Patent Application
20070004660
The present invention relates to the treatment of a cell proliferative disorder or disorders related to FLT3 using a farnesyl transferase inhibitor in combination with an inhibitor of FLT3 tyrosine kinase.
The fms-like tyrosine kinase 3 (FLT3) ligand (FLT3L) is one of the cytokines that affects the development of multiple hematopoietic lineages. These effects occur through the binding of FLT3L to the FLT3 receptor, also referred to as fetal liver kinase-2 (flk-2) and STK-1, a receptor tyrosine kinase (RTK) expressed on hematopoietic stem and progenitor cells. The FLT3 gene encodes a membrane-spanning class III RTK that plays an important role in proliferation, differentiation and apoptosis of cells during normal hematopoiesis. The FLT3 gene is mainly expressed by early myeloid and lymphoid progenitor cells. See McKenna, Hilary J. et al. Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells. Blood. June 2000; 95: 3489-3497; Drexler, H. G. and H. Quentmeier (2004). “FLT3: receptor and ligand.” Growth Factors 22(2): 71-3.
The ligand for FLT3 is expressed by the marrow stromal cells and other cells and synergizes with other growth factors to stimulate proliferation of stem cells, progenitor cells, dendritic cells, and natural killer cells.
Hematopoietic disorders are pre-malignant disorders of these systems and include, for instance, the myeloproliferative disorders, such as thrombocythemia, essential thrombocytosis (ET), angiogenic myeloid metaplasia, myelofibrosis (MF), myelofibrosis with myeloid metaplasia (MMM), chronic idiopathic myelofibrosis (IMF), polycythemia vera (PV), the cytopenias, and pre-malignant myelodysplastic syndromes. See Stirewalt, D. L. and J. P. Radich (2003). “The role of FLT3 in haematopoietic malignancies.” Nat Rev Cancer 3(9): 650-65; Scheijen, B. and J. D. Griffin (2002). “Tyrosine kinase oncogenes in normal hematopoiesis and hematological disease.” Oncogene 21(21): 3314-33.
Hematological malignancies are cancers of the body's blood forming and immune systems, the bone marrow and lymphatic tissues. Whereas in normal bone marrow, FLT3 expression is restricted to early progenitor cells, in hematological malignancies, FLT3 is expressed at high levels or FLT3 mutations cause an uncontrolled induction of the FLT3 receptor and downstream molecular pathway, possibly Ras activation. Hematological malignancies include leukemias, lymphomas (non-Hodgkin's lymphoma), Hodgkin's disease (also called Hodgkin's lymphoma), and myeloma—for instance, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large-cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocyctic leukemia (JMML), adult T-cell ALL, AML with trilineage myelodysplasia (AML/TMDS), mixed lineage leukemia (MLL), myelodysplastic syndromes (MDSs), myeloproliferative disorders (MPD), multiple myeloma, (MM) and myeloid sarcoma. See Kottaridis, P. D., R. E. Gale, et al. (2003). “Flt3 mutations and leukaemia.” Br J Haematol 122(4): 523-38. Myeloid sarcoma is also associated with FLT3 mutations. See Ansari-Lari, Ali et al. FLT3 mutations in myeloid sarcoma. British Journal of Haematology. 2004 September 126(6):785-91.
Acute Myelogenous Leukemia (AML) is the most prevalent form of adult leukemia and represents 15-20% of childhood leukemias. In 2002, in the United States, approximately 11,000 new cases of AML were diagnosed and an estimated 8,000 patients died from AML. See National Cancer Institute SEER database-http://seer.cancer.gov/. Although diagnosis for AML is traditionally based on histological techniques and blood leukocyte count, recent advances in cytogenetic and genetic analysis have revealed that AML is a mixture of distinct diseases that differ in their genetic abnormalities, clinical features and response to therapy. Recent efforts have begun to tailor chemotherapy to the different sub-types of AML (subtypes are based on cytogenetic analysis and immunohistochemical analysis for disease associated protein expression) with some success. Treatment of AML typically occurs in two phases: induction and post-induction therapy. Induction therapy typically consists of three doses of an anthracycline such as daunorubicin followed by i.v. bolus infusion of the cytotoxic cytarabine for 7-10 days. This regime is effective at inducing remission in 70-80% of patient <60 years of age and ˜50% of patients >60. See Burnett, A. K. (2002). “Acute myeloid leukemia: treatment of adults under 60 years.” Rev Clin Exp Hematol 6(1): 26-45; Buchner T., W. Hiddemann, et al. (2002). “Acute myeloid leukemia: treatment over 60. ” Rev Clin Exp Hematol. 6(1):46-59. After remission induction there are several post-induction options including an additional cycle of chemotherapy or bone marrow transplantation. Post-induction treatment choice and success depends on the patient's age and AML sub-type. Despite the advances in diagnosis and treatment of AML over the last decade, the 5 year disease free survival for patients under 65 is only 40% and the 5 year disease free survival of patients over 65 is less than 10% percent. Thus, there remains a significant unmet clinical need for AML particularly in patients over 65. With the increased knowledge of the mechanisms of the different sub-types of AML new tailored treatments for the disease are beginning to immerge with some promising results.
One recent success in relapse and refractory AML treatment is the development and use of farnesyl transferase inhibitors (FTI) for post-induction treatment. Farnesyl transferase inhibitors are a potent and selective class of inhibitors of intracellular farnesyl protein transferase (FPT). FPT catalyses the lipid modification of a host of intracellular proteins, including the small GTPases of the Ras and Rho family and lamin proteins, to direct their localization to the plasma membrane or membrane compartments within the cell.
FTIs were originally developed to prevent post-translational farnesylation and activation of Ras oncoproteins (Prendergast G. C. and Rane, N. (2001) “Farnesyl Transferase Inhibtors: Mechanism and Applications” Expert Opin Investig Drugs. 10(12):2105-16). Recent studies also demonstrate FTI induced inhibition of Nf-kB activation leading to increased sensitivity to induction of apoptosis and downregulation of inflammatory gene expression through suppression of Ras-dependent Nf-kB activation. See Takada, Y., et al. (2004). “Protein farnesyltransferase inhibitor (SCH 66336) abolishes NF-kappaB activation induced by various carcinogens and inflammatory stimuli leading to suppression of NF-kappaB-regulated gene expression and up-regulation of apoptosis.” J Biol Chem 279, 26287-99.
Of particular interest for oncology, FTI inhibition of the oncogenes of the Ras and Rho family leads to growth arrest and apoptosis of tumor cells both in vitro and in vivo. See Haluska P., G. K. Dy, A. A. Adjei. (2002) “Farnesyl transferase inhibitors as anticancer agents.” Eur J Cancer. 38(13):1685-700. From a clinical perspective, myeloid malignancies, particularly AML, represent a significant opportunity for FTI therapy.
As discussed earlier, AML is a disease with very low long-term survival and an elevated rate of chemotherapy-induced toxicity and resistance (particularly in patients >60 years of age). Additionally, the mechanism of proliferation of AML cells relies on the small GTPases of the Ras and Rho family. With the plethora of pre-clinical data supporting the efficacy of FTIs in AML treatment, several clinical trials were initiated with an FTI including; Tipifarnib (Zarnestra™, Johnson and Johnson), BMS-214662, CP-60974 (Pfizer) and Sch-6636 (lonafarnib, Schering-Plough). ZARNESTRA® (also known as R115777 or Tipifarnib) is the most advanced and promising of the FTI class of compounds. In clinical studies of patients with relapsed and refractory AML, Tipifarnib treatment resulted in a ˜30% response rate with 2 patients achieving complete remission. See Lancet J. E., J. D. Rosenblatt, J. E. Karp. (2003) “Farnesyltransferase inhibitors and myeloid malignancies: phase I evidence of Zarnestra activity in high-risk leukemias.” Semin Hematol. 39(3 Suppl 2):31-5. These responses occurred independently of the patients Ras mutational status, as none of the patients in the trial had the Ras mutations that are sometimes seen in AML patients. However, there was a direct correlation of patient responses to their level of MAPkinase activation (a downstream target of both Ras and Rho protein activity) at the onset of treatment, suggesting that the activity of the Ras/MAPkinase pathway, activated by other mechanisms may be a good predictor of patient responses. See Lancet J. E., J. D. Rosenblatt, J. E. Karp. (2003) “Farnesyltransferase inhibitors and myeloid malignancies: phase I evidence of Zarnestra activity in high-risk leukemias.” Semin Hematol. 39(3 Suppl 2): 31-5. Additionally, a recent multicenter Phase II trial in patients with relapsed AML demonstrated complete responses (bone marrow blasts <5%) in 17 of 50 patients and a >50% reduction in bone marrow blasts in 31 of 50 patients. Reviewed in Gotlib, J (2005) “Farnesyltransferase inhibitor therapy in acute myelogenous leukemia.” Curr. Hematol. Rep.;4(1):77-84. Preliminary analysis of genes regulated by the FTI treatment in responders in that trial also demonstrated an effect on proteins in the MAPKinase pathway. This promising result has experts in the field anticipating the use of Tipifarnib in the clinic in the near future.
Recently, another target for the treatment of AML, and a subset of patients with MDS and ALL, has emerged. The receptor tyrosine kinase, FLT3 and mutations of FLT3, have been identified as key player in the progression of AML. A summary of the many studies linking FLT3 activity to disease have been extensively reviewed by Gilliland, D. G. and J. D. Griffin (2002). “The roles of FLT3 in hematopoiesis and leukemia.” Blood 100(5): 1532-42, and Stirewalt, D. L. and J. P. Radich (2003). “The role of FLT3 in haematopoietic malignancies.” Nat Rev Cancer 3(9): 650-65. Greater than 90% of patients with AML have FLT3 expression in blast cells. It is now known that roughly 30-40% of patients with AML have an activating mutation of FLT3, making FLT3 mutations the most common mutation in patients with AML. There are two known types of activating mutations of FLT3. One is a duplication of 4-40 amino acids in the juxtamembrane region (ITD mutation) of the receptor (25-30% of patients) and the other is a point mutation in the kinase domain (5-7% of patients). These receptor mutations cause constituitive activation of multiple signal transduction pathways including Ras/MAPkinase, PI3kinase/AKT, and the STAT pathways. Additionally, the FLT3ITD mutation also has been shown to decrease the differentiation of early myeloid cells. More significantly, patients with the ITD mutation have decreased rates of remission induction, decreased remission times, and poorer overall prognosis. FLT3ITD mutations have also been found in ALL with the MLL gene rearrangement and in a sub-population of MDS patients. The presence of the FLT3ITD mutation in MDS and ALL is also correlated with accelerated disease progression and poorer prognosis in these patients. See Shih L. Y. et al., (2004) “Internal tandem duplication of fins-like tyrosine kinase 3 is associated with poor outcome in patients with myelodysplastic syndrome.” Cancer, 101; 989-98; and Armstrong, S. A. et al., (2004) “FLT3 mutations in childhood acute lymphoblastic leukemia.” Blood. 103: 3544-6. To date, there is no strong evidence that suggests either the kinase domain point mutations or the over expressed wild-type receptor is causative of disease, however, FLT3 expression may contribute to the progression of the disease. This building pre-clinical and clinical evidence has led to the development of a number of FLT3 inhibitors which are currently being evaluated in the pre-clinical and clinical setting.
An emerging strategy for the treatment of AML is the combination of target directed therapeutic agents together or with conventional cytotoxic agents during induction and/or post-induction therapy. Recent proof of concept data has been published that demonstrate the combination of the cytotoxic agents (such as cytarabine or daunorubicin) and FLT3 inhibitors inhibit the growth of AML cells expressing FLT3ITD. See Levis, M., R. Pham, et al. (2004). “In vitro studies of a FLT3 inhibitor combined with chemotherapy: sequence of administration is important to achieve synergistic cytotoxic effects.” Blood 104(4): 1145-50, and Yee K W, Schittenhelm M, O'Farrell A M, Town A R, McGreevey L, Bainbridge T, Cherrington J M, Heinrich M C. (2004) “Synergistic effect of SU11248 with cytarabine or daunorubicin on FLT3ITD-positive leukemic cells.” Blood. 104(13):4202-9.
Accordingly, the present invention provides a synergistic method of treatment comprising co-administration (simultaneous or sequential) of a novel FLT3 kinase inhibitor described herein and a farnesyl transferase inhibitor for the treatment of FLT3 expressing cell proliferative disorders.
A variety of FTase inhibitors are currently known. FTIs appropriate for use in the present invention are the following: WO-97/21701 and U.S. Pat. No. 6,037,350, which are incorporated herein in their entirety, describe the preparation, formulation and pharmaceutical properties of certain farnesyl transferase inhibiting (imidazoly-5-yl)methyl-2-quinolinone derivatives of formulas (I), (II) and (III), as well as intermediates of formula (II) and (III) that are metabolized in vivo to the compounds of formula (I). The compounds of formulas (I), (II) and (III) are represented by
the pharmaceutically acceptable acid or base addition salts and the stereochemically isomeric forms thereof, wherein
WO-97/16443 and U.S. Pat. No. 5,968,952, which are incorporated herein in their entirety, describe the preparation, formulation and pharmaceutical properties of farnesyltransferase inhibiting compounds of formula (IV), as well as intermediates of formula (V) and (VI) that are metabolized in vivo to the compounds of formula (IV). The compounds of formulas (IV), (V) and (VI) are represented by
the pharmaceutically acceptable acid or base addition salts and the stereochemically isomeric forms thereof, wherein
WO-98/40383 and U.S. Pat. No. 6,187,786, which are incorporated herein in their entirety, disclose the preparation, formulation and pharmaceutical properties of farnesyltransferase inhibiting compounds of formula (VII)
the pharmaceutically acceptable acid addition salts and the stereochemically isomeric forms thereof, wherein
WO-98/49157 and U.S. Pat. No. 6,117,432, which are incorporated herein in their entirety, concern the preparation, formulation and pharmaceutical properties of farnesyltransferase inhibiting compounds of formula (VIII)
the pharmaceutically acceptable acid addition salts and the stereochemically isomeric forms thereof, wherein
WO-00/39082 and U.S. Pat. No. 6,458,800, which are incorporated herein in their entirety, describe the preparation, formulation and pharmaceutical properties of farnesyltransferase inhibiting compounds of formula (IX)
or the pharmaceutically acceptable acid addition salts and the stereochemically isomeric forms thereof, wherein
In addition to the famesyltransferase inhibitors of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX) above, other farnesyltransferase inhibitors known in the art include: Arglabin (i.e.1(R)-10-epoxy-5(S),7(S)-guaia-3(4),11(13)-dien-6,12-olide described in WO-98/28303 (NuOncology Labs); perrilyl alcohol described in WO-99/45912 (Wisconsin Genetics); SCH-66336, i.e. (+)-(R)-4-[2-[4-(3,10-dibromo-8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-yl)piperidin-1-yl]-2-oxoethyl]piperidine-1-carboxamide, described in U.S. Pat. No. 5874442 (Schering); L778123, i.e. 1-(3-chlorophenyl)-4-[1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone, described in WO-00/01691 (Merck); compound 2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl]-pentyloxy-3-phenylpropionyl-methionine sulfone described in WO-94/10138 (Merck); and BMS 214662, i.e. (R)-2,3,4,5-tetrahydro-1-(IH-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thienylsulphonyl)-1H-1,4-benzodiazapine-7-carbonitrile, described in WO 97/30992 (Bristol Myers Squibb); and Pfizer compounds (A) and (B) described in WO-00/12498 and WO-00/12499:
FLT3 kinase inhibitors known in the art include: AG1295 and AG1296; Lestaurtinib (also known as CEP 701, formerly KT-5555, Kyowa Hakko, licensed to Cephalon); CEP-5214 and CEP-7055 (Cephalon); CHIR-258 (Chiron Corp.); EB-10 and IMC-EB10 (ImClone Systems Inc.); GTP 14564 (Merk Biosciences UK). Midostaurin (also known as PKC 412 Novartis AG); MLN 608 (Millennium USA); MLN-518 (formerly CT53518, COR Therapeutics Inc., licensed to Millennium Pharmaceuticals Inc.); MLN-608 (Millennium Pharmaceuticals Inc.); SU-11248 (Pfizer USA); SU-11657 (Pfizer USA); SU-5416 and SU 5614; THRX-165724 (Theravance Inc.); AMI-10706 (Theravance Inc.); VX-528 and VX-680 (Vertex Pharmaceuticals USA, licensed to Novartis (Switzerland), Merck & Co USA); and XL 999 (Exelixis USA).
See also Levis, M., K. F. Tse, et al. (2001) “A FLT3 tyrosine kinase inhibitor is selectively cytotoxic to acute myeloid leukemia blasts harboring FLT3 internal tandem duplication mutations.” Blood 98(3): 885-7; Tse K F, et al. (2001) Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor. Leukemia. July; 15(7):1001-10; Smith, B. Douglas et al. Single-agent CEP-701, a novel FLT3 inhibitor, shows biologic and clinical activity in patients with relapsed or refractory acute myeloid leukemia Blood, May 2004; 103: 3669-3676; Griswold, Ian J. et al. Effects of MLN518, A Dual FLT3 and KIT Inhibitor, on Normal and Malignant Hematopoiesis. Blood, July 2004; [Epub ahead of print]; Yee, Kevin W. H. et al. SU5416 and SU5614 inhibit kinase activity of wild-type and mutant FLT3 receptor tyrosine kinase. Blood, September 2002; 100: 2941-294; O'Farrell, Anne-Marie et al. SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood, May 2003; 101: 3597-3605; Stone, R. M. et al. PKC 412 FLT3 inhibitor therapy in AML: results of a phase II trial. Ann Hematol. 2004; 83 Suppl 1:S89-90; and Murata, K. et al. Selective cytotoxic mechanism of GTP-14564, a novel tyrosine kinase inhibitor in leukemia cells expressing a constitutively active Fms-like tyrosine kinase 3 (FLT3). J Biol Chem. 2003 Aug 29; 278(35):32892-8; Levis, Mark et al. Novel FLT3 tyrosine kinase inhibitors. Expert Opin. Investing. Drugs (2003) 12(12) 1951-1962; Levis, Mark et al. Small Molecule FLT3 Tyrosine Kinase Inhibitors. Current Pharmaceutical Design, 2004, 10, 1183-1193.
