(en)This invention discloses and claims a class of indole and benzo(b)thiophene derivatives for use in treating allergy, asthma, rhinitis, dermatitis, B-cell lymphomas, tumors and diseases associated with bacterial, rhinovirus or respiratory syncytial virus (RSV) infections. The compounds of this invention are defined by the Formula (I):









wherein X, Y, Z, R 1 , R 2 and R 3 are as defined in the specification; or a pharmaceutically acceptable salt thereof. In preferred embodiments of this invention it is also disclosed and claimed that the compounds of this invention are capable of modulating T helper (Th) cells, Th1/Th2, and thereby capable of inhibiting the transcription of interleukin-4 (IL-4) message, IL-4 release or IL-4 production.

1.ApplicationNumber: US-94792207-A
1.PublishNumber: US-2008132481-A1
2.Date Publish: 20080605
3.Inventor: ALKAN SEFIK S.
DINERSTEIN Robert J.
HRIB NICHOLAS J.
SUBRAMANIAM ARUN
JURCAK JOHN G.

4.Inventor Harmonized: ALKAN SEFIK S(US)
DINERSTEIN ROBERT J(US)
HRIB NICHOLAS J(US)
SUBRAMANIAM ARUN(US)
JURCAK JOHN G(US)

5.Country: US
6.Claims:
(en)This invention discloses and claims a class of indole and benzo(b)thiophene derivatives for use in treating allergy, asthma, rhinitis, dermatitis, B-cell lymphomas, tumors and diseases associated with bacterial, rhinovirus or respiratory syncytial virus (RSV) infections. The compounds of this invention are defined by the Formula (I):









wherein X, Y, Z, R 1 , R 2 and R 3 are as defined in the specification; or a pharmaceutically acceptable salt thereof. In preferred embodiments of this invention it is also disclosed and claimed that the compounds of this invention are capable of modulating T helper (Th) cells, Th1/Th2, and thereby capable of inhibiting the transcription of interleukin-4 (IL-4) message, IL-4 release or IL-4 production.

7.Description:
(en)This application is a continuation of U.S. application Ser. No. 10/421,597, filed Apr. 23, 2003, now pending, which claims the benefit of U.S. Provisional Application No. 60/375,304, filed Apr. 23, 2002.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to medical methods of treatment of inflammatory disease conditions, such as for example, allergy, asthma, rhinitis, dermatitis, B-cell lymphomas, tumors and diseases associated with bacterial, rhinovirus or respiratory syncytial virus (RSV) infections. More specifically, the present invention relates to treatment of aforementioned inflammatory diseases in a patient by administering to such a patient a compound capable of inhibiting the transcription of interleukin-4 (IL-4) message, IL-4 release or IL-4 production. In particular, the present invention concerns the use of various indole derivatives.
2. Description of the State of the Art
The immune system consists of a sophisticated array of cells and cellular interactions developed to recognize and eradicate a wide variety of microorganisms using one of two general types of immune responses; those responses involving production of antibodies, and those involving cell-mediated responses. Specialized immune cells are involved in each of these different responses, particularly of importance is a class of cell types known as lymphocytes.
Lymphocytes are produced in the bone marrow and one class of lymphocytes is educated in the thymus, and is called T cells. Lymphocytes are further characterized by the presence of specific cell surface markers, generally known as cluster determinants, CDs. Thus, the two major subcategories of T cells are helper T cells (which express the CD4 receptor and hence are CD4 + ) and cytotoxic T cells (which express the CD8 receptor and hence are CD8 + ). Helper T cells enhance or activate the responses of other white blood cells, such as macrophages or B cells, by secreting a variety of local mediators, lymphokines, interleukins and/or cytokines. By contrast, cytotoxic T cells kill cells infected with virus or other intracellular microorganism. T lymphocytes or T cells, when stimulated by antigens presented on major histocompatibility complex (MHC) molecules expressed on antigen-presenting cells (APCs), produce the cytokine interleukin-2 (IL-2) and clonally expand. These CD4 + T helper cells, because they are not polarized yet, can secrete many different types of cytokines, thus are referred to as Th 0 cells. Th 0 cells are capable of differentiation into at least two T cell subsets, each subset expressing new patterns of cytokines involved in different effector functions. Th1 and Th2 cells are distinguished by the array of cytokine genes each cell type expresses and appears to be involved in different types of immune responses.
Th1 cells are important for the eradication of microorganisms, including bacteria, parasites, yeasts and viruses and predominantly mediate cellular immunity. Th1 cells express interferon-gamma (IFN-γ) and lymphotoxin, which can activate microbicidal activity and cytokine production in macrophages. Activation of a Th1 response often includes production of complement-fixing antibodies of the IgG2a isotype, as well as activation of natural killer (NK) cells and cytotoxic CD8 + T cells which express IFN-γ and perforin. However, if uncontrolled, Th1 cells are implicated in the pathogenesis of autoimmune diseases such as type-1 diabetes, rheumatoid arthritis and multiple sclerosis (MS).
Th2 cells are important in the eradication of helminthes and other extracellular parasites and are involved in allergic and atopic reactions. Cytokines produced by Th2 cells can induce airway hyperreactivity as well as production of IgE. Th2 cells express cytokines IL-4, IL-5 and IL-13 and can activate mast cells and eosinophils. Th2 cells stimulate B cells to proliferate and secrete antibodies effectively (humoral immunity).
Evidence indicates that Th1 and Th2 cells and their cytokines, such as IFN-γ and IL-4, can counter regulate each other and try to achieve an immunological balance under physiological conditions. See, for example, Abbas A. K., et al., Nature , (1996), 383, 787-793; Ray A., and Cohn, L. Current Opinion In Investigational Drugs , (2000), 1, 442-448; Robinson D. S., et al., New Engl. J. Med ., (1992), 326, 298-304; Busse W. W., and Lemanske R. F., New Engl. J. Med ., (2001), 344, 350-362. Thus, IL-12 stimulates naive CD4 + cells to differentiate into Th1 cells, whereas IL-4 stimulates naive CD4 + cells to differentiate into Th2 cells. Expression of the transcription factor, GATA-3, is low in naive CD4 + cells, upregulated in cells differentiating into Th2 cells, and downregulated in cells differentiating into Th1 cells. Zhang D. H., et al., J. Biol. Chem ., (1997), 272, 21597-21603, Ouyang, W. et al., Immunity , (1998), 9, 745-755. Polarized populations of Th2 cells can be induced to shift to produce Th1 cytokines when re-activated in vitro in the presence of the Th1-inducing cytokine, IL-12, and anti-IL-4, Heinzel, F. P., et al., J. Exp. Med ., (1993), 177, 1505-1509, and Mocci, S., and Coffman, R. L., J. Immunol ., (1995), 154, 3779-3787, and vice-versa.
Interleukin-4 is a pleiotropic type I cytokine produced by Th2 cells, basophils, eosinophils and mast cells, in response to receptor-mediated activation events. IL-4 is also produced by a specialized subset of T cells, some of which express NK1.1 and appear to be specific for CD-1 (NK T cells), Yoshimoto, T., et al., Science , (1995), 270, 1845-1847. T cells produce IL-4, and mice lacking these cells fail to develop IL-4-dependent airway hypersensitivity upon immunization with ovalbumin in alum.
IL-4 plays a central role in regulating the differentiation of antigen-stimulated naive T cells. IL-4 causes such cells to develop into cells capable of producing IL-4 and a series of other cytokines including IL-5, IL-10 and IL-13 (i.e. Th2-like cells). IL-4 potently suppresses the appearance of IFN-γ-producing CD4 + T cells, e.g., T H 1 cells. A second function of major physiologic importance is IL-4's control of the specificity of immunoglobulin class switching. IL-4 determines that human B cells switch to the expression of IgE and IgG4 and mouse B cells to IgE and IgG1. In IL-4 and IL-4 receptor-alpha knockout mice, as well as in mice that lack Signal Transducer and Activator of Transcription (STAT-6), a principal gene involved in IL-4 receptor signaling, IgE production is diminished by a factor of 100-fold or more. IL-4 receptor knockout mice and STAT-6 knockout mice are also deficient in the development of IL-4-producing T cells in mice infected with the helminthic parasite Nippostrongylus brasiliensis . These physiologic functions of IL-4 give it a preeminent role in the regulation of allergic conditions; it also plays a major role in the development of protective immune responses to helminths and other extracellular parasites. In experimental and clinical situations, IL-4 appears to be capable of ameliorating the effects of tissue-damaging autoimmunity.
IL-4 has a variety of other effects in hematopoietic tissues. IL-4 increases expression of class II MHC molecules in B cells, enhances expression of CD23, upregulates the expression of the IL-4 receptor, and, in association with lipopolysaccharide, allows B cells to express Thy 1. IL-4 also acts as a co-mitogen for B cell growth. Although not a growth factor by itself for resting lymphocytes, IL-4 can substantially prolong the lives of T and B lymphocytes in culture and can prevent apoptosis by factor-dependent myeloid lines that express IL-4 receptors.
IL-4 also has an important role in tissue adhesion and inflammation. IL-4 acts with TNF to induce expression of vascular cell adhesion molecule-1 (VCAM-1) on vascular endothelial cells, and IL-4 down-regulates expression of E-selectin. This shift in balance of expression of adhesion molecules by IL-4 is thought to favor the recruitment of T cells and eosinophils, rather than granulocytes, into a site of inflammation.
IL-4 receptors are present in hematopoietic, endothelial, epithelial, muscle, fibroblast, hepatocyte and brain tissues and are usually expressed at 100 to 5000 sites per cell, in keeping with the broad range of action of this cytokine. IL-4 evokes a wide variety of biological responses by binding to a high affinity receptor complex.
In experimental conditions it has been shown that Th1 and Th2 cells can suppress each other's activities. Numerous clinical studies on asthma patients have linked overexpression of Th2 cytokines with specific disease conditions. In addition, given the role of Th2 cytokines in suppressing the differentiation of Th1 cells, see, for example, Abbas, A. K., et al., Nature , (1996), 383, 787-793; Ray A. and Cohn, L., Current Opinion In Investigational Drugs , (2000), 1, 442-448; Robinson D. S., et al., New Engl. J. Med ., (1992), 326, 298-304; Busse W. W. and Lemanske R. F., New Engl. Med ., (2001), 344, 350-362., the overactivation of Th2 cytokines has additional health risks by predisposing patients to disease conditions associated with low levels of Th1 cells. High local expression of IL-4 is associated with the allergen-induced late nasal response. Thus, high levels of IL-4 is implicated in IgE production, tissue eosinophilia and other events considered to be relevant to allergic inflammation, by IL-4 interacting with its receptor and activating at least two distinct signaling pathways that culminate in the transcription of specific target genes. One pathway involves the activation of STAT-6. See for example, Ghaffar, O., et al., Clin. Exp. Allergy , (2000), 30(1), 86-93, investigated STAT-6 expression in the allergen-induced late nasal response and the effect of local steroid treatment on STAT-6 expression, which demonstrated that topical steroid treatment decreases STAT-6 expression that parallel the reduction in IL-4 expression.
Atopy is an inherited tendency, underlying asthma, rhinitis, and eczema, and generating high non-specific IgE and/or high specific IgE against common antigens. IL-4 and IL-13 are unique cytokines, in that they induce IgE synthesis in B cells and Th2 type differentiation in T cells. Both cytokines exert their biological activities by binding to their functional receptors on target cells. These receptors are thought to be composed as heterodimers, both having the IL-4Rα chain (IL-4Rα) as a component. Among the signal-transducing molecules of IL-4 and IL-13, STAT-6, which is activated by these cytokines and recruits to IL-4Rα, is essential for the biological activities of these cytokines. Izuhara, K. and Shirakawa, T. Int. J. Mol. Med ., (1999), 3(1), 3-10, verified that at least one polymorphism existing in the IL-4Rα gene, Ile50Val, correlates with atopy by both genetic and functional aspects.
Numerous studies demonstrate that in the pathogenesis of asthma, (a disease characterized by airflow obstruction, bronchial hyperresponsiveness and airway inflammation), of other inflammatory diseases, and of allergic diseases involves IL-4 activation and that IL-4 stimulation generates a cascade of interrelated immunoactivation.
IL-4 has been demonstrated as playing a significant role in human allergy, including enhanced predisposition towards allergic reactions. For example, Ghaemmaghami, Amir M., et al., Eur. J. Immunol ., (2001), 31(4), 1211-1216, report that the house dust mite Dermatophagoides pteronyssinus allergen, Der p 1 elicits IgE antibody responses in a significant proportion of patients suffering from dust mite allergy. They have shown that Der p 1 proteolytically cleaves a cell surface molecule involved in the homeostatic control of human IgE synthesis, namely the IL-2 receptor (CD25) on T cells. As a result, these T cells show markedly diminished proliferation and IFN-γ secretion in response to stimulation by anti-CD3 antibody.
IL-4 also plays a role in occupational asthma and other allergic sensitization of the respiratory tract caused by exposure to a variety of chemicals. Allergic sensitization induced by chemicals may take a variety of forms, including allergic contact dermatitis (skin sensitization) and allergic asthma and rhinitis (sensitization of the respiratory tract). It has been demonstrated that whereas contact allergens provoke selective type 1 immune responses in mice, which are characterized by the secretion by draining lymph node cells (LNC) of high levels of the cytokine IFN-γ, chemical respiratory allergens stimulate instead preferential type 2 responses associated with comparatively high levels of IL-4 and IL-10, see, e.g., Dearman, R. J. and Kimber, I., J. Appl. Toxicol ., (2001), 21(2), 153-163. Chemical allergy is a common and important occupational health issue. Cytokine fingerprinting of lymph node cells (LNCs) may be used to distinguish between chemical contact and respiratory allergens, and a recent study has concluded that contact allergens stimulate the selective development of type-1 immune responses associated with the secretion by draining LNCs of IFN-γ, but little IL-4 or IL-10, see Pearlman, David S., J. Allergy Clin. Immunol ., (1999), 104 (4, Pt. 1) S132-S137. In contrast, chemical respiratory allergens induce the appearance of preferential type-2 immune responses characterized by IL-4 and IL-10 production, but comparatively low levels of IFN-γ.
IL-4, as well as histamine, is involved in the mast cell allergic wheal reaction in skin. For example, Saarinen, J. V., et al., Allergy ( Copenhagen ), (2001), 56(1), 58-64, report that examination of biopsies of healthy and wheal skin of sensitive atopic subjects prick-tested with the cow-dander allergen, revealed both the percentage and the number of IL-4-positive mast cells showed significant positive correlation with the wheal size per se, as well as with the ratio of the wheal size by cow allergen to that by histamine control. In addition., tryptase-, chymase-, and IL-4-positive mast cells correlated with total IgE, but not with specific IgE levels, and they concluded that IL-4-positive, but not tryptase- and chymase-positive, mast cells are intimately associated with the extent of the prick-test wheal.
Allergic bronchopulmonary aspergillosis (ABPA) is characterized by a heightened Th2 CD4 + T cell response to Aspergillus fumigatus allergens and a hyper-IgE state compared to atopic asthmatic and cystic fibrosis patients without ABPA. ABPA patients have increased sensitivity to IL-4 stimulation compared to other atopic individuals. The B cells from ABPA patients were significantly more sensitive to IL-4 stimulation compared to atopic and nonatopic patients. IL-4 also stimulated upregulated CD86 + expression on B cells in atopic patients with little effect on nonatopic patients. Khan, S., et al., Int. Arch. Allergy Immunol ., (2000), 123(4), 319-326, hypothesized that one reason for this response is increased sensitivity to IL-4 in ABPA, resulting in increased expression of CD23 and CD86, leading to a positive amplification mechanism which increases Th2 CD4 + T cell responses.
IL-4 also has an important role in the development of allergen-induced airway inflammation and bronchial hyperresponsiveness (BHR). Tanaka, H., et al., Allergol. Int ., (2000), 49(4), 253-261, studied two different inbred IL-4 gene-knockout mice; one was BALB/c, which is known to be a high IgE responder, and the other was C57BL/(6, known to be a low IgE responder and a lower responder to acetylcholine (ACh) in the airways, immunized with antigen and analyzed bronchial responsiveness by measuring ACh and taking bronchoalveolar lavages. This study showed that differences in genetic background can directly influence the pathophysiology of bronchial asthma, including the role of IgE, and that IL-4 has a crucial role in the development of allergen-induced BHR independent of genetic background.
In another study of the role of IL-4 in atopic pathogenesis, it was recently found that BALB/c mice sensitized intranasally with Schistosoma mansoni egg antigen (SEA) mount a Th2 response that initiates allergic rhinitis. See, Okano, M., et al., Allergy ( Copenhagen ), (2000), 55(8), 723-731. The results from this study indicate that IL-4 is necessary for the production of Th2-associated antibodies—in particular, IgE—but IL-4 is not required for either the production of the Th2-associated cytokines IL-5 and IL-10, or the induction of nasal eosinophilia.
In another study of cells obtained from bronchoalveolar lavage (BAL) after segmental allergen challenge, Schroeder, J. T., et al., J. Allergy Clin. Immunol , (2001), 107(2), 265-271, report that human blood basophils secrete high levels of IL-4 following activation with specific allergen, and conclude that basophils, not lymphocytes or eosinophils, migrating to the lung following allergen challenge represent a major source of IL-4.
Human mast cells synthesize and secrete many cytokines of relevance to the pathogenesis of allergic disease such as asthma and rhinitis. In particular, IL-4 and IL-5 are likely to play key roles in the development of the inflammatory response that characterizes these diseases. Immunohistochemical studies on human nasal and bronchial mucosal biopsies suggest that IL-4 and IL-5 may be stored preformed in mast cells. For example, Wilson, S. J., et al., Clin. Exp. Allergy , (2000), 30(4), 493-500, analyzed bronchial mucosa and lung parenchyma from resected lung specimens, and a nasal mucosal biopsy from a patient with active allergic rhinitis, and determined that immunoreactivity for IL-4, but not IL-5, was localized to the granules of mast cells in all tissue samples. Thus, the store of preformed IL-4 within mast cell granules is likely to have an important influence during the initiation and maintenance of the allergic immunological response.
Airway dendritic cells are essential for stimulating naive T cells in response to inhaled antigen and for the development of allergic sensitization. Comparisons of CD1a, IL-4, and IL-4 receptor expression in asthmatics and in controls has revealed that the number of CD1a + cells was positively correlated to the number of IL-4 + cells, and bronchial biopsy serial section studies showed that CD1a + cells express the receptor for IL-4. Bertorelli, G., et al., Allergy ( Copenhagen ), (2000), 55(5), 449-454, concluded that an increased amount of IL-4 may play a physiopathological role in maintaining the dendritic cell pool in vivo. Therefore, because of possible IL-4 activity on antigen-presenting cells in T-cell immune responses to allergens, IL-4 plays an important role in asthma inflammation.
Epidemiological studies and experiments with mouse models suggest that polyaromatic hydrocarbons contained in, among others, diesel exhaust particles, can promote the development of allergy. Because IL-4 organizes allergic responses in vivo, Bommel, H., et al., J. Allergy Clin. Immunol ., (2000), 105(4), 796-802, investigated whether pyrene, a major compound of diesel exhaust particles, can affect the production of IL-4 by assessing IL-4 production in primary human T cells by ELISA and mRNA transcription by Northern blotting and concluded that pyrene induced transcription of IL-4 mRNA and expression of IL-4 protein in primary human T cells. Pyrene, but not related polyaromatic hydrocarbons, enhanced basal transcription of the human and mouse IL-4 promoter, suggesting that pyrene may promote allergic diseases by inducing the production of IL-4.
Production of Th2-type cytokines, IL-4 and IL-5, and tissue eosinophilia are characteristic features of allergic diseases. At 24 h after allergen provocation, CD3 + T-lymphocytes were the principal cell source of IL-4 and IL-5 mRNA transcripts in both atopic asthma and rhinitis. Eosinophils represent an early source of IL-4 which may contribute to Th2-type responses during late nasal responses and ongoing allergic rhinitis. Nouri-Aria, K. T., et al., Clin. Exp. Allergy , (2000), 30(12), 1709-1716, found that in patients with allergic rhinitis, eosinophils, but not T-cells (CD3+ cells), increased in the nasal mucosa at 6 h after allergen challenge. The number of cells expressing IL-4 mRNA and IL-5 mRNA also increased at 6 h. No increases in T-cells, eosinophils or cytokine expression were detected in non-atopic subjects.
Natural exposure to pollen provokes an increase in airway responsiveness in nonasthmatic subjects with seasonal allergic rhinitis. This natural exposure may induce inflammatory cell recruitment and cytokine release, leading to inflammation of the lower airways. Chakir, J., et al., J. Allergy Clin. Immunol ., (2000), 106(5), 904-910, characterized lower airway inflammation in nonasthmatic pollen-sensitive subjects by immunohistochemical tests on bronchial biopsy specimens from subjects with rhinitis who had no past or current history of asthma and evaluated cytokine expression and inflammatory cell numbers and activation both in and out of the pollen season. The authors found that the number of CD4 + , CD8 + , and CD45RO + lymphocyte subpopulations were significantly higher during the pollen season compared with the out-of-season period. The authors concluded that natural pollen exposure was associated with an increase in lymphocyte numbers, eosinophil recruitment, and IL-5 expression in the bronchial mucosa of nonasthmatic subjects with allergic rhinitis.
Topical treatment with glucocorticoids (GCs) is known to decrease eosinophils, but not neutrophils, in patients with allergic rhinitis. Benson, Mikael, et al., J. Allergy Clin. Immunol ., (2000), 106(2), 307-312, studied the differential effects of GC treatment on eosinophils and neutrophils and differential effects on Th1/Th2 cytokines and the neutrophil-associated cytokines IL-1β and TNF-α, by examining levels of eosinophils and neutrophils in nasal fluids from 60 children with seasonal allergic rhinitis treated with a topical GC, determining nasal fluid levels of IFN-γ, IL-4, IL-6, IL-10, IL-1β, TNF-α, IgE and eosinophil cationic protein (ECP). After GC treatment, there was a statistically significant decrease of the Th2 cytokines IL-4, IL-6, and IL-10, as well as ECP and IgE. By contrast, there were no significant changes of the levels of IFN-γ, IL-1β, TNF-α, or neutrophils. The authors concluded that the beneficial effects of topical treatment with GC in patients with allergic rhinitis could be attributed to down-regulation of Th2 cytokines, with an ensuing decrease of eosinophils, ECP, and IgE.
Allergic diseases like atopic rhinitis, bronchial asthma and urticaria are prevalent and their incidence is on the increase. T cells are known to play a fundamental role in allergic diseases through the recognition of antigen, and secretion of Th2-type cytokines like IL-4, IL-5 and IL-13, that not only induce the synthesis of IgE but also recruit effector cells like eosinophils and basophils into the site of allergic inflammation. Recent studies have suggested that in addition to the αβ T-cell receptor (TCR)-bearing cells, the less common γδ TCR bearing cells may play important roles in the development and perpetuation of allergic inflammation as effector and immunoregulatory cells, see Pawankar, R., Clin. Exp. Allergy , (2000), 30(3), 318-323.
IgE is present in airway secretions from human patients with allergic rhinitis and bronchial asthma. Zuberi, R. I., et al., J. Immunol ., (2000), 164(5), 2667-2673, demonstrated that BAL obtained from BALB/c mice first sensitized with OVA in alum systemically and then challenged with nebulized OVA, contained significant amounts of IgE, of which greater than 50% was Ag specific and that the IgE levels in airway secretions remained elevated for more than 15 days after the termination of Ag exposure. Significant amounts of IgE-OVA immune complexes were detected in BAL fluid from the OVA-challenged mice. The IgE immune complexes did not augment the inflammatory response in high affinity IgE receptor (FcεRI)-deficient mice.
The mechanism of cytokine regulation in atopy has been analyzed in terms of the effects of 3 major cytokines, IL-4, IL-12, and IFN-γ on in vitro IgE synthesis of human peripheral blood mononuclear cells (PBMC) from normal individuals and atopic patients. See for example, Liu, Meng, et al., Chin. Med. J . (Beijing, Engl. Ed.), (1999), 112(6), 550-553.
Atopic dermatitis (AD) is a chronic inflammatory skin disease frequently associated with asthma, rhinitis, and food allergy. Lymphocytes producing Th2-type cytokines (such as IL-3, IL-4, and IL-5) have been thought to have a key role in the pathogenesis of the disease. Elevated serum levels of the soluble form of CD30 (sCD30), an activation marker of Th2-cell clones, correlates with disease activity in pediatric patients suffering from AD, and clinical trials have demonstrated that cyclosporin A (CsA) treatment resulted in significant improvement of clinical symptoms in patients affected with AD. Bottari, V., et al., Allergy ( Copenhagen ), (1999), 54(5), 507-510, evaluated the role of CsA in modulating sCD30 release in a group of adult patients affected by severe AD treated with CsA and demonstrated, in parallel with an improvement of clinical symptoms, a significant reduction of serum levels of both IL-4 and sCD30, thus suggesting that CsA can prevent the activation of Th2 cells observed in AD.
Specific allergen immunotherapy (SIT) is effective for treatment of IgE-mediated diseases. While the mechanisms of action still remain unclear, it has been determined that IL-4 and IL-13 are produced in response to specific allergens. Gabrielsson, S., et al., Allergy ( Copenhagen ), (2001), 56(4), 293-300, investigated the effect of SIT on allergen-specific IgE and IgG4 levels, by performing cytokine analysis on blood samples from pollen-sensitized individuals were collected before the pollen season (before treatment) and during the pollen season (after SIT or placebo treatment), by measuring the numbers of IL-4-, IL-13-, IL-10-, and IFN-γ-producing cells in peripheral blood mononuclear cells activated in vitro with allergens and by measuring the serum levels of allergen-specific IgE and IgG4. The numbers of IL-4- and IL-13-producing cells were shown to be increased in the placebo group during the pollen season, an increment which was absent in patients receiving allergen SIT. SIT-treated individuals had increased allergen-specific IgG4, but not in the placebo group. Both groups displayed elevated specific IgE levels during the pollen season. The authors conclude that there was a downregulation of IL-4- and IL-13-producing cells in peripheral blood after SIT, suggesting induction of nonresponsiveness/tolerance or a redistribution of these cells, and that SIT acts on antibody production by increasing the specific IgG4 levels.
There is also evidence that the ratio of Th1 to Th2 cells is highly relevant to the outcome of a wide array of immunologically-mediated clinical diseases including B-cell lymphomas, tumors or infectious diseases including anti-bacterial diseases. For instance, many human diseases including chronic infections (such as with human immunodeficiency virus (HIV) and tuberculosis) and certain metastatic carcinomas, are characterized by a Th1 or Th2 switch, see, e.g., Shearer, G. M. and Clerici, M., Prog. Chem. Immunol ., (1992), 54, 21-43; Clerici, M. and Shearer, G. M., Immunology Today , (1993), 14, 107-111; Yamarnura, M., et al., J. Clin. Invest . (1993), 91, 1005-1010; Pisa, P., et al., Proc. Natl. Acad. Sci. USA , (1992), 89, 77089-7712; and Fauci, A. S., Science (1988), 239, 617-623.
IL-4 is also implicated in other tumor growths. For example, growth stimulation of human head and neck squamous cell carcinoma cell lines by IL-4 has been reported, see Myers, J. N. et al., Clin. Cancer Res ., (1996), 2(1), 127-135. Pancreatic cancer cells have been shown to express IL-13 and IL-4 and their growth is inhibited by Pseudomonas exotoxin coupled to IL-13 and IL-4, see Kornmann M., et al., Anticancer Res ., (1999), 19(1A), 125-131. A recent study has also shown that IL-2 and IL-4 serve as autocrine growth factors in the autonomous proliferation of tumor cells, particularly those that are retrovirally induced, see Hassuneh, M. R., Nagarkatti, P. S., Nagarkatti, M., Blood , (1997), 89(2), 610-620.
Further, evidence has demonstrated IL-4 gene expression is responsible for triggering biological effects across a wide variety if pathophysiological conditions including conditions manifested by dysfunctional leukocytes, T-lymphocytes, e.g., acute and chronic inflammatory disease, auto-immune disorders, rheumatoid arthritis, myasthenia gravis, transplant rejection and Hodgkin's disease. See, e.g., WO 99/21993, published May 6, 1999.
There is also evidence that preexisting asthmatic inflammation may exacerbate certain viral infection, in particular rhinovirus infection. See, e.g., Message S. D., Johnston S. L., Eur. Respir. J . (2001), 18(6), 1013-25; O'Sullivan S., et al., Am. J. Respir. Crit. Care Med ., (2001), 164(4), 560-4; also see, Schwarze, J., Gelfand, E. W., Eur. Respir. J . (2002), 19(2), 341-349; and Imani, F., Proud, D., Griffin, D. E., J. Immunol., (1999), 162(3), 1597-1602. It has been shown that rhinovirus infection induces increased production of IL-8, IL-6, and GM-CSF from epithelial cells, see Subauste, M. C., et al., J. Clin. Invest., (1995), 96(1), 549-557.
It has also been reported that respiratory syncytial virus (RSV) infection has been associated with a Th2 type immune response. See, e.g., Boelen A., et al., J. Med. Virol ., (2002), 66(4), 552-560. There is evidence that RSV-associated airway hyperresponsiveness (AHR) occurs in hosts with allergic responses and that allergic inflammation is diminished when preceded by RSV infection, see Peebles, R. S., Jr., J. Infect, Dis ., (2001), 184(11), 1374-1379. It has also been shown that as the RSV infection progresses, the local production of Th1 and Th2 associated cytokines increases, particularly IL-13. In this study, it was concluded that the primary factor leading to chronic RSV-induced airway dysfunction is due to the inappropriate production of IL-13, see Tekkanat, K. K., et al., J. Immunol ., (2001), 166(5), 3542-3548.
Therefore, there is considerable evidence that allergy and related diseases involve higher levels of Th2 cytokines, and that clinical evidence supports the role of lowering Th2 cytokines correlates with clinical improvement. Therefore, there is considerable need for compounds capable of modulating Th2 cytokines in vivo, and methods to identify such compounds.
All of the references described herein are incorporated herein by reference in their entirety.
SUMMARY OF THE INVENTION
Thus in accordance with the practice of this invention there is provided a method of treating a variety of inflammatory disease conditions. In general, the disease conditions that can be treated in accordance with the practice of this invention include but not limited to allergy, asthma, rhinitis, dermatitis, B-cell lymphomas, tumors and diseases associated with bacterial, rhinovirus or respiratory syncytial virus (RSV) infections. The diseases as disclosed herein can be treated by administering to a patient in need of such treatment a therapeutically effective amount of a compound of the formula I:









