: C, 52.85; H, 5.13; N, 18.92.
From n-pentanol (333 mg, 4.5 mmol) to yield the pure title compound (0.37 g, 40%) which was characterised as follows:
M.p. 174° C.;
UV (MeOH): 238 (4.60), 266 (4.13), 275 (4.13), 322 (3.72); and
Calc. for C14H17ClN4O2 (308.8): C, 54.46; H, 5.55; N, 18.15. Found: C, 54.47; H, 5.66; N, 18.14.
From benzylalcohol (486 mg, 4.5 mmol) and stirring for 72 hours to give the pure title compound as a yellowish powder (240 mg, 24%) which was characterised as follows:
M.p. 199-200° C.;
UV (MeOH): 207 (4.40), 237 (4.56), 265 (4.15), 274 (4.13), 322 (3.74);
Calc. for C16H13ClN4O2 (328.8): C, 58.46; H, 3.99; N, 17.04. Found: C, 58.56; H, 4.04; N, 17.05.
To a degassed suspension of a 2-acetamido-4-alkoxy-6-chloro-pyrido[3,2-d]pyrimidine (0.5 mmol), 2-, 3-, or 4-fluorophenylboronic acid (80 mg, 0.57 mmol) and potassium carbonate (2-4 mmol) in a mixture of dioxane (7.3 ml) and water (1.6 ml) was added tetrakis(triphenylphosphine)palladium(0) (29 mg, 0.025 mmol). The mixture was refluxed (bath temperature 120° C.) for 24 hours. After cooling to room temperature dichloromethane (30 ml) was added and the mixture was washed with a brine solution. The organic layer was separated, dried over Na2SO4 and evaporated in vacuo. The resulting crude material was purified by silica gel flash chromatography. The compound was eluted with the following solvent systems: CH2Cl2 (100 ml), CH2Cl2/MeOH 100:1 (101 ml), 100:2 (102 ml), 100:3 (103 ml). Evaporation of the product fractions afforded 2-amino-4-O-substituted-6-(fluorophenyl)pyrido[3,2-d]pyrimidines as crystal solids in yields varying from 70-85%. In some cases the corresponding 2-acetamidoderivates were detected and also isolated as the faster-moving component. The analytical samples were prepared by recrystallization from ether or methanol. The following compounds were synthesized according to this general procedure:
Analogous to the general procedure with 2-fluorophenylboronic acid (80 mg, 0.57 mmol) to yield the pure title compound (0.657 g, 77%) which was characterised as follows:
M.p. 182° C.;
UV (MeOH): 231 (4.47), 284 (4.29), 348 (3.89); and
Calc. for C15H13FN4O (284.3): C, 63.37; H, 4.61; N, 19.41. Found: C, 62.70; H, 4.65; N, 19.41.
Analogous to the general procedure with 3-fluorophenylboronic acid (80 mg, 0.57 mmol) to yield the pure title compound (0.69 g, 81%) which was characterised as follows:
M.p. 174° C.;
UV (MeOH): 234 (4.43), 292 (4.31), 352 (3.92); and
Calc. for C15H13FN4O (284.3): C, 63.37; H, 4.61; N, 19.41. Found: C, 62.51; H, 4.72; N, 19.10.
Analogous to the general procedure with 4-fluorophenyl boronic acid (80 mg, 0.57 mmol) to give the pure title compound (0.657 g, 77%) which was characterised as follows:
M.p. 188-189° C.;
UV (MeOH): 216 (4.48), 234 (4.44), 287 (4.34), 354 (3.89); and
Calc. for C15H13FN4O (284.3): C, 63.37; H, 4.61; N, 19.41. Found: C, 62.98; H, 4.63; N, 19.67.
Analogous to the general procedure with 2-fluorophenyl boronic acid (80 mg, 0.57 mmol) to yield the pure title compound (0.698 g, 78%) which was characterised as follows:
M.p. 191° C.;
UV (MeOH): 231 (4.49), 284 (4.30), 348 (3.90);
Calc. for C15H13FN4O (298.3): C, 64.42; H, 5.07; N, 18.78. Found: C, 64.15; H, 5.00; N, 18.76.
Analogous to the general procedure with 4-fluorophenyl boronic acid (80 mg, 0.57 mmol) to yield the pure title compound (0.698 g, 78%) which was characterised as follows:
M.p. 185-186° C.;
UV (MeOH): 216 (4.50), 233 (4.46), 287 (4.35), 353 (3.90); and
Calc. for C15H13FN4O (298.3): C, 64.42; H, 5.07; N, 18.78. Found: C, 63.86; H, 5.37; N, 18.46.
Analogous to the general procedure with 3-fluorophenylboronic acid (80 mg, 0.57 mmol) to yield the pure title compound (0.698 g, 78%) which was characterised as follows:
M.p. 200-201° C.;
UV (MeOH): 236 (4.38), 292 (4.29), 352 (3.91),
Calc. for C16H15FN4O (298.3): C, 64.42; H, 5.07; N, 18.78. Found: C, 63.07; H, 5.08; N, 18.06.
Analogous to the general procedure with 4-fluorophenyl boronic acid (80 mg, 0.57 mmol) and isolated from the first fraction on column chromatography to give the pure title compound (0.694 g, 68%) which was characterised as follows:
M.p. 196-197° C.;
UV (MeOH): 239 (4.39), 257 (4.24), 286 (4.30), 334 (3.99); and
Calc. for C18H17FN4O2 (340.4): C, 63.52; H, 5.03; N, 16.46. Found: C, 62.65; H, 4.73; N, 16.40.
Analogous to the general procedure with 4-fluorophenylboronic acid (80 mg, 0.57 mmol) and isolated a the second fraction of column chromatography to give the pure title compound (0.143 g, 16%) which was characterised as follows:
M.p. 191-192° C.;
UV (MeOH): 216 (4.50), 233 (4.46), 287 (4.35), 353 (3.90); and
Calc. for C16H15FN4O (298.3): C, 64.42; H, 5.07; N, 18.78. Found: C, 64.25; H, 5.16; N, 18.68.
Analogous to the general procedure with 2-fluorophenyl boronic acid (80 mg, 0.57 mmol) to give the pure title compound (0.75 g, 80%) which was characterised as follows:
M.p. 147-148° C.;
UV (MeOH): 232 (4.42), 284 (4.28), 348 (3.88); and
Calc. for C17H17FN4O (312.4): C, 65.37; H, 5.49; N, 17.94. Found: C, 64.55; H, 5.56; N, 17.62.
Analogous to the general procedure with 3-fluorophenyl boronic acid (80 mg, 0.57 mmol) to give the pure title compound (0.61 g, 65%) which was characterised as follows:
M.p. 160-161° C.;
UV (MeOH): 236 (4.38), 292 (4.29), 352 (3.91);
Calc. for C17H17FN4O (312.4): C, 65.37; H, 5.49; N, 17.94. Found: C, 64.84; H, 5.65; N, 18.03.
Analogous to the general procedure with 4-fluorophenyl boronic acid (80 mg, 0.57 mmol) and isolated from the first fraction of column chromatography to give the pure title compound (0.16 g, 15%) which was characterised as follows:
M.p. 170° C.;
UV (MeOH): 225 (4.32), 239 (4.39), 257 (4.22), 288 (4.32), 334 (4.00);
Calc. for C19H19FN4O2 (312.4): C, 64.40; H, 5.40; N, 15.81. Found: C, 63.73; H, 5.54; N, 15.50.
Analogous to the general procedure with 4-fluorophenyl boronic acid (80 mg, 0.57 mmol) and isolated from the second fraction of column chromatography to give the pure title compound (0.73 g, 78%) which was characterised as follows:
M.p. 172-173° C.;
UV (MeOH): 218 (4.50), 234 (4.39), 288 (4.35), 352 (3.89);
Calc. for C17H17FN4O (312.4): C, 65.37; H, 5.49; N, 17.94. Found: C, 64.84; H, 5.65; N, 18.03.
Analogous to the general procedure with 2-fluorophenyl boronic acid (80 mg, 0.57 mmol) to give the pure title compound (0.75 g, 78%) which was characterised as follows:
M.p. 165° C.;
UV (MeOH): 232 (4.46), 284 (4.32), 348 (3.93); and
Calc. for C17H17FN4O (312.4): C, 65.37; H, 5.49; N, 17.94. Found: C, 65.60; H, 5.75; N, 18.04.
Analogous to the general procedure with 3-fluorophenyl boronic acid (80 mg, 0.57 mmol) to give the pure title compound (0.75 g, 78%) which was characterised as follows:
M.p. 185° C.;
UV (MeOH): 236 (4.39), 292 (4.31), 352 (3.93);
Calc. for C17H17FN4O (312.4): C, 65.37; H, 5.49; N, 17.94. Found: C, 65.59; H, 5.55; N, 18.00.
Analogous to the general procedure with 4-fluorophenylboronic acid (80 mg, 0.57 mmol) to give the pure title compound (0.806 g, 86%) which was characterised as follows:
M.p. 196° C.;
UV (MeOH): 234 (4.40), 287 (4.34), 353 (3.89); and
Calc. for C17H17FN4O (312.4): C, 65.37; H, 5.49; N, 17.94. Found: C, 64.94; H, 5.42; N, 17.90.
Analogous to the general procedure with 2-fluorophenylboronic acid (80 mg, 0.57 mmol) to give the pure title compound (0.693 g, 74%) which was characterised as follows:
M.p. 159° C.;
UV (MeOH): 233 (4.42), 284 (4.27), 348 (3.89); and
Calc. for C17H17FN4O (312.4): C, 65.37; H, 5.49; N, 17.94. Found: C, 65.60; H, 5.42; N, 17.70.
Analogous to the general procedure with 3-fluorophenyl boronic acid (80 mg, 0.57 mmol) to give the pure title compound (0.646 g, 69%) which was characterised as follows:
M.p. 158-159° C.;
UV (MeOH): 237 (4.39), 292 (4.31), 352 (3.94); and
Calc. for C17H17FN4O (312.4): C, 65.37; H, 5.49; N, 17.94. Found: C, 64.58; H, 5.19; N, 18.04.
Analogous to the general procedure with 4-fluorophenyl boronic acid (80 mg, 0.57 mmol) to yield the pure title compound (0.645 g, 69%) which was characterised as follows:
M.p. 148° C.;
UV (MeOH): 234 (4.37), 287 (4.31), 354 (3.87); and
Calc. for C17H17FN4O (312.4): C, 65.37; H, 5.49; N, 17.94. Found: C, 65.28; H, 5.34; N, 18.03.
Analogous to the general procedure with 2-fluorophenyl boronic acid (80 mg, 0.57 mmol) to give 0.803 g (82%) which was characterised as follows:
M.p. 136-137° C.;
UV (MeOH): 232 (4.43), 284 (4.28), 348 (3.89); and
Calc. for C18H19FN4O (326.4): C, 66.24; H, 5.87; N, 17.17. Found: C, 65.83; H, 5.62; N, 17.14.
Analogous to the general procedure with 3-fluorophenyl boronic acid (80 mg, 0.57 mmol) to give the pure title compound (0.783 g, 80%) which was characterised as follows:
M.p. 142-143° C.;
UV (MeOH): 236 (4.39), 292 (4.30), 351 (3.92); and
Calc. for C18H19FN4O (326.4): C, 66.24; H, 5.87; N, 17.17. Found: C, 65.36; H, 5.72; N, 16.52.
Analogous to the general procedure with 2-fluorophenyl boronic acid (80 mg, 0.57 mmol) to yield the pure title compound (0.748 g, 72%) which was characterised as follows:
M.p. 200-202° C.;
UV (MeOH): 208 (4.45), 232 (4.43), 285 (4.28), 350 (3.90); and
Calc. for C20H15FN4O (346.4): C, 69.36; H, 4.37; N, 16.18. Found: C, 69.16; H, 4.59; N, 16.30.
Analogous to the general procedure with 3-fluorophenyl boronic acid (80 mg, 0.57 mmol) to give the pure title compound (0.717 g, 69%) which was characterised as follows:
M.p. 199-200° C.;
UV (MeOH): 208 (4.43), 235 (4.39), 292 (4.30), 352 (3.92); and
Calc. for C20H15FN4O (346.4): C, 69.36; H, 4.37; N, 16.18. Found: C, 69.07; H, 4.44; N, 15.60.
Analogous to the general procedure with 4-fluorophenyl boronic acid (80 mg, 0.57 mmol) to give the pure title compound (0.81 g, 78%) which was characterised as follows:
M.p. 225° C.;
UV (MeOH): 210 (4.46), 233 (4.43), 287 (4.35), 354 (3.92); and
Calc. for C20H15FN4O (346.4): C, 69.36; H, 4.37; N, 16.18. Found: C, 69.16; H, 4.59; N, 16.30.