The present invention comprises a method of inhibiting FLT3 tyrosine kinase activity or expression or reducing FLT3 kinase activity or expression in a cell or a subject comprising the administration of a FLT3 kinase inhibitor and a farnesyl transferase inhibitor. Included within the present invention is both prophylactic and therapeutic methods for treating a subject at risk of (or susceptible to) developing a cell proliferative disorder or a disorder related to FLT3, the methods comprising generally administering to the subject a prophylactically effective amount of a FLT3 kinase inhibitor and a farnesyl transferase inhibitor. The FLT3 kinase inhibitor and farnesyl transferase inhibitor can be administered as a unitary pharmaceutical composition comprising a FLT3 kinase inhibitor, a farnesyl transferase inhibitor and a pharmaceutically acceptable carrier, or as separate pharmaceutical compositions: (1) a first pharmaceutical composition comprising a FLT3 kinase inhibitor and a pharmaceutically acceptable carrier, and (2) a second pharmaceutical composition comprising a farnesyl transferase inhibitor and a pharmaceutically acceptable carrier.
The invention further encompasses a multiple component therapy for treating or inhibiting onset of a cell proliferative disorder or a disorder related to FLT3 in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of a FLT3 kinase inhibitor, a farnesyl transferase inhibitor and one or more other anti-cell proliferation therapy(ies) including chemotherapy, radiation therapy, gene therapy and immunotherapy.
Other embodiments, features, advantages, and aspects of the invention will become apparent from the detailed description hereafter in reference to the drawing figures.
a-c. A low dose of a FLT3 inhibitor significantly shifts the potency of Tipifarnib in FLT3 dependent cells.
a-d. Single dose combinations of a FLT3 inhibitor Compound (A) and Tipifarnib or Cytarabine synergistically inhibit FLT3-dependent cell line growth.
a-b. Single dose combination of FLT3 inhibitor Compounds B and D with either Tipifarnib or Cytarabine synergistically inhibits MV4-11 cell growth.
a-c. The combination of a FLT3 inhibitor and an FTI synergistically induces apoptosis of MV4-11 cells.
a-d. Dose responses of single agent induction of caspase 3/7 activation and apoptosis of FLT3 dependent MV4-11 cells.
The terms “comprising”, “including”, and “containing” are used herein in their open, non-limited sense.
The present invention comprises a method of inhibiting FLT3 tyrosine kinase activity or expression or reducing FLT3 kinase activity or expression in a cell or a subject comprising the administration of a FLT3 kinase inhibitor and a farnesyl transferase inhibitor.
An embodiment of the present invention comprises a method for reducing or inhibiting FLT3 tyrosine kinase activity in a subject comprising the administration of a FLT3 kinase inhibitor and a farnesyl transferase inhibitor to the subject.
An embodiment of the present invention comprises a method of treating disorders related to FLT3 tyrosine kinase activity or expression in a subject comprising the administration of a FLT3 kinase inhibitor and a farnesyl transferase inhibitor to the subject.
An embodiment of the present invention comprises a method for reducing or inhibiting the activity of FLT3 tyrosine kinase in a cell comprising the step of contacting the cell with a FLT3 kinase inhibitor and a farnesyl transferase inhibitor.
The present invention also provides a method for reducing or inhibiting the expression of FLT3 tyrosine kinase in a subject comprising the step of administering a FLT3 kinase inhibitor and a farnesyl transferase inhibitor to the subject.
The present invention further provides a method of inhibiting cell proliferation in a cell comprising the step of contacting the cell with a FLT3 kinase inhibitor and a farnesyl transferase inhibitor.
The kinase activity of FLT3 in a cell or a subject can be determined by procedures well known in the art, such as the FLT3 kinase assay described herein.
The term “subject” as used herein, refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
The term “contacting” as used herein, refers to the addition of compound to cells such that compound is taken up by the cell.
In other embodiments to this aspect, the present invention provides both prophylactic and therapeutic methods for treating a subject at risk of (or susceptible to) developing a cell proliferative disorder or a disorder related to FLT3.
In one example, the invention provides methods for preventing in a subject a cell proliferative disorder or a disorder related to FLT3, comprising administering to the subject a prophylactically effective amount of (1) a first pharmaceutical composition comprising a FLT3 kinase inhibitor and a pharmaceutically acceptable carrier, and (2) a second pharmaceutical composition comprising a farnesyl transferase inhibitor and a pharmaceutically acceptable carrier.
In one example, the invention provides methods for preventing in a subject a cell proliferative disorder or a disorder related to FLT3, comprising administering to the subject a prophylactically effective amount of a pharmaceutical composition comprising a FLT3 kinase inhibitor, a farnesyl transferase inhibitor and a pharmaceutically acceptable carrier.
Administration of said prophylactic agent(s) can occur prior to the manifestation of symptoms characteristic of the cell proliferative disorder or disorder related to FLT3, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
In another example, the invention pertains to methods of treating in a subject a cell proliferative disorder or a disorder related to FLT3 comprising administering to the subject a therapeutically effective amount of (1) a first pharmaceutical composition comprising a FLT3 kinase inhibitor and a pharmaceutically acceptable carrier, and (2) a second pharmaceutical composition comprising a farnesyl transferase inhibitor and a pharmaceutically acceptable carrier.
In another example, the invention pertains to methods of treating in a subject a cell proliferative disorder or a disorder related to FLT3 comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a FLT3 kinase inhibitor, a farnesyl transferase inhibitor and a pharmaceutically acceptable carrier.
Administration of said therapeutic agent(s) can occur concurrently with the manifestation of symptoms characteristic of the disorder, such that said therapeutic agent serves as a therapy to compensate for the cell proliferative disorder or disorders related to FLT3.
The FLT3 kinase inhibitor and farnesyl transferase inhibitor can be administered as a unitary pharmaceutical composition comprising a FLT3 kinase inhibitor, a farnesyl transferase inhibitor and a pharmaceutically acceptable carrier, or as separate pharmaceutical compositions: (1) a first pharmaceutical composition comprising a FLT3 kinase inhibitor and a pharmaceutically acceptable carrier, and (2) a second pharmaceutical composition comprising a farnesyl transferase inhibitor and a pharmaceutically acceptable carrier. In the latter case, the two pharmaceutical compositions may be administered simultaneously (albeit in separate compositions), sequentially in either order, at approximately the same time, or on separate dosing schedules. On separate dosing schedules, the two compositions are administered within a period and in an amount and manner that is sufficient to ensure that an advantageous or synergistic effect is achieved.
It will be appreciated that the preferred method and order of administration and the respective dosage amounts and regimes for each component of the combination will depend on the agent being administered, their route of administration, the particular tumor being treated and the particular host being treated.
As will be understood by those of ordinary skill in the art, the optimum method and order of administration and the dosage amounts and regime of the FLT3 kinase inhibitor and farnesyl transferase inhibitor can be readily determined by those skilled in the art using conventional methods and in view of the information set out herein.
Generally, the dosage amounts and regime of the FLT3 kinase inhibitor and farnesyl transferase inhibitor will be similar to or less than those already employed in clinical therapies where these agents are administered alone, or in combination with other chemotherapeutics.
The term “prophylactically effective amount” refers to an amount of an active compound or pharmaceutical agent that inhibits or delays in a subject the onset of a disorder as being sought by a researcher, veterinarian, medical doctor or other clinician.
The term “therapeutically effective amount” as used herein, refers to an amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a subject that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
Methods are known in the art for determining therapeutically and prophylactically effective doses for the instant pharmaceutical composition(s). As used herein, the term “composition” is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combinations of the specified ingredients in the specified amounts.
As used herein, the terms “disorders related to FLT3”, or “disorders related to FLT3 receptor”, or “disorders related to FLT3 receptor tyrosine kinase” shall include diseases associated with or implicating FLT3 activity, for example, the overactivity of FLT3, and conditions that accompany with these diseases. The term “overactivity of FLT3” refers to either 1) FLT3 expression in cells which normally do not express FLT3; 2) FLT3 expression by cells which normally do not express FLT3; 3) increased FLT3 expression leading to unwanted cell proliferation; or 4) mutations leading to constitutive activation of FLT3. Examples of “disorders related to FLT3” include disorders resulting from over stimulation of FLT3 due to abnormally high amount of FLT3 or mutations in FLT3, or disorders resulting from abnormally high amount of FLT3 activity due to abnormally high amount of FLT3 or mutations in FLT3. It is known that overactivity of FLT3 has been implicated in the pathogenesis of a number of diseases, including the cell proliferative disorders, neoplastic disorders and cancers listed below.
The term “cell proliferative disorders” refers to unwanted cell proliferation of one or more subset of cells in a multicellular organism resulting in harm (i.e., discomfort or decreased life expectancy) to the multicellular organisms. Cell proliferative disorders can occur in different types of animals and humans. For example, as used herein “cell proliferative disorders” include neoplastic disorders and other cell proliferative disorders.
As used herein, a “neoplastic disorder” refers to a tumor resulting from abnormal or uncontrolled cellular growth. Examples of neoplastic disorders include, but are not limited to, hematopoietic disorders such as, for instance, the myeloproliferative disorders, such as thrombocythemia, essential thrombocytosis (ET), angiogenic myeloid metaplasia, myelofibrosis (MF), myelofibrosis with myeloid metaplasia (MMM), chronic idiopathic myelofibrosis (IMF), polycythemia vera (PV), the cytopenias, and pre-malignant myelodysplastic syndromes; cancers such as glioma cancers, lung cancers, breast cancers, colorectal cancers, prostate cancers, gastric cancers, esophageal cancers, colon cancers, pancreatic cancers, ovarian cancers, and hematoglogical malignancies, including myelodysplasia, multiple myeloma, leukemias and lymphomas. Examples of hematological malignancies include, for instance, leukemias, lymphomas (non-Hodgkin's lymphoma), Hodgkin's disease (also called Hodgkin's lymphoma), and myeloma—for instance, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute promyelocytic leukemia (APL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic neutrophilic leukemia (CNL), acute undifferentiated leukemia (AUL), anaplastic large-cell lymphoma (ALCL), prolymphocytic leukemia (PML), juvenile myelomonocyctic leukemia (JMML), adult T-cell ALL, AML with trilineage myelodysplasia (AML/TMDS), mixed lineage leukemia (MLL), myelodysplastic syndromes (MDSs), myeloproliferative disorders (MPD), and multiple myeloma, (MM).
In a further embodiment to this aspect, the invention encompasses a multiple component therapy for treating or inhibiting onset of a cell proliferative disorder or a disorder related to FLT3 in a subject comprising administering to the subject a therapeutically or prophylactically effective amount of a FLT3 kinase inhibitor, a farnesyl transferase inhibitor and and one or more other anti-cell proliferation therapy(ies) including chemotherapy, radiation therapy, gene therapy and immunotherapy.
As used herein, “chemotherapy” refers to a therapy involving a chemotherapeutic agent. A variety of chemotherapeutic agents may be used in the multiple component treatment methods disclosed herein. Chemotherapeutic agents contemplated as exemplary, include, but are not limited to: platinum compounds (e.g., cisplatin, carboplatin, oxaliplatin); taxane compounds (e.g., paclitaxcel, docetaxol); campotothecin compounds (irinotecan, topotecan); vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine); anti-tumor nucleoside derivatives (e.g., 5-fluorouracil, leucovorin, gemcitabine, capecitabine); alkylating agents (e.g., cyclophosphamide, carmustine, lomustine, thiotepa); epipodophyllotoxins/podophyllotoxins (e.g. etoposide, teniposide); aromatase inhibitors (e.g., anastrozole, letrozole, exemestane); anti-estrogen compounds (e.g., tamoxifen, fulvestrant), antifolates (e.g., premetrexed disodium); hypomethylating agents (e.g., azacitidine); biologics (e.g., gemtuzamab, cetuximab, rituximab, pertuzumab, trastuzumab, bevacizumab, erlotinib); antibiotics/anthracyclines (e.g. idarubicin, actinomycin D, bleomycin, daunorubicin, doxorubicin, mitomycin C, dactinomycin, carminomycin, daunomycin); antimetabolites (e.g., aminopterin, clofarabine, cytosine arabinoside, methotrexate); tubulin-binding agents (e.g. combretastatin, colchicine, nocodazole); topoisomerase inhibitors (e.g., camptothecin). Further useful agents include verapamil, a calcium antagonist found to be useful in combination with antineoplastic agents to establish chemosensitivity in tumor cells resistant to accepted chemotherapeutic agents and to potentiate the efficacy of such compounds in drug-sensitive malignancies. See Simpson W G, The calcium channel blocker verapamil and cancer chemotherapy. Cell Calcium. 1985 December; 6(6):449-67. Additionally, yet to emerge chemotherapeutic agents are contemplated as being useful in combination with the compound of the present invention.
In another embodiment of the present invention, the FLT3 kinase inhibitor and farnesyl transferase inhibitor may be administered in combination with radiation therapy. As used herein, “radiation therapy” refers to a therapy that comprises exposing the subject in need thereof to radiation. Such therapy is known to those skilled in the art. The appropriate scheme of radiation therapy will be similar to those already employed in clinical therapies wherein the radiation therapy is used alone or in combination with other chemotherapeutics.
In another embodiment of the present invention, the FLT3 kinase inhibitor and farnesyl transferase inhibitor may be administered in combination with gene therapy. As used herein, “gene therapy” refers to a therapy targeting on particular genes involved in tumor development. Possible gene therapy strategies include the restoration of defective cancer-inhibitory genes, cell transduction or transfection with antisense DNA corresponding to genes coding for growth factors and their receptors, RNA-based strategies such as ribozymes, RNA decoys, antisense messenger RNAs and small interfering RNA (siRNA) molecules and the so-called 'suicide genes'.
In other embodiments of this invention, the FLT3 kinase inhibitor and farnesyl transferase inhibitor may be administered in combination with immunotherapy. As used herein, “immunotherapy” refers to a therapy targeting particular protein involved in tumor development via antibodies specific to such protein. For example, monoclonal antibodies against vascular endothelial growth factor have been used in treating cancers.
Where one or more additional chemotherapeutic agent(s) are used in conjunction with the FLT3 kinase inhibitor and farnesyl transferase inhibitor, the additional chemotherapeutic agent(s), the FLT3 kinase inhibitor and the farnesyl transferase inhibitor may be administered simultaneously (e.g. in separate or unitary compositions) sequentially in any order, at approximately the same time, or on separate dosing schedules. In the latter case, the pharmaceuticals will be administered within a period and in an amount and manner that is sufficient to ensure that an advantageous and synergistic effect is achieved. It will be appreciated that the preferred method and order of administration and the respective dosage amounts and regimes for the additional chemotherapeutic agent(s) will depend on the particular chemotherapeutic agent(s) being administered in conjunction with the FLT3 kinase inhibitor and farnesyl transferase inhibitor, their route of administration, the particular tumor being treated and the particular host being treated. As will be understood by those of ordinary skill in the art, the appropriate doses of the additional chemotherapeutic agent(s) will be generally similar to or less than those already employed in clinical therapies wherein the chemotherapeutics are administered alone or in combination with other chemotherapeutics.
The optimum method and order of administration and the dosage amounts and regime can be readily determined by those skilled in the art using conventional methods and in view of the information set out herein.
By way of example only, platinum compounds are advantageously administered in a dosage of 1 to 500 mg per square meter (mg/m2) of body surface area, for example 50 to 400 mg/m2, particularly for cisplatin in a dosage of about 75 mg/m2 and for carboplatin in about 300 mg/m2 per course of treatment. Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
By way of example only, taxane compounds are advantageously administered in a dosage of 50 to 400 mg per square meter (mg/m2) of body surface area, for example 75 to 250 mg/m2, particularly for paclitaxel in a dosage of about 175 to 250 mg/m2 and for docetaxel in about 75 to 150 mg/m2 per course of treatment.
By way of example only, camptothecin compounds are advantageously administered in a dosage of 0.1 to 400 mg per square meter (mg/m2) of body surface area, for example 1 to 300 mg/m2, particularly for irinotecan in a dosage of about 100 to 350 mg/m2 and for topotecan in about 1 to 2 mg/m2 per course of treatment.
By way of example only, vinca alkaloids may be advantageously administered in a dosage of 2 to 30 mg per square meter (mg/m2) of body surface area, particularly for vinblastine in a dosage of about 3 to 12 mg/m2, for vincristine in a dosage of about 1 to 2 mg/m2, and for vinorelbine in dosage of about 10 to 30 mg/m2 per course of treatment.
By way of example only, anti-tumor nucleoside derivatives may be advantageously administered in a dosage of 200 to 2500 mg per square meter (mg/m2) of body surface area, for example 700 to 1500 mg/m2. 5-fluorouracil (5-FU) is commonly used via intravenous administration with doses ranging from 200 to 500 mg/m2 (preferably from 3 to 15 mg/kg/day). Gemcitabine is advantageously administered in a dosage of about 800 to 1200 mg/m2 and capecitabine is advantageously administered in about 1000 to 2500 mg/m2 per course of treatment.