wherein
X and Y are the same or different and are independently selected from the group consisting of hydrogen, halogen, nitro, amino, hydroxy, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 perfluoroalkyl, C 1-6 perfluoroalkoxy, phenyl and benzyl, wherein phenyl or benzyl is optionally substituted with one or two substituents each independently selected from C 1-6 alkyl, C 1-6 perfluoroalkyl, halogen, hydroxy or C 1-6 alkoxy or C 1-6 perfluoroalkoxy; Z is N—R or S, wherein R is selected from the group consisting of hydrogen, C 1-6 alkyl, C 1-6 alkylcarbonyl, C 1-6 alkoxycarbonylC 1-6 alkyl, C 1-6 alkylcarbamoyl-C 1-6 alkyl and C 1-6 dialkylcarbamoylC 1-6 alkyl; R 1 is selected from the group consisting of hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 perfluoroalkyl, C 3-8 cycloalkyl, C 3-8 cycloalkylC 1-6 alkyl, C 1-6 alkoxyC 1-6 alkyl, hydroxyC 1-6 alkyl, aminoC 1-6 alkyl, mono- or di-C 1-6 alkyl-aminoC 1-6 alkyl, formyl, C 1-6 alkylcarbonyl, aminoC 1-6 alkylcarbonyl, C 1-6 alkoxycarbonyl, phenyl, diphenylC 1-6 alkyl and phenylC 1-6 alkyl, phenylcarbonyl-C 1-6 alkyl, phenoxyC 1-6 alkyl, wherein phenyl is optionally substituted with one or two substituents each independently selected from C 1-6 alkyl, C 1-6 perfluoroalkyl, halogen, hydroxy or C 1-6 perfluoroalkyl or C 1-6 perfluoroalkoxy; R 3 is










wherein
R 4 is selected from the group consisting of hydrogen, halogen, nitro, amino, hydroxy, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 perfluoroalkyl, C 1-6 perfluoroalkoxy, phenyl and benzyl, wherein phenyl or benzyl is optionally substituted with one or two substituents each independently selected from C 1-6 alkyl, C 1-6 perfluoroalkyl, halogen, hydroxy or C 1-6 perfluoroalkyl or C 1-6 perfluoroalkoxy; and n is an integer from 0 to 4; and R 2 is selected from the group consisting of hydrogen, C 1-6 alkyl, C 1-6 perfluoroalkyl, perfluoroaryl, indanyl, C 1-6 alkoxyC 1-6 alkyl, C 2-6 acyloxyC 1-6 alkyl, C 1-6 alkoxycarbonylC 1-6 alkyl, C 1-6 alkoxycarbonyloxyC 1-6 alkyl, C 3-8 cycloalkyl, C 3-8 cycloalkoxycarbonyloxy-C 1-6 alkyl, adamantyloxycarbonyloxyC 1-6 alkyl, C 3-8 cycloalkoxycarbonyl-C 1-6 alkyl, mono- or di-C 1-6 alkylamino-C 1-6 alkyl, C 3-8 azacycloalkylC 1-6 alkyl, mono- or di-C 1-6 alkylcarbamoyl-C 1-6 alkyl, C 3-8 azacycloalkylcarbonyloxyC 1-6 alkyl, benzylC 1-6 alkylcarbamoylC 1-6 alkyl, mono- or di-C 1-6 alkylcarbamoyloxyC 1-6 alkyl, C 3-8 azacycloalkylcarbonyloxy-C 1-6 alkyl, benzylC 1-6 alkylcarbamoyloxyC 1-6 alkyl, benzylcarbamoyloxyC 1-6 alkyl, C 1-6 alkoxycarbonylamino-oxo-C 1-6 alkyl,










wherein
R 5 is hydrogen or C 1-6 alkyl, R 6 is C 1-6 alkyl, phenyl or tolyl, and A is CH 2 , NH or O.

Any of the compounds as mentioned herein may also be administered as its pharmaceutically acceptable salt whenever such pharmaceutically acceptable salts can be formed. Further, above mentioned compounds may optionally be administered in combination with a pharmaceutically acceptable carrier.
In another aspect of this invention there is also disclosed a method of treating certain diseases including but not limited to allergy, asthma, rhinitis, dermatitis or infectious diseases, such as rhinovirus or respiratory syncytial virus infection. In accordance with this aspect of the invention the method comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound capable of modulating T helper (Th) cells, Th1/Th2, which compound is of the formula (I) as defined hereinabove.
In yet another aspect of this invention there is also provided a method of treating certain disease states including but not limited to allergy, asthma, rhinitis or dermatitis. In this aspect of the invention the method comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound capable of inhibiting the transcription of interleukin-4 (IL-4) message, IL-4 release or IL-4 production, which compound is of the formula (I) as defined herein.
In yet another aspect of this invention there is also disclosed a pharmaceutical composition, which comprises a compound as defined hereinabove. The compound is administered in an amount effective for treating various disease states as described herein. The disease states that may be enumerated include but not limited to allergy, asthma, rhinitis, dermatitis, B-cell lymphomas, tumors and diseases associated with bacterial, rhinovirus or respiratory syncytial virus (RSV) infections. The pharmaceutical composition additionally contains at least one pharmaceutically acceptable carrier.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a bar graph depicting the effects of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) on bronchoalveolar lavage fluid (BALF) cytokines and lung inflammation in mice.
FIG. 2 is a bar graph depicting the inhibition by 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) of allergen-induced lung inflammation in the rat.
FIG. 3A is a graph depicting the effect of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) on antigen-induced early and late bronchial responses.
FIG. 3B is a bar graph depicting the effect of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) (3 mg/kg) on post challenge airway responsiveness.
FIG. 4 is a listing of the sequence, SEQ ID NO:1, a 6.7 kb fragment comprising nucleotides −6635 to +66 of human IL-4 gene promoter.



DETAILED DESCRIPTION OF THE INVENTION
The terms as used herein have the following meanings:
As used herein, the expression “C 1-6 alkyl” includes methyl and ethyl groups, and straight-chained or branched propyl, butyl, pentyl and hexyl groups. Particular alkyl groups are methyl, ethyl, n-propyl, isopropyl and tert-butyl. Derived expressions such as “C 1-6 alkoxy”, “C 1-6 alkoxyC 1-6 alkyl”, “hydroxyC 1-6 alkyl”, “C 1-6 alkylcarbonyl”, “C 1-6 alkoxycarbonylC 1-6 alkyl”, “C 1-6 alkoxycarbonyl”, “aminoC 1-6 alkyl”, “C 1-6 alkylcarbamoylC 1-6 alkyl”, “C 1-6 dialkylcarbamoylC 1-6 alkyl” “mono- or di-C 1-6 alkylaminoC 1-6 alkyl”, “aminoC 1-6 alkylcarbonyl” “diphenylC 1-6 alkyl”, “phenylC 1-6 alkyl”, “phenylcarboylC 1-6 alkyl” and “phenoxyC 1-6 alkyl” are to be construed accordingly.
As used herein, the expression “C 2-6 alkenyl” includes ethenyl and straight-chained or branched propenyl, butenyl, pentenyl and hexenyl groups. Similarly, the expression “C 2-6 alkynyl” includes ethynyl and propynyl, and straight-chained or branched butynyl, pentynyl and hexynyl groups.
As used herein, the expression “C 1-6 perfluoroalkyl” means that all of the hydrogen atoms in said alkyl group are replaced with fluorine atoms. Illustrative examples include trifluoromethyl and pentafluoroethyl, and straight-chained or branched heptafluoropropyl, nonafluorobutyl, undecafluoropentyl and tridecafluorohexyl groups. Derived expression, “C 1-6 perfluoroalkoxy”, is to be construed accordingly.
As used herein, the expression “C 3-8 cycloalkyl” means cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.
As used herein, the expression “C 3-8 cycloalkylC 1-6 alkyl” means that the C 3-8 cycloalkyl as defined herein is further attached to C 1-6 alkyl as defined herein. Representative examples include cyclopropylmethyl, 1-cyclobutylethyl, 2-cyclopentylpropyl, cyclohexylmethyl, 2-cycloheptylethyl and 2-cyclooctylbutyl and the like.
“CsA” means cyclosporin A.
“Halogen” or “halo” means chloro, fluoro, bromo, and iodo.
“IL” means interleukin.
“Luc” means luciferase.
As used herein, the various forms of the term “modulation” are intended to include stimulation (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity).
As used herein, “patient” means a warm blooded animal, such as for example rat, mice, dogs, cats, guinea pigs, and primates such as humans.
As used herein, the expression “pharmaceutically acceptable carrier” means a non-toxic solvent, dispersant, excipient, adjuvant, or other material which is mixed with the compound of the present invention in order to permit the formation of a pharmaceutical composition, i.e., a dosage form capable of administration to the patient. One example of such a carrier is a pharmaceutically acceptable oil typically used for parenteral administration.
The term “pharmaceutically acceptable salts” as used herein means that the salts of the compounds of the present invention can be used in medicinal preparations. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds of this invention include acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, methanesulfonic acid, 2-hydroxyethanesulfonic acid, p-toluenesulfonic acid, fumaric acid, maleic acid, hydroxymaleic acid, malic acid, ascorbic acid, succinic acid, glutaric acid, acetic acid, salicylic acid, cinnamic acid, 2-phenoxybenzoic acid, hydroxybenzoic acid, phenylacetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, carbonic acid or phosphoric acid. The acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate can also be formed. Also, the salts so formed may present either as mono- or di-acid salts and can exist either as hydrated or can be substantially anhydrous. Furthermore, where the compounds of the invention carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g. sodium or potassium salts; alkaline earth metal salts, e.g. calcium or magnesium salts; and salts formed with suitable organic ligands, e.g. quaternary ammonium salts.
The expression “stereoisomers” is a general term used for all isomers of the individual molecules that differ only in the orientation of their atoms in space. Typically it includes mirror image isomers that are usually formed due to at least one asymmetric center, (enantiomers). Where the compounds according to the invention possess two or more asymmetric centers, they may additionally exist as diastereoisomers, also certain individual molecules may exist as geometric isomers (cis/trans). It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present invention.
“Substituted” means substituted by 1 to 2 substituents independently selected from the group consisting of C 1-6 alkyl, C 1-6 perfluoroalkyl, hydroxy, —CO 2 H, an ester, an amide, C 1 -C 6 alkoxy, C 1 -C 6 perfluoroalkoxy, —NH 2 , Cl, Br, I, F, —NH-lower alkyl, and —N(lower alkyl) 2 .
“Therapeutically effective amount” means an amount of the compound which is effective in treating the named disorder or condition.
As used in the examples and preparations that follow, the terms used therein shall have the meanings indicated: “kg” refers to kilograms, “g” refers to grams, “mg” refers to milligrams, “μg” refers to micrograms, “pg” refers to picograms, “mol” refers to moles, “mmol” refers to millimoles, “nmole” refers to nanomoles, “L” refers to liters, “mL” or “ml” refers to milliliters, “μL” refers to microliters, “° C.” refers to degrees Celsius, “R f ” refers to retention factor, “mp” or “m.p.” refers to melting point, “dec” refers to decomposition, “bp” or “b.p.” refers to boiling point, “mm of Hg” refers to pressure in millimeters of mercury, “cm” refers to centimeters, “nm” refers to nanometers, “[α] 20 D ” refers to specific rotation of the D line of sodium at 20° C. obtained in a 1 decimeter cell, “c” refers to concentration in g/mL, “THF” refers to tetrahydrofuran, “DMF” refers to dimethylformamide, “NMP” refers to 1-methyl-2-pyrrolidinone, “brine” refers to a saturated aqueous sodium chloride solution, “M” refers to molar, “mM” refers to millimolar, “μM” refers to micromolar, “nM” refers to nanomolar, “TLC” refers to thin layer chromatography, “HPLC” refers to high performance liquid chromatography, “HRMS” refers to high resolution mass spectrum, “lb” refers to pounds, “gal” refers to gallons, “L.O.D.” refers to loss on drying, “μCi” refers to microcuries, “i.p.” refers to intraperitoneally, “i.v.” refers to intravenously.
In one aspect of the present invention there is provided a method of treating a variety of inflammatory disease states. In general, the disease states that can be treated in accordance with this invention include but not limited to allergy, asthma, rhinitis, dermatitis, B-cell lymphomas, tumors and diseases associated with bacterial, rhinovirus or respiratory syncytial virus (RSV) infections. The diseases as disclosed herein can be treated by administering to a patient in need of such treatment a therapeutically effective amount of a compound of the formula (I):









wherein
X and Y are the same or different and are independently selected from the group consisting of hydrogen, halogen, nitro, amino, hydroxy, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 alkoxy, C 1-6 perfluoroalkyl, C 1-6 perfluoroalkoxy, phenyl and benzyl, wherein phenyl or benzyl is optionally substituted with one or two substituents each independently selected from C 1-6 alkyl, C 1-6 perfluoroalkyl, halogen, hydroxy or C 1-6 alkoxy or C 1-6 perfluoroalkoxy; Z is N—R or S, wherein R is selected from the group consisting of hydrogen, C 1-6 alkyl, C 1-6 alkylcarbonyl, C 1-6 alkoxycarbonylC 1-6 alkyl, C 1-6 alkylcarbamoyl-C 1-6 alkyl and C 1-6 dialkylcarbamoylC 1-6 alkyl; R 1 is selected from the group consisting of hydrogen, C 1-6 alkyl, C 2-6 alkenyl, C 2-6 alkynyl, C 1-6 perfluoroalkyl, C 3-8 cycloalkyl, C 3-8 cycloalkylC 1-6 alkyl, C 1-6 alkoxyC 1-6 alkyl, hydroxyC 1-6 alkyl, aminoC 1-6 alkyl, mono- or di-C 1-6 alkyl-aminoC 1-6 alkyl, formyl, C 1-6 alkylcarbonyl, aminoC 1-6 alkylcarbonyl, C 1-6 alkoxycarbonyl, phenyl, diphenylC 1-6 alkyl and phenylC 1-6 alkyl, phenylcarbonyl-C 1-6 alkyl, phenoxyC 1-6 alkyl, wherein phenyl is optionally substituted with one or two substituents each independently selected from C 1-6 alkyl, C 1-6 perfluoroalkyl, halogen, hydroxy or C 1-6 perfluoroalkyl or C 1-6 perfluoroalkoxy; R 3 is










wherein
R 4 is selected from the group consisting of hydrogen, halogen, nitro, amino, hydroxy, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 perfluoroalkyl, C 1-6 perfluoroalkoxy, phenyl and benzyl, wherein phenyl or benzyl is optionally substituted with one or two substituents each independently selected from C 1-6 alkyl, C 1-6 perfluoroalkyl, halogen, hydroxy or C 1-6 perfluoroalkyl or C 1-6 perfluoroalkoxy; and n is an integer from 0 to 4; and R 2 is selected from the group consisting of hydrogen, C 1-6 alkyl, C 1-6 perfluoroalkyl, perfluoroaryl, indanyl, C 1-6 alkoxyC 1-6 alkyl, C 2-6 acyloxyC 1-6 alkyl, C 1-6 alkoxycarbonylC 1-6 alkyl, C 1-6 alkoxycarbonyloxyC 1-6 alkyl, C 3-8 cycloalkyl, C 3-8 cycloalkoxycarbonyloxy-C 1-6 alkyl, adamantyloxycarbonyloxyC 1-6 alkyl, C 3-8 cycloalkoxycarbonyl-C 1-6 alkyl, mono- or di-C 1-6 alkylamino-C 1-6 alkyl, C 3-8 azacycloalkylC 1-6 alkyl, mono- or di-C 1-6 alkylcarbamoyl-C 1-6 alkyl, C 3-8 azacycloalkylcarbonyloxyC 1-6 alkyl, benzylC 1-6 alkylcarbamoylC 1-6 alkyl, mono- or di-C 1-6 alkylcarbamoyloxyC 1-6 alkyl, C 3-8 azacycloalkylcarbonyloxy-C 1-6 alkyl, benzylC 1-6 alkylcarbamoyloxyC 1-6 alkyl, benzylcarbamoyloxyC 1-6 alkyl, C 1-6 alkoxycarbonylamino-oxo-C 1-6 alkyl,










wherein
R 5 is hydrogen or C 1-6 alkyl, R 6 is C 1-6 alkyl, phenyl or tolyl, and A is CH 2 , NH or O.

Any of the compounds as mentioned herein may also be administered as their pharmaceutically acceptable salts. Further, the compounds of this invention may optionally be administered in combination with a pharmaceutically acceptable carrier.
In another aspect of this invention there is also disclosed a method of treating certain diseases including but not limited to allergy, asthma, rhinitis, dermatitis, infectious diseases, such as rhinovirus or respiratory syncytial virus (RSV) infection. In accordance with this aspect of the invention the method comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound capable of modulating T helper (Th) cells, Th1/Th2, which compound is of the formula (I) as described herein.
As noted above, in this embodiment of the method of this invention, the term “modulation” is intended to mean that the production of a Th2-associated cytokine is inhibited by administering a compound having such an activity thereby diminishing the number of Th2 cells in reference to Th1 cells thus restoring the balance of an immune response.
In yet another aspect of this invention there is also provided a method of treating certain disease states including but not limited to allergy, asthma, rhinitis or dermatitis. In this aspect of the invention the method comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound capable of inhibiting the transcription of interleukin-4 (IL-4) message, IL-4 release or IL-4 production, which compound is of the formula (I).
In an embodiment of the aforementioned methods of this invention, the disease states that are treated are allergy, asthma, rhinitis, dermatitis and rhinovirus or respiratory syncytial virus infections.
In another embodiment encompassing the methods of this invention, the compound of formula (I) of this invention have the following moieties:
X and Y are the same or different and are independently selected from the group consisting of hydrogen, halogen, C 1-3 alkyl, C 1-3 perfluoroalkyl, C 1-3 alkoxy, C 1-3 perfluoroalkoxy, phenyl and benzyl; Z is N—R or S wherein R is selected from the group consisting of hydrogen, C 1-3 alkyl, C 1-3 alkoxycarbonylC 1-3 alkyl and C 1-3 dialkylcarbamoyl-C 1-3 alkyl; R 1 is hydrogen, C 1-6 alkyl, or phenylC 1-6 alkyl; R 3 is














wherein
R 4 is hydrogen or C 1-6 alkyl; and
n is an integer from 0 to 3; and


R 2 is selected from the group consisting of hydrogen, C 1-4 alkyl, perfluoroaryl, indanyl, C 1-3 alkoxyC 1-3 alkyl, C 2-5 acyloxyC 1-3 alkyl, C 1-3 alkoxycarbonylC 1-3 alkyl, C 1-4 alkoxycarbonyloxyC 1-4 alkyl, C 4-6 cycloalkoxycarbonyloxy-C 1-3 alkyl, adamantyloxycarbonyloxy-C 1-3 alkyl, mono- or di-C 1-3 alkylamino-C 1-3 alkyl, C 4-6 azacycloalkyl-C 1-3 alkyl, mono- or di-C 1-3 alkylcarbamoyl-C 1-3 alkyl, C 4-6 azacycloalkylcarbonylC 1-3 alkyl, benzylC 1-3 alkylcarbamoyl-C 1-6 alkyl, mono- or di-C 1-3 alkylcarbamoyloxyC 1-3 alkyl, C 3-8 azacycloalkylcarbonyloxyC 1-6 alkyl, benzylcarbamoyloxyC 1-3 alkyl, C 1-3 alkoxycarbonylamino-carbonylC 1-3 alkyl,

















wherein
R 5 is hydrogen or C 1-3 alkyl,
R 6 is C 1-6 alkyl, phenyl or tolyl, and
A is O,

or a pharmaceutically acceptable salt thereof, optionally in combination with a pharmaceutically acceptable carrier.







In yet another embodiment involving the methods of this invention, the compound of formula (I) of this invention have the following moieties:
X and Y are the same or different and are independently selected from the group consisting of hydrogen, fluorine, chlorine, trifluoromethyl, methoxy, phenyl and benzyl; Z is N—R or S wherein R is selected from the group consisting of hydrogen, methyl, ethoxycarbonylmethyl and diethylcarbamoylmethyl; R 1 is hydrogen, methyl or propyl; R 3 is














wherein
R 4 is hydrogen; and
n is an integer from 0 to 2; and


R 2 is selected from the group consisting of hydrogen, methyl, ethyl, t-butyl, pentafluorophenyl, indanyl, methoxyethyl, ethoxypropyl, acetyloxymethyl, 2,2-dimethylpropionyloxymethyl, pentanoyloxymethyl, methoxycarbonyl-1-ethyl, ethoxycarbonylmethyl, iso-propoxycarbonyloxymethyl, tert-butoxycarbonyloxymethyl, cyclohexanoxycarbonyloxymethyl, adamantyloxycarbonyloxymethyl, diethylaminoethyl, dimethylaminoethyl, piperidinyl-1-ethyl, diethylcarbamoylmethyl, benzylethylcarbamoylmethyl, 2-azetidin-1-yl-2-oxo-ethyl, 2-oxo-2-pyrrolidin-1-yl-ethyl, piperidine-1-carbonyloxymethyl, diethylcarbamoyloxymethyl, benzylcarbamoyloxymethyl, ethoxycarbonylamino-carbonylmethyl,

















wherein
R 5 is hydrogen or methyl,
R 6 is methyl, phenyl or tolyl, and
A is O,

or a pharmaceutically acceptable salt thereof, optionally in combination with a pharmaceutically acceptable carrier.







Several of the compounds used in the method of this invention are known compounds and a few of the compounds used herein are novel. In general, the compounds of this invention can be readily synthesized by any of the procedures known to one skilled in the art. Specifically, the known compounds used in the method of this invention can be made in accordance with the procedures set forth in U.S. Pat. Nos. 5,177,088; 5,189,054; 5,328,920; 5,491,153; and 5,675,018. Each of these patents is herein incorporated by reference in its entirety.
More specifically, the compounds disclosed herein can be synthesized according to the following procedures of Schemes A-H, wherein the X, Y, Z, R 1 , R 2 and R 3 substituents are as defined for Formula (I), above unless otherwise indicated.
In general, for the indole derivatives used in the method of this invention, the starting 3-aminoindole can be prepared following the procedures shown in Scheme A-D.