A mixture of 2-amino-6-(3,4-dimethoxy-phenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one (722 mg, 2.42 mmol), 1,1,1,3,3,3-hexamethyldisilazane (2.6 ml, 12 mmol), piperazine (840 mg, 9.75 mmol), p-toluenesulphonic acid (60 mg, 0.32 mmol) and ammonium sulphate (47 mg, 0.36 mmol) in pyridine (12 ml) is refluxed for 2 days. Upon cooling down to room temperature, the reaction mixture is evaporated with silica gel. The residue is purified by silica gel flash chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 15:85, with 1% triethylamine), affording the pure title compound (439 mg). An impure fraction is purified further by preparative TLC on silica eluting with 20% MeOH and 1% Et3N in CH2Cl2 to give another 140 mg of the title compound (combined yield: 579 mg, 65%).
MS (m/z): 367 ([M+H]+, 100)
To a suspension of the compound of example 313 (36 mg, 98 μmol) in CH2Cl2 (2 ml) and triethylamine (15 μl) is added an appropriate acid chloride (105 μmol). The reaction mixture was stirred at room temperature for 45 minutes. The solvents are evaporated in vacuo and the residue is purified by preparative TLC on silica gel. Elution with 5% MeOH in CH2Cl2 afforded the pure title compounds in yields varying from 55 to 90%, depending on the acid chloride used.
This compound was synthesized using cyclohexanecarbonyl chloride. MS (m/z): 477 ([M+H]+, 100)
This compound was synthesized using propionyl chloride. MS (m/z): 423 ([M+H]+, 100)
This compound was synthesized using hexanoyl chloride. MS (m/z): 465 ([M+H]+, 100).
This compound was synthesized using methoxyacetyl chloride. MS (m/z): 439 ([M+H]+, 100).
This compound was synthesized using methanesulfonyl chloride. MS (m/z): 445 ([M+H]+, 100)
Pyrido[3,2-d]pyrimidine derivatives were first dissolved (10 mM) in dimethylsulfoxide (hereinafter referred as DMSO) and further diluted in culture medium before use for the following in vitro experiments. The commercially available culture medium consisted of RPMI-1640+10% foetal calf serum (FCS). Some pyrido[3,2-d]pyrimidine derivatives described herein were tested in the following mixed lymphocyte reaction (MLR) assay.
Peripheral blood mononuclear cells (hereinafter referred as PBMC) were isolated from heparinized peripheral blood by density gradient centrifugation over Lymphoprep (Nycomed, Maorstua, Norway). Allogeneic PBMC or Eppstein-Barr Virus-transformed human B cells [commercially available under the trade name RPMI1788 (ATCC name CCL156)] which strongly express B7-1 and B7-2 antigens were used as stimulator cells after irradiation with 30 Gy. MLR was performed in triplicate wells. After 5 days incubation at 37° C., 1 μCi [3H]-thymidine was added to each cup. After a further 16 hours incubation, cells were harvested and counted in a β-counter. Inhibition of proliferation by a compound described in some of the present examples was counted while using the formula:
wherein cpm is the thymidine count per minute. The MLR assay is regarded by those skilled in the art as an in vitro analogue of the transplant rejection since it is based on the recognition of allogeneic major histocompatibility antigens on the stimulator leukocytes, by responding lymphocytes. The IC50 value represents the lowest concentration of the pyrido[3,2-d]pyrimidine derivative (expressed in μmole/l) that resulted in a 50% suppression of the MLR. The following IC50 values in the MLR test are mentioned in table 1 below.
Peripheral blood mononuclear cells (herein referred as PBMC), in response to stimulation by lipopolysaccharide (hereinafter LPS), a gram-negative bacterial endotoxin, produce various chemokines, in particular human TNF-α. Inhibition of the activation of PBMC can therefore be measured by the level of suppression of the production of TNF-α by PBMC in response to stimulation by LPS. Inhibition measurement was performed as follows: PBMC were isolated from heparinized peripheral blood by density gradient centrifugation over Lymphoprep (commercially available from Nycomed, Norway). LPS was then added to the PMBC suspension in complete medium (106 cells/ml) at a final concentration of 1 μg/ml. The pteridine derivative to be tested was added at different concentrations (0.1 μM, 1 μM and 10 μM) and the cells were incubated at 37° C. for 72 hours in 5% CO2. The supernatants were collected, then TNF-α concentrations were measured with respectively an anti-TNF-α antibody in a sandwich ELISA (Duo Set ELISA human TNFα, commercially available from R&D Systems, United Kingdom). The calorimetric reading of the ELISA was measured by a Multiskan RC plate reader (commercially available from ThermoLabsystems, Finland) at 450 nm (reference wavelength: 690 nm). Data analysis was performed with Ascent software 2.6. (also from ThermoLabsystems, Finland): a standard curve (recombinant human TNFα) was drawn and the amount (pg/ml) of each sample on the standard curve was determined. The % suppression of human TNFα production by the pyrido[3,2-d]pyrimidine derivatives of the invention was calculated using the formula:
Peripheral blood mononuclear cells (herein referred as PBMC), in response to stimulation by lipopolysaccharide (LPS), a gram-negative bacterial endotoxin, produce various chemokines, in particular human IL-1 β. Inhibition of the activation of PBMC can therefore be measured by the level of suppression of the production of IL-1 β by PBMC in response to stimulation by LPS.
Such inhibition measurement was performed as follows: PBMC were isolated from heparinized peripheral blood by density gradient centrifugation over Lymphoprep (commercially available from Nycomed, Norway). LPS was then added to the PMBC suspension in complete medium (106 cells/ml) at a final concentration of 1 μg/ml. The pteridine derivative to be tested was added at different concentrations (0.1 μM, 1 μM and 10 μM) and the cells were incubated at 37° C. for 72 hours in 5% CO2. The supernatants were collected, then IL-1 β concentrations were measured with an anti-IL-1 β antibody in a sandwich ELISA. The calorimetric reading of the ELISA was measured by a Multiskan RC plate reader (commercially available from ThermoLabsystems, Finland) at 450 nm (reference wavelength: 690 nm). Data analysis was performed with Ascent software 2.6. (also from ThermoLabsystems, Finland): a standard curve (recombinant human IL-1 β) was drawn and the amount (pg/ml) of each sample on the standard curve was determined.
The % suppression of human IL-1 β by the pyrido[3,2-d]pyrimidine derivatives of this invention was calculated using the formula:
Some of the pyrido[3,2-d]pyrimidine derivatives being described in the previous examples have been tested for biological activities according to the methodologies of examples 169 to 171.
The detailed nomenclature of these pyrido[3,2-d]pyrimidine derivatives is shown in the following table 1, which also shows their IC50 values (expressed in μM) in the MLR test of example 169 and in the TNF-α assay of example 170. IC50 values found in the IL-1 assay of example 171 were:
1.8 μM for the derivative of example 42.
The procedure of examples 26 to 36 is repeated, except for the use of other arylamines (as mentioned below for each example) as starting materials, and achieves in good yield the following 2-amino-4-arylamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidines, each time through the corresponding intermediate having the 2-amino group protected in the form of acetamido:
The procedure of examples 26 to 36 is repeated, except for the use of other arylalkylamines (as mentioned below for each example) as starting materials, and achieves in good yield the following 2-amino-4-arylalkylamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidines, each time through the corresponding intermediate having the 2-amino group protected in the form of acetamido:
The procedure of examples 26 to 36 is repeated, except for the use of other alkylamines (as mentioned below for each example) as starting materials, and achieves in good yield the following 2-amino-4-alkylamino-6-(3,4-dimethoxyphenyl)pyrido[3,2-d]pyrimidines, each time through the corresponding intermediate having the 2-amino group protected in the form of acetamido:
A phosphodiesterase-4 (PDE-4) extract was prepared from cultured U937 cells, then cells were lysed and homogenised. Following homogenization, the supernatant was collected by centrifugation and loaded onto a Sephacryl S-200 column. Fractions found to contain PDE-4 activity were used in the subsequent assay procedure.
PDE-4 inhibitory activity of some of the pyrido[3,2-d]pyrimidine derivatives described in the previous examples has been assessed using an isotopic two-step method as follows. The derivative to be tested (in 1% DMSO) was combined with 0.2 μg of PDE-4 enzyme and preincubated for 15 minutes at 25° C. in a buffer containing 50 mM Tris-HCl and 5 mM MgCl2 at pH 7.5. Radiolabelled cyclic [3H]AMP+cAMP was then added to provide a final concentration of 1.01 μM and incubated for 20 minutes at 25° C. Active PDE-4 enzyme hydrolyses the cyclic [3H]AMP into 5′-[3H]AMP. The reaction was terminated by incubating the reaction mixture at 100° C. Snake venom from Crotalus atrox (10 μl of 10 mg/ml) was added for 10 minutes at 37° C. for further hydrolyzing 5′-[3H]AMP into [3H]adenosine by the effect of nucleotidase contained in said snake venom. The reaction was then terminated by the addition of 200 μL of an anion exchange resin (AG1-X2) which binds all charged nucleotides except [3H]adenosine. The resin was allowed to settle for 5 minutes and then 50 μl of the aqueous phase was taken and combined with 0.2 ml of scintillation fluid. The radioactivity of the solution was measured using a liquid scintillation counter.
Table 2 shows IC50 values (expressed in μM), or the percentage inhibition at a certain concentration, of some derivatives of the previous examples which have been tested in this assay.
Huh-5-2 cells [a cell line with a persistent HCV replicon 13891 uc-ubi-neo/NS3-3′/5.1; replicon with firefly luciferase-ubiquitin-neomycin phosphotransferase fusion protein and EMCV-IRES driven NS3-5B HCV polyprotein] was cultured in a RPMI medium (commercially available from Gibco) supplemented with 10% fetal calf serum, 2 mM L-glutamine (commercially available from Life Technologies), 1× non-essential amino acids (commercially available from Life Technologies); 100 IU/ml penicillin, 100 μg/ml streptomycin and 250 μg/ml G418 (Geneticin, commercially available from Life Technologies). Cells were seeded at a density of 7,000 cells per well in 96 well View Plate (commercially available from Packard) in a medium containing the same components as described above, except for G418. Cells were allowed to adhere and proliferate for 24 hours. At that time, the culture medium was removed and serial dilutions of the pyrido[3,2-d]pyrimidine derivatives to be tested were added in a culture medium lacking G418. Interferon-α 2a (500 IU) was included as a positive control. Plates were further incubated at 37° C. and 5% CO2 for 72 hours. Replication of the HCV replicon in Huh-5 cells resulted in luciferase activity in the cells. Luciferase activity was measured by adding 50 μl of 1× Glo-lysis buffer (commercially available from Promega) for 15 minutes followed by 50 μl of the Steady-Glo Luciferase assay reagent (commercially available from Promega). Luciferase activity was measured with a luminometer and the signal in each individual well was expressed as a percentage of the untreated cultures. Parallel cultures of Huh-5-2 cells, seeded at a density of 7,000 cells/well of classical 96-well cell culture plates (commercially available from Becton-Dickinson) were treated in a similar fashion except that no Glo-lysis buffer or Steady-Glo Luciferase reagent was added. Instead the density of the culture was measured by means of the MTS method (commercially available from Promega).
Results in table 3 are expressed by the following data:
Table 3 shows EC50 and CC50 values (expressed in μM, i.e. μmol/l) of a few derivatives tested in this assay.
To a suspension of 2-amino-6-chloro-3H-pyrido[3,2-d]pyrimidin-4-one (2.55 g, 13.0 mmol) in dioxane (190 ml) and water (40 ml), potassium carbonate (7.22 g, 52.2 mmol) and 4-fluorophenylboronic acid (2.02 g, 14.4 mmol) were added and the mixture was purged with nitrogen for 15 minutes. Upon addition of tetrakis (triphenylphosphine)palladium(0) (750 mg, 0.65 mmol), the reaction mixture was heated at reflux temperature under a nitrogen atmosphere for 18 hours. The cooled mixture was filtered through Celite 545 and the filtrate was acidified with 6 M hydrochloric acid till pH 5-6 (as measured with indicator paper). The resulting suspension was kept at 4° C. overnight and the yellow precipitate (389 mg) was filtered off, washed twice with cold water and dried. The filtrate was concentrated under reduced pressure and worked up in the same way to yield another crop of the pure (by TLC on silica plates developed in 15% methanol in dichloromethane) title product (1.58 g) along with an impure fraction. The latter was purified by column chromatography on silica (10% methanol 1% triethylamine in dichloromethane) to afford a further 168 mg of the title compound. The overall yield was 2.14 g (64%) for the title compound which was characterised as follows: MS (m/z): 257 ([M+H]+, 100).
A suspension of 2-amino-4-oxo-6-(4-fluorophenyl)-pyrido-[3,2-d]pyrimidine (1.97 g, 7.69 mmol), piperazine (2.69 g, 31.2 mmol), p-toluenesulfonic acid monohydrate (195 mg, 1.0 mmol), ammonium sulfate (156 mg, 1.2 mmol) and 1,1,1,3,3,3-hexamethyldisilazane (HMDS; 8.5 ml, 40.3 mmol) in pyridine (40 ml) was heated at reflux for 4 days. Another aliquot of piperazine was added and the reaction mixture was heated at reflux for one more day. Upon cooling, the reaction mixture was evaporated with silica gel and purified twice on a silica gel column (15-20% methanol and 1% triethylamine in dichloromethane) to afford the title compound (1.82 g, 73%) which was characterised as follows: MS (m/z): 325 ([M+H]+, 100).