By way of example only, alkylating agents may be advantageously administered in a dosage of 100 to 500 mg per square meter (mg/m2) of body surface area, for example 120 to 200 mg/m2, particularly for cyclophosphamide in a dosage of about 100 to 500 mg/m2, for chlorambucil in a dosage of about 0.1 to 0.2 mg/kg of body weight, for carmustine in a dosage of about 150 to 200 mg/m2, and for lomustine in a dosage of about 100 to 150 mg/m2 per course of treatment.
By way of example only, podophyllotoxin derivatives may be advantageously administered in a dosage of 30 to 300 mg per square meter (mg/m2) of body surface area, for example 50 to 250 mg/m2, particularly for etoposide in a dosage of about 35 to 100 mg/m2 and for teniposide in about 50 to 250 mg/m2 per course of treatment.
By way of example only, anthracycline derivatives may be advantageously administered in a dosage of 10 to 75 mg per square meter (mg/m2) of body surface area, for example 15 to 60 mg/m2, particularly for doxorubicin in a dosage of about 40 to 75 mg/m2, for daunorubicin in a dosage of about 25 to 45 mg/m2, and for idarubicin in a dosage of about 10 to 15 mg/m2 per course of treatment.
By way of example only, anti-estrogen compounds may be advantageously administered in a dosage of about 1 to 100 mg daily depending on the particular agent and the condition being treated. Tamoxifen is advantageously administered orally in a dosage of 5 to 50 mg, preferably 10 to 20 mg twice a day, continuing the therapy for sufficient time to achieve and maintain a therapeutic effect. Toremifene is advantageously administered orally in a dosage of about 60 mg once a day, continuing the therapy for sufficient time to achieve and maintain a therapeutic effect. Anastrozole is advantageously administered orally in a dosage of about 1 mg once a day. Droloxifene is advantageously administered orally in a dosage of about 20-100 mg once a day. Raloxifene is advantageously administered orally in a dosage of about 60 mg once a day. Exemestane is advantageously administered orally in a dosage of about 25 mg once a day.
By way of example only, biologics may be advantageously administered in a dosage of about 1 to 5 mg per square meter (mg/m2) of body surface area, or as known in the art, if different. For example, trastuzumab is advantageously administered in a dosage of 1 to 5 mg/m2 particularly 2 to 4 mg/m2 per course of treatment.
Dosages may be administered, for example once, twice or more per course of treatment, which may be repeated for example every 7, 14, 21 or 28 days.
The FLT3 kinase inhibitor and farnesyl transferase inhibitor can be administered to a subject systemically, for example, intravenously, orally, subcutaneously, intramuscular, intradermal, or parenterally. The FLT3 kinase inhibitor and farnesyl transferase inhibitor can also be administered to a subject locally. Non-limiting examples of local delivery systems include the use of intraluminal medical devices that include intravascular drug delivery catheters, wires, pharmacological stents and endoluminal paving. The FLT3 kinase inhibitor and farnesyl transferase inhibitor can further be administered to a subject in combination with a targeting agent to achieve high local concentration of the FLT3 kinase inhibitor and farnesyl transferase inhibitor at the target site. In addition, the FLT3 kinase inhibitor and farnesyl transferase inhibitor may be formulated for fast-release or slow-release with the objective of maintaining the drugs or agents in contact with target tissues for a period ranging from hours to weeks.
The separate pharmaceutical compositions comprising the FLT3 kinase inhibitor in association with a pharmaceutically acceptable carrier, and the farnesyl transferase inhibitor in association with a pharmaceutically acceptable carrier may contain between about 0.1 mg and 1000 mg, preferably about 100 to 500 mg, of the individual agents compound, and may be constituted into any form suitable for the mode of administration selected.
The unitary pharmaceutical composition comprising the FLT3 kinase inhibitor and farnesyl transferase inhibitor in association with a pharmaceutically acceptable carrier may contain between about 0.1 mg and 1000 mg, preferably about 100 to 500 mg, of the compound, and may be constituted into any form suitable for the mode of administration selected.
The phrases “pharmaceutically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. Veterinary uses are equally included within the invention and “pharmaceutically acceptable” formulations include formulations for both clinical and/or veterinary use.
Carriers include necessary and inert pharmaceutical excipients, including, but not limited to, binders, suspending agents, lubricants, flavorants, sweeteners, preservatives, dyes, and coatings. Compositions suitable for oral administration include solid forms, such as pills, tablets, caplets, capsules (each including immediate release, timed release and sustained release formulations), granules, and powders, and liquid forms, such as solutions, syrups, elixirs, emulsions, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions and suspensions.
The pharmaceutical compositions of the present invention, whether unitary or separate, may be formulated for slow release of the FLT3 kinase inhibitor and farnesyl transferase inhibitor. Such a composition, unitary or separate, includes a slow release carrier (typically, a polymeric carrier) and one, or in the case of the unitary composition, both, of the FLT3 kinase inhibitor and farnesyl transferase inhibitor.
Slow release biodegradable carriers are well known in the art. These are materials that may form particles that capture therein an active compound(s) and slowly degrade/dissolve under a suitable environment (e.g., aqueous, acidic, basic, etc) and thereby degrade/dissolve in body fluids and release the active compound(s) therein. The particles are preferably nanoparticles (i.e., in the range of about 1 to 500 nm in diameter, preferably about 50-200 nm in diameter, and most preferably about 100 nm in diameter).
Farnesyltranferase Inhibitors
Examples of famesyltransferase inhibitors which may be employed in the methods or treatments in accordance with the present invention include the famesyltransferase inhibitors (“FTIs”) of formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX) above.
Preferred FTIs include compounds of formula (I), (II) or (III):
the pharmaceutically acceptable acid or base addition salts and the stereochemically isomeric forms thereof, wherein
In Formulas (I), (II) and (III), R4 or R5 may also be bound to one of the nitrogen atoms in the imidazole ring. In that case the hydrogen on the nitrogen is replaced by R4 or R5 and the meaning of R4 and R5 when bound to the nitrogen is limited to hydrogen, Ar1, C1-6alkyl, hydroxyC1-6alkyl, C1-6alkyloxyC1-6alkyl, C1-6alkyloxycarbonyl, C1-6alkylS(O)C1-6alkyl, C1-6alkylS(O)2C1-6alkyl.
Preferably the substituent R18 in Formulas (I), (II) and (III) is situated on the 5 or 7 position of the quinolinone moiety and substituent R19 is situated on the 8 position when R18 is on the 7-position.
Preferred examples of FTIs are those compounds of formula (I) wherein X is oxygen.
Also, examples of preferred FTIs are those compounds of formula (I) wherein the dotted line represents a bond, so as to form a double bond.
Another group of preferred FTIs are those compounds of formula (I) wherein R1 is hydrogen, C1-6alkyl, C1-6alkyloxyC1-6alkyl, di(C1-6alkyl)aminoC1-6alkyl, or a radical of formula -Alk1-C(═O)—R9, wherein Alk1 is methylene and R9 is C1-8alkylamino substituted with C1-6alkyloxycarbonyl.
Still another group of preferred FTIs are those compounds of formula (I) wherein R3 is hydrogen or halo; and R2 is halo, C1-6alkyl, C2-6alkenyl, C1-6alkyloxy, trihalomethoxy or hydroxyC1-6alkyloxy.
A further group of preferred FTIs are those compounds of formula (I) wherein R2 and R3 are on adjacent positions and taken together to form a bivalent radical of formula (a-1), (a-2) or (a-3).
A still further group of preferred FTIs are those compounds of formula (I) wherein R5 is hydrogen and R4 is hydrogen or C1-6alkyl.
Yet another group of preferred FTIs are those compounds of formula (I) wherein R7 is hydrogen; and R6 is C1-6alkyl or halo, preferably chloro, especially 4-chloro.
Another exemplary group of preferred FTIs are those compounds of formula (I) wherein R8 is hydrogen, hydroxy, haloC1-6alkyl, hydroxyC1-6alkyl, cyanoC1-6alkyl, C1-6alkyloxycarbonylC1-6alkyl, imidazolyl, or a radical of formula —NR11R12 wherein R11 is hydrogen or C1-12alkyl and R12 is hydrogen, C1-6alkyl, C1-6alkyloxy, hydroxy, C1-6alkyloxyC1-6alkylcarbonyl, or a radical of formula -Alk2-OR13 wherein R13 is hydrogen or C1-6alkyl.
Preferred compounds are also those compounds of formula (I) wherein R1 is hydrogen, C1-6alkyl, C1-6alkyloxyC1-6alkyl, di(C1-6alkyl)aminoC1-6alkyl, or a radical of formula -Alk1-C(═O)—R9, wherein Alk1 is methylene and R9 is C1-8alkylamino substituted with C1-6alkyloxycarbonyl; R2 is halo, C1-6alkyl, C2-6alkenyl, C1-6alkyloxy, trihalomethoxy, hydroxyC1-6alkyloxy or Ar1; R3 is hydrogen; R4 is methyl bound to the nitrogen in 3-position of the imidazole; R5 is hydrogen; R6 is chloro; R7 is hydrogen; R8 is hydrogen, hydroxy, haloC1-6alkyl, hydroxyC1-6alkyl, cyanoC1-6alkyl, C1-6alkyloxycarbonylC1-6alkyl, imidazolyl, or a radical of formula —NR11R12 wherein R11 is hydrogen or C1-12alkyl and R12 is hydrogen, C1-6alkyl, C1-6alkyloxy, C1-6alkyloxyC1-6alkylcarbonyl, or a radical of formula -Alk2-OR13 wherein R13 is C1-6alkyl; R17 is hydrogen and R18 is hydrogen.
Especially preferred FTIs are:
Tipifarnib or ZARNESTRA® is an especially preferred FTI.
Further preferred FTIs include compounds of formula (IX) wherein one or more of the following apply:
Another group of preferred FTIs are compounds of formula (IX) wherein =X1—X2—X3 is a trivalent radical of formula (x-1), (x-2), (x-3), (x-4) or (x-9), >Y1-Y2 is a trivalent radical of formula (y-2), (y-3) or (y-4), r is 0 or 1, s is 1, t is 0, R1 is halo, C(1-4)alkyl or forms a bivalent radical of formula (a-1), R2 is halo or C1-4alkyl, R3 is hydrogen or a radical of formula (b-1) or (b-3), R4 is a radical of formula (c-1) or (c-2), R6 is hydrogen, C1-4alkyl or phenyl, R7 is hydrogen, R9 is hydrogen or C1-4alkyl, R10 is hydrogen or -Alk-OR13, R11 is hydrogen and R12 is hydrogen or C1-6alkylcarbonyl and R13 is hydrogen;
Preferred FTIs are those compounds of formula (IX) wherein =X1—X2—X3 is a trivalent radical of formula (x-1) or (x-4), >Y1-Y2 is a trivalent radical of formula (y-4), r is 0 or 1, s is 1, t is 0, R1 is halo, preferably chloro and most preferably 3-chloro, R2 is halo, preferably 4-chloro or 4-fluoro, R3 is hydrogen or a radical of formula (b-1) or (b-3), R4 is a radical of formula (c-1) or (c-2), R6 is hydrogen, R7 is hydrogen, R9 is hydrogen, R10 is hydrogen, R11 is hydrogen and R12 is hydrogen.
Other preferred FTIs are those compounds of formula (IX) wherein =X1—X2—X3 is a trivalent radical of formula (x-2), (x-3) or (x-4), >Y1-Y2 is a trivalent radical of formula (y-2), (y-3) or (y-4), r and s are 1, t is 0, R1 is halo, preferably chloro, and most preferably 3-chloro or R1 is C1-4alkyl, preferably 3-methyl, R2 is halo, preferably chloro, and most preferably 4-chloro, R3 is a radical of formula (b-1) or (b-3), R4 is a radical of formula (c-2), R6 is C1-4alkyl, R9 is hydrogen, R10 and R11 are hydrogen and R12 is hydrogen or hydroxy.
Especially preferred FTI compounds of formula (IX) are:
5-(3-chlorophenyl)-α-(4-chlorophenyl)-α-(1-methyl-1H-imidazol-5-yl)tetrazolo[1,5-a]quinazoline-7-methanamine, especially the (−) enantiomer, and its pharmaceutically acceptable acid addition salts is an especially preferred FTI.
The pharmaceutically acceptable acid or base addition salts as mentioned hereinabove are meant to comprise the therapeutically active non-toxic acid and non-toxic base addition salt forms which the FTI compounds of formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX) are able to form. The FTI compounds of formulas (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX) which have basic properties can be converted in their pharmaceutically acceptable acid addition salts by treating the base form with an appropriate acid. Appropriate acids include, for example, inorganic acids such as hydrohalic acids, e.g. hydrochloric or hydrobromic acid; sulfuric; nitric; phosphoric and the like acids; or organic acids, such as acetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e. butanedioic acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and the like acids.
The FTI compounds of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX) which have acidic properties may be converted in their pharmaceutically acceptable base addition salts by treating the acid form with a suitable organic or inorganic base. Appropriate base salt forms comprise, for example, the ammonium salts, the alkali and earth alkaline metal salts, e.g. the lithium, sodium, potassium, magnesium, calcium salts and the like, salts with organic bases, e.g. the benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids, for example, arginine, lysine and the like.
Acid and base addition salts also comprise the hydrates and the solvent addition forms which the preferred FTI compounds of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX) are able to form. Examples of such forms are e.g. hydrates, alcoholates and the like.
The FTI compounds of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX), as used hereinbefore, encompass all stereochemically isomeric forms of the depicted structural formulae (all possible compounds made up of the same atoms bonded by the same sequence of bonds but having different three-dimensional structures that are not interchangeable). Unless otherwise mentioned or indicated, the chemical designation of an FTI compound should be understood as encompassing the mixture of all possible stereochemically isomeric forms which the compound may possess. Such mixture may contain all diastereomers and/or enantiomers of the basic molecular structure of the compound. All stereochemically isomeric forms of the FTI compounds of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX) both in pure form or in admixture with each other are intended to be embraced within the scope of the depicted formulae.
Some of the FTI compounds of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX) may also exist in their tautomeric forms. Such forms, although not explicitly shown in the above formulae, are intended to be included within the scope thereof.
Thus, unless indicated otherwise hereinafter, the terms “compounds of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX)” and “farnesyltransferase inhibitors of formulae (I), (II), (III), (IV), (V), (VI), (VII), (VIII) or (IX)” are meant to include also the pharmaceutically acceptable acid or base addition salts and all stereoisomeric and tautomeric forms.
Other farnesyltransferase inhibitors which can be employed in accordance with the present invention include: Arglabin, perrilyl alcohol, SCH-66336, 2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3 (S)-methyl]-pentyloxy-3-phenylpropionyl-methionine sulfone (Merck); L778123, BMS 214662, Pfizer compounds A and B described above. Suitable dosages or therapeutically effective amounts for the compounds Arglabin (W098/28303), perrilyl alcohol (WO 99/45712), SCH-66336 (U.S. Pat. No. 5,874,442), L778123 (WO 00/01691), 2(S)-[2(S)-[2(R)-amino-3-mercapto]propylamino-3(S)-methyl]-pentyloxy-3-phenylpropionyl-methionine sulfone (WO94/10138), BMS 214662 (WO 97/30992), Pfizer compounds A and B (WO 00/12499 and WO 00/12498) are given in the published patent specifications or are known to or can be readily determined by a person skilled in the art.
FLT3 Kinase Inhibitors
The FLT3 kinase inhibitors of the present invention comprise compounds Formula I′:
and N-oxides, pharmaceutically acceptable salts, solvates, and stereochemical isomers thereof, wherein:
As used hereafter, the term “compounds of Formula I′” is meant to include also the N-oxides, pharmaceutically acceptable salts, solvates, and stereochemical isomers thereof.
FLT3 Inhibitors of Formula I′—Abbreviations & Definitions
As used in regards to the FLT3 inhibitors of Formula I′, the following terms are intended to have the following meanings:
(Additional abbreviations are provided where needed throughout the Specification.)
As used in regards to the FLT3 inhibitors of Formula I′, the following terms are intended to have the following meanings (additional definitions are provided where needed throughout the Specification):
The term “alkenyl,” whether used alone or as part of a substituent group, for example, “C1-4alkenyl(aryl),” refers to a partially unsaturated branched or straight chain monovalent hydrocarbon radical having at least one carbon-carbon double bond, whereby the double bond is derived by the removal of one hydrogen atom from each of two adjacent carbon atoms of a parent alkyl molecule and the radical is derived by the removal of one hydrogen atom from a single carbon atom. Atoms may be oriented about the double bond in either the cis (Z) or trans (E) conformation. Typical alkenyl radicals include, but are not limited to, ethenyl, propenyl, allyl (2-propenyl), butenyl and the like. Examples include C2-8alkenyl or C2-4alkenyl groups.
The term “Ca-b” (where a and b are integers referring to a designated number of carbon atoms) refers to an alkyl, alkenyl, alkynyl, alkoxy or cycloalkyl radical or to the alkyl portion of a radical in which alkyl appears as the prefix root containing from a to b carbon atoms inclusive. For example, C1-4 denotes a radical containing 1, 2, 3 or 4 carbon atoms.
The term “alkyl,” whether used alone or as part of a substituent group, refers to a saturated branched or straight chain monovalent hydrocarbon radical, wherein the radical is derived by the removal of one hydrogen atom from a single carbon atom. Unless specifically indicated (e.g. by the use of a limiting term such as “terminal carbon atom”), substituent variables may be placed on any carbon chain atom. Typical alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl and the like. Examples include C1-8alkyl, C1-6alkyl and C1-4alkyl groups.
The term “alkylamino” refers to a radical formed by the removal of one hydrogen atom from the nitrogen of an alkylamine, such as butylamine, and the term “dialkylamino” refers to a radical formed by the removal of one hydrogen atom from the nitrogen of a secondary amine, such as dibutylamine. In both cases it is expected that the point of attachment to the rest of the molecule is the nitrogen atom.