In scheme A, in step A1, the substituted 2-fluorobenzonitrile, 1 is reacted with α-aminocarboxylic acid ester, 2 to form the 3-aminoindole derivative, 3. The reaction is generally carried out neat or in the presence of a suitable organic solvent under basic reaction conditions. Suitable base includes, alkaline hydroxides such as sodium or potassium hydroxide, alkaline carbonates such as sodium or potassium carbonate, ammonia or ammonium hydroxide and the like. The reaction is generally carried out at ambient or elevated temperatures in the range of from about 80° C. to 150° C. in an inert atmosphere such as nitrogen or argon.









Scheme B shows another approach to the synthesis of 3-aminoindole derivatives, 3 involving 5 steps starting from the substituted 2-aminobenzoic acid, 4. In this approach, the carboxylic acid group is first converted to the nitrile group in steps B1 and B2. Any of the procedures known in the art to facilitate such conversion can be employed. Thus, for example, in step B1 of Scheme B, the substituted 2-aminobenzoic acid, 4 is treated with activating moieties such as N-hydroxysuccinimide in the presence of a suitable dehydrating agent in a suitable organic solvent. The activated carboxylic acid derivative is then reacted with an amine such as for example tert-butyl amine to form the benzamide derivative, 5. Suitable dehydrating agents include dicyclohexylcarbodiimide, cyanuric chloride and the like. The reaction can be carried out at ambient or sub-ambient temperatures, for example, −20° C. to 25° C.
In step B2, Scheme B, the benzamide, 5 formed in step B1 is further reacted to form the benzonitrile derivative, 6. In general, this can be affected by reacting the benzamide, 5 with a suitable carboxylic acid anhydride such as trifluoroacetic anhydride to form the benzonitrile derivative, 6. The reaction in this step can be carried out in a suitable organic solvent such as dichloromethane at ambient to sub-ambient reaction temperatures, preferably at around 0° C.
In step B3, Scheme B, the benzonitrile derivative, 6 is further reacted with a desired α-halocarboxylic acid ester, 7 in the presence of a suitable base in an organic solvent such as DMF. Suitable bases include alkali hydrides such as sodium hydride or alkyl-alkali compounds such as butyl lithium and the like. The reaction is generally carried out in inert atmospheres, such as nitrogen or argon, and at sub-ambient temperatures, such as for example 0° C., however, ambient to super-ambient reaction temperatures can also be utilized.
In step B4, Scheme B, the addition product, 8 from step B3 is cyclyzed in the presence of a base under inert reaction conditions typically at sub-ambient temperatures such as 0° C. Any of the base that is effective to carry out this cyclization reaction can be employed and such examples include alkaline hydroxides such as sodium or potassium hydroxide, alkaline carbonates such as sodium or potassium carbonate, alkaline alkoxides such as potassium tert-butoxide, ammonia or ammonium hydroxide and the like. Potassium tert-butoxide is particularly preferred.
Finally, in step B5, Scheme B, the starting indole compound, 3 is prepared by cleaving off the trifluoroacetyl group on the amino group of the indole compound, 8. This cleavage can be effected using any of the methods known in the art. For example, the cleavage can be effected by treating the product, 8 from B4 with a base such as potassium carbonate at ambient or super-ambient reaction temperatures; temperature in the range of 50 to 80° C. is particularly preferred.









Scheme C illustrates a synthesis of particularly one type of substituted 3-amino-indole derivative, 3A in which the substituent on the indole ring is phenyl. Various other mono or diaryl substituted 3-amino-indole derivatives can be synthesized following the steps of Scheme C. In step C1, Scheme C, the amino group of the 2-amino-4-chloro-benzonitrile, 9A is protected by acetyl group by treating with acetic anhydride. Various other protective groups can be employed in a similar fashion. In step C2, scheme C, the protected amino compound, 6A is phenylated using a phenylating agent such as phenylboronic acid in the presence of a transition metal catalyst such as palladium acetate. In step C3, Scheme C, the phenylated compound, 6B is reacted with α-halo-carboxylic acid ester using the procedures similar to the ones described for steps B3 and B4 of Scheme B. Interestingly, in this step both addition and cyclization can be carried out in a single step to form the indole derivative, 8B. This combination of reactions can typically be effected in the presence of a suitable base such as potassium tert-butoxide. The reaction is typically carried out in an organic solvent such as NMP, THF or mixtures thereof at temperatures in the range of from about 0° C. to 40° C. Finally, in step C4, Scheme C, the acetyl indole derivative, 8B is deprotected following the procedures of step B5, Scheme B.









Scheme D illustrates another variation of the synthesis of the 3-amino-indole derivatives useful in the synthesis of the compounds of this invention. Steps D3 to D5, Scheme D are similar to the ones described above in Schemes B and C. Thus synthetic procedures as described above can be utilized in steps D3 to D5. However, in some instances the amino group need not be protected and can be reacted straight with the desirable α-halo-carboxylic acid ester to form the 3-amino-indole derivative, 3 following steps D1 and D2, Scheme D. Thus in step D1, Scheme D, the reaction can be carried out by treating the substituted 2-aminobenzonitrile, 9 with α-halocarboxylic acid ester in the presence of a suitable base such as potassium or sodium carbonate to form the product 10, which is subsequently cyclyzed to indole compound, 3 in the presence of a base such as potassium tert-butoxide.









Scheme E illustrates the preparation of various indole compounds used in the method of this invention. In step, E1, Scheme E, the 3-amino-indole derivative, 3 produced in accordance with any one of the Schemes A through D as described herein is reacted with suitable halo substituted R 3 compound in the presence of a suitable organic solvent as described in U.S. Pat. No. 5,328,920. The reaction is typically carried out at ambient to super-ambient temperature conditions, typically in the temperature range of 20° C. to 150° C. Scheme E is particularly suitable for the preparation of compounds of the method of this invention wherein R 2 is C 1-6 alkyl or C 1-6 perfluoroalkyl; C 1-4 alkyl are preferred, ethyl or t-butyl are more preferred.









Scheme F shows various other synthetic approaches to the preparation of the compounds that are used in the method of this invention. In sum, Scheme F depicts two approaches to the preparation of various compounds used in the method of this invention by way of transesterification reactions. Any of the other known transesterification reactions can be utilized for the preparation of these compounds. Finally, Scheme F also illustrates, in step F8, the preparation of N-substituted indoles.
In step F1, Scheme F, the carboxylic acid esters, preferably, the alkyl esters of the indole derivatives, 11 are hydrolyzed using a suitable base such as potassium hydroxide to form the corresponding potassium salt, 12. Typically the hydrolysis is carried out in an aqueous medium, alcoholic medium or a mixture thereof. The reaction is generally carried out at ambient or super-ambient temperatures, typically in the temperature range of from about 25° C. to 80° C.
In step F2, Scheme F, the potassium salt, 12 so produced from step F1, Scheme F is reacted with a suitable halo compound to produce the desirable transesterified indole compound, 11A used in the method of this invention. The reaction is typically carried out in a polar or a non-polar anhydrous solvent. Suitable solvents include ethereal solvent such as tetrahydrofuran or polar solvents such as dimethylformamide (DMF). The reaction can be carried out at ambient or super-ambient temperatures, typically temperature in the range of 30° C. to 100° C. is preferred. The reaction can also be carried out in the presence of a catalyst such as potassium iodide.
Steps F3 and F4 of Scheme F illustrate an alternative approach for the preparation of transesterified indole compound, 11A used in the method of this invention. In step F3, Scheme F, the ester derivative of an indole compound, 11 is hydrolyzed under acidic reaction conditions at ambient, sub-ambient or super-ambient reaction conditions to form the free carboxylic acid derivative, 13. Suitable acid catalysts include mineral acids such as hydrochloric acid or sulfuric acid; organic sulfonic acids such as methanesulfonic acid, trifluoromethanesulfonic acid or para-toluene sulfonic acid; and carboxylic acids such as acetic acid or trifluoroacetic acid and the like. Solid acid catalysts such as Nafion-H® or H-ZSM-5 can also be employed. Typical reaction temperatures are in the range of from about 0° C. to 50° C.
In step F4, Scheme F, the carboxylic acid derivative, 13 produced in step F3, Scheme F is esterified to obtain the desirable indole compound, 11A used in the method of this invention. The conditions for esterification reaction are varied depending upon the ester that is being formed. Such variation in the reaction conditions are readily appreciated by the one skilled in the art.
For example, the esterification reaction is carried out under essentially neutral conditions in the presence of carboxylic acid activation agents where the resulting product contained a hydrolytically susceptible moiety. However, optionally the esterification can be carried out in the presence of a hindered base such as diethylisopropylamine. Suitable esterification activating agents include benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or benzotriazol-1-yloxytris(pyrrolidino)-phosphonium hexafluorophosphate (PYBOP). The reaction is typically carried out in a polar solvent such as 1-methyl-2-pyrrolidinone (NMP) or DMF. The reaction is generally carried out at sub-ambient to ambient temperatures, i.e., in the temperature range of from about −20° C. to 25° C.
Alternatively, in step F4, Scheme F, the esterification reaction can also be carried out under mildly basic conditions in the presence of a suitable catalyst. Suitable base includes cesium carbonate or potassium carbonate. Optional catalysts include potassium iodide. The reaction is typically carried out at a temperature range of from about 40° C. to 80° C.
In yet an alternative approach, Scheme F, steps F5 and F7 illustrate another method for the preparation of the indole compound, 11A used in the method of this invention. In step F5, Scheme F, the carboxylic acid derivative is first esterified to form pentafluorophenyl ester derivative, 14. The reaction is typically carried out in the presence of an organic base such as pyridine and pentafluorophenyl trifluoroacetate as the esterifying agent. The reaction is carried out typically at ambient temperatures such as 20° C. to 30° C.
In step F7, Scheme F, the pentafluorophenyl ester is reacted with desirable esterifying agent to form the desirable indole compound, 11A in the presence of a base such as sodium hydride. Typical solvents suitable for this reaction include NMP or DMF. The reaction is generally carried out at sub-ambient temperatures, typically, in the range of from about −20° C. to 0° C.
In step F8, Scheme F, the indole compound, 11A is substituted at the 1-nitrogen with a R moiety as defined herein to form the desirable indole compound, 15. Any of the known N-substitution reactions can be employed for this purpose. Specifically, N-alkylation can be carried out by reacting the unsubstituted indole compound with a suitable alkylating agent such as iodoalkane or alkyl sulfate. Specific examples of such alkylating agents include iodomethane, iodoethane, dimethyl sulfate, diethyl sulfate, methyl triflate and the like. The reaction is typically carried out in the presence of a base such as potassium tert-butoxide at around 0° C. in a suitable polar solvent such as DMF.









Scheme G illustrates another preferred method for the preparation of a variety of indole compounds used in the method of this invention. In step G1, Scheme G, the desirable substituted 2-amino-benzonitrile is reacted with a-halocarboxylic acid ester such as ethyl bromoacetate in the presence of a suitable base such as sodium bicarbonate and in the presence of a catalyst such as sodium iodide in a suitable organic solvent such as ethanol. The reaction is generally carried out at a temperature range of from about 60° C. to 120° C. Higher boiling solvents can be employed if higher reaction temperatures are desirable. In general, higher temperatures accelerate the reaction rates thus requiring shorter reaction time.
In step G2, Scheme G, the N-substituted benzonitrile derivative, 10 is subjected to a cyclization reaction in the presence of a suitable base such as potassium tert-butoxide in solvents such as THF under inert atmospheres such as nitrogen. Typically, the reaction temperature is maintained at or below ambient temperatures, i.e., around 25° C. by cooling the reaction mixture with any suitable coolant such as ice. The resulting substituted indole derivative, 3 is isolated.
In step G3, Scheme G, the substituted indole derivative, 3 is subjected to N-substitution reaction as described in Scheme E. This can be effected typically by reacting indole derivative, 3 with a suitable halo compound such as 4-chloropyridine to form the corresponding pyridinylamino compound, 11. This substitution reaction is typically carried out in a suitable organic solvent such as NMP. The reaction is typically carried out at elevated temperatures, generally, in the range of from about 80° C. to 120° C.
In step G4, Scheme G, the N-substituted compound, 11 is hydrolyzed to form the potassium salt of the indole compound, 12. This step is similar to the one described above in step F1 of Scheme F. The hydrolysis is preferably carried out using potassium hydroxide in ethanol as the solvent.
In step G5, Scheme G, the potassium salt, 11 is treated with a suitable acid such as hydrochloric acid to produce the free carboxylic acid, 13 at ambient reaction temperatures.









Scheme H shows two procedures for the preparation of benzo(b)thiophene compounds, 16 and 17 that are used in the method of this invention. The starting substituted 3-aminobenzo(b)thiophene, 15 can be prepared by various methods known in the art. For example, as noted above, U.S. Pat. No. 5,328,920 describes a few such procedures.
In step H1, Scheme H, the substituted 3-aminobenzo(b)thiophene, 15 is reacted with a suitable base such as lithium di-isopropylamide to generate in situ anion of compound 15. This reaction is typically carried out at sub-ambient reaction temperatures generally around −120° C. to 0° C. The reaction is also conducted in an inert atmosphere such as nitrogen and in a non-polar solvent such as ethereal solvents including diethyl ether, THF, alkane solvents including heptane, hexane, etc. and aromatic hydrocarbons such as ethylbenzene or combinations thereof. The anion of compound 15 so formed in this step is then reacted with any desirable esters of carbonic acid including dialkyl carbonate such as diethyl carbonate. This affords compound 16.
Alternatively, in step H2, Scheme H, compound 15 is reacted with a suitable base such as alkyl lithium to generate the anion which is reacted with carbon dioxide to form the free carboxylic acid compound 17. The anion of compound 15 is typically formed at sub-ambient reaction conditions as described above and then reacted with dry ice, i.e., the solid form of carbon dioxide.
Specific compounds within the scope of the practice of the methods of the present invention include:
3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 6-trifluoromethyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 6-chloro-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 5-chloro-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 6-fluoro-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 5-fluoro-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 4-fluoro-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, tert-butyl ester, 6-phenyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, tert-butyl ester, 5,6-dimethoxy-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 3-methoxy-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, tert-butyl ester, 3-(2-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 3-(pyrimidin-2-ylamino)-1H-indole-2-carboxylic acid ethyl ester, 5-fluoro-1-methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 4-fluoro-1-methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, tert-butyl ester, 5-chloro-1-methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 6-chloro-1-methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 1-methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid maleate salt, 4-fluoro-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid trifluoroacetate salt, 4-fluoro-1-methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid trifluoroacetate salt, 5-methoxy-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid trifluoroacetate salt, 6-chloro-1-diethylcarbamoylmethyl-3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid ethyl ester, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid pentafluorophenyl ester, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid 2-diethylamino-ethyl ester, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid 2-dimethylamino-ethyl ester, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid 2-piperidin-1-yl-ethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid (S)-1-methoxycarbonyl-ethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid ethoxycarbonylmethyl ester, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid 2-methoxyethyl ester, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid 3-ethoxypropyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid indan-5-yl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid diethylcarbamoylmethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-morpholin-4-yl-2-oxo-ethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-oxo-2-pyrrolidin-1-yl-ethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-azetidin-1-yl-2-oxo-ethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid (benzyl-ethyl-carbamoyl)-methyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid diethylcarbamoyloxy-methyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid benzylcarbamoyloxymethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid piperidine-1-carbonyloxymethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid morpholine-4-carbonyloxymethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-ethoxycarbonylamino-2-oxy-ethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid iso-propoxycarbonyloxy-methyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid 1,1,2-trimethylpropoxy-carbonyloxy-methyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid cyclohexyloxy-carbonyloxy-methyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid adamantan-1-yloxycarbonyloxymethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid acetoxymethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2,2-dimethyl-propionyloxymethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid pentanoyloxymethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-oxo-piperidin-1-ylmethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid (benzoyl-ethoxycarbonylmethyl-amino)-methyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-oxo-pyrrolidin-1-ylmethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid 5-methyl-2-oxo-(1,3)dioxo-4-ylmethyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid (phenyl-(toulene-4-sulfonyl)-amino)-methyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid (benzenesulfonyl-methyl-amino)-methyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid (methyl-(toulene-4-sulfonyl)-amino)-methyl ester, 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid tert-butoxycarbonyloxy-methyl ester, 3-(4-pyridinylamino)-benzo(b)thiophene-2-carboxylic acid ethyl ester, 6-fluoro-3-(4-pyridinylamino)-benzo(b)thiophene-2-carboxylic acid ethyl ester, ethyl 3-((4-pyridyl)amino-N-methyl)-benzo(b)thiophenyl-2-carboxylate, 3-(4-pyridinylamino)-6-trifluoromethyl-benzo(b)thiophene-2-carboxylic acid methane sulfonate salt, 3-(4-pyridinylamino)-benzo(b)thiophene-2-carboxylic acid hydrochloride salt, 3-(4-pyridinylamino)-6-trifluoromethyl-benzo(b)thiophene-2-carboxylic acid ethyl ester hydrochloride salt, 3-(propyl-4-pyridinylamino)-benzo(b)thiophene-2-carboxylic acid ethyl ester, 3-((3-phenylpropanoyl)methylamino)-1H-6-chloro-indole-2-carboxylic acid, 3-((benzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((benzoyl)amino)-6-chloro-1H-indole-2-carboxylic acid, 3-((benzoyl)amino)-6-chloro-1H-indole-2-carboxylic acid 2-dimethylamino-ethyl ester, 3-((4-chlorobenzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((benzoyl)benzylamino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((4-piperidineacyl)methylamino)-6-chloro-1H-indole-2-carboxylic acid, 3-((benzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid ethyl ester, 3-((benzoyl)methylamino)-5,6-dichloro-1H-indole-2-carboxylic acid, 3-((benzoyl)ethylamino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((benzoyl)methylamino)-6-fluoro-1H-indole-2-carboxylic acid, 3-((2-benzylbenzoyl)amino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((3-fluorobenzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((benzoyl)benzylamino)-4,6-dichloro-1H-indole-2-carboxylic acid ethyl ester, 3-((phenylsulfonyl)amino)-4,6-dichloro-1H-indole-2-carboxylic acid ethyl ester, 3-((phenylsulfonyl)amino)-4,6-dichloro-1H-indole-2-carboxylic acid, and 3-((phenylsulfonyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid.

The following compounds are preferred in the practice of the methods of the present invention:
3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, 3-((3-phenylpropanoyl)methylamino)-1H-6-chloro-indole-2-carboxylic acid, 3-((benzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((benzoyl)amino)-6-chloro-1H-indole-2-carboxylic acid, 3-((benzoyl)methylamino)-5,6-dichloro-1H-indole-2-carboxylic acid, 3-((benzoyl)methylamino)-6-fluoro-1H-indole-2-carboxylic acid, 3-((4-chlorobenzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((benzoyl)benzylamino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((benzoyl)ethylamino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((2-benzylbenzoyl)amino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((3-fluorobenzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((4-piperidineacyl)methylamino)-6-chloro-1H-indole-2-carboxylic acid, 3-((phenylsulfonyl)amino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-((phenylsulfonyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 6-trifluoromethyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester, 3-(pyrimidin-2-ylamino)-1H-indole-2-carboxylic acid ethyl ester, 3-((benzoyl)amino)-6-chloro-1H-indole-2-carboxylic acid 2-dimethylamino-ethyl ester, 3-((benzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid ethyl ester, 3-((benzoyl)benzylamino)-4,6-dichloro-1H-indole-2-carboxylic acid ethyl ester, and 3-((phenylsulfonyl)amino)-4,6-dichloro-1H-indole-2-carboxylic acid ethyl ester.

The invention also provides pharmaceutical compositions comprising one or more of the compounds according to this invention in association with a pharmaceutically acceptable carrier. Preferably these compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or suppositories; for oral, parenteral, intranasal, sublingual or rectal administration, or for administration by inhalation or insufflation. Alternatively, the compositions may be presented in a form suitable for once-weekly or once-monthly administration; for example, an insoluble salt of the active compound, such as the decanoate salt, may be adapted to provide a depot preparation for intramuscular injection. An erodible polymer containing the active ingredient may be envisaged. For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Flavored unit dosage forms contain from 1 to 100 mg, for example 1, 2, 5, 10, 25, 50 or 100 mg, of the active ingredient. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
The liquid forms in which the novel compositions of the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles. Suitable dispersing or suspending agents for aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinyl-pyrrolidone or gelatin.
The preferred administration of the pharmaceutical composition of this invention is by an intranasal route. Any of the known methods to administer pharmaceutical compositions by an intranasal route can be used to administer the composition of this invention.
In the treatment of various disease states as described herein, a suitable dosage level is about 0.01 to 250 mg/kg per day, preferably about 0.05 to 100 mg/kg per day, and especially about 0.05 to 20 mg/kg per day. The compounds may be administered on a regimen of 1 to 4 times per day.
BIOLOGICAL EXAMPLES
The following test protocols are used to ascertain the biological properties of the compounds of this invention.
The first method involves adding the compound to a cell comprising a vector which comprises at least 100 contiguous nucleotides of an exogenous human interleukin-4 promoter, operably linked to a gene encoding a detectable protein under conditions to permit expression of the detectable protein, and detecting the detectable protein.
Suitable detectable proteins include proteins which may be measured by standard laboratory techniques such as staining methods, for example, β-galactosidase, immunoassays such as those involving FLAG-tagged proteins, luminescent proteins, such as luciferase or GFP.
The relative amount of detectable protein may be determined directly such as using standard laboratory techniques suitable for the detectable protein, for example, by immunochemistry or luminescence. Alternatively the relative amount of detectable protein may be determined indirectly by measuring the mRNA or cDNA level corresponding to the detectable protein.
It is preferred that the cell used in the present method does not express the detectable protein endogenously. However, in an alternative embodiment, the assay conditions may be modified such that the endogenous protein is not expressed when the compound is added to the cell.
It is preferred that cells used in the present method are cells capable of cell culturing techniques. Commercial sources of suitable cells are well known and include the American Type Culture Collection (ATCC), and other vendors. Examples of suitable cells include standard cultured cells such as Chinese Hamster Ovary cells (CHO) cells, lymphocytes, T cells, naive T cells, Th1 cells, Th2 cells, macrophages, or other cells derived from the immune system. Generally, human cells are preferred. Primary cells may also be used in the present method.
The use of mammalian cells are preferred in setting up assays to identify compounds capable of modulating one or more Th2 cytokines. The use of primate cells are more preferred in such assays, and use of human cells are especially preferred in such assays.
Cells may be derived from any tissue of the body, but it is generally preferred that cells are derived from the immune system, such as lymphocytes, T cells, naive T cells, Th1 cells, Th2 cells, macrophages, or other cells derived from the immune system.
The detectable protein may be a directly measurable or detectable protein, such as described above, for example, β-galactosidase, FLAG-tagged proteins, luciferase or GFP. Alternatively, the detectable protein may be indirectly measurable, such as a protein which initiates a pathway, the initiation of which is detectable. An example of the latter is a transcription factor the expression of which permits detectable up- or down-regulated transcription of a second gene. An indirectly measurable detectable protein may involve a phenotypic change, for example, apoptosis.
In one embodiment of the invention, the level of the detectable protein is compared to the level of the detectable protein in the absence of the compound.
In an alternative embodiment of the invention, the level of the detectable protein is compared to the level of the detectable protein in the presence of a reference compound. Reference compounds may be known modulators of a Th2 cytokine, such as cyclosporin A, FK-506 and rapamycine etc., or may be previously unknown modulators of a Th2 cytokine.
The Th2 cytokines in the present invention are IL-4, IL-5 and IL-13.
In the Biological Examples that follow, the following abbreviations are used:
Al(OH) 3 Aluminum hydroxide Anti-DNP Anti-dinitrophenyl antibody Anti-OVA Anti-ovalbumin antibody BALF Bronchoalveolar lavage fluid CsA Cyclosporin A DMSO Dimethyl sulfoxide DNP-KLH Dinitrophenol-keyhole limpet hemocyanin ED 50 Median effective dose ELISA Enzyme-linked immunosorbent assays GFP Green fluorescent protein IC 50 Inhibitory concentration 50% IFN-γ Interferon-gamma IgE Immunoglobulin E IgG1 immunoglobulin G1 IgG2a immunoglobulin G2a IL-4 Interleukin-4 IL-5 Interleukin-5 IL-13 Interleukin-13 i.n. Intranasal i.p. Intraperitoneal i.v. Intravenous LD 50 Median lethal dose NY-1 Th1 cell line named NY-Th1 NY-2 Th2 cell line named NY-Th2 PC 400 Post carbachol concentration that increases the SR L by 400% over baseline p.o. By mouth (per os) PHA Phytohaemagglutanin R L Resistance in the lung (pulmonary) SD Standard deviation SE Standard error SEM Standard error of mean STAT-6 Signal transducer and activator of transcription-6 SR L Specific resistance in the lung s.c. Subcutaneous Th T helper Th1 T-helper 1 Th2 T-helper 2