A solution of 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (40 mg, 0.12 mmol), 2-naphthoxyacetic acid (34 mg, 0.16 mmol), diisopropylethylamine (hereinafter referred as DIPEA; 0.33 mmol) and O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU; 66 mg, 0.21 mmol) in dry DMF (2 ml) was stirred under an N2 atmosphere for 3 hours at room temperature. The reaction mixture was applied directly onto a plate of silica gel. Developing with 8% methanol in dichloromethane afforded the title compound (21 mg, 28%) which was characterised as follows: MS (m/z): 509 ([M+H]+, 100).
This compound was prepared according to the procedure of example 383, using (3-methylphenoxy)acetic acid and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 54% for the title compound which was characterised as follows: MS (m/z): 473 ([M+H]+, 100).
This compound was prepared according to the procedure of example 383, using 3-chlorophenoxyacetic acid and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 34% for the title compound which was characterised as follows: MS (m/z): 493, 495 ([M+H]+, 100).
This compound was prepared according to the procedure of example 383, using 2,4-dichlorophenoxyacetic acid and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 41% for the title compound which was characterised as follows: MS (m/z): 527, 529 ([M+H]+, 100).
This compound was prepared according to the procedure of example 383, using 4-fluorophenoxyacetic acid and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 9% for the title compound which was characterised as follows: MS (m/z): 477 ([M+H]+, 100).
This compound was prepared according to the procedure of example 383, using 4-bromophenoxyacetic acid and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 17% for the title compound which was characterised as follows: MS (m/z): 537, 539 ([M+H]+, 100).
Triethylamine (15 μl) was added to a solution of 2-amino-4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (35 mg) in dichloromethane (2 ml), followed by trimethylacetyl chloride (13 μl). After stirring for 30 minutes at room temperature, the reaction mixture was applied directly onto a plate of silica gel. Elution with mixtures of dichloromethane and methanol (6-10% MeOH in CH2Cl2) yielded the title compound (41 mg, 95%) which was characterised as follows: MS (m/z): 451 ([M+H]+, 100).
This compound was prepared according to the procedure of example 389, using 4-pentenoyl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 100% for the title compound which was characterised as follows: MS (m/z): 449 ([M+H]+, 100).
This compound was prepared according to the procedure of example 389, using isobutyryl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 79% for the title compound which was characterised as follows: MS (m/z): 437 ([M+H]+, 100).
This compound was prepared according to the procedure of example 389, using tert-butylacetyl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 61% for the title compound which was characterised as follows: MS (m/z): 465 ([M+H]+, 100).
This compound was prepared according to the procedure of example 389, using acryloyl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 100% for the title compound which was characterised as follows: MS (m/z): 421 ([M+H]+, 100).
This compound was prepared according to the procedure of example 389, using dimethylthiocarbamoyl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 72% for the title compound which was characterised as follows: MS (m/z): 454 ([M+H]+, 100).
This compound was prepared according to the procedure of example 389, using dimethylcarbamoyl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 98% for the title compound which was characterised as follows: MS (m/z): 438 ([M+H]+, 100).
A solution of 2-amino-4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (100 mg, 0.27 mmol), N-(tert-butoxycarbonyl)glycine (54 mg, 0.31 mmol), DIPEA (115 μl, 0.70 mmol) and TBTU (140 mg, 0.44 mmol) in dry DMF (3 ml) was stirred under an N2 atmosphere for 4 hours at room temperature. The reaction mixture was applied directly onto a plate of silica gel. Elution with methanol in dichloromethane (6-8% MeOH in CH2Cl2) yielded the title compound (27 mg, 19%) which was characterised as follows: MS (m/z): 524 ([M+H]+, 100).
n-butyl isocyanate (12 μl, 110 μmol) was added to a solution of 2-amino-4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine (35 mg, 96 μmol) in dichloromethane (2 ml). After stirring for approximately 45 min at room temperature, the reaction mixture was applied directly onto a plate of silica gel. Elution with 10% MeOH in CH2Cl2 yielded the title compound (38 mg, 100%) which was characterised as follows: MS (m/z): 466 ([M+H]+, 100).
This compound was prepared according to the procedure of example 397, using n-hexyl isocyanate and 2-amino-4-(N-piperazin-1-yl)-6-(3,4-dimethoxyphenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 97% for the title compound which was characterised as follows: MS (m/z): 494 ([M+H]+, 100).
A suspension of 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine (40 mg, 92 μmol), potassium fluoride (22 mg, 0.37 mmol) and 4-(trifluoromethyl)phenylboronic acid (21 mg, 0.11 mmol) in dioxane (2 ml) and water (0.5 ml) was purged with nitrogen for 15 minutes. Tetrakis (triphenylphosphine)palladium(0) (8 mg, 7 μmol) was added and the reaction mixture was heated at reflux for 1 hour under an N2 atmosphere. Upon cooling, the mixture was applied directly onto a plate of silica gel. Elution with 5% methanol in dichloromethane afforded the title compound (49 mg, 98%) which was characterised as follows: MS (m/z): 543, 545 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-cyanophenylboronic acid, 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine and potassium carbonate (52 mg, 0.37 mmol) as starting materials. Yield: 18% for the title compound which was characterised as follows: MS (m/z): 500, 502 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3-fluorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 80% for the title compound which was characterised as follows: MS (m/z): 493, 495 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3-furanboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 41% for the title compound which was characterised as follows: MS (m/z): 465, 467 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3-thiopheneboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 67% for the title compound which was characterised as follows: MS (m/z): 481, 483 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3,4-difluorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 71% for the title compound which was characterised as follows: MS (m/z): 511, 513 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-chlorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 53% for the title compound which was characterised as follows: MS (m/z): 509, 511 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3-chlorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 79% for the title compound which was characterised as follows: MS (m/z): 509, 511 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-pyridineboronic acid and 2-amino-4-[4-(4-chlorophenoxy-acetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials and a reaction time of 1.5 h. Yield: 79% for the title compound which was characterised as follows: MS (m/z): 476, 478 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3-chloro-4-fluorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 56% for the title compound which was characterised as follows: MS (m/z): 527, 529 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3-pyridineboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 100% for the title compound which was characterised as follows: MS (m/z): 476, 478 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2-methoxy-5-pyridineboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 86% for the title compound which was characterised as follows: MS (m/z): 506, 508 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3,5-dimethylisoxazole-4-boronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials and a reaction time of 2 h. Yield: 53% for the title compound which was characterised as follows: MS (m/z): 494, 496 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 5-indolylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 60% for the title compound which was characterised as follows: MS (m/z): 514, 516 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 5-(dihydroxyboryl)-2-thiophenecarboxylic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials and a reaction time of 30 min. The title compound (4 mg, 8%) was isolated by precipitation and further washing with 10% ethanol in dichloromethane, and was characterised as follows: MS (m/z): 525, 527 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3-cyanophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxy-acetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials and a reaction time of 2 h. Yield: 89% for the title compound which was characterised as follows: MS (m/z): 500, 502 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-hydroxyphenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 100% for the title compound which was characterised as follows: MS (m/z): 491, 493 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2-cyanophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 49% for the title compound which was characterised as follows: MS (m/z): 500, 502 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-(methanesulfonyl)phenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 62% for the title compound which was characterised as follows: MS (m/z): 553, 555 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3-methoxyphenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 72% for the title compound which was characterised as follows: MS (m/z): 505, 507 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3-aminophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxy-acetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials and a reaction time of 1.5 h. Yield: 80% for the title compound which was characterised as follows: MS (m/z): 490, 492 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-fluoro-3-methylphenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 91% for the title compound which was characterised as follows: MS (m/z): 507, 509 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using phenylboronic acid and 2-amino-4-[4-(4-chlorophenoxy-acetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 42% for the title compound which was characterised as follows: MS (m/z): 475, 477 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2-methoxyphenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 88% for the title compound which was characterised as follows: MS (m/z): 505, 507 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2,4-difluorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 85% for the title compound which was characterised as follows: MS (m/z): 511, 513 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2-fluorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 98% for the title compound which was characterised as follows: MS (m/z): 493, 495 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2,3-dichlorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 100% for the title compound which was characterised as follows: MS (m/z): 543, 545 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-methoxyphenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 78% for the title compound which was characterised as follows: MS (m/z): 505, 507 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2,4-dichlorophenylboronic acid (49 mg, 0.26 mmol), 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine and tetrakis (triphenylphosphine)palladium(0) (14 mg) as starting materials and a reaction time of 2 h. Yield: 10% for the title compound which was characterised as follows: MS (m/z): 543, 545 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2,6-difluorophenylboronic acid (54 mg, 0.34 mmol), 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine and tetrakis (triphenylphosphine)palladium(0) (16 mg) as starting materials and a reaction time of 3.5 h. Yield: 100% for the title compound which was characterised as follows: MS (m/z): 511, 513 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2,5-dichlorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 61% for the title compound which was characterised as follows: MS (m/z): 543, 545 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2-chlorophenylboronic acid (29 mg, 0.18 mmol) and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 73% for the title compound which was characterised as follows: MS (m/z): 509, 511 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 5-chloro-2-fluorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 100% for the title compound which was characterised as follows: MS (m/z): 527, 529 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 3,4,5-trifluorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 100% for the title compound which was characterised as follows: MS (m/z): 529, 531 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2,6-dimethylphenylboronic acid (41 mg, 0.28 mmol), 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine and tetrakis (triphenylphosphine)palladium(0) (13 mg) as starting materials and a reaction time of 3 h. Yield: 87% for the title compound which was characterised as follows: MS (m/z): 503, 505 ([M+H]+, 100).
A solution of 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (114 mg, 0.35 mmol), N-(tert-butoxycarbonyl)glycine (137 mg, 0.78 mmol), DIPEA (162 μl, 0.97 mmol) and TBTU (226 mg, 0.70 mmol) in dry dioxane (10 ml) was stirred under an N2 atmosphere for 2 hours at room temperature. The reaction mixture was partitioned between dichloromethane and water and the aqueous layer was extracted three times with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. Purification by chromatography on a column of silica, eluting with 10% methanol in dichloromethane yielded the title compound (108 mg, 64%) which was characterised as follows: MS (m/z): 482 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using N-3-methanesulfonamidephenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 73% for the title compound which was characterised as follows: MS (m/z): 568, 570 ([M+H]+, 100).
A solution of 2-amino-4-(N-piperazin-1-yl)-6-chloro-pyrido[3,2-d]pyrimidine (48 mg, 0.18 mmol), glycolic acid (18 mg, 0.23 mmol), DIPEA (85 μl, 0.49 mmol) and TBTU (100 mg, 0.31 mmol) in dry DMF (2 ml) was stirred under an N2 atmosphere for 5 h at room temperature. The reaction mixture was applied directly onto a column of silica gel packed in 4% methanol in dichloromethane. Elution with the same solvent mixture yielded the title compound (17 mg, 29%) which was characterised as follows: MS (m/z): 323, 325 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-fluorophenylboronic acid and 2-amino-4-[4-(2-hydroxyacetyl)piperazin-1-yl]-6-chloro-pyrido[3,2-d]pyrimidine as starting materials and chromatography in 10% methanol in dichloromethane for purification. Yield: 67% for the title compound which was characterised as follows: MS (m/z): 383 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2,6-dichlorophenylboronic acid (18 mg, 92 μmol), 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine and tetrakis (triphenylphosphine)palladium(0) (12 mg) as starting materials and a reaction time of 4 hours. Yield: 44% for the title compound which was characterised as follows: MS (m/z): 543, 545 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-trifluoromethoxyphenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 100% for the title compound which was characterised as follows: MS (m/z): 559, 561 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2,5-difluorophenylboronic acid (44 mg, 0.28 mmol), 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine and tetrakis (triphenylphosphine)palladium(0) (14 mg, 12 μmol) as starting and a reaction time of 2.5 h. Yield: 100% for the title compound which was characterised as follows: MS (m/z): 511, 513 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-(hydroxymethyl)phenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 100% for the title compound which was characterised as follows: MS (m/z): 505, 507 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 2-chloro-6-fluorophenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials and a reaction time of 2 hours. Yield: 52% for the title compound which was characterised as follows: MS (m/z): 527, 529 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-(N-methylaminocarbonyl)phenyl boronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 85% for the title compound which was characterised as follows: MS (m/z): 532, 534 ([M+H]+, 100).