The term “alkynyl,” whether used alone or as part of a substituent group, refers to a partially unsaturated branched or straight chain monovalent hydrocarbon radical having at least one carbon-carbon triple bond, whereby the triple bond is derived by the removal of two hydrogen atoms from each of two adjacent carbon atoms of a parent alkyl molecule and the radical is derived by the removal of one hydrogen atom from a single carbon atom. Typical alkynyl radicals include ethynyl, propynyl, butynyl and the like. Examples include C2-8alkynyl or C2-4alkynyl groups.
The term “alkoxy” refers to a saturated or partially unsaturated branched or straight chain monovalent hydrocarbon alcohol radical derived by the removal of the hydrogen atom from the hydroxide oxygen substituent on a parent alkane, alkene or alkyne. Where specific levels of saturation are intended, the nomenclature “alkoxy”, “alkenyloxy” and “alkynyloxy” are used consistent with the definitions of alkyl, alkenyl and alkynyl. Examples include C1-8alkoxy or C1-4alkoxy groups.
The term “alkoxyether” refers to a saturated branched or straight chain monovalent hydrocarbon alcohol radical derived by the removal of the hydrogen atom from the hydroxide oxygen substituent on a hydroxyether. Examples include 1-hydroxyl-2-methoxy-ethane and 1-(2-hydroxyl-ethoxy)-2-methoxy-ethane groups.
The term “aralkyl” refers to a C1-6 alkyl group containing an aryl substituent. Examples include benzyl, phenylethyl or 2-naphthylmethyl. It is intended that the point of attachment to the rest of the molecule be the alkyl group.
The term “aromatic” refers to a cyclic hydrocarbon ring system having an unsaturated, conjugated π electron system.
The term “aryl” refers to an aromatic cyclic hydrocarbon ring radical derived by the removal of one hydrogen atom from a single carbon atom of the ring system. Typical aryl radicals include phenyl, naphthalenyl, fluorenyl, indenyl, azulenyl, anthracenyl and the like.
The term “arylamino” refers to an amino group, such as ammonia, substituted with an aryl group, such as phenyl. It is expected that the point of attachment to the rest of the molecule is through the nitrogen atom.
The term “benzo-fused cycloalkyl” refers to a bicyclic fused ring system radical wherein one of the rings is phenyl and the other is a cycloalkyl or cycloalkenyl ring. Typical benzo-fused cycloalkyl radicals include indanyl, 1,2,3,4-tetrahydro-naphthalenyl, 6,7,8,9,-tetrahydro-5H-benzocycloheptenyl, 5,6,7,8,9,10-hexahydro-benzocyclooctenyl and the like. A benzo-fused cycloalkyl ring system is a subset of the aryl group.
The term “benzo-fused heteroaryl” refers to a bicyclic fused ring system radical wherein one of the rings is phenyl and the other is a heteroaryl ring. Typical benzo-fused heteroaryl radicals include indolyl, indolinyl, isoindolyl, benzo[b]furyl, benzo[b]thienyl, indazolyl, benzthiazolyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, and the like. A benzo-fused heteroaryl ring is a subset of the heteroaryl group.
The term “benzo-fused heterocyclyl” refers to a bicyclic fused ring system radical wherein one of the rings is phenyl and the other is a heterocyclyl ring. Typical benzo-fused heterocyclyl radicals include 1,3-benzodioxolyl (also known as 1,3-methylenedioxyphenyl), 2,3-dihydro-1,4-benzodioxinyl (also known as 1,4-ethylenedioxyphenyl), benzo-dihydro-furyl, benzo-tetrahydro-pyranyl, benzo-dihydro-thienyl and the like.
The term “carboxyalkyl” refers to an alkylated carboxy group such as tert-butoxycarbonyl, in which the point of attachment to the rest of the molecule is the carbonyl group.
The term “cyclic heterodionyl” refers to a heterocyclic compound bearing two oxo substituents. Examples include thiazolidinedionyl, oxazolidinedionyl and pyrrolidinedionyl.
The term “cycloalkenyl” refers to a partially unsaturated cycloalkyl radical derived by the removal of one hydrogen atom from a hydrocarbon ring system that contains at least one carbon-carbon double bond. Examples include cyclohexenyl, cyclopentenyl and 1,2,5,6-cyclooctadienyl.
The term “cycloalkyl” refers to a saturated or partially unsaturated monocyclic or bicyclic hydrocarbon ring radical derived by the removal of one hydrogen atom from a single ring carbon atom. Typical cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl and cyclooctyl. Additional examples include C3-8cycloalkyl, C5-8cycloalkyl, C3-12cycloalkyl, C3-20cycloalkyl, decahydronaphthalenyl, and 2,3,4,5,6,7-hexahydro-1H-indenyl.
The term “fused ring system” refers to a bicyclic molecule in which two adjacent atoms are present in each of the two cyclic moieties. Heteroatoms may optionally be present. Examples include benzothiazole, 1,3-benzodioxole and decahydronaphthalene.
The term “hetero” used as a prefix for a ring system refers to the replacement of at least one ring carbon atom with one or more atoms independently selected from N, S, O or P. Examples include rings wherein 1, 2, 3 or 4 ring members are a nitrogen atom; or, 0, 1, 2 or 3 ring members are nitrogen atoms and 1 member is an oxygen or sulfur atom.
The term “heteroaralkyl” refers to a C1-6 alkyl group containing a heteroaryl substituent. Examples include furylmethyl and pyridylpropyl. It is intended that the point of attachment to the rest of the molecule be the alkyl group.
The term “heteroaryl” refers to a radical derived by the removal of one hydrogen atom from a ring carbon atom of a heteroaromatic ring system. Typical heteroaryl radicals include furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, indolyl, isoindolyl, benzo[b]furyl, benzo[b]thienyl, indazolyl, benzimidazolyl, benzthiazolyl, purinyl, 4H-quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalzinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl and the like.
The term “heteroaryl-fused cycloalkyl” refers to a bicyclic fused ring system radical wherein one of the rings is cycloalkyl and the other is heteroaryl. Typical heteroaryl-fused cycloalkyl radicals include 5,6,7,8-tetrahydro-4H-cyclohepta(b)thienyl, 5,6,7-trihydro-4H-cyclohexa(b)thienyl, 5,6-dihydro-4H-cyclopenta(b)thienyl and the like.
The term “heterocyclyl” refers to a saturated or partially unsaturated monocyclic ring radical derived by the removal of one hydrogen atom from a single carbon or nitrogen ring atom. Typical heterocyclyl radicals include 2H-pyrrolyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl, 1,3-dioxolanyl, 2-imidazolinyl (also referred to as 4,5-dihydro-1H-imidazolyl), imidazolidinyl, 2-pyrazolinyl, pyrazolidinyl, tetrazolyl, piperidinyl, 1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl, thiomorpholinyl 1,1 dioxide, piperazinyl, azepanyl, hexahydro-1,4-diazepinyl and the like.
The term “oxo” refers to an oxygen atom radical; said oxygen atom has two open valencies which are bonded to the same atom, most preferably a carbon atom. The oxo group is an appropriate substituent for an alkyl group. For example, propane with an oxo substituent is either acetone or propionaldehyde. Heterocycles can also be substituted with an oxo group. For example, oxazolidine with an oxo substituent is oxazolidinone.
The term “squaryl” refers to a cyclobutenyl 1,2 dione radical.
The term “substituted,” refers to a core molecule on which one or more hydrogen atoms have been replaced with one or more functional radical moieties. Substitution is not limited to a core molecule, but may also occur on a substituent radical, whereby the substituent radical becomes a linking group.
The term “independently selected” refers to one or more substituents selected from a group of substituents, wherein the substituents may be the same or different.
The substituent nomenclature used in the disclosure of the FLT3 inhibitors of Formula I′ was derived by first indicating the atom having the point of attachment, followed by the linking group atoms toward the terminal chain atom from left to right, substantially as in:
(C1-6)alkylC(O)NH(C1-6)alkyl(Ph)
or by first indicating the terminal chain atom, followed by the linking group atoms toward the atom having the point of attachment, substantially as in:
Ph(C1-6)alkylamido(C1-6)alkyl
either of which refers to a radical of the formula:
Additionally, lines drawn into ring systems from substituents indicate that the bond may be attached to any of the suitable ring atoms.
When any variable (e.g. R4) occurs more than one time in any embodiment of the FLT3 inhibitors of Formula I′, each definition is intended to be independent.
Embodiments of FLT3 Inhibitors of Formula I′
In an embodiment of the FLT3 inhibitors of Formula I′: N-oxides are optionally present on one or more of: N-1 or N-3 (when X is N) (see
Preferred embodiments of the the FLT3 inhibitors of Formula I′ are compounds of Formula I′ wherein one or more of the following limitations are present:
Other preferred embodiments of the FLT3 inhibitors of Formula I′ are compounds of Formula I′ wherein one or more of the following limitations are present:
Still other preferred embodiments of the FLT3 inhibitors of Formula I′ are compounds of Formula I′ wherein one or more of the following limitations are present:
Particularly preferred embodiments of the FLT3 inhibitors of Formula I′ are compounds of Formula I′ wherein one or more of the following limitations are present:
Most particularly preferred embodiments of the FLT3 inhibitors of Formula I′ are compounds of Formula I′ wherein one or more of the following limitations are present:
The FLT3 inhibitors of Formula I′ may also be present in the form of pharmaceutically acceptable salts.
For use in medicines, the salts of the compounds of the FLT3 inhibitors of Formula I′ refer to non-toxic “pharmaceutically acceptable salts.” FDA approved pharmaceutically acceptable salt forms (Ref. International J. Pharm. 1986, 33, 201-217; J. Pharm. Sci., 1977, January, 66(1), p1) include pharmaceutically acceptable acidic/anionic or basic/cationic salts.
Pharmaceutically acceptable acidic/anionic salts include, and are not limited to acetate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, chloride, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, glyceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, pamoate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, teoclate, tosylate and triethiodide. Organic or inorganic acids also include, and are not limited to, hydriodic, perchloric, sulfuric, phosphoric, propionic, glycolic, methanesulfonic, hydroxyethanesulfonic, oxalic, 2-naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, saccharinic or trifluoroacetic acid.
Pharmaceutically acceptable basic/cationic salts include, and are not limited to aluminum, 2-amino-2-hydroxymethyl-propane-1,3-diol (also known as tris(hydroxymethyl)aminomethane, tromethane or “TRIS”), ammonia, benzathine, t-butylamine, calcium, calcium gluconate, calcium hydroxide, chloroprocaine, choline, choline bicarbonate, choline chloride, cyclohexylamine, diethanolamine, ethylenediamine, lithium, LiOMe, L-lysine, magnesium, meglumine, NH3, NH4OH, N-methyl-D-glucamine, piperidine, potassium, potassium-t-butoxide, potassium hydroxide (aqueous), procaine, quinine, sodium, sodium carbonate, sodium-2-ethylhexanoate (SEH), sodium hydroxide, triethanolamine (TEA) or zinc.
The FLT3 inhibitors of the present invention includes within its scope prodrugs of the compounds of Formula I′. In general, such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into an active compound. Thus, in the methods of treatment of the present invention, the term “administering” shall encompass the means for treating, ameliorating or preventing a syndrome, disorder or disease described herein with a FLT3 inhibitor of Formula I′ specifically disclosed or a compound, or prodrug thereof, which would obviously be included within the scope of the invention albeit not specifically disclosed for certain of the instant compounds. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described in, for example, “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.
One skilled in the art will recognize that the FLT3 inhibitors of Formula I′ may have one or more asymmetric carbon atoms in their structure. It is intended that the present invention include within its scope single enantiomer forms of the FLT3 inhibitors of Formula I′, racemic mixtures, and mixtures of enantiomers in which an enantiomeric excess is present.
The term “single enantiomer” as used herein defines all the possible homochiral forms which the compounds of Formula I′ and their N-oxides, addition salts, quaternary amines or physiologically functional derivatives may possess.
Stereochemically pure isomeric forms may be obtained by the application of art known principles. Diastereoisomers may be separated by physical separation methods such as fractional crystallization and chromatographic techniques, and enantiomers may be separated from each other by the selective crystallization of the diastereomeric salts with optically active acids or bases or by chiral chromatography. Pure stereoisomers may also be prepared synthetically from appropriate stereochemically pure starting materials, or by using stereoselective reactions.
The term “isomer” refers to compounds that have the same composition and molecular weight but differ in physical and/or chemical properties. Such substances have the same number and kind of atoms but differ in structure. The structural difference may be in constitution (geometric isomers) or in an ability to rotate the plane of polarized light (enantiomers).
The term “stereoisomer” refers to isomers of identical constitution that differ in the arrangement of their atoms in space. Enantiomers and diastereomers are examples of stereoisomers.
The term “chiral” refers to the structural characteristic of a molecule that makes it impossible to superimpose it on its mirror image.
The term “enantiomer” refers to one of a pair of molecular species that are mirror images of each other and are not superimposable.
The term “diastereomer” refers to stereoisomers that are not mirror images.
The symbols “R” and “S” represent the configuration of substituents around a chiral carbon atom(s).
The term “racemate” or “racemic mixture” refers to a composition composed of equimolar quantities of two enantiomeric species, wherein the composition is devoid of optical activity.
The term “homochiral” refers to a state of enantiomeric purity.
The term “optical activity” refers to the degree to which a homochiral molecule or nonracemic mixture of chiral molecules rotates a plane of polarized light.
The term “geometric isomer” refers to isomers that differ in the orientation of substituent atoms in relationship to a carbon-carbon double bond, to a cycloalkyl ring or to a bridged bicyclic system. Substituent atoms (other than H) on each side of a carbon-carbon double bond may be in an E or Z configuration. In the “E” (opposite sided) configuration, the substituents are on opposite sides in relationship to the carbon-carbon double bond; in the “Z” (same sided) configuration, the substituents are oriented on the same side in relationship to the carbon-carbon double bond. Substituent atoms (other than hydrogen) attached to a carbocyclic ring may be in a cis or trans configuration. In the “cis” configuration, the substituents are on the same side in relationship to the plane of the ring; in the “trans” configuration, the substituents are on opposite sides in relationship to the plane of the ring. Compounds having a mixture of “cis” and “trans” species are designated “cis/trans”.
It is to be understood that the various substituent stereoisomers, geometric isomers and mixtures thereof used to prepare compounds of the present invention are either commercially available, can be prepared synthetically from commercially available starting materials or can be prepared as isomeric mixtures and then obtained as resolved isomers using techniques well-known to those of ordinary skill in the art.
The isomeric descriptors “R,” “S,” “E,” “Z,” “cis,” and “trans” are used as described herein for indicating atom configuration(s) relative to a core molecule and are intended to be used as defined in the literature (IUPAC Recommendations for Fundamental Stereochemistry (Section E), Pure Appl. Chem., 1976, 45:13-30).
The FLT3 inhibitors of Formula I′ may be prepared as individual isomers by either isomer-specific synthesis or resolved from an isomeric mixture. Conventional resolution techniques include forming the free base of each isomer of an isomeric pair using an optically active salt (followed by fractional crystallization and regeneration of the free base), forming an ester or amide of each of the isomers of an isomeric pair (followed by chromatographic separation and removal of the chiral auxiliary) or resolving an isomeric mixture of either a starting material or a final product using preparative TLC (thin layer chromatography) or a chiral HPLC column.
Furthermore, the FLT3 inhibitors of Formula I′ may have one or more polymorph or amorphous crystalline forms and as such are intended to be included in the scope of the invention. In addition, some of the FLT3 inhibitors of Formula I′ may form solvates, for example with water (i.e., hydrates) or common organic solvents. As used herein, the term “solvate” means a physical association of a compound of the present invention with one or more solvent molecules. This physical association involves varying degrees of ionic and covalent bonding, including hydrogen bonding. In certain instances the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid.
The term “solvate” is intended to encompass both solution-phase and isolatable solvates. Non-limiting examples of suitable solvates include ethanolates, methanolates, and the like.
It is intended that the present invention include within its scope solvates of the FLT3 inhibitors of Formula I′ of the present invention. Thus, in the methods of treatment of the present invention, the term “administering” shall encompass the means for treating, ameliorating or preventing a syndrome, disorder or disease described herein with a FLT3 inhibitor of Formula I′ specifically disclosed or a compound, or solvate thereof, which would obviously be included within the scope of the invention albeit not specifically disclosed for certain of the instant compounds.
The FLT3 inhibitors of Formula I′ may be converted to the corresponding N-oxide forms following art-known procedures for converting a trivalent nitrogen into its N-oxide form. Said N-oxidation reaction may generally be carried out by reacting the starting material of Formula I′ with an appropriate organic or inorganic peroxide. Appropriate inorganic peroxides comprise, for example, hydrogen peroxide, alkali metal or earth alkaline metal peroxides, e.g. sodium peroxide, potassium peroxide; appropriate organic peroxides may comprise peroxy acids such as, for example, benzenecarboperoxoic acid or halo substituted benzenecarboperoxoic acid, e.g. 3-chlorobenzenecarboperoxoic acid, peroxoalkanoic acids, e.g. peroxoacetic acid, alkylhydroperoxides, e.g. t-butyl hydroperoxide. Suitable solvents are, for example, water, lower alcohols, e.g. ethanol and the like, hydrocarbons, e.g. toluene, ketones, e.g. 2-butanone, halogenated hydrocarbons, e.g. dichloromethane, and mixtures of such solvents.
Some of FLT3 inhibitors of Formula I′ may also exist in their tautomeric forms. Such forms although not explicitly indicated in the present application are intended to be included within the scope of the present invention.