IL-4 Luciferase Assay in Jurkat Cells
An IL-4 luciferase assay may be used to screen for compounds which modulate levels of IL-4 transcription. Several luciferase assays have been developed to screen compounds that are effective to transcriptionally modulate expression of the gene and thereby affect the level of the protein encoded by the gene which is expressed by the cell. For example, a transcriptional assay method is used to show that a 11 base pair DNA sequence motif is involved in the human IL-4 gene that is responsible for the T-cell activation signals, see Abe, E., et al., Proc. Natl. Acad. Sci. USA , (1992), 89, 2864-2868. It has also been shown that nuclear factor-IL-6 (NF-IL-6) is involved in transcriptional activation of the human IL-4 promoter in T cells by screening cDNA expression library from Jurkat T cells and isolating a cDNA encoding NF-IL-6, see Davydov, I. V., et al., J. Immunol ., (1995), 155(11), 5273-5279.
The contribution to T cell specific expression by other promoter sites has been assessed in a transient expression assay with IL-13 promoter constructs linked to a luciferase gene, focusing initially on the core binding factor (CBF) site, which is footprinted in vivo upon T cell activation, see Taylor, D. S., et al., J. Biol. Chem ., (1996), 271(14), 14020-14027. It has been reported recently that human IL-4 promoter exists in multiple allelic forms that exhibit distinct transcriptional activities in IL-4-positive T cells, see Song, Z. et al., J. Immunol., (1996), 156(2), 424-429. More recently, U.S. Pat. Nos. 5,863,733; and 5,976,793 disclose methods of transcriptionally modulating gene expression and of discovering chemicals capable as gene expression modulators.
Isolation of the Human IL-4 Gene Promoter:
A 6.7 kb fragment comprising nucleotides −6635 to +66 of the IL-4 gene promoter ( FIG. 4 ; SEQ ID NO:1) was isolated from a human genomic P-1 library, clone F0178, obtained from BIOS Laboratories (New Haven, Conn.) using an IL-4 specific, 5′ end biased, probe (Arai et al., J. of Immunol ., (1989), 142, 274-282). The promoter construct was generated by ligating two EcoRI restriction fragments of 5.5 and 1.2 KBs respectively. A HindIII restriction site was added to the 3′ end of the 1.2 KB EcoRI fragment and cloned into a similar site on the multiple cloning region on pGL3Neo. Similarly, a XhoI restriction site was added to the 5′ end of the 5.5 KB EcoRI fragment and cloned into the XhoI site on pGL3Neo. The IL-4 promoter construct was verified by sequencing.
Jurkat cells are grown in RPMI 1640, Gibco/BRL, catalog no: 11875-093; 10% FBS Gibco/BRL, cat no: 16000-096; 1% antibiotic/antimycotic, Gibco/BRL, cat no: 15240-096) and transfected with the above DNA following the protocol described by Staynov et al., Proc. Natl. Acad. Sci. USA , (1995), 92, 3606-3610, and selected against 800 μg/ml of G418 (Geneticin, Gibco/BRL, cat no: 10131-035). Monoclonal lines, 1F7, F8 and C5 are derived from the stable transfected polyclonal population by the limiting dilution method and used for the assay. The monoclonal cell lines are cultured in the above described medium in the presence of 400 μg/ml G418.
The IL-46.7 Luc monoclonal lines are grown in 150 cm vented T-flasks seeded at 100,000 cells/ml for two days. The cells are harvested, resuspended in fresh media and plated at a density of 50,000 cells/well in 120 μL volume, in a 96 well plate and grown for 16 hours. Test compounds are added to the culture (in DMSO final concentration of 0.5%) in replicates of 3 or 4 in a 15 μL volume. Typically, dose response curves are generated using three (10, 1 and 0.1 μM) or five concentrations (10, 1, 0.1, 0.01 and 0.001 μM). The cultures are then stimulated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA, Sigma, cat no: P-8139); and 1.0 μM calcium ionophore (A 23187 , Sigma, cat no: C-7522); in 25 μL of media for 8 hours. The stimulation process is terminated by the addition of 50 μL s of 1× lysis buffer (25 mM Tris Phosphate, pH 7.8, 2.0 mM DTT, 2.0 mM EDTA, 100 ml/L glycerol, 1.0% Triton X-100 (v/v)). The cells are incubated in the presence of lysis buffer for 5 minutes, pipetted up and down, 5×, to ensure complete lysis. A 100 μL aliquot of the cell lysate is transferred to white luminometer plates (Dynatech) and assayed for luciferase activity with a Dynatech luminometer. The substrate, luciferase reagent (470 μM luciferin, 530 μM ATP, 270 μM Coenzyme A, and 33.3 mM DTT) is dissolved in 2× luciferase assay buffer (20 mM Tricine, 1.07 mM (MgCO 3 ) 4 Mg(OH) 2 .5H2O, 2.67 mM Mg SO 4 , 0.1 mM EDTA, pH 7.8).
All experimental plates included non-induced and induced control wells. Additionally, CsA (Cyclosporin A, Sigma Cat no: C-3662) at concentrations of 1, 0.3, 0.1, 0.03 & 0.01 μM is included on the plates as a positive control.
ANALYSIS OF RESULTS: A five-six fold induction (determined by the ratio of stimulated to non stimulated) is seen with 6.7IL-4-Luc. The results generated from the assay are expressed as percent activity.







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×
100




The primary assay measured the effects of the ability of a compound to modulate the level of a reporter gene, luciferase, linked to a human IL-4 promoter transfected into a human Jurkat cell line. Using this assay, 250 positive compounds were found capable of modulating levels of IL-4.
Additional assays may be used to further characterize the ability of the compound to modulate other Th2 cytokines for example, IL-13, or alternatively, to characterize the ability of the compound to modulate Th1 cytokines, for example, IFN-γ.
In Table 1, effects of the compound of the present invention on gene expression of Jurkat cells transfected with IL-4, IL-13 and IFN-γ are presented.





TABLE 1



Inhibition of IL-4 and IL-13 gene expression in Jurkat

cell line by the compound of the present invention







6.7-IL-4
2.1-IL-13 -
2.7-IFN-γ -


Luc (% of
Luc (% of
Luc (% of

Compound
control)
control)
control)



3-(4-pyridinylamino)-
62 ± 29
50 ± 12
100 ± 15

1H-indole-2-carboxylic

acid (Example 28)

CsA (IC 50 , μM)
0.06 ± 0.01
0.06 ± 0.001
0.06 ± 0.001



























IFN-γ Luc Assay:
The human IFN-γ promoter fragment, comprising nucleotides −3218 to +128, was cloned into pGL3NEO (SstI/Hind III) to give IFN-γ Luc. Jurkat cells were (grown in RPMI 1640, 10% FBS, 1% antibiotic/antimycotic) transfected with the above DNA (protocol described by Staynov et al., Proc. Natl. Acad. Sci. USA , (1995), 92, 3606-3610, and selected against 800 μg/ml of G418 (Geneticin). Monoclonal lines were derived from the stable transfected polyclonal population and used for the assay. The monoclonal cell lines were cultured in the above described medium in the presence of 400 μg/ml G418.
Jurkats containing 2.3IFN-γ Luc (monoclonal lines) were plated at a density of 50,000 cells in 120 μl per well, and grown for 16 hours. The cultures were then stimulated with 10 ng/ml of phorbol 12-myristate 13-acetate (PMA) and 1.0 μm calcium ionophore A 23187 in 10 μl of media for 8 hours. The stimulation process was terminated by the addition of 50 μls of 1× lysis buffer (25 mM Tris Phosphate, pH 7.8, 2.0 mM DTT, 2.0 mM EDTA, 100 ml/L glycerol, 1.0% Triton X-100 (v/v)). The cells were incubated in the presence of lysis buffer for 5 minutes, pipetted up and down, 5×, to ensure complete lysis. A 100 μL aliquot of the cell lysate was transferred to white luminometer plates (Dynatech) and assayed for luciferase activity with a Dynatech luminometer. The substrate, luciferase reagent (7.1 mg luciferin, 14.6 mg ATP, 10.4 mg Coenzyme A, and 250 mg DTT, in 50 ml volume) was dissolved in 2× luciferase assay buffer (14.33 g Tricine, 2.07 g (MgCO 3 ) 4 Mg(OH) 2 .5H2O, 2.63 g Mg SO 4 , 0.15 g EDTA in 2 L of water, and pH was adjusted to 7.8).
In order to use this assay to screen for agonists or antagonists from a chemical library, desired doses of a compound were added directly along with PMA/A 23187 mix.
Greater than twenty fold induction (determined by the ratio of stimulated to non stimulated) was seen with IFN-γ Luc. The results generated from the HTPS will be expressed as a percentage of the control at given concentrations (viz.: 0.1 μM, 1 μM, 10 μM).
REFERENCES: Staynov, D. Z., Cousins, D. J., and Lee, T. K., Proc. Natl. Acad. Sci. USA , (1995), 92, 3606-3610.
IL-13 Luc Assay
The human IL-13 promoter fragment comprising nucleotides −2154 to +53 (as described by Dulganov et al., Blood , (1996), 87, 3316-3326), was cloned into pGL3NEO (KpNI/Hind III) to give 2.1IL-13 Luc. Jurkat cells were (grown in RPMI 1640, 10% FBS, 1% antibiotic/antimycotic) transfected with the above DNA (protocol described by Staynov et al., Proc. Natl. Acad. Sci. USA , (1995), 92, 3606-3610) and selected against 800 μg/ml of G418 (Geneticin). Monoclonal lines were derived from the stable transfected polyclonal population and used for the assay. The monoclonal cell lines were cultured in the above described medium in the presence of 400 μg/ml G418.
Jurkats containing 2.1IL-13 Luc (monoclonal lines) were plated at a density of 50,000 cells in 120 μL per well, and grown for 16 hours. The cultures were then stimulated with 10 ng/ml of Phorbol 12-myristate 13-acetate (PMA) and 1.0 μM calcium ionophore A 23187 in 10 μL of media for 8 hours. The stimulation process was terminated by the addition of 50 μL s of 1× lysis buffer (25 mM Tris Phosphate, pH 7.8, 2.0 mM dithiothritol (DTT), 2.0 mM ethylenediamine tetra-acetic acid (EDTA), 100 ml/L glycerol, 1.0% Triton X-100 (v/v)). The cells were incubated in the presence of lysis buffer for 5 minutes, pipetted up and down, 5×, to ensure complete lysis. A 100 μL aliquot of the cell lysate was transferred to white luminometer plates (Dynatech) and assayed for luciferase activity with a Dynatech luminometer. The substrate, luciferase reagent (7.1 mg luciferin, 14.6 mg ATP, 10.4 mg Coenzyme A, and 250 mg DTT, in 50 ml volume) was dissolved in 2× luciferase assay buffer (14.33 g Tricine, 2.07 g (MgCO 3 ) 4 Mg(OH) 2 .5H2O, 2.63 g Mg SO 4 , 0.15 g EDTA in 2 L of water, and pH was adjusted to 7.8).
In order to use this assay to screen for agonists or antagonists from a chemical library, desired doses of a compound were added directly along with PMA/A 23187 mix.
Greater than fifteen fold induction (determined by the ratio of stimulated to non stimulated) was seen with IL-13 Luc. The results generated from the high throughput screening (HTS) will be expressed as a percentage of the control at given concentrations (viz.: 0.1 μM, 1 μM, 10 μM).
REFERENCES: Staynov, D. Z., Cousins, D. J., and Lee, T. K. Proc. Natl. Acad. Sci. USA , (1995), 92, 3606-3610.
The second method involves adding the compound to a primary human cell secreting detectable cytokines in particular IL-4 protein under conditions to permit expression of the detectable protein, and detecting the detectable protein.
Generation and Use of Human Primary Th1 and Th2 Cells:
The methods described herein permit polarization of primary human peripheral blood cells into Th1 and Th2 cells and generation of sufficiently large quantities of Th1 and Th2 cells, e.g., billions of cells, to allow drug screening. Briefly, CD4 + CD45RA + T cells, isolated from donor peripheral blood mononuclear cells of healthy donors, are educated in the presence of anti-CD3/CD28 antibodies and various growth factors to obtain either a Th1-like or Th2-like phenotypes as determined by the known cytokine profiles of these two cell types. Primary human Th1 cells secreted IFN-γ and IL-2 cytokines while primary human Th2 cells secreted IL-4, IL-5, IL-13 and basal amounts of IFN-γ that are measurable by enzyme linked immuno absorption assay (ELISA).
Generation and Maintenance of Th1 and Th2 Cell Lines from Human Blood:
Peripheral blood mononuclear cells (PBMC) are prepared from human blood by Ficoll-Hypacque (Pharmacia). The cells are washed two times in Hanks balanced salt solution (HBSS) and counted.
Naïve T cells (CD4 + /CD45RA) are prepared by negative selection method using following antibodies and Dynabeads: anti-CD8, anti-HLA-DR, (where HLA means human leucocyte antigen) anti-CD14, anti-CD16, anti-CD19, anti-CD45RO antibodies (all from R&D Systems, 1-2 μg/ml) are added to the cells, and the cells are kept on ice for 30 min. The cells are washed two times in HBSS, and goat anti-mouse Ig-beads, (bead:target ratio, 5:1) are added. The cells and beads are slowly rotated at 4° C. for 20 min. (following the protocol supplied from Dynal). The supernatant is removed and fresh goat anti-mouse Ig-beads are added to the supernatant, which is slowly rotated at 4° C. for 20 min. The cells are counted and phenotypically characterized by flow cytometric analysis. Typically after negative selection purity of CD4 + /CD45RA + cells were 96%. The CD8 + , CD14 + , CD16 + CD19 + and CD45RO + cell contamination were below detectable levels (<0.1%)
Generation of Th1/Th2 cells: Twenty four well plates are coated with 300 μL anti-CD3 Ab (10 μg/ml, in PBS 1-2 h at 37° C. or overnight at 4° C.), and washed two times with PBS. CD14 + /CD45RA + cells at a density of 10 6 /ml in RPMI, is prepared and added to RPMI medium containing 10% Hyclone serum/glutamine and 1-2 μg/ml anti-CD28 Ab. The cells are split in two aliquots. For Th1 cells: anti-IL-4 Ab (1-2 μg/ml), IFN-γ (1 μg/ml, or 5000 units/ml) are added to the wells. For Th2 cells: IL-4 (100-200 ng/ml), anti-IFN-γ neutralizing Ab (1-2 μg/ml), anti-IL-12 neutralizing Ab (1-2 μg/ml) are added into the wells. The cells are incubated for 2-3 days at 37° C. The cells are transferred to fresh wells and grown in 200 units/ml IL-2 for 2 weeks by feeding them every other day with IL-2 containing medium and splitting the cultures 1:2 or 1:3 as needed. The degree of polarization is determined by intracellular staining of cells with anti-IFN-γ and anti-IL-4 antibodies from Pharmingen, and using flow cytometry from Becton Dickinson (BD), according to manufacturers description. The cells are stimulated with PHA and alloantigens at day 14 and 28 as described below. Eight days after the second allostimulation the cells can be used for compound screening or frozen for future use. For compound testing, large quantities of Th1 and Th2 cells derived from a given donor (NYTh1 and NYTh2) were expanded and frozen as aliquots in liquid nitrogen. The cells are thawed and cultured as and when needed. The thawed cells are stimulated with alloantigen/PHA combination. The cells are used for compound screening after 8-10 days in culture.
Re-stimulation by allogeneic cells: Human PBMCs, to be used as alloantigens, are isolated from freshly drawn blood by Ficoll separation. Cells are resuspended in medium containing 50 μg/ml mitomycin C and incubated for 40 minutes at 37° C. and then washed extensively to remove all of the Mitomycin C. Cells are counted and diluted to a final concentration of 2×10 6 /ml in medium containing 10 μg/ml PHA and 200 units/ml IL-2. Th1 or Th2 cells are (3×10 5 cells/ml) mixed with above PBMC in 1:1 ratio in 24 well plates and incubated at 37° C. The cells so formed can be used for compound testing typically after 8 to 10 days.
Compound Testing:
Ninetysix well Falcon tissue culture plates (Fisher Scientific) are coated with 100 microliters per well of 10 μg/ml anti-CD3 (PharMingen) antibody in phosphate buffered saline (PBS) and stored at 4° C. one day before the experiment.
On the day of assay, anti-CD3 antibody coated plates are washed with PBS once to remove all soluble anti-CD3 antibody. T cells are centrifuged and washed with 1640 RPMI cell culture medium (Gibco) without IL-2, counted, and resuspended to a final concentration of 1×10 6 cells/ml. Stock solutions of the compounds of this invention (10 mM concentration) are diluted to a final concentration of 10 μM in 0.1% DMSO (dimethylsulfoxide) and titrated using 3-fold dilutions over 7 rows for IC 50 determinations. All compounds and controls are assayed in triplicates. Control wells consist of 0.25% ethanol, 0.1% DMSO, anti-CD3 and anti-CD28 stimulated cells (served as positive control) or unstimulated cells (as negative control) in triplicate. Cyclosporin A (CsA) in ethanol (at 1 μM concentration), titrated over seven rows, is included as a plate control within the first plate of each assay. 1 μg/ml of anti-CD28 antibody (R&D Systems) as a costimulus is added to all wells and 100 microliters of 1×10 5 cells is pipetted into each well.
Cells are incubated at 37° C. in a 5% CO 2 atmosphere. After 48 hours, supernatants are removed for ELISA testing. Percent control is measured by subtracting the background from the mean OD of triplicates and is compared to the mean OD of controls minus the background i.e. (x OD of compound-background)/(x OD controls-background) X100 IL-5 (PharMingen), IL-4 (R&D Systems), IL-13 (R&D Systems) and IFN-γ (R&D Systems) are measured by ELISA kits and results expressed as % Control. After 48 hours, with the remaining cells a “viability test” is performed using a non-radioactive MTS assay kit purchased from Promega. Because the viable cells continue to proliferate, the results are expressed as “proliferation” in the tables. (Toxic compounds inhibited the proliferation of Th cells).
In Table 2, effects of a compound of the present invention, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, on human primary Th2 cells are presented. Th2 cells were activated with anti-CD3 and anti-CD28, as described in methods section in the absence or presence of the compounds of this invention. Supernatants were collected for cytokine measurements by standard ELISA 48 hr after stimulation. The cell numbers are determined by MTS Assay Kit (Promega) by incubating the remaining cells for additional 20 hrs. Standard deviations of triplicate cultures are given.





TABLE 2



Effects of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example

28) on Cytokine Expression and proliferation of Human Th2 cells









Proliferation
IL-5
DL-4
IFN-γ
IL-13


(% control)
(% control)
(% control)
(% control)
(% control)













μM
mean
SD
mean
SD
mean
SD
mean
SD
mean
SD















10
71.55
0.07
53.90
3.96
30.80
4.95
75.00
10.32
68.40
4.10

3
72.05
0.64
70.40
0.00
36.40
0.00
83.50
7.07
73.40
0.71

1
77.25
1.06
70.70
2.12
42.55
2.90
89.25
5.30
75.15
1.77

0.3
80.60
3.68
70.80
7.21
45.00
0.00
92.10
2.26
80.60
0.00

0.1
86.70
1.84
78.60
5.37
59.75
1.77
91.75
0.21
82.30
0.85

0.03
85.05
6.86
85.20
0.00
65.30
0.00
91.60
2.97
86.40
0.00

0.01
89.50
8.34
102.45
3.46
102.15
3.61
96.90
1.70
91.05
1.91




































In Table 3, combined data from 10 different experiments is presented. Th2 cells were activated as described above. Standard deviations were less than 15% and hence not included. As control CsA, as a nonselective cytokine inhibitor was used. The compound of this invention, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, selectively inhibited IL-4, but not IFN-γ and cell proliferation, whereas CsA inhibited IL-4, IFN-γ and cell proliferation.





TABLE 3



Summary of effects of 3-(4-pyridinylamino)-1H-indole-2-carboxylic

acid (Example 28) on human Th2 cells











IL-13

IFN-γ
Prol.
IFN-γ/

Human primary Th2
IL-4 IC 50
IC 50
IL-5 IC 50
IC 50
IC 50
IL-4

cells treated with:
(μM)
(μM)
(μM)
(μM)
(μM)
Ratio











3-(4-pyridinylamino)-
0.24
≈10
≅10
>10
>10
>41

1H-indole-2-

carboxylic acid (n = 10)

Cyclosporin A (CsA)
0.006
0.008
0.008
0.008

1.0

(n = 25)































In Table 4 effect of a compound of this invention, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, on human primary NYTh1 cells is presented. NYTh1 cells are activated with anti-CD3 and anti-CD28, as described in Tables 1 and 2. The data from 8 different experiments were combined, all of which used 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid. Standard deviations were less than 15% and hence not included. CsA, a non-specific immunosuppressive drug was used as control. These experiments demonstrated that a compound of this invention had no effect on Th1 cells.





TABLE 4



Summary of effects of 3-(4-pyridinylamino)-1H-indole-2-carboxylic

acid (Example 28) on human Th1cells









IFN-γ
Prolifer-



Human primary Th1 cells
IC 50
ation
IFN-γ/


treated with
(μM)
IC 50 (μM)
IL-4 Ratio











3-(4-pyridinylamino)-1H-
>10
>10
—


indole-2-carboxylic acid


(n = 8)


Cyclosporin A
0.006
0.005
1.0































In Table 5 effect of several close analogs of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid on human primary Th2 cells are presented. The cells were activated with anti-CD3 and anti-CD28, as described in methods section in the absence or presence of several of the compounds of the present invention and results were given as IC 50 in μM. Standard deviations were less than 15% and hence not included. As can be seen various compounds of the present invention inhibit the expression of IL-4 selectively and have a similar selectivity profile as that of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid.





TABLE 5



Summary of Activity and Selectivity Profile

of the Compounds of the Present Invention









IL-4
Prolif.
IL-5
IFN-γ
IL-13


IC 50
IC 50
IC 50
IC 50
IC 50

Compound #
μM
μM
μM
μM
μM










Example 11
>10
>10
>10
>10
>10

Example 24A
2.0
>10
>10
>10
>10

Example 28
0.24
>10
>10
>10
~10

(maleate salt)

Example 76
4.0
>10
>10
>10
>10

Example 77
3.6
>10
>10
>10
>10

Example 78
0.3
>10
>10
>10
>10

Example 79
5.0
>10
>10
>10
>10

Example 80
6.0
>10
>10
>10
>10

Example 81
6.0
>10
7.0
9.5
>10

Example 82
0.15
>10
>10
>10
>10

Example 83
1.0
>10
>10
>10
>10

Example 84
1.0
>10
>10
>10
>10

Example 85
0.9
>10
>10
>10
>10

Example 86
1.0
>10
>10
>10
>10

Example 87
0.6
>10
2.0
>10
>10

Example 88
2.5
>10
>10
>10
>10

Example 89
2.5
4.0
8
>10
>10

Example 90
1.0
>10
>10
>10
>10

Example 91
1.0
>10
>10
>10
>10

Example 92
0.45
>10
>10
>10
>10















































In Table 6 the effect of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid on steady state IL-4 mRNA level is presented. Human Th2 cells were activated with anti-CD3 and anti-CD28 (as described in methods) in the presence of 10 μM solution of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid or 100 nM Cyclosporin A (CsA) for 18 hours. RNAs were isolated from the harvested cells and analyzed for IL-4 message by Taqman. GAPDH message was used as an internal standard for normalization. The percent activity was calculated by treating the values from not drug treated, anti-CD3 and anti-CD28 stimulated Th2 cells as 100%. Table 6 summarizes data from two independent experiments. In the second experiment IL-4 protein levels were also determined by standard ELISA in culture supernatants as described earlier. These results demonstrate that 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid inhibits IL-4 mRNA in primary human T cells.





TABLE 6



Human Th2 IL-4 transcriptional assay









IL-4


RNA
RNA
PROTEIN

Treatment of human primary
Exp #1
Exp #2
Exp #2

Th2 cells for 18 hrs
% activity
% activity
pg/ml








Unstimulated
6
24
3.2 +/− 0.0

Stimulated: CD3/CD28
100
100
625.0 +/− 23

Stimulated: CD3/CD28 +
44
45
286.2 +/− 19

3-(4-pyridinylamino)-1H-

indole-2-carboxylic

acid (Example 28)

Stimulated: CD3/CD28 +
12
ND
ND

Cyclosporin A



ND—not determined.

































A third assay utilized in the present invention involves an in vitro cytotoxicity assay. This assay is used to further characterize the compounds in order to assess toxic activities of compounds on contact inhibited, non-dividing 3T3 fibroblasts. Other toxicity assays may be used.
Any compound killing 3T3 fibroblast cells was omitted from further studies.
Contact inhibited Swiss 3T3 fibroblasts are purchased from ATCC. The cells are maintained in 1640 RPMI supplemented with 10% fetal bovine serum (Gibco).
On day of assay, cells are detached from flasks by trypsinization, washed in medium and counted. 100 microliters of 2.5×10 4 cells per well are plated into Falcon brand 96 well tissue culture plates and cells incubated at 37° C. in presence of 5% CO 2 for 2 days or until confluent.
On day 2 of assay, 75 microliters of media is added to each well and 25 μL of test compounds are added at concentrations ranging from 10 to 0.01 μM. All compound dilutions are assayed in triplicate. Controls include 10 μM methotrexate (Sigma), 10 μM actinomycin D (Sigma) and 10 μM cyclosporin A (Sigma). Cells are returned to the incubator for 2 days.
After 2 day incubation, the 3T3 cells are assayed for viability using an MTS Assay Kit (Promega). Media is added to blank wells for background calculations. After 2 hours incubation in kit reagent, the optical density at 490 nm for each compound and control is determined by plate reader.
Results are expressed as percent cytotoxicity, i.e., (mean OD of compound wells−background OD)/(mean OD of controls−background).
Drugs which are used clinically may be used as controls in this assay, and include drugs which are expected to have no effect on 3T3 cells, for example, steroids, cyclosporin A, metotraxate, etc. or drugs which have known toxic effects, for example, Actinomycin D.
In Table 7, effect of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid and certain known standard compounds on the viability of quiescent 3T3 cells are presented. As expected, actinomycin D was the only compound which effected the viability of non-dividing cells. Note that starting concentration of CsA was 1 μM. 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid exhibited no toxic effects on these cells (Various other compounds of the present invention are also negative in this assay).