A solution of 2-amino-4-{4-[N-(tert-butoxycarbonyl)-glycyl]-piperazin-1-yl}-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (60 mg, 0.12 mmol) in dichloromethane (5 ml) was cooled to 0° C. Trifluoroacetic acid (2.2 ml, 28.3 mmol) was added slowly via a syringe. After 5 minutes, the ice bath was removed and the reaction mixture was stirred for a further 60 minutes, whereupon it was diluted with dichloromethane (20 ml) and treated with saturated sodium hydrogen carbonate solution until basic pH of the aqueous layer was reached. The layers were separated and the aqueous layer was extracted three times with dichloromethane. The combined organic layers are dried over magnesium sulfate, filtered and concentrated under reduced pressure. Purification by chromatography on a plate of silica, eluting with 20% methanol in dichloromethane yielded the title compound (11 mg, 24%) which was characterised as follows: MS (m/z): 382 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using p-tolylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials and a reaction time of 2 hours. Yield: 98% for the title compound which was characterised as follows: MS (m/z): 489, 491 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-acetylphenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials. Yield: 80% for the title compound which was characterised as follows: MS (m/z): 517, 519 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-aminomethylphenylboronic acid hydrochloride (24 mg, 0.13 mmol), 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]-pyrimidine and tetrakis(triphenylphosphine)palladium(0) (19 mg, 17 μmol) as starting materials. After 1 hour, one more aliquot of boronic acid derivative and palladium catalyst were added and the reaction was allowed to proceed for a further 60 minutes. Yield: 57% for the title compound which was characterised as follows: MS (m/z): 504, 506 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-(cyclopropylaminocarbonyl)phenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials and purification by chromatography in 8% methanol in dichloromethane. Yield: 71% for the title compound which was characterised as follows: MS (m/z): 558, 560 ([M+H]+, 100).
This compound was prepared according to the procedure of example 399, using 4-(acetamido)phenylboronic acid and 2-amino-4-[4-(4-chlorophenoxyacetyl)piperazin-1-yl]-6-chloro-pyrido-[3,2-d]pyrimidine as starting materials and purification by chromatography in 8-10% methanol in dichloromethane. Yield: 48% for the title compound which was characterised as follows: MS (m/z): 532, 534 ([M+H]+, 100).
Ethyl isocyanate (12 μl, 150 μmol) was added to a suspension of 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (43 mg, 130 μmol) in a mixture of dichloromethane (2 ml) and DMF (2 ml). After stirring for 30 minutes at room temperature (the reaction mixture then becomes clear), the whole mixture was applied directly onto a column of silica gel packed in 10% methanol in dichloromethane. Elution with the same solvent mixture yielded the title compound (16 mg, 31%) which was characterised as follows: MS (m/z): 396 ([M+H]+, 100).
This compound was prepared according to the procedure of example 450, using n-butyl isocyanate and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 26% for the title compound which was characterised as follows: MS (m/z): 424 ([M+H]+, 100).
This compound was prepared according to the procedure of example 450, using methyl isocyanate and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 24% for the title compound which was characterised as follows: MS (m/z): 382 ([M+H]+, 100).
This compound was prepared according to the procedure of example 450, using 1-adamantyl isocyanate and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 35% for the title compound which was characterised as follows: MS (m/z): 502 ([M+H]+, 100).
This compound was prepared according to the procedure of example 450, using cyclopentyl isocyanate and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials, HPLC grade acetonitrile as solvent and a reaction time of 2.5 hours. After 60 minutes following the start of the reaction, two additional equivalents of cyclopentyl isocyanate were added, followed by further two equivalents of cyclopentyl isocyanate and dry dioxane (1 ml) after another 60 minutes. Yield: 27% for the title compound which was characterised as follows: MS (m/z): 436 ([M+H]+, 100).
This compound was prepared according to the procedure of example 450, using 4-chlorophenyl isocyanate and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials and dry DMF (2 ml) as solvent. Yield: 20% for the title compound which was characterised as follows: MS (m/z): 478, 480 ([M+H]+, 100).
A suspension of 2-amino-4-oxo-6-(4-fluorophenyl)-pyrido-[3,2-d]-pyrimidine (51 mg, 0.19 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 55 μl, 0.36 mmol) in dry DMF (10 ml) was homogenized by brief sonication and stirred under an N2 atmosphere for 15 minutes. (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP; 119 mg, 0.27 mmol) and 1-(2-phenoxyethyl)piperazine (90 mg, 0.43 mmol) were added and the reaction mixture was stirred for 18 hours, whereupon it was partitioned between dichloromethane (50 ml) and water (50 ml). The organic layer was concentrated under reduced pressure. Purification by chromatography on a column of silica, eluting with 10% methanol in dichloromethane yielded the title compound (84 mg, ˜100%; lyophilization was necessary to completely remove DMF) which was characterised as follows: MS (m/z): 445 ([M+H]+, 100).
A suspension of 2-amino-4-oxo-6-(4-fluorophenyl)-pyrido-[3,2-d]-pyrimidine (21 mg, 80 μmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU; 48 μl, 0.31 mmol) in HPLC grade acetonitrile (5 ml) was homogenized by brief sonication and stirred under an N2 atmosphere for 15 minutes. (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP; 59 mg, 0.13 mmol) and 3-amino-1-Boc-piperidine hydrochloride (43 mg, 0.18 mmol) were added and the reaction mixture was stirred for 4 hours, whereupon it was partitioned between dichloromethane (25 ml) and water (25 ml). The aqueous layer was extracted two times with dichloromethane and the combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. Purification by chromatography on a column of silica, eluting with 10% methanol in dichloromethane yielded the title compound (12 mg, 34%) which was characterised as follows: MS (m/z): 439 ([M+H]+, 100).
This compound was prepared according to the procedure of example 457, using 1-(benzyloxycarbonyl)piperazine and 2-amino-4-oxo-6-(4-fluorophenyl)-pyrido-[3,2-d]-pyrimidine as starting materials, a reaction time of 1.5 hour and the following work-up: the reaction mixture was partitioned between dichloromethane (25 ml) and water (25 ml). The aqueous layer was extracted three times with dichloromethane and the combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. Purification by chromatography on a column of silica, eluting with 10% methanol in dichloromethane yielded the title compound (60 mg, 82%) which was characterised as follows: MS (m/z): 459 ([M+H]+, 100).
This compound was prepared according to the procedure of example 457, using 2-(piperazin-1-yl)-acetic acid N-(2-phenylethyl)amide and 2-amino-4-oxo-6-(4-fluorophenyl)-pyrido-[3,2-d]-pyrimidine as starting materials and dry dioxane as solvent. Yield: 95% for the title compound which was characterised as follows: MS (m/z): 486 ([M+H]+, 100).
This compound was prepared according to the procedure of example 457, using 1-(4-chlorophenyl)piperazine dihydrochloride, 2-amino-4-oxo-6-(4-fluorophenyl)-pyrido-[3,2-d]-pyrimidine and DBU (212 μl, 1.39 mmol) as starting materials and a reaction time of 4 hours. Yield: 34% for the title compound which was characterised as follows: MS (m/z): 435, 437 ([M+H]+, 100).
This compound was prepared according to the procedure of example 457, using 4-amino-1-Boc-piperidine and 2-amino-4-oxo-6-(4-fluorophenyl)-pyrido-[3,2-d]-pyrimidine as starting materials and dry dioxane as solvent. Yield: 43% for the title compound which was characterised as follows: MS (m/z): 439 ([M+H]+, 100).
DIPEA (32 μl, 0.19 mmol) was added to a nitrogen purged solution of 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (53 mg, 0.16 mmol) in dry DMF (2 ml). Methoxyacetyl chloride (15 μl, 0.16 mmol) was dissolved in 0.5 ml of dry DMF and this solution was added dropwise to the above mixture. After stirring for 30 minutes at room temperature, the reaction mixture was applied directly onto a column of silica gel packed in 10% methanol in dichloromethane. Elution with the same solvent mixture yielded the title compound (31 mg, 49%) which was characterised as follows: MS (m/z): 397 ([M+H]+, 100).
A suspension of 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (46 mg, 0.14 mmol) and DIPEA (140 μl, 0.83 mmol) in dry dioxane (5 ml) was brought under an N2 atmosphere and homogenized by brief sonication. Diethylcarbamyl chloride (90 μl, 0.70 mmol) was added and the mixture was stirred at room temperature. Further aliquots of DIPEA and diethylcarbamyl chloride were added at the time points of 30 and 60 minutes. After a total reaction time of 1.5 hour, the clear reaction mixture was applied directly onto a column of silica gel packed in 10% methanol in dichloromethane. Elution with the same solvent mixture yielded the title compound (47 mg, 79%) which was characterised as follows: MS (m/z): 424 ([M+H]+, 100).
This compound was prepared according to the procedure of example 463, using dimethylcarbamyl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 58% for the title compound which was characterised as follows: MS (m/z): 396 ([M+H]+, 100).
This compound was prepared according to the procedure of example 463, using diisopropylcarbamyl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials and a total reaction time of 2.5 hours. Yield: 82% for the title compound which was characterised as follows: MS (m/z): 452 ([M+H]+, 100).
This compound was prepared according to the procedure of example 463 using 4-morpholinocarbonyl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials and a total reaction time of 45 minutes without the additional aliquots of acyl chloride and base. Yield: 69% for the title compound which was characterised as follows: MS (m/z): 438 ([M+H]+, 100).
A suspension of 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (68 mg, 0.21 mmol) and DIPEA (42 μl, 0.25 mmol) in dry dioxane (5 ml) was brought under an N2 atmosphere and homogenized by brief sonication. A solution of 2-(4-chlorophenyl)-3-methylbutyryl chloride (47 μl, 0.23 mmol) in 0.5 ml of dry dioxane was added carefully. The reaction mixture was stirred for approximately 30 minutes and applied directly onto a column of silica gel packed in 10% methanol in dichloromethane. Elution with the same solvent mixture yielded the title compound (29 mg, 27%) which was characterised as follows: MS (m/z): 519, 521 ([M+H]+, 100).
This compound was prepared according to the procedure of example 467 using 2-chloropropionyl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 28% for the title compound which was characterised as follows: MS (m/z): 415, 417 ([M+H]+, 100).
This compound was prepared according to the procedure of example 467, using 4-chlorophenyl chloroformate and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 72% for the title compound which was characterised as follows: MS (m/z): 479, 481 ([M+H]+, 100).
This compound was prepared according to the procedure of example 467, using methyl malonyl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials and the following work-up: the reaction mixture was partitioned between dichloromethane and water. The aqueous layer was extracted three times with dichloromethane and the combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. Purification by RP-HPLC, eluting with methanol/water 50:50+0.1% trifluoroacetic acid afforded the title compound (61 mg, 69%) which was characterised as follows: MS (m/z): 425 ([M+H]+, 100).
A solution of 2-amino-4-[4-(2-chloropropionyl)piperazin-1-yl]-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (21 mg, 51 μmol), 4-chlorophenol (10 mg, 78 μmol) and anhydrous potassium carbonate (7 mg, 51 μmol) in HPLC grade acetone (4 ml) was stirred at reflux temperature under an N2 atmosphere for 3 days with stepwise addition of further potassium carbonate (25 mg, 0.18 mmol) and 4-chlorophenol (95 mg, 0.72 mmol). The reaction mixture was partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. Purification on a column of silica eluting with 10% methanol in dichloromethane afforded the title compound (12 mg, 46%) which was characterised as follows: MS (m/z): 507, 509 ([M+H]+, 100).
This compound was prepared according to the procedure of example 434, using 2-(4-chlorophenoxy)-2-methylpropionic acid and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials, a reaction time of 2 hours and purification on a column of silica eluting with 10% methanol in dichloromethane. Lyophilization was used to remove DMF from the chromatographically purified material. Yield: 59% for the title compound which was characterised as follows: MS (m/z): 521, 523 ([M+H]+, 100).
This compound was prepared according to the procedure of example 434, using 3-(4-chlorophenoxy)propionic acid and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials, dry dioxane (5 ml) as solvent, a reaction time of 1 hour and purification on a column of silica eluting with 10% methanol in dichloromethane. Yield: 64% for the title compound which was characterised as follows: MS (m/z): 507, 509 ([M+H]+, 100).
This compound was prepared according to the procedure of example 471, using phenol (77 mg, 0.82 mmol), potassium carbonate (85 mg, 0.61 mmol) and 2-amino-4-[4-(2-chloropropionyl)piperazin-1-yl]-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials. Yield: 70% for the title compound which was characterised as follows: MS (m/z): 473 ([M+H]+, 100).
A suspension of 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (45 mg, 0.14 mmol) and DIPEA (140 μl, 0.83 mmol) in dry dioxane (10 ml) was brought under an N2 atmosphere and homogenized by brief sonication. A solution of chloroacetic acid 4-chlorobenzyl ester (100 mg, 0.46 mmol) in 1 ml of dry dioxane was added and the reaction mixture was stirred at reflux temperature under an N2 atmosphere for 21 hours. The reaction mixture was partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. Purification on a column of silica eluting with 10% methanol in dichloromethane afforded the title compound (31 mg, 44%) which was characterised as follows: MS (m/z): 507, 509 ([M+H]+, 100).