Preparation of FLT3 Inhibitors of Formala I′
During any of the processes for preparation of the FLT3 inhibitors of Formula I′, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups, such as those described in Protecting Groups, P. Kocienski, Thieme Medical Publishers, 2000; and T. W. Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd ed. Wiley Interscience, 1999. The protecting groups may be removed at a convenient subsequent stage using methods known in the art.
FLT3 inhibitors of Formula I′ can be prepared by methods known to those who are skilled in the art. The following reaction schemes are only meant to represent examples of the invention and are in no way meant to be a limit of the invention.
FLT3 inhibitors of Formula I′ can be prepared by methods known to those who are skilled in the art. The following reaction schemes are only meant to represent examples of the invention and are in no way meant to be a limit of the invention.
The FLT3 inhibitors of Formula I′, wherein X, B, G, Q, Z, R1, R2, and R3 are as defined in Formula I′, may be synthesized as outlined by the general synthetic route illustrated in Scheme 1. In the first step, treatment of a piperidinyl ester II′ with a strong base such as lithium hexamethyldisilazide in solvent such as tetrahydrofuran (THF) followed by addition of an appropriate chloroquinazoline/quinoline III′ at a temperature of −78° C. to 25° C. can provide the substituted piperidine IV′. Treatment of IV′ to decarboxylation conditions, such as LiCl in DMSO/H2O at a temperature of 100° C. to 200° C. or KOH in MeOH at a temperature of 25° C. to 200° C., followed by deprotection of the amine protecting group (PG) under standard conditions known to those skilled in the art can provide piperidine V′. The final step can involve reaction of piperidine V′ with an appropriate acylating/alkylating reagent VI′, wherein LG may be an appropriate leaving group such as Br, Cl, I, imidazole, or p-nitrophenoxy, to provide the desired final product I′. These reactions are generally performed in the presence of a solvent, such as methylene chloride, and a base, such as diisopropylethylamine, at a temperature of 0° C. to 150° C., preferably from 0° C.-25° C. The 4-chloroquinazolines or quinolines III′ are either commercially available or can be prepared as outlined in Scheme 5. The acylating reagents VI′ are either commercially available or, wherein Q is a direct bond and Z is NH or N(alkyl), can be prepared as illustrated in Scheme 1. Treatment of an appropriate R3BZH, wherein Z is NH or N(alkyl), with an appropriate acylating reagent such as carbonyldiimidazole, thiophosgene, or p-nitrophenylchloroformate in the presence of a base such as triethylamine can provide VI′. Many R3BZH reagents are either commercially available or can be prepared by a number of known methods (e.g. Tet Lett 1995, 36,
An alternative method to prepare the piperidine intermediate V′, wherein X is N and R1 and R2 are defined as in Formula I′, is illustrated in Scheme 2. Treatment of isonipecotic acid with an appropriate amino protecting group can provide the N-protected piperidine VII′. Transformation of the carboxylic acid to the primary amide and subsequent dehydration under standard conditions can provide the cyano piperidine VIII′. Treatment of piperidine VIII′ with an appropriate aniline IX′ utilizing a Friedel Crafts reaction with a Lewis acid, such as BF3 Et2O, can provide the substituted aniline X′. Formation of the quinazoline ring can be accomplished by treating aniline X′ with a reagent such as formamide at a temperature of 100° C. to 200° C. and subsequent deprotection of the amino protecting group under standard conditions can provide the desired piperidine V′.
The FLT3 inhibitors of Formula I′, wherein Q is a direct bond, Z is NH or N(alkyl), and G, X, R1, R2, and R3 are defined as in Formula I′, can be prepared by the reaction sequence outlined in Scheme 3. Treatment of piperidine V′, prepared by the method outlined in Scheme 1, with an acylating agent such as phosgene, thiophosgene, or carbonyldiimidazole, wherein LG is Cl or imidazole, and an organic base such as diisopropylethylamine can provide intermediate XI′, which upon treatment with an appropriate R3BZH can provide the final compound I′. Alternatively compound I′, wherein Z is NH, can be obtained via direct treatment of piperidine V′ with an appropriate isocyanate or isothiocyanate (R3—B—N═C═G). The isocyanates are either commercially available or can be prepared by a known method (J. Org Chem, 1985, 50, 5879-5881).
The FLT3 inhibitors of Formula I′, where Q is a direct bond, B is phenyl or heteroaryl, G is , Z is NH or N(alkyl), R3 is phenyl or heteroaryl, and X, R1, and R2 are defined as in Formula I′, can be prepared by the reaction sequence outlined in Scheme 4. Treatment of a piperidine V′, which can be prepared as described in Scheme 1, with an appropriate iodoarylamide acylating agent XII′, wherein LG is an appropriate leaving group, for instance, bromide, chloride, or p-nitrophenoxide, can provide the iodoaryl XIII′. Reaction of iodoaryl XIII′ with an appropriate aryl boronic acid or aryl boronic ester (R is H or alkyl) in the presence of a palladium catalyst such as bis(triphenylphosphine)palladium dichloride in a solvent such as toluene at a temperature of 50° C. to 200° C. can provide the final product I. The iodoaryl acylating agents are either commercially available or prepared as outlined in Scheme 1 while the boronic acids/boronic esters are either commercially available or prepared by known methods (Synthesis 2003, 4, 469-483; Organic letters 2001, 3, 1435-1437).
Preparation of an appropriate chloroquinazoline III′ can be accomplished by the reaction sequence illustrated in Scheme 5. Starting from a corresponding anthranilic acid XIV′, treatment with a reagent such as formamidine acetate in a solvent such as ethanol can provide quinazolone XV′. Subsequent treatment of XV′ with a chlorinating agent, such as oxalyl chloride in DMF in a solvent such as dichloroethane, can provide the desired chloroquinazoline III′. The anthranilic acids are either commercially available or can prepared by known methods (WO9728118).
FLT3 inhibitors of Formula I′, wherein R1 is —CC(CH2)nR3, G is O, and X, B, Q, Z, Ra, R2, and R3 are defined as in Formula I′, can be prepared by the sequence outlined in Scheme 6. Treatment of the appropriate iodo substituted piperidine V′, which can be prepared as described in Scheme 1, with an appropriate reagent VI′ can provide the iodoaryl intermediate XVI′. Reaction of XVI′ with an appropriate alkynyl alcohol in the presence of a palladium catalyst such as bis(triphenylphosphine)palladium dichloride, a copper catalyst such as copper(I) iodide, a base such as diethyl amine and a solvent such as dimethylformamide at a temperature of 25° C. to 150° C. can provide the alkynyl alcohol XVII′. Conversion of the alcohol XVII′ to an appropriate leaving group known by those skilled in the art such as a mesylate followed by an SN2 displacement reaction of XVIII′ with an appropriate nucleophilic heterocycle, heteroaryl, amine, alcohol, sulfonamide, or thiol can provide the final compound I′. If Ra nucleophile is a thiol, further oxidation of the thiol can provide the corresponding sulfoxides and sulfones. If Ra nucleophile is an amino, acylation of the nitrogen with an appropriate acylating or sulfonylating agent can provide the corresponding amides, carbamates, ureas, and sulfonamides. If the desired Ra is COORy or CONRwRx, these can be derived from the corresponding hydroxyl group. Oxidation of the hydroxyl group to the acid followed by ester or amide formation under conditions known in the art can provide examples wherein Ra is COORy or CONRwRx. One could prepare the compounds where R2 is —CC(CH2)nRa utilizing the same reaction sequence with the appropriate 7-iodoaryl quinazoline or quinoline.
FLT3 inhibitors of Formula I′, wherein R1 is phenyl or heteroaryl, G is O, and X, B, Q, Z, R2, and R3 are defined as in Formula I′, can also be prepared as outlined in Scheme 7. Treatment of compound XIX′, which can be prepared by decarboxylation of previously described compound IV′, with an appropriate aryl boronic acid or aryl boronic ester (R is H or alkyl) in the presence of a palladium catalyst such as bis(triphenylphosphine)palladium dichloride in a solvent such as toluene at a temperature of 50° C. to 200° C. can provide aryl intermediate XX′. Deprotection of the amine protecting group known to those skilled in the art under standard conditions can provide the piperidine XXI′, which can then be acylated or alkylated using reagent VI′ to provide the final compound I′. The boronic acids/boronic esters are either commercially available or prepared by known methods (Synthesis 2003, 4, 469-483; Organic letters 2001, 3, 1435-1437). One could prepare the compounds where R2 is phenyl or heteroaryl utilizing the same reaction sequence with the appropriate 7-iodo quinazoline or quinoline.
FLT3 inhibitors of Formula I′, wherein R1 is —CHCH(CH2)nRa, G is O, and X, B, Q, Z, Ra, R2, and R3 are defined as in Formula I′, can be prepared by the sequence outlined in Scheme 8. Treatment of the appropriate iodo substituted piperidine V′, which can be prepared as described in Scheme 1, with an appropriate reagent VI′ can provide the iodoaryl intermediate XVI′. Reaction of XVI′ with an appropriate vinylstannane XXII′ in the presence of a palladium catalyst such as bis(triphenylphosphine)palladium dichloride and a solvent such as dimethylformamide at a temperature of 25° C. to 150° C. can provide the alkenyl alcohol XXIII′. Conversion of the alcohol XXIII′ to an appropriate leaving group known by those skilled in the art such as a mesylate followed by an SN2 displacement reaction of XXIV′ with an appropriate nucleophilic heterocycle, heteroaryl, amine, alcohol, sulfonamide, or thiol can provide the final compound I′. If Ra nucleophile is a thiol, further oxidation of the thiol can provide the corresponding sulfoxides and sulfones. If Ra nucleophile is an amino, acylation of the nitrogen with an appropriate acylating or sulfonylating agent can provide the corresponding amides, carbamates, ureas, and sulfonamides. If the desired Ra is COORy or CONRwRx, these can be derived from the corresponding hydroxyl group. Oxidation of the hydroxyl group to the acid followed by ester or amide formation under conditions known in the art can provide examples wherein Ra is COORy or CONRwRx. The corresponding cis olefin isomers of Formula I can be prepared by the same method utilizing the appropriate cis vinyl stannane. Reduction of the olefin moiety under known conditions can provide the saturated compounds where R1 is —CH2CH2(CH2)nRa. One could prepare the compounds where R2 is —CHCH(CH2)nRa utilizing the same reaction sequence with the appropriate 7-iodo quinazoline or quinoline.
FLT3 inhibitors of Formula I′, wherein R2 is —Y(CH2)nRa, Y is O, S, NH, or N(alkyl), G is O, and X, B, Q, Z, Ra, R1, and R3 are defined as in Formula I′, can be prepared by the sequence outlined in Scheme 9. Treatment of compound XXV′, which can be prepared as described in Scheme 1, with a base such as hydroxide ion or potassium t-butoxide in the presence of a suitable Ra(CH2)nYH at a temperature of 25° C. to 150° C. in a solvent such as THF can provide the substituted XXVI′. Deprotection of the amine protecting group known to those skilled in the art under standard conditions can provide the piperidine XXVII′, which can then be acylated or alkylated using reagent VI′ to provide the final compound I′. One could prepare the compounds where R1 is —Y(CH2)nRa utilizing the same reaction sequence with the appropriate 6-halogenated substituted quinazoline or quinoline. A related synthetic route to intermediate quinazoline/quinoline XXVI′ is also outlined in Scheme 9. Treatment of compound IV′, which can be prepared as described in Scheme 1, with a base such as KOH in the presence of a suitable Ra(CH2)nYH at a temperature of 25° C. to 150° C. in a solvent mixture such as dioxane/water, can provide the substituted intermediate XXVI′. Compounds of formula I′ where R2 is —ORc or Rbb can be prepared by the same reaction sequence outlined in Scheme 9 using an appropriate —ORc or Rbb in the SnAr step.
An alternative method to prepare FLT3 inhibitors of Formula I′, wherein R2 is —Y(CH2)nRa, Y is O, S, NH, or N(alkyl), G is O, and X, B, Q, Z, Ra, R1, and R3 are defined as in Formula I′, is the sequence outlined in Scheme 10. Treatment of compound XXV′, which can be prepared as described in Scheme 1, with a base such as hydroxide ion or potassium t-butoxide in the presence of a suitable PG1O(CH2)nYH, where PG1 is an appropriate alcohol protecting group, at a temperature of 25° C. to 150° C. in a solvent such as THF can provide the substituted XXVIII′. Deprotection of the PG1 group known to those skilled in the art under standard conditions can provide intermediate XXIX′. Conversion of the alcohol XXIX′ to an appropriate leaving group known by those skilled in the art such as a mesylate followed by an SN2 displacement reaction of XXX′ with an appropriate nucleophilic heterocycle, heteroaryl, amine, alcohol, sulfonamide, or thiol can provide compound XXXI′. If Ra nucleophile is a thiol, further oxidation of the thiol can provide the corresponding sulfoxides and sulfones. If Ra nucleophile is an amino, acylation of the nitrogen with an appropriate acylating or sulfonylating agent can provide the corresponding amides, carbamates, ureas, and sulfonamides. If the desired Ra is COORy or CONRwRx, these can be derived from the corresponding hydroxyl group. Oxidation of the hydroxyl group to the acid followed by ester or amide formation under conditions known in the art can provide examples wherein Ra is COORy or CONRwRx. Deprotection of the amine protecting group known to those skilled in the art under standard conditions can provide the piperidine XXXII′, which can then be acylated or alkylated using reagent VI′ to provide the final compound I′. One could prepare the compounds where R1 is —Y(CH2)nRa utilizing the same reaction sequence with the appropriate 6-halogenated substituted quinazoline or quinoline.
An alternative method to prepare FLT3 inhibitors of Formula I′, wherein R2 is —Y(CH2)nRa, Y is O, S, NH, or N(alkyl), G is O, and X, B, Q, Z, Ra, R1, and R3 are defined as in Formula I′, is the sequence outlined in Scheme 11. Removal of the amine protecting group known to those skilled in the art under standard conditions of compound XXV′, which can be prepared as described in Scheme 1, can provide the piperidine XXXIII′, which can then be acylated or alkylated using reagent VI′ to provide compound XXXIV′. Treatment of XXXIV′ with a base such as hydroxide ion or potassium t-butoxide in the presence of a suitable Ra(CH2)nYH at a temperature of 25° C. to 150° C. in a solvent such as THF can provide the final compound I′. One could prepare the compounds where R1 is —Y(CH2)nRa utilizing the same reaction sequence with the appropriate 6-halogenated substituted quinazoline or quinoline.
FLT3 inhibitors of Formula I′, wherein R1 and R2 are —Y(CH2)nRa, Y is O, S, NH, or N(alkyl), G is O, and X, B, Q, Z, Ra, and R3 are defined as in Formula I′, can be prepared by the sequence outlined in Scheme 12. Treatment of compound XXXV′, which can be prepared as described in Scheme 1, with a base such as hydroxide ion or potassium t-butoxide in the presence of a suitable Ra(CH2)nYH at a temperature of 25° C. to 150° C. in a solvent such as THF can provide the substituted XXXVI′. A subsequent SnAr reaction of compound XXXVI′ with a base such as hydroxide ion or potassium t-butoxide in the presence of another Ra(CH2)nYH at a temperature of 25° C. to 150° C. in a solvent such as DMSO can provide the substituted XXXVII′. Deprotection of the amine protecting group known to those skilled in the art under standard conditions can provide the piperidine XXXVIII′, which can then be acylated or alkylated using reagent VI′ to provide the final compound I′. One could also prepare compounds where R1 is —ORc or with an appropriate Rbb such as alkoxy using the same reaction sequence in Scheme 12.
Representative FLT3 inhibitors of Formula I′ synthesized by the afore-mentioned methods are presented hereafter. Examples of the synthesis of specific compounds are presented thereafter. Preferred compounds are numbers 73, 74, 85, 152, 157, 158, 163, 178, 183, 197,207, and 209; particularly preferred are numbers 73,74, 157, 178, and 207.
To a solution of 4-isopropoxyaniline (9.06 g, 60.0 mmol) in DCM (120 mL) and pyridine (30 mL) was added 4-nitrophenyl chloroformate (10.9 g, 54.0 mmol) portionwise with stirring over ˜1 min with brief ice-bath cooling. After stirring at rt for 1 h, the homogeneous solution was diluted with DCM (300 mL) and washed with 0.6 M HCl (1×750 mL) and 0.025 M HCl (1×1 L). The organic layer was dried (Na2SO4) and concentrated to give the title compound as a light violet-white solid (16.64 g, 98%). 1H-NMR (300 MHz, CDCl3) δ 8.26 (m, 2H), 7.40-7.28 (m, 4H), 6.98 (br s, 1H), 6.87 (m, 2H), 4.50 (heptet, J=6.0 Hz, 1H), 1.33 (d, J=6.0 Hz, 6H). LC/MS (ESI): calcd mass 316.1. found 633.2 (2MH)+.