TABLE 7



Lack of in vitro cytotoxicity of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28)









Compounds
Medium
Example 28
Actinomycin D
Methotrexate
Dexamethasone
Cyclosporin A

μM
% control
% control
% control
% control
% control
% control











10
100
99.1
16.8
92.4
60.5
99.8

3
100
109.7
29.7
93.8
60.7
103.0

0.1
100
110.7
28.2
98.9
64.2
104.0

0.03
100
111.1
33.9
100.0
67.8
105.0

0.01
100
111.8
67.2
102.8
71.6
105.5

0.003
100
1116.3
74.9
105.2
77.9
114.4

0.001
100
124.0
79.0
113.2
100.0
121.0































In Vivo Experiments
In vivo immunological assays or disease models may also be used to further characterize the ability of a compound to modulate Th2 cytokine levels. Such assays are well known in the art, and numerous protocols have been published. The following are examples of in vivo assays that may be used to further characterize the ability of a compound to modulate Th2 cytokine levels and thus demonstrate the efficacy of IL-4 inhibition.
Unless stated otherwise, in all of the following in vivo experiments a maleate salt of the 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) was used.
In-Vivo Ovalbumin (OVA) Mouse Model:
This model is designed to determine the ability of a test article to modulate an antigen-induced pulmonary inflammatory cell accumulation in sensitized Balb/c mice from Charles River Laboratories. The mice are given a two week time period from arrival date prior to sensitizations.
Sensitizations and Challenge
On days 0, 7, 14, each animal is injected intraperitoneal (i.p.) with a 0.2 ml Ovalbumin Hydragel solution. Each animal receives 10 μg of Ovalbumin in 0.2 mL of Hydragel, aluminum hydroxide adsorptive gel containing 2% aluminum oxide. On Day 21, the animals are dosed with test compounds 30-60 minutes prior to 5% Ovalbumin aerosolized challenge. On day 22, BALF samples are collected from one group of animals and used for cytokine analysis. On day 24, BALF is obtained from a second group of animals and used for differential cell counts.
Methods and Experimental Design
Test System: Female Babl/cJ mice (Jackson Laboratories), approximately 6-9 weeks old, each weighing approximately 20 to 25 grams, are divided into ten animals per group.
Acclimation/Quarantine: Following arrival of the mice, the animals are assessed as to their general health by a member of the veterinary staff or suitable designee. Prior to experiments, animals are housed for a minimum of 7 days in order to acclimate them to the laboratory environment and to observe them for the development of infectious disease. Any animals considered unacceptable for use in this study are replaced with animals of similar age and weight from the same vendor.
Animal Husbandry: All animals are housed (group or individual) in compliance with USDA guidelines. The animal room environment is controlled, with targeted conditions: Temperature 18° C. to 26° C., relative humidity 30% to 70%, 12 hours artificial light and 12 hours dark. Temperatures and relative humidity are monitored daily.
All animals have access to Harlan Teklad Rodent Diet®, ad libitum, or any other acceptable Lab Chow, checked daily and added or replaced as needed. Feeders are designed to reduce soiling, bridging and scattering. Water is provided to the animals ad libitum.
Sensitization Regimen: Mice are sensitized by an intraperitoneal injection of 10 μg ovalbumin (OVA) mixed in 4 mg aluminum hydroxide Al(OH) 3 gel suspension in 0.2 ml sterile saline on Days 0, 7 and 14. This suspension is prepared one hour before intraperitoneal injection into each mouse. The animals are ready for OVA challenge on Day 21.
Antigen Challenge: Animals are exposed to aerosolized OVA on day 21 (5% w/v in sterile saline) for 20 minutes. The aerosol is generated by a PARI-Master nebulizer, the outlet of which is connected to a small Plexiglas® chamber containing the animals. Bronchoalveolar lavage is performed on Day 22.
Method of Study Performance: On day 24, 72 hours following aerosol OVA exposure, a separate group of animals are anesthetized with urethane (0.15-0.2 gm/kg) and the trachea is exposed and cannulated. Lungs are lavaged with 2×0.5 ml Hank's balanced salt solution without Ca 2+ and Mg 2+ (HBSS; Gibco, Grand Island, N.Y.) containing 0.1% EDTA. Lavage fluid is recovered after 30 sec by gentle aspiration and pooled for each animal. Samples are centrifuged at 2000 rpm for 15 minutes at 5° C. After centrifugation, individual supernatants are stored frozen at −80° C. The resulting pellet is resuspended in 0.5 ml of HBSS containing 0.1% EDTA. Total cells and eosinophils are determined using a Technicon Hl and cytoslides, respectively.
Administration of the Test/Control Article: The test article and vehicle dosing preparations are administered once to each anesthetized mouse intranasally using a 200 μl pipette at 30-60 minutes prior to OVA challenge. Each animal receives 50 μl (25 μl/nostril) at each time point. Method of Euthanasia: Urethane overdose at in-life completion.
Statistical Analysis: Total cell and eosinophil number from various treatment groups is compared using an ANOVA followed by a multiple comparison test.
Results of mouse experiments are presented in Table 8. Compounds administered 30 minutes before aerosol ovalbumin challenge in ova sensitized mice. BALF harvested 24 hours after aerosol challenge. N=6/group. 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid inhibited both IL-4 and IL-13 levels in the BALF significantly in a dose dependent manner.





TABLE 8



In vivo Allergen-induced Lung Inflammation in the Mouse. Effect

of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example

28) on Bronchoalveolar Lavage Fluid (BALF) Cytokines







Dose (mg/kg)
IL-4 pg/ml
IL-13 pg/ml

Compound
(intranasal)
(±SEM)
(±SEM)



Vehicle
—
1220.4 ± 207.7
120.2 ± 19.8

Cyclosporin A
30
144.4 ± 35.7
15.9 ± 2.4

Example 28
30
123.9 ± 48.7
15.1 ± 7.1

Example 28
10
414.3 ± 115.5
59.9 ± 12.7

Example 28
3
835.1 ± 40.4
ND



pg = picogram;

ND—not determined




























FIG. 1 summarizes results from one representative experiment (out of three) in which in addition to BALF cytokines, lung eosinophilia is also determined. Mice (12 animals per group) are sensitized with 0.2 ml of an AlOH 3 hydrogel suspension (2% AlOH 3 ) containing 50 μg/ml ovalbumin on days 0, 7 and 14. On day 21, mice are dosed with various dose levels of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28); 3, 10 & 30 mg/kg, 30 min prior to a 20 min challenge with 5% ovalbumin (OVA). The glucocorticosteroid budesonide (10 mg/kg) is included as a control. All compounds are given in 0.5% methylcellulose/0.2% Tween 80, intranasally (50 μl). After the OVA challenge for 24 hours, the mice underwent bronchoalveolar lavage and IL-4 and IL-13 levels in the BALF are determined by ELISA. Another group of mice underwent bronchoalveolar lavage (72 hours after treatment) to determine the number of the eosinophils (*p<0.05, **p<0.01). As shown in FIG. 1 , 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) significantly inhibited allergen-induced IL-4 and IL-13 levels in the BALF, with ED 50 values of approximately 10 mg/kg for each cytokine. Although 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid is a relatively poor inhibitor of IL-13 in vitro, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid inhibited IL-13 almost as well as IL-4 in vivo.
Table 9 summarizes effects of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) and various other compounds of this invention on the OVA-induced IL-4 and eosinophilia in BALF. Compounds are given (30 mg/kg), intranasally, once, 30 min before OVA challenge unless noted otherwise. BAL fluids are collected 24 hours after OVA challenge and the cytokine levels are measured by standard ELISAs. From a parallel set of animals, BAL are collected at 48 hours and the number of eosinophils are determined. Various salts of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid including the maleate salt, methanesulfonic acid salt (EF, Exp. #146) and potassium salt (ZD, Exp. #146) are tested as summarized in Table 9.





TABLE 9



In vivo allergen-induced lung inflammation in

the mouse: Summary of in vivo effects of different

salts and analogs of 3-(4-pyridinylamino)-1H-indole-2-carboxylic

acid (Example 28)











Dose
IL-4% ctl
Eos % ctl

Exp #
Compound #
Route
(mg/kg)
At 24 hours
At 72 hours










109
Example 24A
i.n.
30
3
8


(maleate salt)

130
Example 24A
i.p.
12
50


(maleate salt)

140
Example 28
i.n.
12
50
Not done


(maleate salt)

146
Example 28
i.n.
10
57
53


(maleate salt)

146
Example 28
i.n.
30
34
28


(maleate salt)

146
Example 28-EF
i.n.
30
27
42

146
Example 28-ZD
i.n.
30
49.6
37.4

115
Example 28
i.p.
30
43
Not done


(maleate salt)

109
Example 90
i.n.
30
37
71



i.n. = intranasal;

i.p. = intraperitoneal; and

p.o. = per os (by mouth)











































Table 10 summarizes effects of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid on OVA induced IgE in mice. Female mice (5-8 week age, Balb/c, Charles River) were used in this study. Ovalbumin (Sigma) was adsorbed to Aluminum Hydroxide Adsorptive Gel, Inter, and injected 10 μg/0.2 ml/mouse intraperitoneally on experimental day 0 and day 7. Dexamethasone (Sigma) and 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) were in 10% HPCD (Hydroxypropyl-β-Cyclodextrin, Aldrich). Dexamethasone (10 mg/kg), 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) (3 and 10 mg/kg) and 10% HPCD (0.2 ml/mouse) are injected into mice intraperitoneally once a day from day 6 to day 14. They were bled at day 15. Serum ovalbumin specific IgE levels were determined by standard ELISA using a goat anti-mouse IgE antibody labeled with alkaline phosphatase. The anti-OVA titers were determined by malting serial dilutions of the test sera and comparing with vehicle control.





TABLE 10



Inhibition of allergen specific IgE antibody

by 3-(4-pyridinylamino)-1H-indole-2-carboxylic

acid (Example 28) in mice







Treatment
Anti-OVA IgE
SE










Vehicle
150.8
52.9


Dexamethasone
27.4
9.4


Example 28 (3 mg/kg)
41.8
8.5


Example 28 (10 mg/kg)
45.6
15.7






























The effect of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid on OVA-induced lung inflammation was also studied in rats. Rat model was very similar to the mouse models described earlier. Briefly, the lung inflammatory model described by Haczku et al. was used with minor modifications (Haczku A., et al., Immunology , (1996), 88, 247-251). Male Brown Norway rats, weighing 160-200 grams were sensitized with intraperitoneal injection of 1 ml of a suspension containing ovalbumin (1 mg) and Al(OH) 3 (100 mg) on days 1, 2 and 3. Three weeks later, the rats were treated with vehicle or drugs via intraperitoneal injections. One hour later, the rats were challenged with ovalbumin inhalation (1% solution, 10 mg/ml in sterilized saline) for 20 minutes in a 10 liter Plexiglas chamber which was connected to a DeVilbiss (Ultra-NEB 99) nebulizer with a carrier air flow of 1 liter/minute.
Results of dose response studies of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid on eosinophil influx into the bronchoalveolar fluid of ovalbumin sensitized and challenged Brown Norway rats are depicted as bar graphs in FIG. 2 . Single dose of cyclosporin A (CsA) and dexamethasone (Dex) were also included as reference standards. 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid, administered with intraperitoneal injection, inhibited the influx of eosinophil in a dose dependent fashion with ED 50 of 10.07 mg/kg.
An In Vivo Model of Humoral Immunity in Mice.
The purpose of this assay was to determine if IL-4 inhibitors had an effect on normal antibody response, which is known to be dependent on T helper cells that regulate B cells to make antibody. This model demonstrates the effect of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid on its general immunosuppressive activities in mice.
Female Balb/c mice, each weighing approximately 20 to 25 grams each, aged 5 to 8 weeks are used. Each experiment uses 30 mice.
Immunization of Mice:
The antigen for immunization is 2,4-dinitrophenyl (DNP) or 2,4,6-trinitrophenyl (TNP) conjugated to keyhole limpet haemocyanin (KLH). The antigen (100 μg/mouse) is adsorbed to an adjuvant (colloidal aluminum hydroxide) and given i.p. to animal in a total volume of 0.2-0.3 ml. The method described herein for production of immune response is similar to the methods used by other investigators (see, for example, Wilder, J. A., et al., J. Immunol ., (1996), 156, 146-152; and Leenaars et al., Immunology , (1997), 90, 337-343.
Protocol and Compound Treatment:
Animals are randomly divided into 5 groups of 6 mice each. Group 1 is a positive control group, Groups 2, 3, and 4 are test compound groups, and Group 5 is a vehicle control group. Compounds may be given by i.n., i.p., p.o., s.c., i.m. or i.v. administration. Generally, compounds of this invention are administered daily to mice, starting one to two days prior to immunization until 7 days after immunization (9-10 times). On day 8 after the immunization, mice are bled by cardiac puncture under anesthesia (isoflurane), and the serum samples are stored at −20° C. until required for antibody analysis. The anesthetized animals are euthanized with overdosage of sodium pentobarbital at the end of experiment. Statistical analysis of the data are performed using ANOVA.
Results presented in Table 11 demonstrate Th2 cytokine selectivity of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid in vivo. Mice are immunized with a hapten carrier conjugate, DNP-KLH, and anti-DNP antibody levels are determined in a isotype specific manner as described above. Treatment with 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid caused a marked reduction of IgG1 anti-DNP antibody levels (partially Th2, IL-4-dependent) but had no significant effect on IgG2a antibody level (Th1, IFN-γ dependent). CsA used as control, at 10 mg/kg in this study, completely inhibited both IgG2a and IgG1 antibodies.





TABLE 11



Differential effect of 3-(4-pyridinylamino)-1H-indole-2-carboxylic

acid (Example 28) on different isotypes of anti-hapten antibodies






IgG2a Anti-DNP Antibody
IgGl Anti-DNP Antibody


Titer
Titer







Animal #
Vehicle
Example 28
Vehicle
Example 28









1
31.40
30.59
123.14
72.42

2
30.64
38.60
149.30
185.22

3
38.46
14.09
205.93
19.83

4
24.21
17.45
236.97
40.19

5
27.64
30.21
158.70
83.11

6
—
27.98
132.12
37.83

Mean +/− SE
30.47 +/−
26.49 +/−
167.69 +/−
73.10 +/−


2.37
3.72
18.19
24.38

Inhibition
—
13%
—
56.5%






































Sheep Asthma Model:
The following in vivo model demonstrates that a compound capable of modulating a Th2 cytokine, specifically IL-4, is also capable of improving pulmonary function and reducing pulmonary resistance in vivo. The following pulmonary function studies are performed in sheep allergic to Ascaris suum , as previously described by Abraham (Abraham, W. M., “Sheep Models of Allergic Bronchoconstriction” in “Allergy and Allergic Diseases,” Kay, A. B., Ed. Blackwell Science—Oxford, (1997), 1045-1055). The sheep used in this study had previously demonstrated early and late airway responses and airway hyperresponsiveness to carbachol following inhalation challenge with Ascaris suum extract. Briefly, transpulmonary pressure, pulmonary resistance (R L ), specific lung resistance (SR L airway hyperreactivity, airway responsiveness, are determined.
Methods
Animal Preparation: All animals demonstrated both early and late airway responses to inhalation challenge with Ascaris suum antigen. Venous blood samples (˜5 ml) are obtained from a peripheral leg vein or the jugular vein for pharmacokinetic data. Measurement of Airway Mechanics: The unsedated sheep are restrained in a cart in the prone position with their heads immobilized. After topical anesthesia of the nasal passages with 2% lidocaine solution, a balloon catheter is advanced through one nostril into the lower esophagus. The animals are incubated with a cuffed endotracheal tube through the other nostril using a flexible fiberoptic bronchoscope as a guide. (The cuff of the endotracheal tube is inflated only for the measurement of airway mechanics and during aerosol challenges to prevent undue discomfort. This procedure has no effect on airway mechanics). Pleural pressure is estimated with the esophageal balloon catheter (filled with one ml of air) which is positioned 5-10 cm from the gastroesophageal junction. In this position the end expiratory pleural pressure ranges between −2 and −5 cm H 2 0. Once the balloon is placed, it is secured so that it remains in position for the duration of the experiment. Lateral pressure in the trachea is measured with a sidehole catheter (inner dimension, 2.5 mm) advanced through and positioned distal to the tip of the endotracheal tube. Transpulmonary pressure, the difference between tracheal and pleural pressure, is measured with a differential pressure transducer catheter system. For the measurement of pulmonary resistance (R L ), the proximal end of the endotracheal tube is connected to a pneumotachograph (Fleisch, Dyna Sciences, Blue Bell, Pa.). The signals of flow and transpulmonary pressure is recorded on an oscilloscope recorder which is linked to a computer for on-line calculation of R L from transpulmonary pressure, respiratory volume (obtained by digital integration) and flow. Analysis of 5-10 breaths is used for the determination of R L . Immediately after the measurement of R L , thoracic gas volume (V tg ) is measured in a constant volume body plethysmograph to obtain specific lung resistance (SR L =R L ·V tg ) in L×cm H 2 0/LPS.
Aerosol Delivery Systems: Aerosols of Ascaris suum extract (diluted 20:1 with phosphate buffered saline; 82,000 PNU/ml) are generated using a disposable medical nebulizer (Raindrop®, Puritan Bennett), which produces an aerosol with a mass median aerodynamic diameter of 3.2 μm (geometric standard deviation, 1.9) as determined by a 7 stage Andersen cascade impactor. The output from the nebulizer is directed into a plastic t-piece, one end of which is connected to the inspiratory port of a Harvard respirator. To better control aerosol delivery, a dosimeter consisting of a solenoid valve and a source of compressed air (20 psi) is activated at the beginning of the inspiratory cycle of the Harvard respirator system for 1 s. The aerosol is delivered at a tidal volume of 500 ml and a rate of 20 breaths per minute for 20 minutes. Each sheep is challenged with an equivalent dose of antigen (400 breaths) in the control and drug trial. Carbachol aerosols are also generated with the nebulizer system described above.
Dose Response Curves to Inhaled Carbachol: For the carbachol dose response curves, measurements of SR L is repeated immediately after inhalation of buffer and after each administration of 10 breaths of increasing concentrations of carbachol solution (0.25%, 0.5%, 1.0%, 2.0% and 4.0% w/v). To assess airway responsiveness, the cumulative carbachol dose in breath units (BU) that increases SR L 400% over the post-buffer value (i.e. PC 400 ) is calculated from the dose response curve. One breath unit is defined as one breath of a 1% w/v carbachol solution.
Experimental Protocol
Baseline dose response curves to aerosol carbachol are obtained 1-3 days prior to antigen challenge. On occasions at least 2 weeks apart baseline values of specific lung resistance (SR L ) are obtained and then the sheep are administered saline (control) or drug (at doses of 3 mg of test compound, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28), in NaOH/saline) 30 min before antigen challenge. For these studies the compound, 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28), is administered by aerosol. Then the sheep are challenged with Ascaris suum antigen. Measurements of SR L are obtained immediately after challenge, hourly from 1-6 h after challenge and on the half-hour from 6½-8 h after challenge. Measurements of SR L are obtained 24 h after antigen challenge followed by the 24 h post challenge carbachol dose response curve. For the initial studies, drug trials are compared to the animal's historical control data.
For the evaluation of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) baseline values of SR L are obtained and then the sheep are administered either vehicle or drug (3 mg/kg) as an aerosol in PEG400 vehicle. SR L is remeasured and 30 minutes after dosing animals are challenged by aerosol with Ascaris suum antigen. Measurements of SR L are obtained immediately after challenge, hourly from 1 to 6 hours after challenge, and on the half-hour from 6.5 to 8 hours after challenge.
The results presented in FIG. 3 represent the mean ±sem from 2 sheep per group. FIG. 3A depicts the effect of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) on antigen-induced early and late bronchial responses. 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) (3 mg/kg) is given 30 min before antigen challenge as an aerosol. It did not affect the acute increase in specific lung resistance (SR L ) but blocked the late response compared to control.
FIG. 3B depicts the effect of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) (3 mg/kg) on post challenge airway responsiveness. In the control trial, PC 400 (the amount of carbachol that causes a 400% increase in SR L ) decreased after antigen challenge (i.e., the sheep became hyperresponsive). Treatment with 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (Example 28) prevented this effect. All values are mean ±SE for 2 sheep. Responses are compared to historical control values (control) and vehicle controls.
This invention is further illustrated by the following examples of compounds used herein which are provided for illustration purposes and in no way limit the scope of the present invention.
EXAMPLES
General
General Analytical Techniques Used for the Characterization: A variety of analytical techniques were used to characterize the compounds used in the method of this invention, which included the following:
The phrase “concentrated in vacuo or rotary evaporated” indicates rotary evaporation using a Buchi apparatus at 20-60° C. and 15-30 torr using a KNF Neuberger diaphragm pump. Room temperature is abbreviated as “rt”.
Preparative reversed phase HPLC was carried out on a Rainin SD1 unit using a Dynamax C 18 column (60 A spherical 13 μm particles). The mobile phase consisted of acetonitrile/buffer mixtures, with buffer composed of distilled water, acetonitrile, and trifluoroacetic acid (TFA) in the ratio listed in the experimental procedures.
1 H NMR spectra were recorded on a Varian Gemini 300, Unity 300, Unity 400, or Unity 500 spectrometers with chemical shifts (δ) reported in ppm relative to tetramethylsilane (0.00 ppm) or chloroform (7.26 ppm) as a reference. Signals were designated as s (singlet), d (doublet), t (triplet), q (quartet), p (pentuplet), m (multiplet), br (broad).
Chromatographic (flash) purifications on silica gel were done using pre-packed Isco or Biotage cartridges (32-63 μm, 60 A).
Mass spectra (MS) were obtained on a Finnigan MAT Model TSQ 700 Mass Spectrometer System by chemical ionization at 120 eV using methane (CI, 120 eV). The protonated molecular ion designated as (M + +1) is given in parentheses.
Liquid chromatography with mass spectral analysis (LC/MS): HPLC: column: 50×4.6 mm, Hypersil BDS C18 3u. Mobile Phase: A=water with 0.05% trifluoroacetic acid, B=acetonitrile with 0.05% trifluoroacetic acid, flow rate=1.0 ml/min, gradient=5% B to 100% B in 3 min, stay 100% for 2 min. Mass Spectrometry: Lct API LC/Orthogonal Time of Flight Mass Spectrometer and Masslynx Data System from Micromass. Ionization mode=electrospray (ESI), Source temperature=120° C., Desolvation temperature=250° C., Cone voltage=25 volt, Acquisition mass range m/z from 145 to 1000. Values were determined for the protonated molecular ions (M + +1).
Example 1









Scheme A, Step A1: 3-Amino-6-trifluoromethyl-1H-indole-2-carboxylic acid, ethyl ester
Under nitrogen, stir and heat a mixture of 2-fluoro-4-(trifluoromethyl)benzonitrile (1.0 g, 5.29 mmol), ethyl glycinate hydrochloride (886 mg, 6.3 mmol), potassium carbonate (1.46 g, 6.3 mmol) and 1-methyl-2-pyrrolidinone (20 mL) at 115-120° C. After six hours add potassium tert-butoxide (700 mg, 6.2 mmol), and stir at ambient temperature for 2 h. Quench into ice/water, extract the aqueous mixture with ether, wash the extract with water and dry it with magnesium sulfate. Filter and concentrate under vacuum to yield the crude product. Chromatograph the crude product on silica gel (10 g, Sepack cartridge) with dichloromethane as eluent to afford 332 mg (23%) of the title compound. Identical on TLC (silica gel, dichloromethane) to the compound disclosed in U.S. Pat. No. 5,189,054.
Example 2









Scheme A, Step A1: 3-Amino-6-fluoro-1H-indole-2-carboxylic acid, ethyl ester
Under nitrogen, stir and heat a mixture of 2,4-difluorobenzonitrile (1.14 g, 8.2 mmol), ethyl glycinate hydrochloride (1.5 mg, 10.7 mmol), potassium carbonate (3.4 g, 24.6 mmol) and 1-methyl-2-pyrrolidinone (20 mL). After 2 h allow the reaction to cool to 40° C. and add potassium tert-butoxide (300 mg, 2.7 mmol). Allow the reaction to stir and for 1 h and then add additional potassium tert-butoxide (500 mg, 4.4 mmol). Cool to room temperature overnight, pour the reaction mixture onto ice. Add water to dissolve the solids in the reaction flask and add to the reaction quench. Filter off the precipitated brown solid and extract the filtrate with ethyl acetate. Wash the extract with water (2×'s), brine and dry over magnesium sulfate. Filter and concentrate under vacuum to give a residue, which is triturated with dichloromethane to afford a solid. Collect the solid, concentrate the filtrate and chromatograph the solid on silica gel (10 g, Sepack cartridge) with dichloromethane as the eluent. Collect the appropriate fractions and concentrate to obtain a solid. Combine with the previously collected solid to provide 136.7 mg (7.5%) of the title compound: MS 223 (M+H).
Example 3









Scheme B: 3-Amino-5-chloro-1H-indole-2-carboxylic acid, ethyl ester
Step B1: 2-Amino-N-tert-butyl-5-chlorobenzamide
Stir and cool to 0° C. a solution of 2-amino-5-chlorobenzoic acid (1.21 g, 10 mmol) and N-hydroxysuccinimide (1.38 g, 12 mmol) in dichloromethane (12 mL). Add, dropwise, a 1M solution of dicyclohexylcarbodiimide in dichloromethane (12 mL, 12 mmol), and stir at 0° C. for 1 h. Add tert-butyl amine (1.74 g, 23.79 mmol) by syringe and stir at ambient temperature overnight. Filter the reaction mixture and wash the filtrate with aqueous sodium bicarbonate. Dry the organic phase with MgSO 4 and chromatograph over silica gel eluting with 4% ethyl acetate/dichloromethane to afford 1.74 g of the title compound: MS 227 (M+H), m.p. 126-127° C.
Step B2: N-(4-Chloro-2-cyanophenyl)-2,2,2-trifluoroacetamide
Stir and cool to 0° C. a solution 2-amino-N-tert-butyl-5-chlorobenzamide (5.07 g, 22.36 mmol) in dichloromethane (200 mL), and add, dropwise trifluoroacetic anhydride. Stir at ambient temperature overnight and concentrate under reduced pressure. Partition between chloroform and aqueous sodium bicarbonate, and collect the organic layer. Dry the extract with MgSO 4 , filter and evaporate the solvent. Triturate the residue with heptane and collect the white solid to obtain 4.80 g (82%) of the title compound: MS 249 (M+H), m.p. 105-107° C.
Step B3: ((4-Chloro-2-cyanophenyl)-(2,2,2-trifluoro-ethanoyl)amino)-acetic acid ethyl ester
Under nitrogen, stir and cool to 0° C. a solution of N-(4-chloro-2-cyano-phenyl)-2,2,2-trifluoroacetamide (4.80 g, 19.31 mmol) in dimethylformamide (50 mL), and add NaH (0.7 g, 29.1 mmol). Stir the reaction for 1 h at ambient temperature and add ethyl bromoacetate (4.3 mL, 38.77 mmol) by means of a syringe. Stir at 50° C. overnight and then pour the reaction into aqueous ammonium chloride-ethyl acetate. Wash the organic layer with brine (2×'s), dry over MgSO 4 , filter and evaporate to obtain an oil. Chromatograph the oil eluting with 20% ethyl acetate/heptane and then 30% ethyl acetate/heptane. Concentrate the appropriate fractions and obtain 6.0 g of a solid MS 335 (M+H), m.p. 71-73° C.
Step B4: 3-Amino-5-chloro-1-(2,22-trifluoro-ethanoyl)-1H-indole-2-carboxylic acid ethyl ester
Under nitrogen, stir and cool to 0° C. a solution [(4-chloro-2-cyanophenyl)-(2,2,2-trifluoro-ethanoyl)amino]-acetic acid ethyl ester (93.34 g, 10 mmol) in tetrahydrofuran (20 mL) and add, dropwise, a 1M solution of potassium tert-butoxide in tetrahydrofuran (12 mL, 12 mmol). Stir the reaction at ambient temperature for 2 h and then partition between aqueous ammonium chloride/ethyl acetate. Separate the organic layer, dry over MgSO 4 , filter and concentrate to a solid. Triturate the solid with heptane, filter the solid and air dry to obtain 2.86 g (86%) of the title compound: MS 335 (M+H), m.p. 249-251° C.
Step B5: 3-Amino-5-chloro-1H-indole-2-carboxylic acid, ethyl ester
Heat at 70° C. and stir a mixture of 3-amino-5-chloro-1-(2,2,2-trifluoro-ethanoyl)-1H-indole-2-carboxylic acid ethyl ester (3.34 g, 10 mmol), ethanol (50 mL), and potassium carbonate (1.50 g, 10.85 mmol). After 1.75 h, add water (20 mL), and after another hour add additional potassium carbonate (0.58 g, 4.20 mmol). Cool the reaction mixture to ambient temperature and partition between water/ethyl acetate. Separate the organic layer, dry over MgSO 4 , filter and concentrate to afford 1.70 g of the title compound as a solid. MS 239 (M+H).
Example 4