A suspension of 2-amino-4-[4-(aminoacetyl)-piperazin-1-yl]-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (70 mg, 0.18 mmol) and DIPEA (37 μl, 0.22 mmol) in dry dioxane (5 ml) was brought under an N2 atmosphere and homogenized by brief sonication. 4-Chlorobenzoyl chloride (26 μl, 0.20 mmol) was added and the reaction mixture was stirred for approximately 30 minutes. Purification by chromatography on silica eluting with 7-10% methanol in dichloromethane yielded the title compound (48 mg, 51%) which was characterised as follows: MS (m/z): 520, 522 ([M+H]+, 100).
This compound was prepared according to the procedure of example 434, using N-Boc-4-chlorophenylalanine and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials, dry dioxane (5 ml) as solvent, a reaction time of 3.5 hours and the following work-up: the reaction mixture was partitioned between dichloromethane and water and the aqueous layer is extracted three times with dichloromethane. The combined organic layers are dried over magnesium sulfate, filtered and concentrated under reduced pressure. Purification by chromatography on a column of silica, eluting with 10% methanol in dichloromethane yielded the title compound (73 mg, ˜100%) which was characterised as follows: MS (m/z): 606, 608 ([M+H]+, 100).
This compound was prepared according to the procedure of example 476, using benzoyl chloride and 2-amino-4-(N-piperazin-1-yl)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine as starting materials and purification on a column of silica eluting with 10% methanol in dichloromethane. Yield: 94% for the title compound which was characterised as follows: MS (m/z): 486 ([M+H]+, 100).
Trifluoroacetic acid (TFA; 14 ml) was added via a syringe to a solution of 2-amino-4-[1-(tert-butoxycarbonyl)piperid-4-ylamino]-6-(4-fluorophenyl)-pyrido-[3,2-d]pyrimidine (333 mg, 0.76 mmol) in dichloromethane (30 ml) cooled to 0° C. under an N2 atmosphere. After 5 minutes, the ice bath was removed and the reaction mixture was stirred for a further 25 minutes, whereupon it was diluted with dichloromethane (30 ml) and treated with a saturated sodium hydrogen carbonate solution (250 ml). The layers were separated and the aqueous layer was extensively extracted with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was used in the next reaction step without further purification.
A suspension of 2-amino-4-(piperid-4-ylamino)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (87 mg, 0.26 mmol) and DIPEA (52 μl, 0.31 mmol) in dry dioxane (10 ml) was brought under an N2 atmosphere and homogenized by brief sonication. 4-Chlorophenoxyacetyl chloride (62 mg, 0.30 mmol) was added and the reaction mixture was stirred under an N2 atmosphere for 30 minutes. The reaction mixture was partitioned between dichloromethane and water. The aqueous layer was extracted with dichloromethane and the combined organic layers were dried over magnesium sulfate and concentrated under reduced pressure. Purification on a column of silica eluting with 10% methanol in dichloromethane afforded the title compound (39 mg, 30%) which was characterised as follows: MS (m/z): 507, 509 ([M+H]+, 100).
This compound was prepared according to the procedure of example 457, using (R)-1-Boc-3-methylpiperazine and 2-amino-4-oxo-6-(4-fluorophenyl)-pyrido-[3,2-d]-pyrimidine as starting materials and a reaction time of 2 hours. Yield: 22% for the title compound which was characterised as follows: MS (m/z): 439 ([M+H]+, 100).
Trifluoroacetic acid (TFA; 1 ml) was added via a syringe to a solution of (R)-4-[2-amino-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidin-4-yl]-3-methylpiperazine-1-carboxylic acid tert-butyl ester (18 mg, 41 μmol) in dichloromethane (2 ml) cooled to 0° C. under an N2 atmosphere. After 5 minutes, the ice bath was removed and the reaction mixture was stirred for a further 25 minutes, whereupon the volatiles were removed under reduced pressure. The residue was dissolved in dichloromethane (2 ml) and brought under an N2 atmosphere. DIPEA (200 μl, 1.21 mmol) and a solution of 4-chlorophenoxyacetyl chloride (12 mg, 57 μmol) in dichloromethane (2 ml) were added and the resulting mixture was after 30 minutes applied onto a column of silica packed in 6% methanol in dichloromethane. Elution with the same solvent mixture yielded the title compound (10 mg, 48%) which was characterised as follows: MS (m/z): 507, 509 ([M+H]+, 100).
This compound was prepared according to the procedure of example 457, using (1S,4S)-2-Boc-2,5-diazabicyclo[2.2.1]heptane and 2-amino-4-oxo-6-(4-fluorophenyl)-pyrido-[3,2-d]-pyrimidine as starting materials and a reaction time of 2.5 hours. Yield: 77% for the title compound which was characterised as follows: MS (m/z): 437 ([M+H]+, 100).
This compound was prepared according to the procedure of example 482, using (S,S)-5-[2-amino-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidin-4-yl]-2,5-diaza-bicyclo[2.2.1]heptane-2-carboxylic acid tert-butyl ester and 4-chlorophenoxyacetyl chloride as starting materials. Yield: 55% for the title compound which was characterised as follows: MS (m/z): 505, 507 ([M+H]+, 100).
This compound was prepared according to the procedure of example 480, using 2-amino-4-(piperid-4-ylamino)-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine and phenoxyacetyl chloride as starting materials and a final purification step by preparative thin layer chromatography on silica eluting with 7% methanol in dichloromethane. Yield: 19% for the title compound which was characterised as follows: MS (m/z): 473 ([M+H]+, 100).
This compound was prepared according to the procedure of example 457, using 1-benzyloxycarbonyl-(S)-3-methylpiperazine and 2-amino-4-oxo-6-(4-fluorophenyl)-pyrido-[3,2-d]-pyrimidine as starting materials and a reaction time of 4 hours. The extraction step was omitted; instead, solvent was removed under reduced pressure and the residue was taken in dichloromethane and applied onto a plate of silica. Eluting with 10% methanol in dichloromethane yielded the title compound (19 mg, 21%) which was characterised as follows: MS (m/z): 473 ([M+H]+, 100).
This compound was prepared according to the procedure of example 457, using (R)-1-benzoyl-3-methylpiperazine hydrochloride, 2-amino-4-oxo-6-(4-fluorophenyl)-pyrido-[3,2-d]-pyrimidine and DBU (138 μl, 0.90 mmol) as starting materials, a reaction time of 21 hours and a final purification step by preparative thin layer chromatography on silica eluting with 7% methanol in dichloromethane. Yield: 46% for the title compound which was characterised as follows: MS (m/z): 443 ([M+H]+, 100).
This compound was prepared according to the TFA treatment as described for the procedure of example 482, using (S)-[2-{4-[2-amino-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidin-4-yl]-piperazin-1-yl}-1-(4-chlorobenzyl)-2-oxoethyl]-carbamic acid tert-butyl ester as starting material and a total reaction time of 1 hour. The crude product was purified by preparative thin layer chromatography on silica eluting with 5% methanol 1% triethylamine in dichloromethane. Yield: 36% for the title compound which was characterised as follows: MS (m/z): 506, 508 ([M+H]+, 100).
This compound was prepared according to the procedure of example 482, using (S)-4-[2-amino-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidin-4-yl]-3-methylpiperazine-1-carboxylic acid tert-butyl ester and 4-chlorophenoxyacetyl chloride as starting materials and a final purification step by preparative thin layer chromatography on silica eluting with 7% methanol in dichloromethane. Yield 61% for the title compound which was characterised as follows: MS (m/z): 507, 509 ([M+H]+, 100).
A suspension of 2-amino-4-[4-(methoxycarbonylacetyl)piperazin-1-yl]-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (128 mg, 0.30 mmol), 4-chloroaniline (200 mg, 1.54 mmol) and DIPEA (55 μl, 033 mmol) in 15 ml of dry dioxane was stirred at reflux temperature under an N2 atmosphere for 5 days. The reaction mixture was partitioned between dichloromethane (50 ml) and water (50 ml). The aqueous layer was extracted twice with dichloromethane and the combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. Chromatography on silica eluting with mixtures of dichloromethane and methanol (5-10% methanol) afforded the crude title product (46 mg, 29%) for the title compound which was characterised as follows: MS (m/z): 520, 522 ([M+H]+, 100).
To a solution of 2-amino-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (example 20; 0.9 mmol, 1 g) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 9.16 mmol, 1.368 mL) in DMF (80 mL) were added (benzotriazol-1-yloxy)tris-(dimethylamino)phosphonium hexafluorophosphate (BOP, 7.63 mmol, 3.375 g) and piperazine (22.89 mmol, 1.972 g). The reaction mixture was stirred at room temperature for 16 hours after which the solvent was evaporated in vacuo. The residue was adsorbed on silica and purified by silica gel column chromatography (the mobile phase being a dichloromethane/methanol/triethylamine mixture, in a ratio gradually ranging from 98:1:1 to 95:4:1) yielding the title compound (734 mg, yield 54%), which was characterized by its mass spectrum as follows: MS (m/z): 265 ([M+H]+, 100).
To a solution of 2-amino-4-piperazino-6-chloro-pyrido[3,2-d]pyrimidine (2.77 mmol, 734 mg) and N,N-diisopropylethylamine (6.10 mmol, 1 mL) in dioxane (120 mL) and methanol (30 mL) was added a solution of 4-chlorophenoxyacetyl chloride (3.05 mmol, 625 mg) in dioxane (30 mL). The reaction mixture was stirred at room temperature for 16 hours. Then, the solvents were evaporated in vacuo and the residue was purified by means of silica gel column chromatography (the mobile phase being a dichloromethane/methanol mixture, in a ratio gradually ranging from 99:1 to 97:3) yielding the pure title compound (906 mg, yield 75%), which was characterized by its mass spectrum as follows: MS (m/z): 433 ([M+H]+, 100).
To a solution of 2-amino-4-(N-(4-chlorophenoxyacetyl)-piperazin-1-yl)-6-chloro-pyrido[3,2-d]pyrimidine (0.23 mmol, 100 mg) and potassium fluoride (0.58 mmol, 33 mg) in dioxane (8 mL) and water (3 mL) was added the appropriate arylboronic or heteroarylboronic acid, or ester thereof (0.25 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.01 mmol, 13 mg). The reaction mixture was heated at 90° C. for 4 to 7 hours (following the course of the reaction by thin layer chromatography), cooled down to room temperature and extracted with dichloromethane. The organic layer was evaporated in vacuo and the residue was purified by silica gel column chromatography (the mobile phase being a dichloromethane/methanol mixture, in a ratio gradually ranging from 99:1 to 97:3) yielding the pure, corresponding compounds (yields ranging from 45 to 91%).
The following compounds were synthesized and characterised according to this procedure:
This compound was obtained from 3-chloro-4-ethoxyphenylboronic acid in 62% yield and was characterized by its mass spectrum as follows: MS (m/z): 553 ([M+H]+, 100).
This compound was obtained from 4-ethoxyphenylboronic acid in 69% yield and was characterized by its mass spectrum as follows: MS (m/z): 519 ([M+H]+, 100).
This compound was obtained from 4-methylthiophenylboronic acid in 61% yield and was characterized by its mass spectrum as follows: MS (m/z): 521 ([M+H]+, 100).
This compound was obtained from 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol in 58% yield and was characterized by its mass spectrum as follows: MS (m/z): 521 ([M+H]+, 100).
This compound was obtained from 2,3-dihydro-1-benzofuran-5-ylboronic acid in 52% yield and was characterized by its mass spectrum as follows: MS (m/z): 517 ([M+H]+, 100).
This compound was obtained from 3-methylphenylboronic acid in 69% yield and was characterized by its mass spectrum as follows: MS (m/z): 489 ([M+H]+, 100).
This compound was obtained from 2-[(4-cyanomethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane in 62% yield and was characterized by its mass spectrum as follows: MS (m/z): 514 ([M+H]+, 100).
This compound was obtained from 2-[(3-methoxymethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane in 67% yield and was characterized by its mass spectrum as follows: MS (m/z): 519 ([M+H]+, 100).
This compound was obtained from 2-[(3-methoxy-4-benzyloxymethyl)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane in 63% yield (0.25 mmol, 86 mg) and was characterized by its mass spectrum as follows: MS (m/z): 611 ([M+H]+, 100).
This compound was obtained from 3,5-dimethyl-4-methoxyphenylboronic acid in 73% yield and was characterized by its mass spectrum as follows: MS (m/z): 533 ([M+H]+, 100).
This compound was obtained from 2-[(4-cyanomethoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane in 76% yield and was characterized by its mass spectrum as follows: MS (m/z): 530 ([M+H]+, 100).
This compound was obtained from 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl acetate in 45% yield and was characterized by its mass spectrum as follows: MS (m/z): 533 ([M+H]+, 100).
This compound was obtained from 2,4-dimethoxyphenylboronic acid in 89% yield and was characterized by its mass spectrum as follows: MS (m/z): 535 ([M+H]+, 100).