To a mixture of isonipecotic acid (39.0 g, 302 mmol) in MeOH (300 mL) was bubbled HCl gas. The flask was tightly capped and stirred at rt for 1.5 h, at which point the homogeneous solution was concentrated, taken up in DCM (2×125 mL), and repeatedly concentrated under reduced pressure to give a white solid largely free of MeOH. To this was added TEA (43.6 mL, 313 mmol) and DCM (80 mL), and this slurry was stirred on an ice bath while a solution of (Boc)2O (60.9 g, 279 mmol) in DCM (100 mL) was added dropwise with stirring over 10 min at 0° C. After 1 h stirring at 0° C., the ice bath was removed and the slurry was stirred at rt overnight. The slurry was then diluted with ether (700 mL), washed with 0.5M NaH2PO4 (1×400 mL), 4 M NaCl (1×450 mL), dried (Na2SO4), and concentrated under reduced pressure to provide the title compound as a clear light amber oil that crystallized upon standing (65.3 g, 96%). 1H-NMR (300 MHz, CDCl3) δ 4.10-3.95 (br m, 2H), 3.69 (s, 3H), 2.92-2.75 (br m, 2H), 2.45 (m, 1H), 1.93-1.82 (m, 2H), 1.70-1.55 (m, 2H), 1.46 (s, 9H).
c. 4-(6,7-Dimethoxy-quinazolin-4-yl)-piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester
To a mixture of piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (17.1 g, 70.5 mmol), as prepared in the previous step, and 4-chloro-6,7-dimethoxyquinazoline (15.0 g, 67.0 mmol) (Oakwood Products, Inc.) immersed in a −78° C. bath was added 1.08 M LiHMDS/THF (71 mL, 77 mmol) in ˜20 mL portions under argon via syringe along the sides of the flask (to allow cooling of the hindered base before reaction with the ester). Following completion of LiHMDS/THF addition, the reaction was allowed to sit in the −78° C. bath for 2-3 min before removing the cold bath and allowing the mixture to stir with gradual warming to rt. After 18 h stirring at rt, and an additional 2 d sitting at rt, the mixture was quenched with 0.5 M NaH2PO4 (150 mL) and extracted with DCM (1×150 mL and 1×100 mL). The organic layers were combined, dried (Na2SO4), and concentrated under reduced pressure to provide the crude title compound as a translucent yellow oil that was used in the next step without further purification (33 g, “114%” crude yield). A small sample was purified by flash chromatography (1:1 hex/EtOAc) for characterization. 1H-NMR (400 MHz, CDCl3) δ 9.11 (s, 1H), 7.34 (s, 1H), 7.29 (s, 1H), 4.05 (s, 3H), 3.96 (s, 3H), 3.76-3.67 (m, 2H), 3.62-3.49 (m, 2H), 3.61 (s, 3H), 2.50-2.36 (br s, 4H), 1.46 (s, 9H). LC/MS (ESI): calcd mass 431.2. found 432.2 (MH)+.
A mixture of crude 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (33 g), as prepared in the previous step, MeOH (100 mL), and KOH pellets (26 g, 400 mmol assuming 87% w/w water) was stirred at reflux (100° C. oil bath) for 1 h, at which point the translucent reddish-amber solution was allowed to cool to rt and diluted with water (100 mL) and 6 M HCl (100 mL). The solution was stirred at 100° C. for 10 min (Caution: Initial vigorous bubbling), allowed to cool to rt, diluted with 2.5 M NaOH (90 mL) and extracted with DCM (1×150 mL; 1×50 mL). The organic layers were combined, dried (Na2SO4), and concentrated under reduced pressure to afford the title compound as a beige powder (13.95 g, 76% from 4-chloro-6,7-dimethoxyquinazoline). 1H-NMR (300 MHz, DMSO-d6) δ 8.98 (s, 1H), 7.48 (s, 1H), 7.32 (s, 1H), 3.98 (s, 3H), 3.96 (s, 3H), 3.69 (m, 1H), 3.05 (m, 2H), 2.84-2.71 (m, 2H), 1.88-1.65 (m, 4H). LC/MS (ESI): calcd mass 273.2. found 274.2 (MH)+.
To a mixture of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (1.86 g, 6.80 mmol), prepared essentially as described in the previous step, and (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester (2.28 g, 7.22 mmol), prepared essentially as described in Example 1a, in CH3CN (13 mL) was added DIEA (1.24 mL, 7.50 mmol). The homogeneous solution was refluxed for 4 h, allowed to cool to rt, shaken with 1 M K2CO3, and extracted with EtOAc (2×25 mL). The organic layers were combined, washed with 0.5 M NaH2PO4 (1×50 mL), 4 M NaCl (1×25 mL), dried three times (Na2SO4), and concentrated under reduced pressure to give crude title compound as a beige semisolid (3.5 g). Flash chromotography (3:4→1:2 hex/acetone) afforded the title compound as an off-white foam (2.21 g, 72%). 1H-NMR (300 MHz, CDCl3) 9.08 (s, 1H), 7.34 (s, 1H), 7.28-7.22 (m, 3H), 6.83 (m, 2H), 6.46 (br s, 1H), 4.47 (heptet, J=6.1 Hz, 1H), 4.27 (br m, 2H), 4.07 (s, 3H), 4.06 (s, 3H), 3.59 (tt, J=11.0 Hz, 3.7 Hz, 1H), 3.15 (td, J=12.8 Hz, 2.4 Hz, 2H), 2.22-2.06 (m, 2H), 2.04-1.92 (m, 2H), 1.31 (d, J=6.1 Hz, 6H). LC/MS (ESI): calcd mass 450.2. found 451.3 (MH)+. Anal. Calcd for C25H30N4O4: C, 66.65; H, 6.71; N, 12.44. Found: C, 66.41; H, 6.68; N, 12.22.
To a solution of 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide (1.41 g, 3.14 mmol), as prepared in the preceding step, in dry CH3CN (3.0 mL) was added 1.70 M HCl/ CH3CN (2.0 mL, 3.4 mmol) in one portion at rt. The slightly translucent solution was swirled once, allowed to sit at rt for 30 min, and then stored overnight in a desiccator at −30° C. to initiate crystal formation. (The 1.70 M HCl/ CH3CN solution was formed by briefly bubbling dry HCl gas into a tared graduated cylinder containing 8.3 mL dry CH3CN.) The vial was then allowed to sit at rt for 1 d. The resulting crystals were washed with CH3CN (3×10 mL), dried under reduced pressure, and powdered to provide, after additional drying at 80° C. under reduced pressure, the title compound as a yellow powder (463 mg, 30%). 1H-NMR (300 MHz, DMSO-d6) 9.16 (s, 1H), 8.44 (br s, 1H), 7.72 (s, 1H), 7.49 (s, 1H), 7.35 (m, 2H), 6.80 (m, 2H), 4.50 (heptet, J=6.0 Hz, 1H), 4.29 (br m, 2H), 4.12-4.00 (m, 1H), 4.05 (s, 3H), 4.03 (s, 3H), 3.16-3.01 (m, 2H), 1.97-1.80 (br m, 4H), 1.23 (d, J=6.0 Hz, 6H). LC/MS (ESI): free base calcd mass 450.2. found 451.3 (MH)+. Anal. Calcd for C25H30N4O4.1.33 HCl.0.71 water.0.18 CH3CN: C, 58.69; H, 6.46; N, 11.28; Cl, 9.05. Found: C, 58.98; H, 6.41; N, 11.39; Cl, 9.05. Karl Fischer: 2.46% water.
The title compound was prepared from 4-iodoaniline essentially as described in Example 1a, except the reaction was stirred at rt for 3 h. The homogeneous solution was then partitioned with DCM and aq HCl essentially as described in Example 1a, except a heavy precipitate formed in the organic layer. Filtration of the organic layer provided the title compound as an off-white solid (8.50 g, 61%). 1H-NMR (400 MHz, CDCl3) 8.30 (m, 2H), 7.68 (m, 2H), 7.39 (m, 2H), 7.30-7.20 (m, 2H), 6.98 (br s, 1H).
To a mixture of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (5.18 g, 19.0 mmol), prepared as described in Example 1d, and (4-iodo-phenyl)-carbamic acid 4-nitro-phenyl ester (8.00 g, 20.8 mmol), prepared as described in the preceding step, in DCM (20 mL) was added DIEA (3.44 mL, 20.8 mmol) with stirring at rt. After stirring at rt for 5 min, CHCl3 (20 mL) was added to thin the slurry, and after stirring for 4 h at rt, the greenish mixture was washed with 0.1 M NaOH (208 mL), and the resulting precipitate in the organic layer was filtered. The filter cake was dissolved in 92:8 DCM/MeOH (250 mL) and washed with water (1×50 mL) and 0.1 M NaOH (1×200 mL). The organic layer was then dried (Na2SO4), concentrated under reduced pressure, and the resulting greyish solid was triturated with hot toluene (1×20 mL) and filtered. The filter cake was washed with toluene (2×20 mL) to provide, after drying of the filter cake, the title compound as an off-white solid (7.84 g, 80%). Nmr reveals a single ˜15 mol % impurity. A sample was purified to homogeneity by flash chromatography. 1H-NMR (300 MHz, CDCl3) 9.07 (s, 1H), 7.58 (m, 2H), 7.35 (s, 1H), 7.25 (s, 1H), 7.17 (m, 2H), 6.49 (br s, 1H), 4.32-4.22 (m, 2H), 4.07 (s, 3H), 4.06 (s, 3H), 3.60 (m, 1H), 3.24-3.11 (m, 2H), 2.23-2.07 (m, 2H), 2.05-1.94 (m, 2H). LC/MS (ESI): calcd mass 518.1. found 519.2 (MH)+.
To a −78° C. solution of 1.85 M phosgene in toluene (15.8 mL, 29.3 mmol) and DCM (32 mL) was added 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (4.00 g, 14.6 mmol), prepared as described in Example 1d, in one portion with stirring, followed immediately by the rapid addition of DIEA (2.66 mL, 16.1 mmol) along the walls of the flask over ˜5 sec. The flask was sealed and stirred at −78° C. for another 5 min before placing the flask in an ice bath with stirring at 0° C. for 30 min. The opaque easily stirred slurry was then poured into a mixture of DCM (70 mL), 0.5 M trisodium citrate (60 mL), and ice (60 mL), and partitioned. The aqueous layer was extracted with DCM (1×50 mL) and the organic layers combined, dried (Na2SO4), and concentrated under reduced pressure to give the crude title compound as an orange solid. Purification by flash chromatography (7:1→4:1 DCM/acetone) afforded the title compound as abeige solid (2.50 g, 51%). 1H-NMR (300 MHz, CDCl3) 9.07 (s, 1H), 7.35 (s, 1H), 7.23 (s, 1H), 4.58-4.47 (m, 2H), 4.072 (s, 3H), 4.068 (s, 3H), 3.65 (tt, J=10.9 Hz, 4.0 Hz, 1H), 3.46-3.33 (m, 1H), 3.28-3.14 (m, 1H), 2.30-2.06 (m, 2H), 2.06-1.95 (m, 2H). LC/MS (ESI): calcd mass 335.1. found 336.1 (MH)+.
b. 4-(6,7-Dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-imidazol-1-yl-phenyl)-amide
4-(6,7-Dimethoxy-quinazolin-4-yl)-piperidine-1-carbonyl chloride (16.5 mg, 0.05 mmol), prepared as described in Example 3a, was dissolved in anhydrous THF (2 mL) and to it was added 4-imidazol-1-yl-phenylamine (12 mg, 0.075 mmol) followed by DIEA (14 μL, 0.075 mmol) and the mixture was stirred at 65° C. for 3 h. It was then concentrated in vacuo and the residue was purified by Preparative TLC (silica gel, 5 % MeOH/DCM) to obtain 2 mg (5%) of pure 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-imidazol-1-yl-phenyl)-amide. 1H-NMR (300 MHz, CDCl3) δ 9.04 (s, 2H), 7.63 (m, 3H), 7.55-7.40 (m, 5H), 7.33 (s, 1H), 4.37 (m, 2H), 4.07 (s, 6H), 3.76-3.58 (m, 2H), 3.14 (m, 2H), 2.18-1.90 (m, 3H). LC/MS (ESI): calcd mass 458.2. found 459.5 (MH)+.
To a solution of 4-isopropylaniline (3.02 g, 22.3 mmol) in DCM (40 mL) and pyridine (10 mL) was added 4-nitrophenyl chloroformate (4.09 g, 20.3 mmol) portionwise with stirring over ˜30 sec with brief ice-bath cooling. After stirring at rt for 1 h, the homogeneous solution was diluted with DCM (100 mL) and washed with 0.6 M HCl (1×250 mL), 0.025 M HCl (1×400 mL), water (1×100 mL), and 1 M NaHCO3 (1×100 mL). The organic layer was dried (Na2SO4) and concentrated to give the title compound as a light peach-colored solid (5.80 g, 95%). 1H-NMR (300 MHz, CDCl3) δ 8.28 (m, 2H), 7.42-7.32 (m, 4H), 7.22 (m, 2H), 6.93 (br s, 1H), 2.90 (h, J=6.9 Hz, 1H), 1.24 (d, J=6.9 Hz, 6H). LC/MS (ESI): calcd mass 300.1, 601.3 (2MH)+.
A mixture of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (18.8 mg, 68.9 μmol), as prepared in Example 1d, and (4-isopropyl-phenyl)-carbamic acid 4-nitro-phenyl ester 21.3 mg, 71.0 μmol), as prepared in the preceding step, was stirred in CH3CN (250 μL) at 80° C. for 4 h. The reaction was then partitioned with DCM (4 mL) and 1 M K2CO3 (4 mL), and the organic layer was dried (Na2SO4) and concentrated under reduced pressure. Flash chromatography of the residue (EtOAc) provided the title compound (21.5 mg, 72%). 1H-NMR (300 MHz, CDCl3) δ 9.08 (s, 1H), 7.34 (s, 1H), 7.31-7.25 (m, 3H), 7.16 (m, 2H), 6.37 (br s, 1H), 4.32-4.22 (m, 2H), 4.07 (s, 3H), 4.06 (s, 3H), 3.60 (tt, 1H), 3.18 (td, 2H), 2.87 (heptet, 1H), 2.24-2.08 (m, 2H), 2.04-1.94 (m, 2H), 1.23 (d, 6H). LC/MS (ESI): calcd mass 434.2. found 435.3 (MH)+.
A solution of 1.03 M LiHMDS/THF (11.5 mL, 11.8 mmol) was treated dropwise with a solution of piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-ethyl ester (2.79 g, 10.9 mmol) (WO 2003064413) in THF (6 mL) over 5 min at 0° C. with stirring under argon. After stirring 30 min at 0° C., the dark yellow homogeneous solution was treated dropwise with a solution of 4-chloroquinoline (1.615 g, 9.88 mmol) in THF (5 mL) over 1-2 min at 0° C. with stirring. The ice bath was then removed and the reaction was stirred at rt overnight, then refluxed for two hours. After cooling to rt, 1 M KOH (aq) (44 mL, 44 mmol) was added and the reaction refluxed for 30 min. Dioxane (22 mL) was added to the bilayer, and the reaction was refluxed an additional 30 min. After cooling to rt, the bilayer was treated dropwise with 12 N HCl (7.4 mL, 89 mmol HCl) (Caution: exotherm) and then refluxed for 30 min under air. The light amber bilayer was allowed to cool to rt, made basic by the addition of 2.5 M NaOH (50 mL), and extracted with DCM (1×50 mL) and 4:1 DCM/MeOH (1×50 mL). The organic layers were combined, dried (Na2SO4), and concentrated to give a residue that was shown by LC/MS to contain the title compound as a minor component and the ethyl ester intermediate as the major component. The ethyl ester intermediate was stirred with KOH pellets (2.4 g, 37 mmol) in MeOH (10 mL) at 100° C. (oil bath) for 3 h, allowed to cool to rt, treated cautiously with 6 M HCl (aq) (10 mL) and water (10 mL), and stirred at 100° C. for 20 min. After cooling to rt, the homogeneous solution was brought to pH>12 with 2.5 M NaOH and extracted with 9:1 DCM/MeOH (2×50 mL). The organic layers were combined, dried (Na2SO4), and concentrated under reduced pressure. Flash chromatography (85:15 DCM/MeOH saturated with NH3) afforded the title compound as a white semisolid (702 mg, 34%). 1H-NMR (300 MHz, CDCl3) δ 8.86 (d, 1H), 8.11 (m, 2H), 7.70 (m, 1H), 7.56 (m, 1H), 7.30 (d, 1H), 3.46 (tt, 1H), 3.27 (m, 2H), 2.91 (td, 2H), 2.02-1.92 (m, 2H), 1.87 (br s, 1H), 1.85-1.69 (m, 2H). LC/MS (ESI): calcd mass 212.1. found 213.1 (MH)+.
A solution of 4-piperidin-4-yl-quinoline (21.1 mg, 99.5 μmol), as prepared in the previous step, (4-isopropyl-phenyl)-carbamic acid 4-nitro-phenyl ester (33.2 mg, 111 μmol), as prepared in Example 4a, and DIEA (18 μL, 109 μmol) in DMSO (100 μL) was stirred at 100° C. for 14 h. The reaction was then allowed to cool to rt, shaken with 2 M K2CO3 (aq) (2 mL), and extracted with DCM (2×2 mL). The organic layers were combined, dried (Na2SO4), and concentrated under reduced pressure. Flash chromatography of the residue (3:4 hex/acetone) provided the title compound (12 mg, 32%). 1H-NMR (300 MHz, CDCl3) δ 8.88 (d, 1H), 8.14 (m, 2H), 7.74 (m, 1H), 7.61 (m, 1H), 7.30 (m, 2H), 7.28 (d, 1H), 7.17 (m, 2H), 6.38 (br s, 1H), 4.36-4.26 (m, 2H), 3.58 (m, 1H), 3.16 (td, 2H), 2.87 (heptet, 1H), 2.13-2.03 (m, 2H), 1.95-1.79 (m, 2H), 1.23 (d, 6H). LC/MS (ESI): calcd mass 373.2. found 374.2 (MH)+.
Prepared essentially as described for Example 5b using 1.4 eq (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester, as prepared in Example 1a. Flash chromatography (3:4 hex/acetone) provided the title compound (9 mg, 31%). 1H-NMR (300 MHz, CDCl3) δ 8.87 (d, 1H), 8.12 (m, 2H), 7.72 (m, 1H), 7.59 (m, 1H), 7.26 (m, 3H), 6.84 (m, 2H), 6.45 (br s, 1H), 4.48 (heptet, 1H), 4.35-4.25 (m, 2H), 3.55 (tt, 1H), 3.12 (td, 2H), 2.10-2.00 (m, 2H), 1.92-1.76 (m, 2H), 1.31 (d, 6H). LC/MS (ESI): calcd mass 389.2. found 390.2 (MH)+.