Scheme B: 3-Amino-5-fluoro-1H-indole-2-carboxylic acid, ethyl ester
Step B1: 2-Amino-N-tert-butyl-5-fluorobenzamide
Stir and cool to 0° C. a solution of 2-amino-5-fluorobenzoic acid (7.75 g, 50 mmol), N-hydroxysuccinimide (6.9 g, 60 mmol) and dimethylaminopyridine (1.0 g) in dichloromethane (200 mL). Add, dropwise, a 1M solution of dicyclohexylcarbodiimide in dichloromethane (60 mL, 60 mmol), and stir at 0° C. for 1 h. Add tert-butyl amine (20 mL, 190.3 mmol) by syringe and stir at 0° C. for 3 h and then at ambient temperature overnight. Pour onto a pad of silica gel and elute with 5% ethyl acetate/dichloromethane. Combine like fractions and concentrate to obtain a crude solid. Triturate the solid with heptane and filter to obtain 7.7 g (73%) of the title compound: MS 211 (M+H), m.p. 77-78° C.
Steps B2-B5: 3-Amino-5-fluoro-1H-indole-2-carboxylic acid, ethyl ester
Follow the procedure of Example 3 (Steps B2-B5) to obtain 3-amino-5-fluoro-1H-indole-2-carboxylic acid, ethyl ester: MS 223 (M+H), TLC (silica gel, 4% ethyl acetate/dichloromethane) R f =0.50.
Example 5









Scheme B, Steps B3-B5: 3-Amino-1H-indole-2-carboxylic acid, tert-butyl ester
Follow the procedure of Example 3 (Steps B3-B5) but start with N-(2-cyano-phenyl)-2,2,2-trifluoroacetamide (synthesis described in J. Fluorine Chem. 1981, 18, (2), 185-95) and tert-butyl bromoacetate to obtain the title compound: MS 310 (M+H), TLC (silica gel, dichloromethane/methanol, 7:1) R f =0.38.
Example 6









Scheme B, Steps B1-B5: 3-Amino-5-methoxy-1H-indole-2-carboxylic acid, tert-butyl ester
Follow the procedure of Example 3 but start with 2-amino-5-methoxy benzoic acid, and in Step B3 use tert-butyl bromoacetate instead of ethyl bromoacetate to obtain the title compound: MS 263 (M+H), m.p. 146-147° C.
Example 7









Scheme B, Steps B3-B5: 3-Amino-4-fluoro-1H-indole-2-carboxylic acid, tert-butyl ester
Follow the procedure of Example 5, but start with 3-fluoro-N-(2-cyano-phenyl)-2,2,2-trifluoroacetamide (synthesis described in J. Fluorine Chem. 1981, 18, (2), 185-95) and tert-butyl bromoacetate to obtain the title compound: MS 251 (M+H), TLC (silica gel, dichloromethane/methanol, 7:1) R f =0.38.
Example 8









Scheme C: 3-Amino-6-phenyl-1H-indole-2-carboxylic acid, ethyl ester
Step C1: N-(5-Chloro-2-cyanophenyl)acetamide
Heat on a steam bath a mixture of 2-amino-4-chlorobenzonitrile (20.0 g, 131 mmol) and acetic anhydride (80 mL) for 40 min. Add water (300 mL) and stir for 1 h. Filter the resulting solid, wash it with water and dry it under vacuum at 40° C. to obtain 22.9 g of product. Recrystallize a 12.8 g sample from ethanol-ethyl acetate and obtain 9.83 g of the title compound: MS 195 (M+H), TLC (silica gel, ethyl acetate/heptane1:1) R f =0.38.
Step C2: N-(2-cyanophenyl-5-phenyl)acetamide
Stir at ambient temperature a mixture of N-(5-Chloro-2-cyanophenyl)-acetamide (0.389 g, 2.0 mmol), phenylboronic acid (0.366 g, 3.0 mmol), palladium (II) acetate (8.8 mg), 2-(dicyclohexylphosphino)biphenyl (28.0 mg), potassium fluoride (0.348 g, 5.99 mmol) and tetrahydrofuran (5 mL) overnight. Pour the reaction mixture into an aqueous solution of potassium carbonate (30 mL) and extract with ethyl acetate (2×25 mL). Combine the extracts and wash with water and brine, dry over MgSO 4 and concentrate under vacuum to obtain a tan solid. Triturate the solid with ether and collect 0.365 g (77%) of the title compound as an off-white powder: MS 237 (M+H), m.p. 168-170° C.
Step C3: 3-Amino-1-(ethanoyl)-6-phenyl-1H-indole-2-carboxylic acid ethyl ester
Cool to 4° C. a solution of N-(2cyanophenyl-5-phenyl)acetamide (15.0 g, 63.5 mmol), in 1-methyl-2-pyrrolidinone (150 mL) and add dropwise, a 1M solution of potassium tert-butoxide in tetrahydrofuran (77.0 mL, 77.0 mmol). Age the reaction at 0° C. for 30 min and then add ethyl bromoacetate (12.0 g, 72.1 mmol). Stir at room temperature overnight and quench the reaction into water (600 mL). Extract the aqueous mixture with ether (3×300 mL), combine the extracts, wash the extract with water and brine and dry over MgSO 4 . Filter and concentrate the extracts under vacuum to obtain 20.6 g of a dark oil. Chromatograph on a Waters Prep Pak silica gel cartridge (500 g) and elute with a step gradient of 5%, 10% and 20% respectively of ethyl acetate/dichloromethane. Concentrate fractions containing starting material and obtain a yellow solid. Triturate the solid with ether and filter to afford 2.61 g of a white solid.
Combine the filtrate from above with fractions from the chromatography that contain product and concentrate to afford 16.2 g of a dark oil. Chromatograph the oil on a Waters Prep Pak silica gel cartridge and elute with a step gradient of 2%, 5% and 10% respectively of ethyl acetate/dichloromethane. Combine like fractions and concentrate to obtain 7.33 g of the title compound: MS 323 (M+H), TLC (silica gel, 20% ethyl acetate/dichloromethane) R f =0.67.
Step C4: 3-Amino-6-phenyl-1H-indole-2-carboxylic acid, ethyl ester
Reflux a mixture of 3-amino-1-(ethanoyl)-6-phenyl-1H-indole-2-carboxylic acid ethyl ester (7.3 g, 22.6 mmol) 20% aqueous potassium carbonate (100 mL) and ethanol (100 mL) for 1.75 h. Let the reaction stand at ambient temperature overnight and dilute with water (500 mL). Cool at 0° C. and collect a purple solid. Wash the solid with water and dry at 40° C. under vacuum and obtain 5.7 g of a purple solid. Triturate the solid with dichloromethane (40 mL) filter and wash the solid with dichloromethane and ethyl acetate, and then dry at 40° C. under vacuum to obtain 2.5 g of the title compound as a solid: ESI/MS 281 (M+H), HPLC: R t =1.83 min., m.p. 181-183° C.
Example 9









Scheme D: 3-Amino-5,6-dimethoxy-1H-indole-2-carboxylic acid, ethyl ester
Step D1: (2-Cyano-4,5-dimethoxy-phenylamino)-acetic acid ethyl ester
Under N 2 suspend 2-amino-4,5-dimethoxybenzonitrile (2.0 g, 12.25 mmol) in ethanol (50 mL), and add sodium bicarbonate (3.09 g, 36.78 mmol) in one portion. By syringe, slowly add ethyl bromoacetate (2.72 mL, 24.5 mmol), and then sodium iodide (10 mg). Heat the reaction at 65° C. and then cool to ambient temperature. Filter the resulting solid and wash with ethyl acetate to dissolve the organics. Concentrate the filtrate to obtain 0.66 g (23%) of the title compound; MS 265 (M+H).
Step D2: 3-Amino-5,6-dimethoxy-1H-indole-2-carboxylic acid, ethyl ester
Stir under N 2 a solution of (2-cyano-4,5-dimethoxy-phenylamino)-acetic acid ethyl ester (0.124 g, 0.47 mmol) in tetrahydrofuran (6 mL) and add, dropwise, 1M potassium tert-butoxide in tetrahydrofuran (0.66 mL, 0.66 mmol). Stir for 1 h at ambient temperature and pour into ice/water. Extract with ethyl acetate, dry the extract over MgSO 4 , filter and concentrate under vacuum to a solid. Chromatograph on a Sep Pak silica gel cartridge (2 g) eluting with dichloromethane and then 2:1 heptane-ethyl acetate. Combine like fractions and concentrate to provide 61.9 mg (49%) of the title compound as a solid: MS 265 (M+H).
Example 10









Scheme D: 3-Amino-6-chloro-1H-indole-2-carboxylic acid, ethyl ester
Step D4: 3-Amino-1-(ethanoyl)-6-chloro-1H-indole-2-carboxylic acid ethyl ester
Stir and cool to 0° C. a mixture of N-(5-chloro-2-cyanophenyl)acetamide (see Example 8, 1.0 g, 5.14 mmol) and THF (10 mL) and add potassium tert-butoxide (6.2 mL, of a 1M solution in THF) and stir at 0° C. for 30 min. Add bromoethyl acetate (0.6 mL) in one portion. Stir at room temperature for 2 h. Quench the reaction with water (40 mL) and stir with ethyl acetate. Separate the layers and reextract the aqueous with ethyl acetate. Combine the organic extracts and wash with water and brine. Dry over MgSO 4 and concentrate under reduced pressure to obtain a brown solid. Triturate the solid with ether and collect a beige solid. Chromatograph the solid on silica gel with 2% ethyl acetate/dichloromethane followed by 10% ethyl acetate/dichloromethane to obtain 0.65 g (45%) of the title compound as a beige solid: MS 281 (M+H), HPLC: R t =3.20 min., TLC (silica gel, 10% ethyl acetate/dichloromethane) R f =0.53.
Step D5: 3-Amino-6-chloro-1H-indole-2-carboxylic acid, ethyl ester
Add 1N ethanolic potassium hydroxide solution (100 mL) to 3-amino-1-(ethanoyl)-6-chloro-1H-indole-2-carboxylic acid ethyl ester (3.5 g, 12.5 mmol) and permit the reaction to stand for 20 min. Pour the resulting slurry into water at 5° C. with rapid stirring. Age at 0° C. for 10 min., collect the solid, wash it with water dry it at 40° C. under vacuum and obtain 2.42 g (81%) of the title compound: MS 239 (M+H).
Examples 11-21
Scheme E, Step E1: Table 12 illustrates 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid esters of the present invention, which can be synthesized similar to the method disclosed in U.S. Pat. No. 5,328,920. Accordingly, reaction of the appropriate 3-amino-indole-2-carboxylate with 4-chloropyridine hydrochloride in 1-methyl-2-pyrrolidinone produces the desired compounds. Changes of reaction times and temperatures from the referenced procedure are noted in the table. Table 13 lists the corresponding physical properties.





TABLE 12



Reaction Conditions




























Temp.
Time

Example
X
Y
Z
R
R 2
° C.
hours












11
H
H
NH
H
C 2 H 5
165 c
6

12
6-CF 3
H
NH
H
C 2 H 5
80
18

13
6-Cl
H
NH
H
C 2 H 5
100
5

14
5-Cl
H
NH
H
C 2 H 5
100
4.5

15
6-F
H
NH
H
C 2 H 5
80
d

16
5-F
H
NH
H
C 2 H 5
100
2

17
4-F
H
NH
H
C(CH 3 ) 3
75
16

18
6-
H
NH
H
C 2 H 5
100
d


phenyl

19
H
H
NH
H
C(CH 3 ) 3
100
2

20
5-
6-
NH
H
C 2 H 5
80
6


OCH 3
OCH 3

21
5-
H
NH
H
C(CH 3 ) 3
80
3.5


OCH 3



d = overnight about 16-20 h












































TABLE 13



Physical Properties










Retention





Time



MS
Minutes


Example
(M + H)
(HPLC)
mp° C.





11
282

248-250


12
350


13
316
1.90
258-261(dec)


14
316


15
300


16
300


17
328
1.58
210-212


18
358

241-243


19
310


20
342
1.20


21
340
1.13
217-219



































Example 22









Scheme E, Step E1: 3-(2-Pyridinylamino)-1H-indole-2-carboxylic acid, ethyl ester
Heat a mixture of ethyl 3-aminoindole-2-carboxylate (250 mg, 1.2 mmol) and 2-bromopyridine (1.0 mL, 10 mmol) at 150° C. for 1 h. Cool the reaction and partition between aqueous ammonium chloride and ethyl acetate. Separate the organic layer and concentrate to afford a crude mixture. Flash chromatograph the mixture on silica gel eluting with 25% ethyl acetate/dichloromethane to afford 50 mg of the title compound: MS 282 (M+H).
Example 23









Scheme E, Step E1: 3-(Pyrimidin-2-ylamino)-1H-indole-2-carboxylic acid ethyl ester
Heat a mixture of ethyl 3-aminoindole-2-carboxylate (204 mg, 1.0 mmol) and 2-chloropyrimidine (1.2 g, 10.5 mmol) at 100° C. for 16 h. Cool the reaction and add aqueous ammonium chloride and ethyl acetate. Filter off excess 2-chloropyrimidine, separate the organic layer, dry with MgSO 4 , filter and concentrate to afford a crude mixture. Flash chromatograph the mixture on silica gel eluting with 25% ethyl acetate/dichloromethane to afford 50 mg of the title compound: MS 283 (M+H), TLC (silica gel, 25% ethyl acetate/dichloromethane) R f =0.44.
Example 24









Scheme F, Step F8: 5-Fluoro-1-Methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic ethyl ester
Stir at 0° C., a solution of 5-fluoro-3-(4-pyridinylamino)-1H-indole-2-carboxylic ethyl ester (0.195 mg, 0.65 mmol) in dimethylformamide (3.0 mL) and add a solution of 1M potassium tert-butoxide in tetrahydrofuran (0.8 mL, 0.8 mmol). Stir for 0.5 to 1 h at 0° C. and add iodomethane (0.05 mL, 0.78 mmol). After 1 h, add saturated aqueous ammonium chloride solution (5 mL) and ethyl acetate (10 mL). Separate the layers and reextract with ethyl acetate. Combine the organic layers and wash with water (3×'s), brine and dry with MgSO 4 . Concentrate the extract to obtain 60 mg (30%) of the title compound as a brown solid: ESI/MS 314 (M+H); HPLC: R t =1.50 min.
Example 24A









Scheme F, Step F8: 1-Methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic ethyl ester
The procedure of Example 24 is essentially repeated in this example except that the starting material used is 3-(4-pyridinylamino)-1H-indole-2-carboxylic ethyl ester. This compound is also described in the U.S. Pat. No. 5,328,920. The maleate salt of this compound exhibits a m.p. of 169°-170° C. (dec.).
Example 25









Scheme F, Step F8: 4-Fluoro-1-methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic tert-butyl ester
Follow the procedure of Example 24 but start with 4-fluoro-3-(4-pyridinylamino)-1H-indole-2-carboxylic tert-butyl ester, but substitute tetrahydrofuran for dimethylformamide to obtain the title compound: ESI/MS 342 (M+H); HPLC: R t =1.41 min.
Example 26









Scheme F, Step F8: 5-Chloro-1-methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic ethyl ester
Follow the procedure of Example 24 starting with 5-chloro-3-(4-pyridinylamino)-1H-indole-2-carboxylic ethyl ester and obtain the title compound: ESI/MS 330 (M+H); HPLC: R t =1.58 min.
Example 27









Scheme F, Step F8: 6-Chloro-1-methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic ethyl ester
Follow the procedure of Example 24 starting with 6-chloro-3-(4-pyridinylamino)-1H-indole-2-carboxylic ethyl ester and obtain the title compound: ESI/MS 330 (M+H); HPLC: R t =1.64 min, m.p. 154-155° C.
Example 28









Scheme G: 3-(4-Pyridinylamino)-1H-indole-2-carboxylic acid
Step G1: (2-Cyanophenylamino)acetic acid ethyl ester
Charge a mixture of 10.0 kg (84.6 mol) of anthranilonitrile, 21.2 kg (127.0 mol, 1.5 equiv.) of ethyl bromoacetate, 12.0 kg (143.0 mol, 1.7 equiv.) of NaHCO 3 , 10.0 g (catalytic) of sodium iodide and 33.0 L of ethanol to a 30-gal reactor. Heat the reaction mixture to and hold at 78-80° C. under nitrogen. Monitor the progress of the reaction by HPLC (Column—150×3.9 mm Waters Symmetry C18, 5 micron; mobile phase—60% acetonitrile/40% 0.1% TFA in water; flow rate—1.0 mL/min; wavelength—225 nm; R T : anthranilonitrile—1.8 min (2-cyanophenylamino)acetic acid ethyl ester—2.7 min). Typically achieve, a 70-72% conversion in about 40-44 h. Allow the mixture to cool to 60° C. and filter warm to remove the inorganic solids. Wash the filter cake with 10.0 L of warm (70° C.) ethanol. Cool the filtrate to and hold at −20° C. for 4 h. Filter off the solid that formed and wash with 6.0 L of cold (−20° C.) ethanol and air dry to give 11.02 kg, 64.0% yield of the title compound: HPLC analysis 100% pure.
Step G2: 3-Amino-1H-indole-2-carboxylic acid ethyl ester
Charge a total of 30.0 L of tetrahydrofuran to a 30-gal reactor under nitrogen. Add a total of 4.0 kg (32.72 mol, 1.045 equiv.) of potassium tert-butoxide. Observe an exotherm to 25° C. from 20° C. Cool the mixture to 20° C. Dissolve a total of 6.40 kg (31.3 mol) of (2-cyanophenylamino)acetic acid ethyl ester in 30.0 L of tetrahydrofuran and add over 5 h at 20-25° C. Stir the reaction mixture at 20-23° C. for 18 h. Monitor the progress of the reaction by HPLC (Column—150×3.9 mm Waters Symmetry C18, 5 micron; mobile phase—60% acetonitrile/40% 0.1% TFA in water; flow rate—1.0 mL/min; wavelength—225 nm; R T : 3-amino-1H-indole-2-carboxylic acid ethyl ester—1.5 min; (2-cyanophenylamino)acetic acid ethyl ester—2.7 min). Typically achieve, a 97% conversion. Concentrate the mixture at 30° C./50 torr to a solid residue. Add a total of 60.0 L of water to the residue and stir the mixture for 2 h at 23° C. Filter off the solid and wash with 60.0 L of water and air dry. Further dry the solid in a forced air oven at 55° C. for 24 h to give 4.45 kg, 69.5% yield, of the title compound: HPLC analysis 79.2% pure.
Step G3: 3-(4-Pyridinylamino)-11H-indole-2-carboxylic acid ethyl ester
Charge a total of 4.0 kg (19.56 mol) of 3-amino-1H-indole-2-carboxylic acid ethyl ester, 4.10 kg (27.33 mol, 1.4 equiv.) of 4-chloropyridine hydrochloride and 8.0 L of 1-methyl-2-pyrrolidinone to a 30-gal reactor and heat to 100° C. under nitrogen. Monitor the progress of the reaction by HPLC (Column—150×3.9 mm Waters Symmetry C18, 5 micron; mobile phase—60% acetonitrile/40% 0.1% TFA in water; flow rate—1.0 mL/min; wavelength—225 nm; R T : 3-amino-1H-indole-2-carboxylic acid ethyl ester—1.5 min; 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid ethyl ester—6.0 min; 4-chloropyridine—1.1 min). Typically achieve, a 99% conversion within 1 h. Allow the mixture to cool to 20° C. and add a total of 55.0 L of water. Extract the mixture with a total of 20.0 L of ethyl acetate and separate the layers. Adjust the pH of the aqueous phase was to pH=9.3 using 25% sodium hydroxide solution. Filter off the solid that forms and wash with 45.0 L of water and air dry. Further air dry the solid in a forced air oven at 50° C. for 24 h to give 4.20 kg, 77% yield, of the title compound: HPLC analysis 95.8% pure.
Step G4: 3-(4-Pyridinylamino)-1H-indole-2-carboxylic acid potassium salt
Charge a mixture of 7.50 kg (26.66 mol) of 3-amino-1H-indole-2-carboxylic acid ethyl ester, 37.5 L of ethanol and 37.5 L of 4M KOH to a 30-gal reactor and heat to 75° C. over 30 min. Monitor the progress of the reaction by HPLC (Column—150×3.9 mm Waters Symmetry C18, 5 micron; mobile phase—20% acetonitrile/80% 0.1% TFA in water; flow rate—1.0 ml/min; wavelength—225 nm; R T : 3-amino-1H-indole-2-carboxylic acid potassium salt—3.3 min; 3-amino-1H-indole-2-carboxylic acid ethyl ester—6.0 min). Typically achieve, a 100% conversion within 3 h. Cool the mixture to 25° C. over 2 h and concentrate at 50-60° C./50 torr to a residue. Add a total of 37.5 L of water and heat the mixture to and hold at 85° C. for 30 min. Cool the mixture to 10° C. over 2 hours, and then further cool to and hold at 5° C. for 2 hours. Filter off the solid that forms, wash with a total of 10 L of 4M KOH solution and air dry. Further dry the solid at 35° C. in a forced air oven for 72 h to give 10.9 kg, 140% yield of the title compound: HPLC analysis 70.7% pure.
Step G5: 3-(4-Pyridinylamino)-1H-indole-2-carboxylic acid
Charge a total of 10.9 kg (37.4 mol, 140% yield for ester hydrolysis) of 3-amino-1H-indole-2-carboxylic acid potassium salt and 75.0 L of water to a 30-gal glass lined reactor. Extract the stirring mixture with 75.0 L (3×25.0 L) of ethyl acetate. Adjust the pH of the aqueous phase to pH=6.0 by the addition of 7.0 L of 6N HCl at 20-22° C. and monitor the purity of the product by HPLC (Column—150×3.9 mm Waters Symmetry C18, 5 micron; mobile phase—20% acetonitrile/80% 0.1% TFA in water; flow rate—1.0 mL/min; wavelength—225 nm; R T : 3-amino-1H-indole-2-carboxylic acid—3.3 min). Stir the mixture for 45 min and the measure pH to be pH=6.0. Filter off the solid that forms, wash with a total of 50 L of water and air dry. Further dry the solid at 35° C. in a forced air oven for 24 hours to give 6.15 kg, 65.5% yield of 3-amino-1H-indole-2-carboxylic acid. HPLC analysis showed the compound to be 93.8% pure. Charge a total of 6.15 kg (24.28 mol) of the product, 11.3 kg (97.35 mol, 4.0 equiv.) of maleic acid, 62.0 L of methanol and 6.2 L of water was to a 30-gal reactor and heat to and hold at 70° C. for 45 min under nitrogen. Cool the mixture to 30° C. over 1.5 hours and concentrate at 60° C./50 torr to a wet solid. Add a total of 20.0 L of IPA and concentrate the mixture at 60° C./50 torr to a solid. Repeat this process. Add a total of 50.0 L of IPA to the residue and heat the mixture to and hold at 70° C. for 60 min, then cool to 20° C. over 16 h. Cool the mixture and hold at 2° C. for 1 hour. Monitor the purity of the product by HPLC (Column—150×3.9 mm Waters Symmetry C18, 5 micron; mobile phase—20% acetonitrile/80% 0.1% TFA in water; flow rate—1.0 mL/min; wavelength—225 nm; R T : 3-amino-1H-indole-2-carboxylic acid—3.3 min; maleic acid—1.2 min). Filter off the solid, wash with 10.0 L of cold (5° C.) EPA and dry at 35° C. in a forced air oven to give 5.4 kg, 60.6% yield of 3-amino-1H-indole-2-carboxylic acid maleate salt: HPLC analysis 99.0% pure.
Charge a total of 7.9 kg (5.4 kg+2.5 kg from two runs) of 3-amino-1H-indole-2-carboxylic acid maleate salt and 50.0 L of 4M KOH to a 30-gal reactor and heat to and hold at 70° C. Add a total of 12.0 L of ethanol until the mixture becomes homogenous at 70° C. Cool the mixture to 20° C. over 2 h. Further cool the mixture to and hold at 4° C. for 1 hour. Monitor the purity of the product by HPLC (Column—150×3.9 mm Waters Symmetry C18, 5 micron; mobile phase—20% acetonitrile/80% 0.1% TFA in water; flow rate—1.0 mL/min; wavelength—225 nm; R T : 3-amino-1H-indole-2-carboxylic acid potassium salt—3.3 min). Filter off the solid and wash with 10.0 L of 4M KOH (22.5% solution) and dry under nitrogen to give 9.4 kg, 151% yield of 3-amino-1H-indole-2-carboxylic acid potassium salt HPLC analysis 97.4% pure.
Charge a total of 18.1 kg (9.4 kg+8.7 kg from two runs) 3-amino-1H-indole-2-carboxylic acid potassium salt and 100.0 L of to a 30-gal reactor. Adjust the pH of the mixture to pH=5.95 by the addition of 13.0 L of 6N HCl at 23-27° C. Stir the mixture for 1.5 h at 22-25° C. Filter off the solid that forms and wash with 60.0 L of water. Monitor the purity of the product by HPLC (Column—150×3.9 mm Waters Symmetry C18, 5 micron; mobile phase—20% acetonitrile/80% 0.1% TFA in water; flow rate—1.0 mL/min; wavelength—225 nm; R T : 3-amino-1H-indole-2-carboxylic acid—3.3 min). Dry the solid at 60° C./50 torr to give 10.2 kg, 41.1% yield of the title compound: HPLC analysis 98.4% pure.
Analysis: Calculated for C 14 H 11 N 3 O 2 .1.21H 2 O: % C 61.15%; % H 4.91%; % N 15.28%. Found, % C 60.83%; % H 5.05%; % N 15.37%.
Example 29