This compound was obtained from 2,5-dimethoxyphenylboronic acid in 91% yield and was characterized by its mass spectrum as follows: MS (m/z): 535 ([M+H]+, 100).
This compound was obtained from 3-amino-4-methylphenylboronic acid in 84% yield and was characterized by its mass spectrum as follows: MS (m/z): 504 ([M+H]+, 100).
This compound was obtained from 4-ethylphenylboronic acid in 72% yield and was characterized by its mass spectrum as follows: MS (m/z): 503 ([M+H]+, 100).
This compound was obtained from 4-methoxy-3-methylphenylboronic acid in 68% yield and was characterized by its mass spectrum as follows: MS (m/z): 519 ([M+H]+, 100).
This compound was obtained from in (4-ethoxycarbonylphenyl)boronic acid in 77% yield and was characterized by its mass spectrum as follows: MS (m/z): 547 ([M+H]+, 100).
This compound was obtained from 4-methylthiophene-2-boronic acid in 59% yield and was characterized by its mass spectrum as follows: MS (m/z): 495 ([M+H]+, 100).
This compound was obtained from 2-methylphenylboronic acid in 81% yield and was characterized by its mass spectrum as follows: MS (m/z): 489 ([M+H]+, 100).
This compound was obtained from 4-benzyloxy-3-fluorophenylboronic acid in 86% yield and was characterized by its mass spectrum as follows: MS (m/z): 599 ([M+H]+, 100).
This compound was obtained from 4-amino-3-methoxyphenylboronic acid in 54% yield and was characterized by its mass spectrum as follows: MS (m/z): 520 ([M+H]+, 100).
This compound was obtained from 4-acetoxycarbonyl-3-methoxyphenyl boronic acid pinacol ester in 69% yield and was characterized by its mass spectrum as follows: MS (m/z): 563 ([M+H]+, 100).
To a solution of 2-amino-4-(N-(4-chlorophenoxyacetyl)-piperazin-1-yl)-6-chloro-pyrido[3,2-d]pyrimidine (0.2 mmol, 86 mg) in dioxane (6 ml) was added the appropriate arylboronic acid (0.22 mmol). A solution of potassium carbonate (170 mg) in water (2 ml) was added to the first solution. Tetrakis(triphenylphosphine)palladium(0) (40 mg) was added and the reaction mixture was heated at 75° C. overnight. The solvents were evaporated in vacuo and the residue was purified by preparative thin layer chromatography, the mobile phase being a mixture of dichloromethane/methanol mixture (in a ratio from 90:10) yielding the pure title compounds (yields ranging from 45 to 75%) which were characterised as follows.
This compound was obtained from 3,4-dimethylphenyl boronic acid in 69% yield and was characterized by its mass spectrum as follows: MS (m/z): 503 ([M+H]+, 100.
This compound was obtained from 3-chloro-4-methoxyphenyl boronic acid in 71% yield and was characterized by its mass spectrum as follows: MS (m/z): 539 ([M+H]+, 100.
This compound was obtained from 4-fluoro-3-ethoxyphenyl boronic acid in 62% yield and was characterized by its mass spectrum as follows: MS (m/z): 537 ([M+H]+, 100).
This compound was obtained from 4-methoxycarbonyl-3-methoxyphenyl boronic acid pinacol ester in 78% yield and was characterized by its mass spectrum as follows: MS (m/z): 563 ([M+H]+, 100).
This compound was obtained from 4-isobutylphenyl boronic acid in 83% yield and was characterized by its mass spectrum as follows: MS (m/z): 531 ([M+H]+, 100).
This compound was obtained from 4-methoxy-3-hydroxyphenyl boronic acid pinacol ester in 59% yield and was characterized by its mass spectrum as follows: MS (m/z): 521 ([M+H]+, 100).
This compound was obtained from 4-isopropoxyphenyl boronic acid in 77% yield and was characterized by its mass spectrum as follows: MS (m/z): 533 ([M+H]+, 100).
To a solution of chromane-2-carboxylic acid (0.91 mmol, 161 mg) and N,N-diisopropylethylamine (0.91 mmol, 150 μL) in dioxane (25 mL) was added O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU, 0.91 mmol, 291 mg). After 5 minutes of stirring at room temperature, 2-amino-4-piperazino-6-chloro-pyrido[3,2-d]pyrimidine (0.75 mmol, 200 mg) was added whereupon the mixture was sonicated for 1 minute. The reaction mixture was stirred for 16 hours at room temperature, the solvent was evaporated in vacuo and the crude residue was purified by means of silica gel column chromatography (the mobile phase being a dichloromethane/methanol mixture, in a ratio gradually ranging from 99:1 to 97:3) yielding the pure title compound (234 mg, yield 73%), which was characterized by its mass spectrum as follows: MS (m/z): 425 ([M+H]+, 100).
To a solution of [4-(2-amino-6-chloropyrido[3,2-d]pyrimidin-4-yl)piperazin-1-yl]chroman-2-ylmethanone (0.12 mmol, 50 mg) and potassium fluoride (0.29 mmol, 17 mg) in dioxane (4 mL) and water (1.5 mL) was added the appropriate arylboronic acid (0.13 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.006 mmol, 7 mg). The reaction mixture was heated at 90° C. for 24 hours, cooled down to room temperature and extracted with dichloromethane. The organic layer was evaporated in vacuo and the residue was purified by silica gel column chromatography (the mobile phase being a dichloromethane/methanol mixture, in a ratio gradually ranging from 99:1 to 97:3) yielding the pure, corresponding compounds (yield 53%) which were characterised as follows.
This compound was obtained from 4-fluorophenylboronic acid in 53% yield and was characterized by its mass spectrum as follows: MS (m/z): 485 ([M+H]+, 100).
This compound was obtained from p-tolylboronic acid in 53% yield and was characterized by its mass spectrum as follows: MS (m/z): 481 ([M+H]+, 100).
To a solution of 2-carboxamide-3-amino-6-chloropyridine (11.6 mmol, 2.0 g) and potassium carbonate (29.1. mmol, 4.03 g) in dioxane (240 mL) and water (90 mL) was added 4-fluorophenylboronic acid (12.8 mmol, 1.79 g) and tetrakis (triphenylphosphine)palladium(0) (0.58 mmol, 673 mg). The reaction mixture was heated at 90° C. for 6 hours, cooled down to room temperature and extracted with dichloromethane. The organic layer was evaporated in vacuo and the residue was purified by silica gel column chromatography (the mobile phase being a dichloromethane/methanol mixture of 99.75:0.25) yielding the pure title compound (2.237 g, yield 83%) which was characterized by its mass spectrum as follows: MS (m/z): 232 ([M+H]+, 100).
A suspension of 2-carboxamide-3-amino-6-(4-fluorophenyl)pyridine (1.3 mmol, 300 mg) and the appropriate ortho ester (5 mL) was heated at 140° C. for 24 hours. After cooling down the reaction mixture, the solids formed were filtered off, rinsed with diethyl ether and dried to the air (yields ranging from 58% to 89%). The following compounds were synthesized according to this procedure and were used as such without further purification:
This compound was obtained from ethyl ortho-acetate in 58% yield and was characterized by its mass spectrum as follows: MS (m/z): 256 ([M+H]+, 100).
This compound was obtained from ethyl ortho-propionate in 63% yield and was characterized by its mass spectrum as follows: MS (m/z): 270 ([M+H]+, 100).
This compound was obtained from ethyl ortho-benzoate in 89% yield and was characterized by its mass spectrum as follows: MS (m/z): 318 ([M+H]+, 100).
To a suspension of 2-carboxamide-3-amino-6-(4-fluorophenyl)pyridine (4.3 mmol, 1.0 g) in o-xylene (10 mL) was added 2-chloro-1,1,1-triethoxyethane (4.7 mmol, 935 mg). The reaction mixture was heated at 145° C. for 4 hours, cooled down and the solids formed were filtered off, rinsed with diethyl ether and dried to the air (1.035 g, yield 87%). The resulting title compound was characterized by its mass spectrum as follows: MS (m/z): 290 ([M+H]+, 100).
To a solution of 2-substituted-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one (1.0 mmol) and N,N-diisopropylethylamine (2.2 mmol, 377 μL) in toluene (10 mL) was added POCl3 (1.5 mmol, 137 μL). The reaction mixture was stirred at 110° C. for 5 hours, cooled down to room temperature and extracted with dichloromethane and 2N hydrogen chloride. The organic phase was dried over magnesium sulfate and reduced in vacuo. The residue was dissolved in dichloromethane (25 mL) and piperazine (4.0 mmol, 86 mg), followed by N,N-diisopropylethylamine (2.0 mmol, 342 μL) were added. The reaction mixture was stirred at room temperature for 16 hours whereupon the solvent was evaporated in vacuo. The residue was dissolved in dichloromethane (25 mL) and m-tolyl isocyanate (2.0 mmol, 258 μL) was added. The mixture was stirred at room temperature for 6 hours. Then, the solvent was evaporated in vacuo and the crude reaction mixture was purified by means of silica gel column chromatography (the mobile phase being a dichloromethane/methanol mixture, in a ratio gradually ranging from 99:1 to 97:3) yielding the corresponding pure title compounds (yields ranging from 54% to 91%) which were characterised as follows.
This compound was obtained in 85% yield and was characterized by its mass spectrum as follows: MS (m/z): 457 ([M+H]+, 100).
This compound was obtained in 54% yield and was characterized by its mass spectrum as follows: MS (m/z): 471 ([M+H]+, 100).
This compound was obtained in 91% yield and was characterized by its mass spectrum as follows: MS (m/z): 519 ([M+H]+, 100).
To a solution of 2-chloromethyl-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidin-4(3H)-one (2.9 mmol, 850 mg) and N,N-diisopropylethylamine (3.2 mmol, 552 μL) in toluene (40 mL) was added POCl3 (4.1 mmol, 376 μL). The reaction mixture was stirred at 110° C. for 3 hours, cooled down to room temperature and extracted with dichloromethane and 2N hydrogen chloride. The organic phase was dried over magnesium sulfate and reduced in vacuo. The residue was dissolved in 1,4-dioxane (80 mL) and tert-butyl 1-piperazinecarboxylate (3.2 mmol, 601 mg), followed by N,N-diisopropylethylamine (6.5 mmol, 1.1 mL) were added. The reaction mixture was stirred at room temperature for 16 hours whereupon the solvent was evaporated in vacuo. The residue was purified by means of silica gel column chromatography (the mobile phase being a dichloromethane/methanol mixture, in a ratio gradually ranging from 99.5:0.5 to 99:1) yielding the pure title compound (1.115 g, yield 83%) which was characterized by its mass spectrum as follows: MS (m/z): 458 ([M+H]+, 100).
To a solution of 2-chloromethyl-4-[N-(tert-butoxycarbonyl)piperazin-1-yl]-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (0.44 mmol, 200 mg) in dichloromethane (20 mL) was added trifluoroacetic acid (20 mL). The reaction mixture was stirred at room temperature for 1 hour after which the solvents were evaporated in vacuo. The residue was dissolved in dichloromethane (20 mL) and N,N-diisopropylethylamine (7.9 mmol, 1.3 mL) followed by the appropriate isocyanate (0.87 mmol) were added. The mixture was stirred at room temperature for 3 hours, the solvents were evaporated in vacuo and the residue was purified by silica gel column chromatography (the mobile phase being a dichloromethane/methanol mixture, in a ratio gradually ranging from 99:1 to 97:3) yielding the corresponding, pure title compounds (yields ranging from 81% to 87%) which were characterised as follows.
This compound was obtained from m-tolyl isocyanate in 87% yield and was characterized by its mass spectrum as follows: MS (m/z): 491 ([M+H]+, 100).
This compound was obtained from 3-methylbenzyl isocyanate in 81% yield and was characterized by its mass spectrum as follows: MS (m/z): 505 ([M+H]+, 100).
To a solution of 2-chloromethyl-4-[N-(3-methylphenylcarbamoyl)piperazin-1-yl]-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (0.10 mmol, 50 mg) in DMF (1 mL) was added the appropriate amine. The reaction mixture was stirred at room temperature for 20 hours whereupon the solvent was evaporated in vacuo. The residue was purified by preparative liquid chromatography (Waters Delta 600, XBridge™ Prep C18 5 μm 19×150 mm, using a gradient of water/acetonitrile (0.1% triethylamine) as mobile phase) yielding the corresponding pure compounds (yields ranging from 23% to 69%) which were characterised as follows.
This compound was obtained from dimethylamine (40% in water) in 69% yield. and was characterized by its mass spectrum as follows: MS (m/z): 500 ([M+H]+, 100).
This compound was obtained from methoxyethylamine in 24% yield and was characterized by its mass spectrum as follows: MS (m/z): 530 ([M+H]+, 100).
This compound was obtained from cyclopropylamine and was characterized by its mass spectrum as follows: MS (m/z): 512 ([M+H]+, 100).