A mixture of 4-hydroxyquinazoline (2.56 g, 17.5 mmol) and POCl3 (8.0 mL, 88 mmol) was stirred at 140° C. (oil bath) for 10 min. The homogeneous light amber solution was then allowed to cool to rt before concentrating under reduced pressure at 70° C. The translucent residue was dissolved in DCM (25 mL), and the homogeneous yellow solution was partitioned with ice and 1 M NaHCO3 to pH˜6 (paper) (˜20 mL aq layer). The organic layer was dried twice (Na2SO4), filtered through a 0.22 micron filter, and concentrated under reduced pressure (bath <40° C.) to provide the title compound as a yellow solid (2.53 g, 88%). 1H-NMR (300 MHz, CDCl3) δ 9.07 (s, 1H), 8.30 (ddd, 1H), 8.11 (m, 1H), 8.00 (m, 1H), 7.77 (m, 1H).
A mixture of 4-chloroquinazoline (2.02 g, 12.3 mmol), prepared as described in the preceding step, and piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (3.13 g, 12.8 mmol), as prepared in Example 1b, was treated with 1.08 M LiHMDS/THF in one portion by syringe at 0° C. with stirring under argon. After stirring for an additional 5 min at 0° C., the ice bath was removed and the homogeneous amber solution was stirred at rt for 4.5 h. The reaction was quenched with 1 M NaH2PO4 (30 mL) and extracted with DCM (2×30 mL). The organic layers were combined, dried (Na2SO4), and concentrated under reduced pressure to give the crude title compound as a clear amber syrup (4.98 g). 1H-NMR (300 MHz, CDCl3) δ 9.29 (s, 1H), 8.06 (m, 2H), 7.87 (m, 1H), 7.59 (m, 1H), 3.72-3.52 (m, 4H), 3.60 (s, 3H), 2.50-2.40 (br m, 4H), 1.46 (s, 9H). LC/MS (ESI): calcd mass 371.2, found 372.2 (MH)+.
A mixture of crude 4-quinazolin-4-yl-piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (4.58 g), as prepared in the previous step, DMSO (7.5 mL), and 10 M KOH (aq) (7.5 mL) was vigorously stirred at 100° C. for 12 h. After cooling to rt, the reaction was cautiously treated with 6 M HCl (18.4 mL) (gas evolution!) and water (19 mL), and the mixture with heavy precipitate was stirred at 100° C. for 10 min. The resulting amber translucent solution was allowed to cool to rt, made basic with 2.5 M NaOH (20 mL) and water (10 mL), shaken to dissolve the DMSO into the aqueous milieu, and extracted with DCM (2×75 mL). The organic layers were combined, dried (Na2SO4), and concentrated under reduced pressure to give the impure title compound as an amber translucent syrup (2.63 g, “100%” crude yield from 4-chloroquinazoline). 1H-NMR (300 MHz, CDCl3) δ 9.27 (s, 1H), 8.17 (dd, 1H), 8.06 (m, 1H), 7.89 (m, 1H), 7.65 (m, 1H), 3.75 (m, 1H), 3.45-3.35 (m, 2H), 3.04-2.92 (m, 2H), 2.1-1.8 (m, 5H). LC/MS (ESI): calcd mass 213.1. found 214.0 (MH)+.
Prepared essentially as described for Example 5b using 4-piperidin-4-yl-quinazoline, as described in the previous step, and stirring at 100° C. for 100 min. Flash chromatography (1:4 hex/EtOAc) afforded the title compound as a beige solid (23.3 mg, 54%). 1H-NMR (300 MHz, CDCl3) δ 9.26 (s, 1H), 8.18 (m, 1H), 8.08 (m, 1H), 7.91 (m, 1H), 7.67 (m, 1H), 7.28 (m, 2H), 7.16 (m, 2H), 6.40 (br s, 1H), 4.33-4.24 (m, 2H), 3.78 (tt, 1H), 3.17 (td, 2H), 2.87 (heptet, 1H), 2.23-1.97 (m, 4H), 1.23 (d, 6H). LC/MS (ESI): calcd mass 374.2. found 375.2 (MH)+.
Prepared essentially as described for Example 7d, using (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester, as prepared in Example 1a. Flash chromatography (1:4 hex/EtOAc) afforded the title compound as a beige solid (27.6 mg, 55%). 1H-NMR (300 MHz, CDCl3) δ 9.26 (s, 1H), 8.18 (m, 1H), 8.08 (m, 1H), 7.91 (m, 1H), 7.67 (m, 1H), 7.25 (m, 2H), 6.84 (m, 2H), 6.36 (br s, 1H), 4.48 (heptet, 1H), 4.32-4.23 (m, 2H), 3.78 (tt, 1H), 3.16 (td, 2H), 2.22-1.96 (m, 4H), 1.32 (d, 6H). LC/MS (ESI): calcd mass 390.2. found 391.2 (MH)+.
A mixture of 4-isopropylaniline (17.7 mg, 131 μmol), CaCO3 (33.1 mg, 331 μmol) (10 micron powder), and CH3CN (240 μL) was stirred in an ice bath for 2-3 min before adding bromoacetyl bromide (10.3 μL, 119 μmol) dropwise over 10-15 s with stirring at 0° C. After an additional 2-3 min stirring at 0° C., the ice bath was removed and the slurry was stirred at rt for 30 min. Then 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (35.1 mg, 129 μmol), as prepared in Example 1d, was added in one portion and the mixture was stirred at 100° C. for 40 min. The reaction was then allowed to cool to rt, quenched with 2 M K2CO3 (2 mL), and extracted with DCM (2×2 mL). The organic layers were combined, dried (Na2SO4), and concentrated under reduced pressure. Flash chromatography of the residue (1:1 hex/acetone) provided the title compound (30.3 mg, 57%). 1H-NMR (300 MHz, CDCl3) δ 9.11 (s, 2H), 7.51 (m, 2H), 7.33 (s, 1H), 7.25 (s, 1H), 7.19 (m, 2H), 4.05 (s, 6H), 3.41 (tt, 1H), 3.21 (s, 2H), 3.18-3.10 (m, 2H), 2.88 (heptet, 1H), 2.51 (td, 2H), 2.24 (qd, 2H), 2.02-1.92 (m, 2H), 1.22 (d, 6H). LC/MS (ESI): calcd mass 448.3. found 449.3 (MH)+.
Prepared essentially as described for Example 9, using 4-isopropoxyaniline. Flash chromatography (1:1 hex/acetone) provided the target compound (20.3 mg, 39%). 1H-NMR (300 MHz, CDCl3) δ 9.12 (s, 1H), 9.08 (br s, 1H), 7.49 (m, 2H), 7.35 (s, 1H), 7.25 (s, 1H), 6.87 (m, 2H), 4.51 (heptet, 1H), 4.07 (s, 6H), 3.42 (tt, 1H), 3.21 (s, 2H), 3.20-3.11 (m, 2H), 2.53 (td, 2H), 2.25 (qd, 2H), 2.03-1.93 (m, 2H), 1.33 (d, 6H). LC/MS (ESI): calcd mass 464.2. found 465.2 (MH)+.
A mixture of 5-iodoanthranilic acid (9.96 g, 37.9 mmol) and formamidine acetate (4.20 g, 40.3 mmol) (adapted from J. Org. Chem. 51:616, 1986) in absolute EtOH (80 mL) was refluxed under air for 2 h. The smoky amber solution with heavy white precipitate was then concentrated under reduced pressure at 90° C., and residual protic solvent was removed with toluene rotary evaporation (2×100 mL) at 90° C. The resulting sticky tan solid was treated with a thick white slurry of Vilsmeier-Haack reagent in one portion under air at rt. [The Vilsmeier-Haack reagent was prepared by the addition of a solution of oxalyl chloride (10.9 mL, 125 mmol) in DCE (44 mL) to a solution of DMF (6.7 mL, 87 mmol) in DCE (21 mL) dropwise over 10 min at 0° C. with vigorous stirring. The ice bath was removed immediately following completion of oxalyl chloride addition, and the white slurry was stirred at “rt” for 5 min before transfer to the crude 4-hydroxy-6-iodo-quinazoline intermediate.] The reaction was then refluxed under air (oil bath 110° C.) for 1 h 15 min, and the resulting homogeneous brown solution was allowed to cool to rt, at which point a heavy precipitate formed. The reaction was poured into ice water (300 mL) and extracted with DCM (3×250 mL). The opaque organic layers were combined, dried (Na2SO4), and filtered to provide a clear red amber filtrate. Concentration under reduced pressure, followed by toluene rotary evaporation at 90° C. to remove potentially reactive volatiles, afforded the title compound as a tan powder (8.41 g, 94% from iodoanthranilic acid) suitable for treatment with LiHMDS in the next step. 1H-NMR (300 MHz, CDCl3) δ 9.07 (s, 1H), 8.67 (dd, 1H), 8.22 (dd, 1H), 7.81 (d, 1H).
Prepared essentially as described in Example 1c using 4-chloro-6-iodo-quinazoline, as prepared in the preceding step, 1.1 eq LiHMDS/THF and 1.1 eq piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester, as prepared in Example 1b, and stirring at rt for 14 h following enolate formation at −78° C. The homogeneous brown solution was worked up as described in Example 1c to provide the impure crude title compound as a very dark brown thick oil (14.97 g). 1H-NMR (300 MHz, CDCl3) δ 9.28 (s, 1H), 8.41 (d, 1H), 8.10 (dd, 1H), 7.80 (d, 1H), 3.8-3.5 (m, 4H), 3.66 (s, 3H), 2.45-2.35 (m, 4H), 1.46 (s, 9H). LC/MS (ESI): calcd mass 497.1. found 398.0 (MH−Boc)+.
A mixture of 4-(6-iodo-quinazolin-4-yl)-piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (14.21 g, 28.6 mmol), prepared as described in the preceding step, LiCl (2.38 g, 56.1 mmol), water (1.54 mL, 85.8 mmol), and DMSO (14 mL) was stirred at 150° C. under air for 3 h in a 500 mL flask fitted with a lightly capped Liebig condenser to minimize loss of reagent water while allowing gas escape. The reaction was then allowed to cool to rt, 2 M HCl (aq) (100 mL) was added, and the mixture was stirred at 100° C. for 10 min (Caution: Gas evolution). The reaction was cooled on an ice bath, 2.5 M NaOH (100 mL) was added, and the reaction was extracted with DCM (1×250 mL and 1×50 mL). The organic layers were combined, dried (Na2SO4), and concentrated to provide a 60:40 mixture of the title compound and its methyl ester, contaminated with DMSO, as a dark green oil (10.5 g). This material was resubjected to Krapchow decarboxylation conditions using LiCl (2.41 g, 63 mmol), water (1.54 mL, 85.8 mmol), and DMSO (4 mL) (˜7 mL total DMSO) for an additional 5 h at 150° C. After a total of 8 h at 150° C., the reaction was allowed to cool to rt, and 3 M HCl (100 mL) was added (gas evolution) and the reaction stirred at 100° C. for 15 min. The reaction was then stirred at 0° C. while 2.5 M NaOH (120 mL) was added slowly over ˜30 s to pH>12 (paper), and the cream-colored opaque slurry was extracted with 9:1 DCM/MeOH (4×100 mL). The combined organic layers were dried (Na2SO4) and concentrated under reduced pressure to provide the title compound as a clear dark green oil contaminated with DMSO and an aromatic impurity (5.97 g). 1H-NMR (300 MHz, CDCl3) δ 9.27 (s, 1H), 8.52 (d, 1H), 8.12 (dd, 1H), 7.78 (d, 1H), 3.68-3.55 (m, 1H), 3.36-3.27 (m, 2H), 2.92 (td, 2H), 2.1-1.8 (m, 5H). LC/MS (ESI): calcd mass 339.0. found 340.1 (MH)+.
A solution of impure 6-iodo-4-piperidin-4-yl-quinazoline (4.00 g, “11.8 mmol”), as prepared in the preceding step, in CHCl3 (20 mL) was treated with (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester (4.10 g, 13.0 mmol), prepared as described in Example 1a, in one portion at rt under air. DIEA (2.15 mL, 13.0 mmol) was then added in one portion, and residual nitrophenyl ester and DIEA was transferred to the reaction with additional CHCl3 (20 mL). After 8 h rt stirring, the reaction was washed in succession with 1 M NaH2PO4 (50 mL) and 2 M K2CO3 (1×50 mL). The organic phase was filtered, the filter cake was washed with DCM (2×10 mL), and the combined filtrates were dried (Na2SO4) and concentrated under reduced pressure. Flash chromatography of the residue (1:2 hex/EtOAc) afforded the title compound as a beige foam (2.58 g, 42%). 1H-NMR (300 MHz, CDCl3) δ 9.26 (s, 1H), 8.52 (d, 1H), 8.13 (dd, 1H), 7.80 (d, 1H), 7.25 (m, 2H), 6.83 (m, 2H), 6.33 (br s, 1H), 4.48 (heptet, 1H), 4.32-4.22 (m, 2H), 3.68 (tt, 1H), 3.17 (td, 2H), 2.21-1.92 (m, 4H), 1.32 (d, 6H). LC/MS (ESI): calcd mass 516.1. found 517.2 (MH)+.
A mixture of 4-(6-iodo-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide (1.056 g, 2.05 mmol), as prepared in Example 11d, CuI (3.9 mg, 20.5 μmol), trans-PdCl2[P(C6H5)3]2 (26.8 mg, 38.2 μmol), propargyl alcohol (139 μL, 2.36 mmol), and diethylamine (3.4 mL) was flushed with a stream of argon for 30 s, and then quickly sealed and vigorously stirred at rt under argon for 5 h. The resulting dark brown bilayer was concentrated under reduced pressure at rt, dissolved in DCM (10 mL), and vigorously shaken with 0.75 M EDTA (tetrasodium salt) (1×2 mL). The light green aqueous layer was extracted with DCM (1×10 mL), the organic layers were combined, dried (Na2SO4), and concentrated to give a beige foam soluble in 9:1 EtOAc/DCM (˜5 mL). Flash chromatography (1:9 hex/EtOAc→EtOAc) provided the title compound as a yellow foam (825 mg, 91%). 1H-NMR (300 MHz, CDCl3) δ 9.24 (s, 1H), 8.26 (d, 1H), 8.01 (d, 1H), 7.87 (dd, 1H), 7.25 (m, 2H), 6.85 (m, 2H), 6.33 (br s, 1H), 4.59 (d, 2H), 4.48 (heptet, 1H), 4.32-4.23 (m, 2H), 3.71 (m, 1H), 3.22-3.10 (m, 2H), 2.21-1.94 (m, 5H), 1.32 (d, 6H). LC/MS (ESI): calcd mass 444.2. found 445.2 (MH)+.
A solution of 4-[6-(3-hydroxy-prop-1-ynyl)-quinazolin-4-yl]-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide (816 mg, 1.84 mmol), as prepared in Example 12, and DIEA (350 μL, 2.12 mmol) in DCM (13 mL) was treated with methanesulfonyl chloride (157 μL, 2.02 mmol) dropwise over 1 min with stirring at 0° C. under positive argon pressure. The ice bath was immediately removed, and the reaction was stirred at rt for 1 h 15 min. Flash chromatographic purification of the crude reaction mixture (1:9 hex/EtOAc→EtOAc) afforded the title compound (896 mg, 93%). 1H-NMR (300 MHz, CDCl3) δ 9.27 (s, 1H), 8.31 (d, 1H), 8.04 (d, 1H), 7.89 (dd, 1H), 7.26 (m, 2H), 6.85 (m, 2H), 6.34 (br s, 1H), 5.15 (s, 2H), 4.49 (heptet, 1H), 4.33-4.23 (m, 2H), 3.73 (m, 1H), 3.25-3.11 (m, 2H), 3.18 (s, 3H), 2.22-1.94 (m, 4H), 1.32 (d, 6H). LC/MS (ESI): calcd mass 522.2. found 523.3 (MH)+.
A solution of methanesulfonic acid 3-{4-[1-(4-isopropoxy-phenylcarbamoyl)-piperidin-4-yl]-quinazolin-6-yl}-prop-2-ynyl ester (180.0 mg, 345 μmol), as prepared in the previous step, in CH3CN (0.5 mL) was treated with diethylamine (79 μL, 759 μmol) very rapidly by syringe in 1 portion with stirring at rt, and the pale yellow solution was allowed to stir at rt for 2 h. Purification of the crude reaction with a flash silica column (1:2 hex/acetone) afforded the title compound as an off-white foam (136 mg, 79%). 1H-NMR (300 MHz, CDCl3) δ 9.22 (s, 1H), 8.21 (d, 1H), 7.99 (d, 1H), 7.88 (dd, 1H), 7.25 (m, 2H), 6.84 (m, 2H), 6.38 (br s, 1H), 4.48 (heptet, 1H), 4.32-4.22 (m, 2H), 3.78-3.65 (m, 1H), 3.70 (s, 2H), 3.16 (td, 2H), 2.68 (q, 4H), 2.21-1.94 (m, 4H), 1.31 (d, 6H), 1.15 (t, 6H). LC/MS (ESI): calcd mass 499.3. found 500.5 (MH)+. A select fraction of this material was submitted for combustion analysis: Anal. Calcd for C30H37N5O2.0.18 water: C, 71.65; H, 7.49; N, 13.93. Found: C, 71.7; H, 7.55; N, 13.92.