Salt formation: 1-Methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid Maleate
Under N 2 , stir a mixture of 1-methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid ethyl ester (preparation described in Example 24A, also see U.S. Pat. No. 5,328,920, 0.5 g, 1.69 mmol) lithium hydroxide hydrate (0.106 g, 2.53 mmol) and water-ethanol, 1:4 at 50° C. for 4 h. Remove a portion of the ethanol and store the reaction mixture at approximately 5° C. overnight. Filter the resulting solid, wash with water, dry at 40° C. at high vacuum to obtain 0.41 g of a white solid. Suspend the solid in methanol and add maleic acid (0.346 g) and let stand for 0.5 h. Filter and concentrate the filtrate to obtain a tan solid. Recrystallize the solid from methanol/ethyl acetate and obtain 0.19 g (29%) of the title compound: MS 224 (—CO 2 , M+H), m.p. 192-194° C. (dec.).
Analysis: Calculated for C 15 H 13 N 3 O 2 .C 4 H 4 O 4 : 59.53% ; C, 4.47%; H, 10.96%; N, Found: 59.47%; C, 4.56%; H, 10.86%; N.
Example 30









Scheme F, Step F3: 4-Fluoro-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid Trifluoroacetate Salt
Stir at ambient temperature for 3 h a solution of 4-fluoro-3-(4-pyridinylamino)-1H-indole-2-carboxylic tert-butyl ester (Example 17, 0.15 g, 0.458 mmol) in 35% trifluoroacetic acid/dichloromethane. Concentrate the reaction under vacuum to obtain a gray solid. Dry the solid at 40° C. under vacuum and then triturate with hot ethyl acetate. Allow to cool to ambient temperature and collect a light gray powder. Dry the powder at 45° C. under vacuum for 2 h and obtain 0.15 g (88%) of the title compound: MS 272 (M+H), m.p. 231-234° C. (dec.)
Example 31









Scheme F, Step F3: 4-Fluoro-1-Methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid Trifluoroacetate Salt
Follow the procedure of Example 30 starting with 4-fluoro-1-methyl-3-(4-pyridinylamino)-1H-indole-2-carboxylic tert-butyl ester (Example 25) to obtain the title compound as off-white powder: MS 286 (M+H), m.p. 197-199° C.
Example 32









Scheme F, Step F3: 5-Methoxy-3-(4-pyridinylamino)-1H-indole-2-carboxylic acid Trifluoroacetate Salt
Follow the procedure of Example 30 starting with 5-methoxy-3-(4-pyridinylamino)-1H-indole-2-carboxylic tert-butyl ester (Example 21) to obtain the title compound as on-white powder: ESI/MS 284 (M+H), m.p. 211-213° C.
Example 33









Scheme F, Step F8: 1-Ethoxycarbonylmethyl-3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid ethyl ester
Under nitrogen, stir and cool to 0° C. a mixture of ethyl 3-(4-pyridinylamino)indole-2-carboxylate (see U.S. Pat. No. 5,328,920; 0.20 g, 0.711 mmol) in dimethylformamide (5 mL). Add a 1M solution of potassium tert-butoxide in tetrahydrofuran (0.75 mL, 0.75 mmol) in one portion, stir for 10 min, and then add ethyl bromoacetate (0.12 g, 0.72 mmol) in one portion. Stir at 0° C. for 50 min and quench the reaction into water (30 mL). Extract the aqueous mixture with ethyl acetate (2×20 mL), combine the extracts, wash with water (25 mL), brine (25 mL) and dry over MgSO 4 . Concentrate under vacuum, chromatograph the residue on a Redisep silica gel cartridge (10 g) eluting with 5% methanol/dichloromethane to 20% methanol/dichloromethane. Combine non-homogeneous fractions concentrate and chromatograph again as above. Combine all homogenous fractions and concentrate to obtain 0.13 g (50%) of the title compound as a white solid: MS 368 (M+H).
Example 34









Scheme F, Step F8: 6-Chloro-1-diethylcarbamoylmethyl-3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid ethyl ester
Under nitrogen, stir and cool to 0° C. a mixture of ethyl 6-chloro-3-(4-pyridinylamino)indole-2-carboxylate (0.50 g, 1.58 mmol) in dimethylformamide (5 mL). Add a 1M solution of potassium tert-butoxide in tetrahydrofuran (1.7 mL, 1.7 mmol) over a 2 min period, and stir for 10 min. Add 2-chlor-N,N-diethylacetamide (0.22 mL, 1.6 mmol) in one portion and stir at ambient temperature overnight. Quench the reaction into water (35 mL), and extract the aqueous mixture with ethyl acetate (2×20 mL). Combine the extracts, wash with water (25 mL), brine (25 mL) and dry over MgSO 4 . Concentrate under vacuum, and chromatograph the residue on a Redisep silica gel cartridge (10 g) eluting with 5% methanol/dichloromethane to 20% methanol/dichloromethane. Combine all homogenous fractions and concentrate to obtain a solid. Recrystallize the solid from ethyl acetate and obtain 0.1 g of the title compound as a white solid: MS 429 (M+H), m.p. 212-214° C.
Example 35









Scheme F, Step F5: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid pentafluorophenyl ester
Stir under N 2 a suspension of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (0.2 g, 0.78 mmol) in dimethylformamide, add pyridine (0.13 mL, 1.6 mmol) and then add dropwise pentafluorophenyl trifluoroacetate (0.31 g, 1.13 mmol). Stir the reaction overnight at ambient temperature and quench into water. Extract the aqueous mixture with ethyl acetate, wash with 5% aqueous potassium carbonate, brine and dry over MgSO 4 . Concentrate under vacuum to afford a crude solid, which is triturated with ether to obtain 0.13 g (41%) of the title compound: MS 420 (M+H).
Example 36









Scheme F, Step F7: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-diethylamino-ethyl ester
Stir under N 2 at 0° C. a suspension of NaH (29 mg, 0.72 mmol of 60% oil dispersion) in 1-methyl-2-pyrrolidinone (15 mL), and slowly add N,N-diethylethanolamine (0.095 mL, 0.72 mmol). Stir for 10 min and then add dropwise 3-(pyridin-4-ylamino)-1H-indole-2-carboxylic acid pentafluorophenyl ester (100 mg, 0.24 mmol). Allow the reaction to reach ambient temperature and stir overnight. Quench into water and extract with ethyl acetate. Wash the extract with water, brine, dry (MgSO 4 ), filter and concentrate under vacuum to obtain crude product. Chromatograph on silica gel (10 g, Sepack cartridge) and use 1:1 methanol/ethyl acetate as eluent. Combine like fractions and concentrate under vacuum to obtain 26.3 mg (31%) of the titled compound: MS 353 (M+H).
Example 37









Scheme F, Step F7: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-dimethylamino-ethyl ester
Follow the procedure of Example 36 starting with N,N-dimethyl-ethanolamine to obtain the title compound as a solid: MS 325 (M+H).
Example 38









Scheme F, Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-piperidin-1-yl-ethyl ester
Stir under N 2 at 0° C. a mixture of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (0.25 g, 1 mmol), benzotriazol-1-yloxytris(pyrrolidino)-phosphonium hexafluorophosphate (0.52 g, 1 mmol), diethylisopropylamine (0.175 mL, 1 mmol) and 1-methyl-2-pyrrolidinone (4.0 mL). Add 1-piperidineethanol (0.50 mL, 3.8 mmol) in one portion and stir at ambient temperature for 18.5 h. Pour the reaction into 5% aqueous potassium carbonate and extract with ethyl acetate (2×20 mL). Wash the combined extract with water (20 mL), brine (20 mL), dry over MgSO 4 , filter and concentrate under vacuum to a dark residue. Chromatograph over silica gel (Redisep cartridge, 30 g) and elute with a step gradient of 10% methanol/dichloromethane (150 mL) to 20% methanol/dichloromethane. Concentrate like fractions under vacuum to an oil. Dry the oil under high vacuum at ambient temperature and obtain 0.14 g of the title compound as a tan foam: MS 365 (M+H), TLC (silica gel, 0.5/9.5/90 ammonium hydroxide/methanol/dichloromethane) R f =0.25.
Example 39









Scheme H, Step H1: 3-(4-Pyridinylamino)-benzo(b)thiophene-2-carboxylic acid, ethyl ester
Cool to −78° C. a solution of anhydrous tetrahydrofuran (25 mL) and 3-(4-pyridyl)amino)benzo(b)thiophene HCl (see U.S. Pat. No. 5,328,920; 298 mg, 1.09 mmol) and add a 2 molar solution of lithium di-isopropylamide in heptane/tetrahydrofuran/ethylbenzene (1.9 mL, 3.8 mmol). After 2 h, add diethyl carbonate (1.2 mL, 9.89 mmol). Stir the reaction for another 3 h while the temperature rose to −40° C. Remove the cold bath and after 30 min quench the reaction by the addition of water (10 mL). Extract the reaction with ethyl acetate. Separate the organic layer, wash with brine, dry over sodium sulfate, filter and concentrate. Purify the residue on an ISCO RediSep 10-gram silica gel cartridge, with ethyl acetate-50% heptane and ethyl acetate as eluents. Combine product containing fractions and concentrate to give ethyl 3-[(4-pyridyl)amino]-benzo[b]thiophenyl-2-carboxylate as a tan solid (289 mg, 86%): ESI/MS 299 (M+H); 1 H-NMR δ 1.41-1.43 (m, 3H), 4.11-4.43 (m, 2H), 6.81 (2H), 7.23-7.52 (m, 1H), 7.60 (d, J=9 Hz, 1H), 7.84 (d, J=9 Hz, 1H), 8.36 (br d, 2H), 8.43 (s, 1H, NH); 13 C-NMR δ 14.29, 61.44, 112.61, 113.92, 123.36, 124.11, 125.15, 127.86, 132.53, 139.49, 141.75, 149.68, 150.36, 164.53.
Example 40









Scheme H, Step H1: 6-Fluoro-3-(4-pyridinylamino)-benzo[b]thiophene-2-carboxylic acid, ethyl ester
Cool to −78° C. a solution of anhydrous tetrahydrofuran (20 mL) and 6-fluoro-3-[4-pyridyl)amino]benzo[b]thiophene (see U.S. Pat. No. 5,177,088; 181 mg, 0.741 mmol) and add a 2 molar solution of lithium di-isopropylamide in heptane/tetrahydrofuran/ethylbenzene (0.74 mL, 1.5 mmol). After 2 h, add diethyl carbonate (0.8 mL, 6.59 mmol). Stir the reaction for another 1 h, allow to warm slowly to room temperature and continue to stir overnight. Quench the reaction by the addition of water (10 mL). Remove the solvents on a rotary evaporator and take up the residue in ethyl acetate. Wash the ethyl acetate with water and brine, dry over sodium sulfate, filter and concentrate. Purify the residue on an ISCO RediSep 10-gram silica gel cartridge, with ethyl acetate-50% heptane and ethyl acetate-0.5% NH 4 OH as eluents. Combine product containing fractions and concentrate to give ethyl 6-fluoro-3-[(4-pyridyl)amino]benzo[b]thiophenyl-2-carboxylate: ESI/MS 317 (M+H); 1 H-NMR δ 1.40-1.43 (m, 3H); 4.35-4.42 (m, 2H); 6.80 (br d, 2H); 7.04-7.10 (m, 1H); 7.48-7.57 (m, 2H); 8.37 (br s, 2H); 8.43 (s, 1H, NH); 13 C-NMR δ 14.3; 61.5; 109.2 (J CF =25 Hz); 112.7 (J CF =4 Hz); 113.4; 113.7 (J CF =25 Hz); 126.7 (J CF =9 Hz); 129.0; 140.9 (J CF =11 Hz); 141.4; 149.5; 150.5; 162.4 (J CF =260 Hz); 164.1; 19 F-NMR (282 MHz) δ-110.8.
Example 41









Scheme H, Step H1: Ethyl 3-[(4-pyridyl)amino-N-methyl]benzo[b]thiophenyl-2-carboxylate
Under nitrogen, cool to −76° C. a solution of anhydrous tetrahydrofuran (4 mL) and 3-[4-pyridyl)amino-N-methyl]benzo[b]thiophene HCl (see U.S. Pat. No. 5,328,920; 50 mg, 0.18 mmol) and add a 2 molar solution of lithium di-isopropylamide in heptane/tetrahydrofuran/ethylbenzene (0.28 mL, 0.54 mmol). After 20 min, replace the dry ice/acetone bath with an ice/water bath. After 1 h, add diethyl carbonate (0.27 mL, 2.23 mmol) and stir the reaction for 4 h at this temperature and then overnight at room temperature. Quench the reaction by the addition of water (3 mL). Extract the reaction with ether, and wash the extract with brine, dry it over sodium sulfate, filter and concentrate. Purify the residue on an ISCO RediSep 4 gram silica gel cartridge, with ethyl acetate-3% methanol-NH 4 OH as eluent. Combine product containing fractions and concentrate to give ethyl 3-[(4-pyridyl)amino-N-methyl]benzo[b]thiophenyl-2-carboxylate as a tan solid (30 mg, 53%): ESI/MS 313 (M+H); 1 H-NMR δ 1.26 (t, 3H, J=9 Hz), 3.38 (s, 3H, Me), 4.30 (q, 2H, J=6 Hz), 6.42 (br s, 2H), 7.57 (m, 3H), 7.90 (d, 1H, J=9 Hz), 8.21 (br s, 2H); 13 C-NMR δ 14.1, 38.2, 61.6, 107.7, 123.3, 123.4, 125.2, 127.9, 135.8, 139.3, 142.6, 149.9, 153.5, 161.2.
Example 42









Scheme H, Step H2: 3-(4-Pyridinylamino)-6-trifluoromethyl-benzo[b]thiophene-2-carboxylic acid Methane sulfonate salt
Under nitrogen, cool to −78° C. a solution of anhydrous tetrahydrofuran (60 mL) and 3-(4-pyridinylamino)-6-trifluoromethylbenzo[b]thiophene (see U.S. Pat. No. 5,328,920; 0.47 g, 1.6 mmol) and add a 2.5 molar solution in hexanes of n-butyl lithium (1.3 mL, 3.25 mmol) by syringe. Stir the reaction for 2 h, pour it into a beaker containing 140 g of dry ice and 10 mL of anhydrous tetrahydrofuran. Stir the reaction for 1 h, add water (50 mL), and basify to pH 13-14 with 10% aqueous sodium hydroxide. Extract the basic solution with ether (2×10 mL) and acidify the aqueous portion with 3% aqueous HCl. At about pH 7 a white solid precipitates from solution. Collect the solid and dry overnight under vacuum to afford 0.35 g (65%) of the free base of the title compound. React a portion of the free base with methane sulfonic acid and obtain 59.0 mg of the title compound: ESI/MS 339 (M+H); HPLC: R t =1.38 min. (95/5/0.1 (A) 5/95/0.1 (B) Water/CAN/Formic Acid YMC ODS-A 2×50 mm 1 mL/min Gradient Composition: 100% A for 0.1 min Linear Gradient to 100% B at 2 min Hold until 3.5 min.)
Example 43









Scheme H, Step H2: 3-(4-Pyridinylamino)-benzo[b]thiophene-2-carboxylic acid Hydrochloride salt
Follow the procedure of Example 42 starting with 3-(4-pyridinylamino)-6-benzo[b]thiophene (see U.S. Pat. No. 5,328,920 for synthesis). Form the hydrochloride salt in methanol and ethereal HCl to obtain the title compound as white solid: ESI/MS 271 (M+H).
Example 44









Scheme H, Step H1: 3-(4-Pyridinylamino)-6-trifluoromethyl-benzo[b]thiophene-2-carboxylic acid ethyl ester Hydrochloride salt
Follow the procedure of Example 40 starting with 3-(4-pyridinylamino)-6-trifluoromethylbenzo[b]thiophene (see U.S. Pat. No. 5,328,920 for synthesis). Form the hydrochloride salt in ethereal HCl and obtain the title compound as an off-white solid: MS 367 (M+H), TLC (silica gel, ethyl acetate/methanol/ammonium hydroxide) R f =0.80.
Example 45









Scheme H, Step H1: 3-(Propyl-4-pyridinylamino)benzo[b]thiophene-2-carboxylic acid ethyl ester
Follow the procedure of Example 40 starting with 3-(propyl-4-pyridinylamino)benzo[b]thiophene (see U.S. Pat. No. 5,328,920 for synthesis). Obtain the title compound as a tan solid: ESI/MS 341 (M+H); HPLC: R t =1.53 min. (95/5/0.1 (A) 5/95/0.1 (B) Water/CAN/Formic Acid YMC ODS-A 2×50 mm 1 mL/min Gradient Composition: 100% A for 0.1 min Linear Gradient to 100% B at 2 min Hold until 3.5 min.)
Example 46









Scheme F, Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid (S)-1-methoxycarbonyl-ethyl ester
Stir at 0° C. a mixture of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (3.0 g, 12 mmol), methyl (S)-(−)-lactate (2.3 mL, 24 mmol), 4-methylmorpholine (2.6 mL, 24 mmol) and DMF (50.0 mL). Add benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP, 5.32 g, 12 mmol) in one portion and stir at ambient temperature overnight. Add brine (50 mL) and extract with ethyl acetate (3×200 mL). Concentrate under vacuum to an oil. Chromatograph over silica gel (5 g) and elute with dichloromethane, EtOAC, and MeOH. Concentrate like fractions under vacuum to an oil. Treat the oil with HCl in ether (10 mL) add additional ether (50 mL) and concentrate under vacuum to an oil. Treat the oil with 5% aqueous NaHCO 3 and extract with EtOAc. Dry the extract over Na 2 SO 4 , filter and concentrate to an oil. Scratch the oil with a glass rod in the presence of ether to obtain a sticky solid. Add CH 3 CN and then ether and obtain of the title compound as a yellow solid, 140 mg: MS 340 (M+H), R t =2.73 min. (95/5/0.1 (A) 5/95/0.1 (B) Water/CAN/Formic Acid YMC ODS-A 2×50 mm 1 mL/min Gradient Composition: 100% A for 0.1 min Linear Gradient to 100% B at 2 min Hold until 3.5 min.)
Example 47









Scheme F, Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-methoxy-ethyl ester
Follow the procedure of Example 46 but cool the reaction to −10 to −15° C. substituting 2-methoxy ethanol for methyl (S)-(−)-lactate and purify by column chromatography on silica gel eluting with 15% MeOH/dichloromethane to obtain the title compound: MS 312 (M+H), TLC (silica gel, 15% MeOH/dichloromethane) R f =0.53.
Example 48









Scheme F, Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 3-ethoxy-propyl ester
Follow the procedure of Example 47 substituting 2-ethoxy ethanol for 2-methoxy ethanol and purify by column chromatography on silica gel eluting with 8.5% MeOH/dichloromethane to obtain the title compound: MS 340 (M+H), TLC (silica gel, 8.5% MeOH/dichloromethane) R f =0.30.
Example 49









Scheme F, Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid ethoxycarbonylmethyl ester
Follow the procedure of Example 46 substituting ethyl glycolate for methyl (S)-(−)-lactate and NMP for DMF purify by column chromatography on silica gel eluting with dichloromethane, EtOAc and 1% MeOH/dichloromethane to obtain the title compound: MS 340 (M+H), ), R t =1.19 min. (95/5/0.1 (A) 5/95/0.1 (B) Water/CAN/Formic Acid YMC ODS-A 2×50 mm 1 ml/min Gradient Composition: 100% A for 0.1 min Linear Gradient to 100% B at 2 min Hold until 3.5 min.) TLC R f =0.51 (3:1 EtOAc:CH 3 OH, silica gel).
Example 50









Scheme F, Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid indan-5-yl ester
Follow the procedure of Example 49 substituting 5-indanol for ethyl glycolate and benzotriazol-1-yloxytris(pyrrolidino)-phosphonium hexafluorophosphate (PYBOP) for BOP. Purify by column chromatography on silica gel eluting with EtOAc to obtain the title compound: MS 370 (M+H), R t =1.35 min. (95/5/0.1 (A) 5/95/0.1 (B) Water/CAN/Formic Acid YMC ODS-A 2×50 mm 1 mL/min Gradient Composition: 100% A for 0.1 min Linear Gradient to 100% B at 2 min Hold until 3.5 min.); TLC Rf=0.52 (EtOAc, silica gel).
Example 51









Scheme F, Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid diethylcarbamoylmethyl ester
Stir at 50° C. for 10 min. a mixture of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (2.0 g, 8 mmol), KI (10 mg) and Cs 2 CO 3 (2.8 g, 8.6 mmol) in DMF (125 mL). Allow to cool to ambient temperature and then cool in an ice bath. Add 2-chloro-N,N-diethylacetamide (1.077 g, 0.9 mmol) and allow to warm to ambient temperature. Add a brine solution to the reaction and extract with EtOAc (3×). Wash the extract with brine and water, dry (MgSO 4 ), filter and concentrate under vacuum to give a semi-solid. Flash chromatograph over silica gel eluting first with EtOAc and 10% MeOH/EtOAc and obtain 0.317 g the title compound as a solid: MS 367 (M+H); HPLC: R t =4.75 min. (95/5/0.1 (A) 5/95/0.1 (B) Water/CAN/Formic Acid YMC ODS-A 2×50 mm 1 mL/min Gradient Composition: 100% A for 0.1 min Linear Gradient to 100% B at 2 min Hold until 3.5 min).
Example 52









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-morpholin-4-yl-2-oxo-ethyl ester
Intermediate: 4-(Chloroacetyl)morpholine
Utilize a Quest 205 Parallel Synthesizer and to a mixture of morpholine (2.6 g, 30 mmol), dichloromethane (45 mL) and 2M K 2 CO 3 slowly add chloroacetylchloride (3.73 g, 33 mmol) in dichloromethane (10 mL). Agitate the reaction overnight and filter off the organic layer. Extract the aqueous with dichloromethane (20 mL) and add it to the previously collected organic layer. Dry the organic phase (MgSO 4 ) and concentrate under vacuum to obtain the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-morpholin-4-yl-2-oxo-ethyl ester
Follow the procedure of Example 51 substituting 4-(chloroacetyl)morpholine for 2-chloro-N,N-diethylacetamide. Collect the precipitate that forms in the EtOAc extract, wash with ether to obtain the title compound as a solid: MS 381 (M+H); HPLC: R t =2.48 min. (95/5/0.1 (A) 5/95/0.1 (B) Water/CAN/Formic Acid YMC ODS-A 2×50 mm 1 mL/MIN Gradient Composition: 100% A for 0.1 min Linear Gradient to 100% B at 2 min Hold until 3.5 min).
Example 53









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-oxo-2-pyrrolidin-1-yl-ethyl ester
Intermediate: 1 Choroacetlpyrrolidine
Follow the procedure of Example 53, substituting pyrrolidine for morpholine and obtain the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-oxo-2-pyrrolidin-1-yl-ethyl ester
Follow the procedure of Example 51 substituting 1-(chloroacetyl)pyrrolidine for 2-chloro-N,N-diethylacetamide. Collect the precipitate that forms in the EtOAc extract, wash with ether to obtain the title compound as a solid: MS 365 (M+H); HPLC: R t =2.58 min. (95/5/0.1 (A) 5/95/0.1 (B) Water/CAN/Formic Acid YMC ODS-A 2×50 mm 1 mL/min Gradient Composition: 100% A for 0.1 min Linear Gradient to 100% B at 2 min Hold until 3.5 min.).
Example 54









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid (benzyl-ethyl-carbamoyl)-methyl ester
Intermediate: 2-Chloro-N-ethyl-N-(phenylmethyl)acetamide
Follow the procedure of Example 53, substituting N-benzyl-N-ethyl amine for morpholine and obtain the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid (benzyl-ethyl-carbamoyl)-methyl ester
Follow the procedure of Example 51 substituting 2-chloro-N-ethyl-N-(phenylmethyl)acetamide for 2-chloro-N,N-diethylacetamide. Collect the precipitate that forms in the EtOAc extract, wash with ether to obtain the title compound as a yellow solid: MS 429 (M+H); HPLC: R t =2.72 min. (95/5/0.1 (A) 5/95/0.1 (B) Water/CAN/Formic Acid YMC ODS-A 2×50 mm 1 mL/min Gradient Composition: 100% A for 0.1 min Linear Gradient to 100% B at 2 min Hold until 3.5 min).
Example 55









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-azetidin-1-yl-2-oxo-ethyl ester
Intermediate: 1-(chloroacetyl)azetidine
To a stirring solution of chloroacetylchloride and K 2 CO 3 (4.14 g, 30 mmol) in dichloromethane-water (45-15 mL), add slowly by syringe azetidine (2.8 g, 30 mmol). After addition, stir overnight at ambient temperature. Separate the organic layer and wash with H 2 O (twice) and brine (twice), and then concentrate under reduced pressure to give the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-azetidin-1-yl-2-oxo-ethyl ester
Follow the procedure of Example 51 substituting 1-(chloroacetyl)azetidine for 2-chloro-N,N-diethylacetamide. Collect the precipitate that forms in the EtOAc extract, wash with ether to obtain the title compound as a yellow solid: MS 351 (M+H); HPLC: R t =2.52 min. (95/5/0.1 (A) 5/95/0.1 (B) Water/CAN/Formic Acid YMC ODS-A 2×50 mm 1 mL/min Gradient Composition: 100% A for 0.1 min Linear Gradient to 100% B at 2 min Hold until 3.5 min).
Example 56