To a solution of 2-amino-6-chloro-pyrido[3,2-d]pyrimidin-4(3H)-one (example 20, 100 mg, 0.51 mmol) in DMF (20 ml) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 115 μl, 0.76 mmol), benzotriazol-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate (BOP, 0.66 mmol, 292 mg) and piperazine (1.53 mmol, 131 mg). The solution was stirred for 3 hours at room temperature. The solvents were evaporated in vacuo and the crude residue was purified by flash chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 3:97 with 0.5% aq. NH3 solution), yielding the title compound as a white powder (85 mg, 63%) which was characterised as follows: MS (m/z): 266 ([M+H]+, 100).
To a solution of 2-amino-4-(N-piperazin-1-yl)-6-chloro-pyrido(3,2-d)pyrimidine (85 mg, 0.32 mmol) in DMF (10 ml) was added m-tolyl isocyanate (0.35 mmol, 46 μl). The reaction was stirred at room temperature overnight. The solvents were evaporated and the crude residue was further purified by flash chromatography, the mobile phase being a mixture of methanol/dichloromethane (in a ratio ranging from 2:98 to 3:97), yielding the pure title compound as a white solid (91 mg, 72%) which was characterised as follows MS (m/z): 398 ([M+H]+, 100).
To a solution of 2-amino-4-[(3-methylphenyl carbamoyl)-piperazin-1-yl]-6-chloro-pyrido[3,2-d]pyrimidine (70 mg, 0.18 mmol) in dioxane (15 ml) and water (3 ml) was added potassium carbonate (0.53 mmol, 72 mg), the appropriate arylboronic or heteroarylboronic acid (0.23 mmol) and tetrakis(triphenylphosphine)palladium (8.8 μmol, 10 mg). The reaction was refluxed for 2 hours. The solvents were evaporated and the crude residue was purified by silica gel flash chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio gradually ranging from 2:98 to 3:97), yielding the pure title compounds in yields ranging from 64 to 75% which were characterised as follows.
This compound was obtained from 4-pyridylboronic acid and was characterised as follows MS (m/z): 441 ([M+H]+, 100)
This compound was obtained from 4-cyanophenylboronic acid and was characterised as follows: MS (m/z): 465 ([M+H]+, 100).
This compound was obtained from 3-fluorophenylboronic acid and was characterised as follows:
MS (m/z): 458 ([M+H]+, 100)
This compound was obtained from 3-cyanophenylboronic acid and was characterised as follows: MS (m/z): 465 ([M+H]+, 100)
This compound was obtained from 2-cyanophenylboronic acid_and was characterised as follows:_MS (m/z): 465 ([M+H]+, 100)
This compound was obtained from 2-fluorophenylboronic acid and was characterised as follows: MS (m/z): 457 ([M+H]+, 100)
This compound was obtained from 4-trifluoromethylphenylboronic acid and was characterised as follows:
MS (m/z): 508 ([M+H]+, 100).
This compound was obtained from 2-chlorophenylboronic acid and was characterised as follows:
MS (m/z): 474 ([M+H]+, 100)
This compound was obtained from 2,6-dimethylphenylboronic acid and was characterised as follows:
MS (m/z): 467 ([M+H]+, 100).
This compound was obtained from 2,4,6-trifluorophenylboronic acid and was characterised as follows:
MS (m/z): 494 ([M+H]+, 100).
To a solution of 4-(N-piperazin-1-yl)-6-chloro-pyrido[3,2-d]-pyrimidine (example 65; 450 mg; 1.8 mmol) in dichloromethane (30 ml) was added m-tolyl isocyanate (1 ml). The reaction mixture was stirred for 16 hours. The solvents were evaporated in vacuo. The crude residue was purified by silica gel flash chromatography, the mobile phase being a mixture of dichloromethane and methanol (in a ratio of 95:5), yielding the title compound as a white solid (598 mg, 87%) and was characterised as follows:
MS (m/z): 383 ([M+H]+, 100).
A solution of 4-[(3-methylphenyl carbamoyl)-piperazin-1-yl]-6-chloro-pyrido[3,2-d]pyrimidine (0.2 mmol, 77 mg), the appropriate arylboronic or heteroarylboronic acid (0.3 mmol) and potassium fluoride (46 mg, 0.8 mmol) in dioxane (8 ml) and water (2 ml) was degassed for 30 minutes. Then, tetrakis(triphenylphosphine)palladium (30 mg) was added and the reaction mixture was refluxed for 3 hours. The solvents were evaporated and the residue was further purified by flash chromatography on silica, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 4:96), yielding the title compounds as white powders, in yields ranging from 50 to 70%, which were characterised as follows:
This compound was obtained from 4-cyanophenylboronic acid and was characterised as follows: MS (m/z): ([M+H]+, 100).
This compound was obtained from 3-cyanophenylboronic acid and was characterised as follows: MS (m/z): ([M+H]+, 100).
This compound was obtained from 3-methyl-4-fluoro-phenylboronic acid and was characterised as follows:
MS (m/z): 457 ([M+H]+, 100).
This compound was obtained from 2,4-difluorophenylboronic acid and was characterised as follows:
MS (m/z): 461 ([M+H]+, 100).
This compound was obtained from 2-cyanophenylboronic acid and was characterised as follows:
MS (m/z): 449 ([M+H]+, 100).
This compound was obtained from 4-fluorophenylboronic acid and was characterised as follows: MS (m/z): 443 ([M+H]+, 100)
This compound was obtained from 3-fluoro-4-ethoxy-phenylboronic acid and was characterised as follows: MS (m/z): 487 ([M+H]+, 100)
This compound was obtained from 3-chloro-4-ethoxyphenyl boronic acid and was characterised as follows: MS (m/z): 503 ([M+H]+, 100)
This compound was synthesized from 3,4,5-trifluorophenylboronic acid and was characterised as follows:
MS (m/z): 479 ([M+H]+, 100).
This compound was obtained from 2-thienylboronic acid and was characterised as follows:
MS (m/z): 431 ([M+H]+, 100).
This compound was obtained from 2-furylboronic acid and was characterised as follows:
MS (m/z): 414 ([M+H]+, 100).
A mixture of 3-amino-6-(4-fluorophenyl)-pyridine-2-carboxylic acid amide (0.92 g, 4.0 mmol) and triphosgene (0.60 g, 2.0 mmol) in 10 ml dioxane was heated under reflux for 2 h. After cooling to room temperature, the precipitate was collected by filtration and washed with diethyl ether. The title compound was obtained (0.98 g, 95%) as a yellowish solid and was characterised as follows:
MS (m/z): 258.2 ([M+H]+, 100).
A suspension of 2,4-dihydroxy-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (0.52 g, 2.0 mmol) in phosphorus oxychloride (10 ml) and diisopropylethylamine (1 ml) was refluxed for 6 hours. After concentration under reduced pressure, the residue was extracted with dichloromethane (100 ml) and washed with ice water till pH=6-7. The combined organic layers were dried over MgSO4, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel flash chromatography, the mobile phase being a mixture of methanol and dichloromethane, in a ratio of 1:50, yielding the title compound as a white solid (400 mg, 68%) and was characterised as follows:
MS (m/z): 295.2 ([M+H]+, 100).
A mixture of 2,4-dichloro-6-(4-fluorophenyl)-pyrido[3,2-d]pyrimidine (0.35 g, 1.2 mmol) and 1-Boc-piperazine (0.28 g, 1.5 mmol) in dioxane (20 ml) was stirred at room temperature for 1 hour. After concentration under reduced pressure, the residue was purified by silica gel flash chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 1:100), yielding the title compound as a white solid (0.52 g, 98%) which was characterised as follows:
MS (m/z): 444.2 ([M+H]+, 100).
A mixture of 2-chloro-6-(4-fluorophenyl)-4-(4-Boc-piperazino)-pyrido[3,2-d]pyrimidine (133 mg, 0.3 mmol) and pyrrolidine (2.0 ml) in dioxane (10 ml) was heated under reflux for 4 hours. After concentration under reduced pressure, the residue was purified by silica gel flash chromatography, the mobile phase being a mixture of methanol and dichloromethane, in a ratio of 1:25, yielding the title compound as a yellow solid (140 mg, 97%) which was characterised as follows:
MS (m/z): 479.1 ([M+H]+, 100)
A similar procedure as for the synthesis of the compound of previous example was followed, using cyclopentylamine instead of pyrrolidine. The pure title compound was isolated in 95% yield as a yellow solid which was characterised as follows:
MS (m/z): 493.1 ([M+H]+, 100)
A mixture of 2-chloro-6-(4-fluorophenyl)-4-(4-Boc-piperazino)-pyrido[3,2-d]pyrimidine (400 mg, 0.9 mmol) and methylamine in THF (2 ml, 8 mmol) was heated in a sealed tube at 90° C. for 3 hours. After cooling to room temperature, the solvents were removed under reduced pressure. The residue was purified by silica gel flash chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 1:20), yielding two compounds:
2-methylamino-6-(4-fluorophenyl)-4-(4-Boc-piperazino)-pyrido[3,2-d]pyrimidine (237 mg, 60%), which was characterised as follows:
MS (m/z): 439.1 ([M+H]+, 100); and
2,4-dimethylamino-6-(4-fluorophenyl)pyrido[3,2-d]pyrimidine (89 mg, 35%) which was characterised as follows:
MS (m/z): 284.2 ([M+H]+, 100)
To a solution of 2-pyrrolidino-6-(4-fluorophenyl)-4-(4-Boc-piperazino)-pyrido[3,2-d]pyrimidine (30 mg, 0.06 mmol) in dichloromethane (1 ml), was added trifluoroacetic acid (1 ml). The resulting mixture was stirred at room temperature for 30 minutes. After concentration under reduced pressure, the residue was dissolved in dichloromethane (5 ml). Then, diisopropylethylamine (1 ml) and m-tolyl isocyanate (1 ml) were added respectively. The mixture was stirred at room temperature for 30 min. The solvents were removed under reduced pressure and residue was purified by silica gel flash chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 1:30), yielding the title compound as a white solid (30 mg, 98%) which was characterised as follows:
MS (m/z): 512.2 ([M+H]+, 100)
This compound was synthesized from 2-cyclopentylamino-6-(4-fluorophenyl)-4-(4-Boc-piperazino)-pyrido[3,2-d]pyrimidine using the procedure from previous example, yielding the title compound as a white solid in 95% yield which was characterised as follows:
MS (m/z): 526.2 ([M+H]+, 100).
To a solution of 2-pyrrolidino-6-(4-fluorophenyl)-4-(4-Boc-piperazino)-pyrido[3,2-d]pyrimidine (25 mg, 0.05 mmol) in dichloromethane (1 ml), was added trifluoroacetic acid (1 ml) was added. The resulting mixture was stirred at room temperature for 30 minutes. After concentration under reduced pressure, the residue was dissolved in dichloromethane (5 ml). Then, diisopropylethylamine (1 ml) and 4-chlorophenoxyacetyl chloride (0.6 mmol) were added respectively. The mixture was stirred at room temperature for 30 minutes. The solvents were removed under reduced pressure and residue was purified by silica gel flash chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 1:30), yielding the title compound as a white solid (25 mg, 89%) which was characterised as follows:
MS (m/z): 561.2 ([M+H]+, 100)
This compound was synthesized from 2-cyclopentylamino-6-(4-fluorophenyl)-4-(4-Boc-piperazino)-pyrido[3,2-d]pyrimidine using the procedure described for example 572. The title compound isolated in 91% yield was characterised as follows:
MS (m/z): 547.2 ([M+H]+, 100).
This compound was synthesized from 2-methylamino-6-(4-fluorophenyl)-4-(4-Boc-piperazino)-pyrido[3,2-d]pyrimidine using the procedure described for example 572, yielding the title compound as a yellow solid (75% yield) which was characterised as follows:
MS (m/z): 507.1 ([M+H]+, 100).
To a solution of 2-acetylamino-4-(1,2,4-triazolyl)-6-(4-fluorophenyl)-pyrido(3,2-d)pyrimidine (163 mg, 0.5 mmol) in dioxane (20 ml) was added [(R)-3-Boc-aminopyrrolidine (93 mg, 0.5 mmol). The reaction mixture was stirred at 50° C. overnight, yielding crude 2-acetylamino-4-[(R)-3-Boc-amino-pyrrolidin-1-yl]-6-(4-fluorophenyl)-pyrido(3,2-d)pyrimidine. In order to deprotect the acetyl group, the solvents were evaporated in vacuo and the crude residue (containing crude 2-acetylamino-4-[(R)-3-Boc-amino-pyrrolidin-1-yl]-6-(4-fluorophenyl)-pyrido(3,2-d)pyrimidine) was redissolved in a mixture of dichloromethane and ethanol (in a ratio of 80:20, 10 ml). A sodium ethoxide solution (0.2 N solution) was added till pH=12 and the reaction mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo and the crude residue was purified by preparative thin layer chromatography on silica, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 10:90), yielding the title compound as a white powder (89 mg, 42%) which was characterised as follows:
MS (m/z): 425 ([M+H]+, 100).
This compound was synthesized in a similar way as described for example 575, using (S)-3-Boc-amino-pyrrolidine as reagent, and was characterised as follows:
MS (m/z): 425 ([M+H]+, 100).
This compound was synthesized using the procedure of example 575, using (S)-1-Boc-3-aminopyrrolidine as a reagent and was characterised as follows:
MS (m/z): 425 ([M+H]+, 100)
To a solution of 2-amino-4-[(S)-3-Boc-amino-pyrrolidin-1-yl]-6-(4-fluorophenyl)-pyrido(3,2-d)pyrimidine from example 576 (100 mg, 0.24 mmol) in dichloromethane (10 ml) was added a mixture of dichloromethane/trifluoroacetic acid (3 ml, 1:1). The reaction mixture was stirred for 30 minutes at room temperature. The solvents were evaporated in vacuo. The crude residue was redissolved in dichloromethane (5 ml) and diisopropylethylamine (30 μl) and m-tolylisocyanate (30 μl) were added. The reaction was stirred for 2 hours at room temperature. The solvents were evaporated in vacuo and the crude residue was further purified by thin layer preparative thin layer chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 10:90), yielding the pure title compound as a white powder (66 mg, 61%) which was characterised as follows:
MS (m/z): 458 ([M+H]+, 100).
This compound was synthesized using similar methods as described in example 578, using 2-amino-4-[(R)-3-Boc-amino-pyrrolidin-1-yl]-6-(4-fluoro-phenyl)-pyrido(3,2-d)pyrimidine from example 575 as the starting material, and was characterised as follows:
MS (m/z): 458 ([M+H]+, 100).
This compound was synthesized using similar methods as described in example 578, using 2-amino-4-[(S)-3-Boc-amino-pyrrolidin-1-yl]-6-(4-fluorophenyl)-pyrido(3,2-d)pyrimidine from example 577 as the starting material, and was characterised as follows:
MS (m/z): 458 ([M+H]+, 100).
To a solution of N-Boc-piperidine-4-carboxylic acid (1 g, 4.36 mmol) in dry dichloromethane (15 ml) was added 1-hydroxybenzotriazole (HOBT, 600 mg, 4.36 mmol) and dicyclohexylcarbodiimide (900 mg, 4.36 mmol). This solution was stirred for 15 minutes. Then, m-toluidine (4.36 mmol, 0.45 ml) was added and the reaction was stirred overnight at room temperature. The solvents were evaporated in vacuo and the crude residue was redissolved in ethylacetate (10 ml). The precipitate was filtered off and further purified by silica gel flash chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 2:98) yielding the pure title compound (1.15 g, 83%) which was characterised as follows:
MS (m/z): 319 ([M+H]+, 100)
To a solution of m-tolylcarbamoyl-piperidine-1-carboxylic acid tert-butyl ester (1 mmol, 438 mg) in dichloromethane (20 ml) was added a mixture of dichloromethane and trifluoroacetic acid (5 ml, ratio 1:1). The reaction mixture was stirred at room temperature for 90 minutes. The solvents were evaporated in vacuo. The residue was redissolved in dioxane and a few drops of triethylamine were added. The solvents were evaporated and the residue was resuspended in diethyl ether. A white precipitate was formed, which was filtered off yielding the pure title compound (200 mg, 92%) which was characterised as follows:
MS (m/z): 219 ([M+H]+, 100).
To a solution of 2-acetylamino-4-(1,2,4-triazolyl)-6-(4-fluorophenyl)-pyrido(3,2-d)pyrimidine (163 mg, 0.5 mmol) in dioxane (20 ml) was added piperidine-4-carboxylic acid m-tolylamide (93 mg, 0.5 mmol). The reaction mixture was stirred at 50° C. overnight, yielding crude 2-acetylamino-4-[piperidinyl-4-carboxylic acid m-tolylamide)-6-(4-fluorophenyl)-pyrido(3,2-d)pyrimidine. In order to deprotect the acetyl group, the solvents were evaporated in vacuo and the crude residue was redissolved in a mixture of dichloromethane and ethanol (in a ratio of 80:20, 10 ml). A sodium ethoxide solution (0.2 N solution) was added till pH=12 and the reaction mixture was stirred overnight at room temperature. The solvents were evaporated in vacuo and the crude residue was purified by preparative thin layer chromatography on silica, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 10:90), yielding the title compound as a white powder (72 mg, 32%) which was characterised as follows:
MS (m/z): 457 ([M+H]+, 100).
To a solution of N-Boc-4-hydroxy-piperidine (402 mg, 2 mmol) in dioxane (40 ml) was added NaH (70 mg of a 60% dispersion; 2.2 mmol). The solution was stirred for 30 minutes at room temperature. Then, 2-acetylamino-4-(1,2,4-triazolyl)-6-(4-fluorophenyl)-pyrido(3,2-d)pyrimidine (326 mg, 1 mmol) was added and the resulting solution was stirred at room temperature for 24 hours. The precipitate was filtered off, redissolved in dichloromethane and extracted several times with a 0.1 N HCl solution and water. The combined organic layers were dried over sodium sulfate, and evaporated in vacuo yielding the crude compound, which was used for further reaction without any purification, and was characterised as follows:
MS (m/z): 482 ([M+H]+, 100)
The crude residue obtained in step (a) was redissolved in dichloromethane (10 ml) and a mixture of dichloromethane and trifluoroacetic acid (1:1 ratio; 5 ml) was added. The reaction was stirred for 1 hour at room temperature. The solvents were evaporated yielding a crude residue, which was redissolved in dichloromethane (25 ml). Diisopropylethylamine (150 μl), and m-tolylisocyanate (100 μl) were added. The reaction was stirred for 4 hours at room temperature. The solvents were evaporated in vacuo and the residue was purified by preparative thin layer chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 10:90), yielding the pure title compound (87 mg, 14%) which was characterised as follows:
MS (m/z): 515 ([M+H]+, 100)
A solution of 2-acetylamino-4-(4-hydroxy-piperidine-1-carboxylic acid m-tolylamide)-6-(4-fluorophenyl)-pyrido(3,2-d)pyrimidine (50 mg, 0.097 mmol) in dioxane/1 M K2CO3 in water (ratio 80:20; 10 ml) was stirred for 48 hours at 80° C. and for 1 week at room temperature. The solvents were evaporated in vacuo and the crude residue was purified by preparative thin layer chromatography, the mobile phase being a mixture of methanol and dichloromethane (in a ratio of 10:90), yielding the pure title compound (42%, 19 mg) which was characterised as follows:
MS (m/z): 473 ([M+H]+, 100)
Some of the pyrido[3,2-d]pyrimidine derivatives being described in the previous examples 381 to 582 have been tested for biological activities according to the methodology of example 319, in particular for their activity in the MLR assay.
The detailed nomenclature of these pyrido[3,2-d]pyrimidine derivatives is shown in the following table 4, which also shows their IC50 values (expressed in μM) in the MLR assay of example 319.
The synthetic procedure of example 397 is repeated, except that n-butyl isocyanate is replaced with the same molar amount of one of the following reactants: methyl isocyanate, ethyl isocyanate, n-propyl isocyanate, n-pentyl isocyanate, n-octyl isocyanate, cyclopentyl isocyanate, cyclohexyl isocyanate, adamantyl isocyanate, benzyl isocyanate, D-α-methylbenzyl isocyanate, L-α-methylbenzyl isocyanate, 2-methylbenzyl isocyanate, 3-methylbenzyl isocyanate, 4-methylbenzyl isocyanate, 2-methoxybenzyl isocyanate, 3-methoxybenzyl isocyanate, 4-methoxybenzyl isocyanate, 2-fluorobenzyl isocyanate, 3-fluorobenzyl isocyanate, 4-fluorobenzyl isocyanate, 2-chlorobenzyl isocyanate, 3-chlorobenzyl isocyanate, 4-chlorobenzyl isocyanate, methyl isothiocyanate, ethyl isothiocyanate, n-propyl isothiocyanate, n-butyl isothiocyanate, n-pentyl isothiocyanate, n-hexyl isothiocyanate, n-octyl isothiocyanate, cyclopentyl isothiocyanate, cyclohexyl isothiocyanate, benzyl isothiocyanate, D-α-methylbenzyl isothiocyanate, L-α-methylbenzyl isothiocyanate, 2-methylbenzyl isothiocyanate, 3-methylbenzyl isothiocyanate, 4-methylbenzyl isothiocyanate, 4-methoxybenzyl isothiocyanate, 2-fluorobenzyl isothiocyanate, 3-fluorobenzyl isothiocyanate, 4-fluorobenzyl isothiocyanate, 2-chlorobenzyl isothiocyanate, 3-chlorobenzyl isothiocyanate and 4-chlorobenzyl isothiocyanate.
In this way the following compounds of the invention are obtained in high yields similar to example 397:
The synthetic procedure of example 450 is repeated, except that ethyl isocyanate is replaced with the same molar amount of one of the following reactants: n-propyl isocyanate, n-pentyl isocyanate, n-hexyl isocyanate, n-octyl isocyanate, cyclohexyl isocyanate, benzyl isocyanate, D-α-methylbenzyl isocyanate, L-α-methylbenzyl isocyanate, 2-methyl benzyl isocyanate, 3-methyl benzyl isocyanate, 4-methylbenzyl isocyanate, 2-methoxybenzyl isocyanate, 3-methoxybenzyl isocyanate, 4-methoxybenzyl isocyanate, 2-fluorobenzyl isocyanate, 3-fluorobenzyl isocyanate, 4-fluorobenzyl isocyanate, 2-chlorobenzyl isocyanate, 3-chlorobenzyl isocyanate, 4-chlorobenzyl isocyanate, methyl isothiocyanate, ethyl isothiocyanate, n-propyl isothiocyanate, n-butyl isothiocyanate, n-pentyl isothiocyanate, n-hexyl isothiocyanate, n-octyl isothiocyanate, cyclopentyl isothiocyanate, cyclohexyl isothiocyanate, benzyl isothiocyanate, D-α-methylbenzyl isothiocyanate, L-α-methylbenzyl isothiocyanate, 2-methylbenzyl isothiocyanate, 3-methylbenzyl isothiocyanate, 4-methylbenzyl isothiocyanate, 4-methoxybenzyl isothiocyanate, 2-fluorobenzyl isothiocyanate, 3-fluorobenzyl isothiocyanate, 4-fluorobenzyl isothiocyanate, 2-chlorobenzyl isothiocyanate, 3-chlorobenzyl isothiocyanate and 4-chlorobenzyl isothiocyanate.
In this way the following compounds of the invention are obtained in yields similar to example 450:
The synthetic procedure of example 536 is repeated, except that 3-methylbenzyl isocyanate is replaced with the same molar amount of one of the following reactants: methyl isocyanate, ethyl isocyanate, n-propyl isocyanate, n-butyl isocyanate, n-pentyl isocyanate, n-hexyl isocyanate, n-octyl isocyanate, cyclohexyl isocyanate, benzyl isocyanate, adamantyl isocyanate, D-α-methylbenzyl isocyanate, L-α-methylbenzyl isocyanate, 2-methylbenzyl isocyanate, 4-methylbenzyl isocyanate, 2-methoxybenzyl isocyanate, 3-methoxybenzyl isocyanate, 4-methoxybenzyl isocyanate, 2-fluorobenzyl isocyanate, 3-fluorobenzyl isocyanate, 4-fluorobenzyl isocyanate, 2-chlorobenzyl isocyanate, 3-chlorobenzyl isocyanate, 4-chlorobenzyl isocyanate, methyl isothiocyanate, ethyl isothiocyanate, n-propyl isothiocyanate, n-butyl isothiocyanate, n-pentyl isothiocyanate, n-hexyl isothiocyanate, n-octyl isothiocyanate, cyclopentyl isothiocyanate, cyclohexyl isothiocyanate, benzyl isothiocyanate, D-α-methylbenzyl isothiocyanate, L-α-methylbenzyl isothiocyanate, 2-methylbenzyl isothiocyanate, 3-methylbenzyl isothiocyanate, 4-methylbenzyl isothiocyanate, 4-methoxybenzyl isothiocyanate, 2-fluorobenzyl isothiocyanate, 3-fluorobenzyl isothiocyanate, 4-fluorobenzyl isothiocyanate, 2-chlorobenzyl isothiocyanate, 3-chlorobenzyl isothiocyanate and 4-chlorobenzyl isothiocyanate.
In this way the following compounds of the invention are obtained in high yields similar to example 536:
| Number | Date | Country | Kind |
|---|---|---|---|
| 0428475.8 | Dec 2004 | GB | national |
This application is a continuation-in-part of International Application No. PCT/EP2005/014187, filed Dec. 29, 2005, which claims the benefit of British patent application No. 0428475.8, filed Dec. 30, 2004, and U.S. provisional application No. 60/693,899, filed Jun. 24, 2005, the disclosures of which are incorporated by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| 60693899 | Jun 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2005/014187 | Dec 2005 | US |
| Child | 11771924 | Jun 2007 | US |