Scheme F, Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 5-methyl-2-oxo-[1,3]-dioxol-4-ylmethyl ester
Follow the procedure of Example 51 substituting 4-(bromomethyl)-5-methyl-1,3-dioxol-2-one (which was prepared in accordance with the procedures set forth in Saari, W. S., et al., J. Med. Chem ., (1984), 27, 713-717) for 2-chloro-N,N-diethylacetamide.
Purify by column chromatography on silica gel eluting with 10% MeOH/EtOAc and then 15% MeOH/EtOAc to obtain the title compound as a pale yellow powder: MS 366 (M+H).
Example 57









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid diethylcarbamoyloxymethyl ester
Intermediate: Chloromethyl diethylcarbamate
To a stirred solution of chloromethyl chloroformate (7.09 g, 5.5 mmol) in heptane (100 μL) at −20° C., add a solution of diethylamine (10.4 g, 14.7 mmol) in heptane (50 mL), dropwise. Maintain the internal temperature between −15° C. to −5° C. and after 1 h quench the reaction with water. Separate the layers and wash the organic phase with 10% aqueous HCl, water and NaHCO 3 solution. Dry the organic layer (MgSO 4 ), filter and concentrate under reduced pressure to obtain 8.3 g of the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid diethylcarbamoyloxymethyl ester
Stir a mixture of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (2.0 g, 8.0 mmol), chloromethyl diethylcarbamate (1.7 g, 10.4 mmol) and K 2 CO 3 in DMF (50 μL) at ambient temperature overnight. Add EtOAc (400 mL), to the reaction mixture, wash the organic layer with brine solution (400 mL) and concentrate to an oil. Purify by flash chromatography over silica gel (40 g) eluting with EtOAc and then 10% MeOH/EtOAc. Collect like fractions and concentrate under vacuum to yield an oil. Crystallize from heptane-ether to obtain 1.1 g (36%) of the title compound as a white powder: MS 383 (M+H).
Example 58









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid piperidine-1-carbonyloxymethyl ester
Intermediate: 1-piperidinecarboxylic acid, chloromethyl ester
Follow the procedure of Example 57, substituting piperidine for diethylamine, obtain the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid piperidine-1-carbonyloxymethyl ester
Follow the procedure of Example 57 Step F4, substituting 1-piperidinecarboxylic acid, chloromethyl ester for chloromethyl diethylcarbamate to obtain the title compound as a solid: MS 395 (M+H).
Example 59









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid morpholine-4-carbonyloxymethyl ester
Intermediate: 4-Morpholinecarboxylic acid, chloromethyl ester
Follow the procedure of Example 57, substituting morpholine for diethylamine and obtain the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid morpholine-4-carbonyloxymethyl ester
Follow the procedure of Example 57, substituting 4-morpholinecarboxylic-acid, chloromethyl ester for chloromethyl diethylcarbamate to obtain the title compound as a solid: MS 397 (M+H).
Example 60









Scheme F, Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-ethoxycarbonylamino-2-oxo-ethyl ester
Follow the procedure of Example 57, substituting N-chloroacetyl urethane for chloromethyl diethylcarbamate. Purify by column chromatography eluting with 10% MeOH/EtOAc and then 15% MeOH/EtOAc. Recrystallize form CH 3 CN to obtain the title compound as a solid: MS 383 (M+H).
Example 61









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid benzylcarbamoyloxymethyl ester
Intermediate: (Phenylmethyl)carbamic acid, chloromethyl ester
Follow the procedure of Example 57, substituting benzylamine for diethylamine and obtain the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid benzylcarbamoyloxymethyl ester
Follow the procedure of Example 57 Step B, substituting (phenylmethyl)carbamic acid chloromethyl ester for chloromethyl diethylcarbamate. Purify by column chromatography eluting with 10% MeOH/EtOAc and then 20% MeOH/EtOAc to obtain the title compound as a solid: MS 417 (M+H).
Example 62









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid isopropoxycarbonyloxymethyl ester
Intermediate: Chloromethyl carbonic acid 1-methyl ester
Stir and cool to −15° C. a solution of chloromethyl chloroformate (3.55 g, 27.5 mmol) in dichloromethane (30 mL) and add a solution of isopropyl alcohol (1.51 g, 2.5 mmol) in pyridine (2.18 g, 2.5 mmol), dropwise to maintain an internal temperature of between −18° C. to −5° C. After 2 h, quench into water, separate the layers and wash the organic phase with 10% aqueous HCl, water, NaHCO 3 solution and water. Dry with MgSO 4 , filter and concentrate to obtain the title compound as 3.6 g of a colorless oil.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid isopropoxycarbonyloxymethyl ester
Follow the procedure of Example 57, substituting chloromethyl carbonic acid 1-methyl ester for chloromethyl diethylcarbamate and add KI (100 mg) to the reaction mixture. Allow the reaction to proceed for 3 hours, and obtain the title compound as a solid: MS 370 (M+H).
Example 63









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid adamantan-1-yloxycarbonyloxymethyl ester
Intermediate: Chloromethyl 1-adamantyl carbonate
Follow the procedure of Example 62, substituting 1-adamantanol for isopropyl alcohol to obtain the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid adamantan-1-yloxycarbonyloxymethyl ester
Follow the procedure of Example 62 substituting chloromethyl 1-adamantyl carbonate for chloromethyl carbonic acid 1-methyl ester, but allow the reaction to proceed overnight. Obtain the title compound as a solid: MS 462 (M+H).
Example 64









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 1,1,2-trimethyl-propoxycarbonyloxymethyl ester
Intermediate: Carbonic acid chloromethyl ester 1,1,2-trimethyl-propyl ester
Follow the procedure of Example 62, substituting 2,3-dimethyl-2-butanol for isopropyl alcohol to obtain the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 1,1,2-trimethyl-propoxycarbonyloxymethyl ester
Follow the procedure of Example 62, substituting carbonic acid chloromethyl ester 1,1,2-trimethyl-propyl ester for chloromethyl carbonic acid 1-methyl ester, but allow the reaction to proceed overnight. Obtain the title compound as a solid: MS 412 (M+H).
Example 65









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid cyclohexyloxycarbonyloxymethyl ester
Intermediate: Carbonic acid chloromethyl ester 1,1,2-trimethyl-propyl ester
Follow the procedure of Example 62, substituting cyclohexanol for isopropyl alcohol to obtain the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid cyclohexyloxycarbonyloxymethyl ester
Follow the procedure of Example 62 substituting chloromethyl cyclohexyl carbonate for chloromethyl carbonic acid 1-methyl ester, but allow the reaction to proceed overnight. Obtain the title compound as a solid: MS 410 (M+H).
Example 66









Scheme F, Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid acetoxymethyl ester
Stir at 50° C. for 10 min. a mixture of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid (1.27 g, 5 mmol) in DMF (25 mL), and add Cs 2 CO 3 (1.63 g, 5 mmol). After 10 min. add bromomethyl acetate (0.95 g, 6.21 mmol) and one minute thereafter quench into aqueous NH 4 Cl solution. Extract with EtOAc and wash the extract with aqueous NaHCO 3 solution. Dry the extract (MgSO 4 ), filter and concentrate under vacuum to give a solid. Triturate the solid with EtOAc and obtain the title compound as a solid: MS 326 (M+H).
Example 67









Scheme F, Step F2: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2,2-dimethyl-propionyloxymethyl ester
Stir at 50° C. a mixture of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid potassium salt (1.0 g, 3.43 mmol), KI (0.16 g) 2,2-dimethyl-propionic acid chloromethyl ester (0.63 g, 4.16 mmol) in DMF (20 mL) for 1.5 h. Cool, and partition between H 2 O-EtOAc. Wash the extract with aqueous NaHCO 3 solution, dry the extract (MgSO 4 ), filter and concentrate under vacuum to afford the crude product. Chromatography the product on a silica gel column eluting with 8% MeOH/EtOAc to obtain 0.64 g of the title compound as a solid; MS 326 (M+H), m.p. 223-224° C., TLC (silica gel, 8% MeOH/EtOAc) R f =0.30.
Example 68









Scheme F, Step F2: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid pentanoyloxymethyl ester
Follow the procedure of Example 67 except substitute pentanoic acid chloromethyl ester for 2,2-dimethyl-propionic acid chloromethyl ester and allow the reaction to proceed at 50° C. overnight. Purify by column chromatography on silica eluting with 5% MeOH/EtOAc to obtain the title compound: MS 368 (M+H).
Example 69









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-oxo-piperidin-1-yl-methyl ester
Intermediate: N-(chloromethyl)-2-piperidinone
The title compound is prepared in accordance with the procedures set forth in Moreira, R. et al., Tet. Lett ., (1994), 35, 7107-7110. Thus, a mixture of 2-piperidinone (1.0 g, 10.09 mmol) and paraformaldehyde (500 mg) is refluxed in anhydrous THF (30 mL) and chlorotrimethylsilane (30 mL) overnight under N 2 . Concentrate the solvent under vacuum to give N-(chloromethyl)-2-piperidone as an oil (1.30 g).
Step F2: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-oxo-piperidin-1-ylmethyl ester
Heat at 50° C. a suspension of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid potassium salt (1.6 g, 5.49 mmol) in anhydrous THF (30 mL) in an oil bath for 20 min. Add a solution of N-(chloromethyl)-2-piperidone (850 mg, 5.76 mmol) in anhydrous THF (5 mL) dropwise. After 30 min, cool the reaction to ambient temperature. Add water (100 mL), and extract with ethyl acetate (3×100 mL). Combine the organic layers, wash with NaHCO 3 (sat), water, brine, dry with MgSO 4 and then concentrate. Purify the residue on an ISCO RediSep 35 gram silica gel cartridge, eluting with 5% methanol in ethyl acetate then 20% methanol in ethyl acetate. Combined product containing fractions and concentrate to give the title compound as a white solid (520 mg, 26%): MS 365 (M+H), TLC (silica gel, MeOH/EtOAc 25:75) R f =0.18, 1 H NMR (DMSO-d6) δ 11.77 (1H, s), 8.46 (1H, s), 8.08 (2H, d, J=5.7 Hz), 7.50 (3H, m), 7.09 (1H, m), 6.58 (2H, d, J=6 Hz), 5.54 (2H, s), 3.27 (2H, br2), 2.23 (2H, brs), 1.66 (4H, brs). 13 C NMR (DMSO-d6) δ 170.00, 160.43, 152.25, 149.44, 135.54, 125.60, 122.96, 121.84, 120.22, 119.87, 118.99, 113.00, 108.59, 70.99, 47.00, 31.99, 22.39, 20.60.
Example 70









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid (benzoyl-ethoxycarbonylmethyl-amino)-methyl ester
Intermediate: N-chloromethyl-ethyl benzamidoacetate
Reflux a mixture of ethyl benzamidoacetate (1.0 g, 4.83 mmol) and paraformaldehyde (450 mg) in chlorotrimethylsilane (40 mL for overnight under N 2 . Concentrate the solvent under vacuum the solvent to give N-chloromethyl-ethyl benzamidoacetate as an oil (1.2 g, 98%).
Step F2: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid (benzoyl-ethoxycarbonylmethyl-amino)-methyl ester
Heat at 50° C. a suspension of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid potassium salt (1.3 g, 4.46 mmol) in anhydrous THF (20 mL) in an oil bath for 20 min under N 2 . Add a solution of N-chloromethyl-ethyl benzamidoacetate (1.2 g, 4.69 mmol) in anhydrous THF (5 mL) dropwise. After 30 min, cool the reaction to ambient temperature. Add water, and extract with ethyl acetate. Wash the organic layer with NaHCO 3 (sat), water, brine, dry with MgSO 4 and then concentrate. Purify the residue on an ISCO RediSep 35 gram silica gel cartridge, eluting with 50% heptane/ethyl acetate (200 mL), 100% ethyl acetate (300 mL) and then 1% methanol/ethyl acetate. Product containing fractions were combined and concentrated to give the title compound as a yellow solid (834 mg): MS 473 (M+H), 1 H NMR (DMSO-d6) δ 11.81 (1H, s), 8.47 (1H, s), 8.10 (2H, d, J=5.8 Hz), 7.38 (8H, m), 7.08 (1H, m), 6.66 (2H, J=6.0 Hz), 5.49 (2H, s), 4.30 (2H, s), 4.09 (2H, m), 1.99 (3H, m).). 13 C NMR (DMSO-d6) δ 171.51, 168.86, 160.30, 152.07, 149.58, 135.72, 134.06, 130.51, 128.46, 127.13, 125.76, 122.65, 122.34, 120.50, 119.90, 118.51, 113.06, 108.89, 74.53, 60.79, 59.74, 47.52, 20.75, 14.07, 13.95.
Example 71









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-oxo-pyrrolidin-1-ylmethyl ester
Intermediate: N-(chloromethyl)-2-pyrrolidinone
Reflux a mixture of 2-pyrrolidinone (1.0 g, 11.75 mmol) and paraformaldehyde (500 mg) in chlorotrimethylsilane (40 mL) for 2 hrs under N 2 . Evaporate the solvent under vacuum to give N-(chloromethyl)-2-pyrrolidinone as an oil (1.55 g, 99%).
Step F2: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid 2-oxo-pyrrolidin-1-yl-methyl ester
Heated at 50° C. a suspension of 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid potassium salt (2.0 g, 6.86 mmol) in anhydrous THF (100 mL) was oil bath for 20 min. Add to it a solution of N-(chloromethyl)-2-pyrrolidinone (1.10 g, 8.23 mmol) in anhydrous THF (5 mL) dropwise. After 3 hrs, cool the reaction to ambient temperature. Add water (100 mL) and extract with ethyl acetate (3×100 mL). Combine the organic layers and wash with NaHCO 3 (sat), water, brine, dry with MgSO 4 and then concentrate. Purify the residue on an ISCO RediSep 10-gram silica gel cartridge, eluting with ethyl acetate and then 5% methanol in ethyl acetate. Combine product containing fractions and concentrate to give the title compound as an off-yellow solid (205 mg): MS 313 (M+H), TLC (silica gel, MeOH/EtOAc 1:1). R f =0.33, 1 H NMR (DMSO-d6) δ 11.78 (1H, s), 8.47 (1H, s), 8.09 (2H, d, J=6.2 Hz), 7.49 (3H, m), 7.09 (1H, m), 6.59 (2H), 5.76 (residual CH 2 Cl 2 ), 5.44 (2H, s), 3.37 (2H, m), 2.22 (2H, m), 1.87 (2H, m).
Example 72









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid [phenyl-(toluene-4-sulfonyl)-amino]-methyl ester
Intermediate: N-chloromethyl-N-phenyl-4-methylbenzenesulfonamide
The title compound is prepared in accordance with the procedures set forth in Iley, J. et al., Bioorg. Med. Chem ., (2000), 8, 1629-1636. Thus, a mixture of N-phenyl-p-toluene sulfonamide (3.0 g, 12.13 mmol) and paraformaldehyde (600 mg) is refluxed in chlorotrimethylsilane (50 mL) for 3 hrs. Concentrate the solvent under vacuum to give N-chloromethyl-N-phenyl-4-methylbenzenesulfonamide as an oil (3.2 g).
Step F2: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid [phenyl-(toluene-4-sulfonyl)-amino]-methyl ester
Under N 2 , at 60° C. dissolve 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid potassium salt (2.2 g, 7.5 mmol) in a mixture of anhydrous THF and DMF (90:10 mL). Cool to 0° C. and add a solution of N-chloromethyl-N-phenyl-4-methylbenzenesulfonamide (2.6 g, 8.80 mmol) in anhydrous THF (5 mL) dropwise. After 20 min add water (100 mL) and then extract with ethyl acetate (4×50 mL). Combine the organic layers, wash with NaHCO 3 (sat), water, brine, dry with Na 2 SO 4 and then concentrate. Purify the residue on an ISCO RediSep 35 gram silica gel cartridge, eluting with ethyl acetate and then 30% methanol/ethyl acetate. Combine product containing fractions and concentrate to a solid. Dissolve the solid in methanol and precipitate with ether to give the title compound as a yellow solid (390 mg): ESI/MS 313 (M+H, 1 H NMR (CD 3 OD) δ 8.05 (2H, 2br d, J=6.5 Hz, J=6.8 Hz), 7.38 (14H, m), 6.61 (2H, br d, J=7.0 Hz), 5.93 (2H, s), 2.44 (3H, s).
Example 73









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid (benzenesulfonyl-methyl-amino)-methyl ester
Intermediate: N-chloromethyl-N-methyl-4-methylbenzenesulfonamide
Reflux a mixture of N-methylbenzenesulfonamide (2.0 g, 11.68 mmol) and paraformaldehyde (600 mg) in chlorotrimethylsilane (40 mL) for 2.5 hrs. Concentrate under vacuum to give N-chloromethyl-N-methyl-4-methylbenzenesulfonamide as a white solid (2.5 g).
Step F2: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid (benzenesulfonyl-methyl-amino)-methyl ester
Under N 2 at 60° C., dissolve 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid potassium salt (2.2 g, 7.55 mmol) in a mixture of anhydrous THF and DMF (150:40 mL). Cool to 0° C. and add N-chloromethyl-N-methyl-4-methylbenzenesulfonamide (2.0 g, 9.10 mmol) in anhydrous THF (10 mL) dropwise. After 20 min., add water (100 mL) and then extract with dichloromethane (3×100 mL). Combine the organic layers wash with NaHCO 3 (sat), water, brine, dry with Na 2 SO 4 and then concentrate. Purify the residue on an ISCO RediSep 35-gram silica gel cartridge, eluting with 50% heptane/ethyl acetate, ethyl acetate and then 10% methanol/ethyl acetate. Combine product containing fractions and concentrate to an oil. Dissolve the oil in methanol and add ether to obtain the title compound as a white solid (800 mg): MS: 437 (M+H), 1 H NMR (DMSO-d6) δ 11.52 (1H, s), 8.29 (1H, s), 8.04 (2H, d, J=5.5 Hz), 7.76 (2H, d, J=7.2 Hz), 7.43 (6H, m), 7.03 (1H, m), 6.46 (2H, d, J=6.0 Hz), 5.60 (2H, s), 2.79 (3H, s).
Example 74









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid [methyl-(toluene-4-sulfonyl)-amino]-methyl ester
Intermediate: N-chloromethyl-N-methyl-4-methylbenzenesulfonamide
Reflux a mixture of N-methyl-4-methylbenzenesulfonamide (2.3 g, 12.4 mmol) and paraformaldehyde (600 mg) in chlorotrimethylsilane (40 mL) for 2 hrs. Concentrate under vacuum to give N-chloromethyl-N-methyl-4-methylbenzenesulfonamide as a white solid (2.8 g).
Step F2: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid [methyl-(toluene-4-sulfonyl)-amino]-methyl ester
Under N 2 at 60° C., dissolve 3-(4-pyridinylamino)-1H-indole-2-carboxylic acid potassium salt (2.0 g, 6.89 mmol) in a mixture of anhydrous THF and DMF (60:10 mL Cool to −5° C., add piperidinomethyl polystyrene HL resin (NOVA #01-64-0212, 30 mg) and follow with a solution of N-chloromethyl-N-methyl-4-methylbenzenesulfonamide (1.9 g, 8.34 mmol) in anhydrous THF (10 mL) dropwise. After 30 min, add water (100 mL) and then extract with dichloromethane (3×50 mL). Combine organic layers, wash with NaHCO 3 (sat), water, brine, dry with MgSO 4 and then concentrate. Purify the residue on an ISCO RediSep 35-gram silica gel cartridge, eluting with 50% heptane/ethyl acetate, ethyl acetate and then 10% methanol/ethyl acetate. Combine product containing fractions and concentrate to a solid (1.1 g). Wash the solid with methanol/ether, and dry to give the title compound as a white solid (720 mg): TLC (silica gel, EtOAc/MeOH, 8:2), R f =0.49, 1 H NMR (DMSO-d6) δ 11.47 (1H, s), 8.32 (1H, s), 8.09 (2H, d, J=5.7 Hz), 7.63 (2H, d, J=8.3 Hz), 7.48 (1H, d, J=8.3 Hz), 7.34 (2H, m), 7.14 (3H, m), 6.52 (2H, d, J=6.0 Hz), 5.61 (2H, s), 2.83 (3H, s), 2.05 (3H, s).
Example 75









Scheme F: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid tert-butoxycarbonyloxymethyl ester
Intermediate: tert-Butyl chloromethyl carbonate
Follow the procedure of Example 62, substituting tert-butanol for isopropyl alcohol to obtain the title compound.
Step F4: 3-(Pyridin-4-ylamino)-1H-indole-2-carboxylic acid tert-butoxycarbonyloxymethyl ester
Follow the procedure of Example 62 substituting tert-butylchloromethyl carbonate for chloromethyl carbonic acid 1-methyl ester, but allow the reaction to proceed overnight. Obtain the title compound as a solid: MS 384 (M+H), HPLC: R t =2.94 min.
Example 76









3-((3-Phenylpropanoyl)methylamino)-1H-6-chloro-indole-2-carboxylic acid
Stir at room temperature for overnight a solution of 3-((3-phenylpropanoyl)methylamino)-1H-6-chloro-indole-2-carboxylic acid ethyl ester (213 mg, 0.55 mmol) and lithium hydroxide (35 mg, 0.83 mmol) dissolved in a solvent mixture of 5 mL of THF and 5 mL of water. Dilute the reaction mixture with water (˜10 mL) and wash with ethyl acetate to remove any organic impurities. Acidify the aqueous layer with 1N hydrochloric acid to precipitate the title compound. Isolate the title compound by filtration and dry at 60° C. under vacuum, yield 197 mg (99% yield); mp 220°-221° C.
Example 77









3-((Benzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid
Following the procedures as set forth in Example 11 of the U.S. Pat. No. 5,675,018, the title compound is prepared; mp 275° C.
Example 78









3-((Benzoyl)amino)-6-chloro-1H-indole-2-carboxylic acid
Following the procedures as set forth in Example 4 of the U.S. Pat. No. 5,675,018, the title compound is prepared; mp 205°-210° C. (dec).
Example 79









3-((Benzoyl)amino)-6-chloro-1H-indole-2-carboxylic acid 2-dimethylamino-ethyl ester
Following the procedures as set forth in Example 5 of the U.S. Pat. No. 5,675,018, the title compound is prepared.
Example 80









3-((4-chlorobenzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid
Following the procedures as set forth in Example 28Q of the U.S. Pat. No. 5,675,018, the title compound is prepared; mp 279°-283° C. Anal. Calcd for C 17 H 11 Cl 3 N 2 O 3 : C, 51.35; H, 2.79: N, 7.04; Found: C, 51.30; H, 2.81; N, 7.00.
Example 81









3-((Benzoyl)benzylamino)-4,6-dichloro-1H-indole-2-carboxylic acid
Following the procedures as set forth in Example 17 of the U.S. Pat. No. 5,675,018, the title compound is prepared; mp 266°-267° C.
Example 82









3-((4-Piperidineacyl)amino)-6-chloro-1H-indole-2-carboxylic acid
Following the procedures as set forth in the U.S. Pat. No. 5,675,018, the title compound is prepared.
Example 83









3-((Benzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid ethyl ester
The title compound is prepared following the procedures as set forth in Example 10 of the U.S. Pat. No. 5,675,018; mp 143°-144° C.
Example 84









3-((Benzoyl)methylamino)-5,6-dichloro-1H-indole-2-carboxylic acid
The title compound is prepared following the procedures as set forth in Example 41c of the U.S. Pat. No. 5,675,018.
Example 85









3-((Benzoyl)ethylamino)-4,6-dichloro-1H-indole-2-carboxylic acid
The title compound is prepared following the procedures as set forth in Example 15 of the U.S. Pat. No. 5,675,018; mp 254°-256° C.
Example 86









3-((Benzoyl)methylamino)-6-fluoro-1H-indole-2-carboxylic acid
The title compound is prepared following the procedures as set forth in Example 4L of the U.S. Pat. No. 5,675,018; mp 142°-143° C. Anal. Calcd for C 11 H 13 FN 2 O 3 : C, 55.00; H, 5.45: N, 11.66; Found: C, 55.09; H, 5.19; N, 11.63.
Example 87









3-((2-Benzylbenzoyl)amino)-4,6-dichloro-1H-indole-2-carboxylic acid
The title compound is prepared following the procedures as set forth in Example 21 of the U.S. Pat. No. 5,675,018; mp 234°-235° C. Anal. Calcd for C 23 H 16 Cl 2 N 2 O 3 : C, 62.88; H, 3.67: N, 6.38; Found: C, 63.04; H, 4.05; N, 5.97.
Example 88









3-((3-Fluorobenzoyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid
The title compound is prepared following the procedures as set forth in Example 28j of the U.S. Pat. No. 5,675,018; mp 270°-280° C. Anal. Calcd for C 17 H 11 Cl 2 FN 2 O 3 : C, 53.57; H, 2.91: N, 7.35; Found: C, 53.54; H, 3.15; N, 7.24.
Example 89









3-((Benzoyl)benzylamino)-4,6-dichloro-1H-indole-2-carboxylic acid ethyl ester
The title compound is prepared following the procedures as set forth in Example 16 of the U.S. Pat. No. 5,675,018; mp 174°-175° C.
Example 90









3-((Phenylsulfonyl)amino)-4,6-dichloro-1H-indole-2-carboxylic acid ethyl ester
The title compound is prepared following the procedures as set forth in Example 29 of the U.S. Pat. No. 5,675,018; mp 245°-247° C.
Example 91









3-((Phenylsulfonyl)amino)-4,6-dichloro-1H-indole-2-carboxylic acid
The title compound is prepared following the procedures as set forth in Example 30 of the U.S. Pat. No. 5,675,018; mp 229°-235° C.
Example 92









3-((Phenylsulfonyl)methylamino)-4,6-dichloro-1H-indole-2-carboxylic acid
The title compound is prepared following the procedures as set forth in Example 32 of the U.S. Pat. No. 5,675,018; mp 286°-290° C.
Although the invention has been illustrated by certain of the preceding examples, it is not to be construed as being limited thereby; but rather, the invention encompasses the generic area as hereinbefore disclosed. Various modifications and embodiments can be made without departing from the spirit and scope thereof.