Prepared essentially as described in Example 13b, using piperidine (10.9 mg, 63%). 1H-NMR (400 MHz, CDCl3) δ 9.23 (s, 1H), 8.24 (d, 1H), 7.99 (d, 1H), 7.89 (dd, 1H), 7.28-7.23 (m, 2H), 6.88-6.82 (m, 2H), 6.36 (br s, 1H), 4.49 (heptet, 1H), 4.32-4.24 (m, 2H), 3.72 (tt, 1H), 3.52 (s, 2H), 3.16 (td, 2H), 2.62 (br s, 4H), 2.18-2.05 (m, 2H), 2.05-1.95 (m, 2H), 1.68 (m, 4H), 1.49 (br m, 2H), 1.32 (d, 6H). LC/MS (ESI): calcd mass 511.3. found 512.4 (MH)+.
Prepared essentially as described in Example 13b, using morpholine. Flash chromatography (1:2 hex/acetone) afforded the title compound as a white foam (148.9 mg, 87%). 1H-NMR (300 MHz, CDCl3) δ 9.23 (s, 1H), 8.23 (d, 1H), 8.00 (d, 1H), 7.88 (dd, 1H), 7.25 (m, 2H), 6.85 (m, 2H), 6.31 (br s, 1H), 4.49 (heptet, 1H), 4.32-4.23 (m, 2H), 3.84-3.66 (m, 5H), 3.58 (s, 2H), 3.18 (td, 2H), 2.69 (m, 4H), 2.22-2.05 (m, 2H), 2.05-1.94 (m, 2H), 1.32 (d, 6H). LC/MS (ESI): calcd mass 513.3. found 514.5 (MH)+. A select fraction of this material was submitted for combustion analysis: Anal. Calcd for C30H35N5O3.0.20 water: C, 69.66; H, 6.9; N, 13.54. Found: C, 69.58; H, 6.81; N, 13.49.
Prepared essentially as described for Example 9 using 4-piperidin-4-yl-quinazoline, prepared as described in Example 7c. Flash chromatography (1:4 hex/EtOAc) provided the title compound (19.3 mg, 34%). 1H-NMR (300 MHz, CDCl3) δ 9.30 (s, 1H), 9.12 (br s, 1H), 8.17 (m, 1H), 8.08 (m, 1H), 7.91 (m, 1H), 7.67 (m, 1H), 7.52 (m, 2H), 7.21 (m, 2H), 3.61 (tt, 1H), 3.22 (s, 2H), 3.19-3.10 (m, 2H), 2.89 (heptet, 1H), 2.53 (td, 2H), 2.25 (qd, 2H), 2.00 (m, 2H), 1.24 (d, 6H). LC/MS (ESI): calcd mass 388.2. found 389.4 (MH)+.
A mixture of 2-chloro-5-nitropyridine (7.12 g, 45.0 mmol) and cyclobutanol (3.40 g, 47.2 mmol) in THF (30 mL) was vigorously stirred at 0° C. while NaH (1.18 g, 46.7 mmol) was added in three portions over ˜10-20 s under air (Caution: Extensive gas evolution). Reaction residue was rinsed down with additional THF (5 mL), followed by stirring under positive argon pressure in the ice bath for 1-2 more minutes. The ice bath was then removed and the brown homogeneous solution was stirred at “rt” for 1 h. The reaction was concentrated under reduced pressure at 80° C., taken up in 0.75 M EDTA (tetrasodium salt) (150 mL), and extracted with DCM (1×100 mL, 1×50 mL). The combined organic layers were dried (Na2SO4), concentrated, taken up in MeOH (2×100 mL) and concentrated under reduced pressure at 60° C. to provide the title compound as a thick dark amber oil that crystallized upon standing (7.01 g, 80%). 1H NMR (300 MHz, CDCl3) δ 9.04 (dd, J=2.84 and 0.40 Hz, 1H), 8.33 (dd, J=9.11 and 2.85 Hz, 1H), 6.77 (dd, J=9.11 and 0.50 Hz, 1H), 5.28 (m, 1H), 2.48 (m, 2H), 2.17 (m, 2H), 1.87 (m, 1H), 1.72 (m, 1H).
A flask containing 10% w/w Pd/C (485 mg) was gently flushed with argon while slowly adding MeOH (50 mL) along the sides of the flask, followed by the addition in ˜5 mL portions of a solution of 2-cyclobutoxy-5-nitro-pyridine (4.85 g, 25 mmol), as prepared in the previous step, in MeOH (30 mL). (Caution: Large scale addition of volatile organics to Pd/C in the presence of air can cause fire.) The flask was then evacuated one time and stirred under H2 balloon pressure for 2 h at rt. The reaction was then filtered, and the clear amber filtrate was concentrated, taken up in toluene (2×50 mL) to remove residual MeOH, and concentrated under reduced pressure to provide the crude title compound as a translucent dark brown oil with a faint toluene smell (4.41 g, “108%” crude yield). 1H NMR (300 MHz, CDCl3) δ 7.65 (d, J=3.0 Hz, 1H), 7.04 (dd, J=8.71 and 2.96 Hz, 1H), 6.55 (d, J=8.74 Hz, 1H), 5.04 (m, 1H), 2.42 (m, 2H), 2.10 (m, 2H), 1.80 (m, 1H), 1.66 (m, 1H). LC-MS (ESI): calcd mass 164.1. found 165.2 (MH+).
A mixture of 6-cyclobutoxy-pyridin-3-ylamine (4.41 g, assume 25 mmol), as prepared in the previous step, and CaCO3 (3.25 g, 32.5 mmol) (10 micron powder) was treated with a homogeneous solution of 4-nitrophenyl chloroformate (5.54 g, 27.5 mmol) in toluene (28 mL) in one portion at rt, and was stirred at “rt” (reaction warmed spontaneously) for 2 h. The reaction mixture was then directly loaded onto a flash silica column (95:5 DCM/MeOH→9:1 DCM/MeOH) to afford 5.65 g of material, which was further purified by trituration with hot toluene (1×200 mL) to provide the title compound (4.45 g, 54%). 1H NMR (400 MHz, CDCl3) δ 8.28 (m, 2H), 8.12 (d, 1H), 7.81 (m, 1H), 7.39 (m, 2H), 6.85 (br s, 1H), 6.72 (d, 1H), 5.14 (m, 1H), 2.45 (m, 2H), 2.13 (m, 2H), 1.84 (m, 1H), 1.68 (m, 1H). LC-MS (ESI): calcd mass 329.1, found 330.1 (MH+).
A mixture of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (114.1 mg, 418 μmol), as prepared in Example 1d, (6-cyclobutoxy-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester (151 mg, 459 μmol), as prepared in the preceding step, and DCM (818 μL) was treated with TEA (63 μL, 455 μmol) in one portion, and stirred under air at 45° C. for 30 min. The reaction mixture was then directly applied to a flash silica column (3:4 hex/acetone) to provide the title compound as a foam (141.1 mg, 73%). This material was taken up in 2 M K2CO3 (2 mL) and extracted with DCM (2×2 mL). The combined organic layers were dried (Na2SO4), concentrated, and repurified with a silica flash column (9:2 EtOAc/acetone) to provide analytically pure title compound as an off-white foam (84.4 mg, 44%). 1H NMR (300 MHz, CDCl3) δ 9.08 (s, 1H), 7.97 (d, 1H), 7.77 (dd, 1H), 7.34 (s, 1H), 7.26 (s, 1H), 6.67 (d, 1H), 6.39 (br s, 1H), 5.11 (m, 1H), 4.32-4.22 (m, 2H), 4.07 (s, 3H), 4.06 (s, 3H), 3.60 (tt, 1H), 3.18 (td, 2H), 2.51-2.37 (m, 2H), 2.24-1.94 (m, 6H), 1.89-1.57 (m, 2H). LC-MS (ESI): calcd mass 463.2. found 464.3 (MH+). Anal. Calcd for C25H29N5O4: C, 64.78; H, 6.31; N, 15.11. Found: C, 64.64; H, 6.24; N, 15.04.
Alternatively, 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (6-cyclobutoxy-pyridin-3-yl)-amide (Example 17d) can be prepared similarly to the procedure given for Example 51:
1H NMR (300 MHz, CDCl3) δ 9.08 (s, 1H), 8.02 (d, J=2.85 Hz, 1H), 7.82 (dd, J=8.64 and 2.69 Hz, 1H), 7.37 (s, 1H), 7.26 (s, 1H), 6.68 (d, J=8.83 Hz, 1H), 6.49 (s, 1H), 5.10 (m, 1H), 4.29 (m, 2H), 4.08 (s, 3H), 4.07 (s, 3H), 3.61 (m, 1H), 3.18 (td, J=12.87 and 2.88 Hz, 2H), 2.43 (m, 2H), 1.95-2.22 (m, 6H), 1.58-1.87 (m, 2H). LC-MS (ESI): calcd mass 463.2. found 464.4 (MH+).
A mixture of 4-morpholinoaniline (1.01 g, 5.68 mmol) and CaCO3 (743 mg, 7.42 mmol) (10 micron powder) was treated with a solution of 4-nitrophenyl chloroformate (1.49 g, 7.39 mmol) in DCM (7.5 mL) in one portion under air on an ice bath. The thick, easily stirred reaction slurry was stirred for 1-2 min on the ice bath before stirring at rt for 1 h. The slurry was then diluted with 9:1 DCM/MeOH (7.5 mL) and directly applied to a flash silica column (95:5 DCM/MeOH) to provide 0.7 g of material. This was further purified by trituration with hot toluene (25 mL) to afford the title compound as a light olive green powder (444 mg, 23%). 1H NMR (300 MHz, CDCl3) δ 8.28 (m, 2H), 7.42-7.31 (m, 4H), 6.95-6.85 (m, 3H), 3.86 (m, 4H), 3.13 (m, 4H).
A mixture of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (111.4 mg, 408 μmol), prepared as described in Example 1d, but with purification by silica flash chromatography (9:1 DCM/MeOH saturated with NH3), (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester (147 mg, 429 μmol), prepared as described in the previous step, and DCM (700 μL) was treated with TEA (63 μL, 449 μmol) in one portion at rt. The homogeneous amber solution was stirred at rt for 3.5 h, diluted with DCM (1.3 mL), and washed with 2 M K2CO3 (2 mL). The aqueous layer was extracted with DCM (2×2 mL), the organic layers were combined, dried (Na2SO4), and concentrated, and the residue was purified with silica flash chromatography (1:1 DCM/acetone) to afford the title compound (167.1 mg, 86%). 1H-NMR (300 MHz, CDCl3): δ 9.08 (s, 1H), 7.34 (s, 1H), 7.31-7.24 (m, 3H), 6.88 (m, 2H), 6.31 (br s, 1H), 4.32-4.22 (m, 2H), 4.07 (s, 3H), 4.06 (s, 3H), 3.86 (m, 4H), 3.59 (m, 1H), 3.23-3.07 (m, 6H), 2.24-2.07 (m, 2H), 2.05-1.93 (m, 2H). LC/MS (ESI): calcd mass 477.2, found 478.3 (MH+). Select fractions of this material were combined (112.5 mg) and submitted for combustion analysis: Anal. Calcd for C26H31N5O4: C, 65.39; H, 6.54; N, 14.67. Found: C, 65.26; H, 6.58; N, 14.51.
Alternatively, the following procedure can be used to prepare 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-morpholin-4-yl-phenyl)-amide (Example 18b):
Prepared as described in Example 3b except that 4-morpholin-4-yl-phenylamine was used in place of 4-imidazol-1-yl-phenylamine. Purification by Preparative TLC (silica gel, 5% MeOH/DCM) yielded 7.3 mg (31%) of pure 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-morpholin-4-yl-phenyl)-amide. LC/MS (ESI): calcd mass 477.2. found 478.5 (MH)+.
(4-Piperidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester
Prepared essentially as described in Example 18a, using 4-piperidinoaniline and toluene solvent. Silica flash chromatography (5:2 hex/EtOAc→EtOAc→9:1 DCM/MeOH) provided the target compound as a grey powder (1.416 g, 73%). 1H-NMR (300 MHz, CDCl3): δ 8.28 (m, 2H), 7.39 (m, 2H), 7.31 (m, 2H), 6.93 (m, 2H), 6.82 (br s, 1H), 3.17-3.09 (m, 4H), 1.77-1.66 (m, 4H), 1.63-1.54 (m, 2H). LC/MS (ESI): calcd mass 341.1. found 342.2 (MH+).
Prepared essentially as described in Example 18b using (4-piperidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester, as prepared in the previous step. Purification of the reaction mixture with silica flash chromatography (12:1 EtOAc/acetone→95:5 EtOAc/MeOH) provided the title compound as a light pink foam (91.3 mg, 46%). 1H-NMR (300 MHz, CDCl3): δ 9.08 (s, 1H), 7.34 (s, 1H), 7.27-7.21 (m, 3H), 6.90 (m, 2H), 6.30 (s, 1H), 4.30-4.22 (m, 2H), 4.07 (s, 3H), 4.06 (s, 3H), 3.59 (m, 1H), 3.21-3.04 (m, 6H), 2.21-2.08 (m, 2H), 2.03-1.94 (m, 2H), 1.75-1.66 (m, 4H), 1.60-1.51 (m, 2H). LC/MS (ESI): calcd mass 475.3. found 476.5 (MH)+. Anal. Calcd for C27H33N5O3: C, 68.19; H, 6.99; N, 14.73. Found: C, 67.96; H, 6.93; N, 14.58.
Alternatively, the following procedure can be used to prepare 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-piperidin-1-yl-phenyl)-amide (Example 19b):
Prepared as described in Example 3b except that 4-piperidin-1-yl-phenylamine was used in place of 4-imidazol-1-yl-phenylamine. Purification by Preparative TLC (silica gel, 5% MeOH/DCM) yielded 7.6 mg (32%) of pure 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-piperidin-1-yl-phenyl)amide. 1H-NMR (300 MHz, CDCl3): 9.07 (s, 1H), 7.34 (s, 1H), 7.31-7.23 (m, 3H), 6.98 (m, 2H), 6.42 (bs, 1H), 4.28 (m, 2H), 4.06 (s, 6H), 3.58 (m, 1H), 3.25-3.00 (m, 6H), 2.23-2.05 (m, 2H), 1.98 (m, 2H), 1.75 (m, 4H), 1.58 (m, 2H). LC/MS (ESI): calcd mass 475.3. found 476.5 (MH)+.
This was prepared as described in Example 3b except that 4-(4-methyl-piperazin-1-yl)-phenylamine was used in place of 4-imidazol-1-yl-phenylamine. Purification by Preparative TLC (silica gel, 5% MeOH/DCM) yielded 14.5 mg (30%) of pure 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid [4-(4-methyl-piperazin-1-yl-phenyl)]-amide. 1H-NMR (300 MHz, CDCl3): 9.07 (s, 1H), 7.32 (s, 1H), 7.30-7.22 (m, 3H), 6.88 (d, 2H), 6.39 (s, 1H), 4.27 (m, 2H), 4.06 (s, 6H), 3.58 (m, 1H), 3.23-3.13 (m, 4H), 2.63 (m, 4H), 2.38 (s, 3H), 2.25-2.04 (m, 4H), 1.98 (m, 2H). LC/MS (ESI): calcd mass 490.3. found 491.5 (MH)+.
This was prepared as described in Example 3b except that 4-cyclohexyl-phenylamine was used in place of 4-imidazol-1-yl-phenylamine. Purification by Preparative TLC (silica gel, 5% MeOH/DCM) yielded 20.4 mg (43%) of pure 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-cyclohexyl-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): 9.08 (s, 1H), 7.37 (s, 1H), 7.29 (s, 1H), 7.26 (m, 2H), 7.13 (d, 2H), 6.42 (s, 1H), 4.27 (m, 2H), 4.07 (s, 6H), 3.60 (m, 1H), 3.13 (m, 2H), 2.45 (m, 1H), 2.23-2.05 (m, 2H), 1.98 (m, 2H), 1.89-1.60 (m, 6H), 1.36 (m, 4H). LC/MS (ESI): calcd 474.3. found 475.4 (MH)+.
This was prepared as described in Example 3b except that 4-hydroxymethyl-phenylamine was used in place of 4-imidazol-1-yl-phenylamine. Purification by Preparative TLC (silica gel, 5% MeOH/DCM) yielded 11.2 mg (27%) of pure 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-hydroxymethyl-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): 9.05 (s, 1H), 7.35 (d, 3H), 7.28 (d, 3H), 6.64 (s, 1H), 4.78 (bs, 1H), 4.62 (s, 2H), 4.29 (m, 2H), 4.07 (s, 6H), 3.60 (m, 1H), 3.16 (m, 2H), 2.22-2.04 (m, 2H), 2.04-1.80 (m, 2H). LC/MS (ESI): calcd 422.2. found 423.3 (MH)+.
This was prepared as described in Example 3b except that 1H-indol-5-ylamine was used in place of 4-imidazol-1-yl-phenylamine. Purification by Preparative TLC (silica gel, 5% MeOH/DCM) yielded 12.4 mg (29%) of pure 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (1H-indol-5-yl)-amide. 1H-NMR (300 MHz, CDCl3): 9.08 (s, 1H), 8.29 (bs, 1H), 7.61 (s, 1H), 7.36 (s, 1H), 7.32-7.25 (m, 2H), 7.19-7.10 (m, 2H), 6.48 (m, 2H), 4.31 (m, 2H), 4.07 (s, 6H), 3.60 (m, 1H), 3.16 (m, 2H), 2.25-2.08 (m, 2H), 2.00 (m, 2H). LC/MS (ESI): calcd 431.2. found 432.3 (MH)+.
This was prepared as described in Example 3b except that benzothiazol-6-ylamine was used in place of 4-imidazol-1-yl-phenylamine. Purification by Preparative TLC (silica gel, 5% MeOH/DCM) yielded 10.3 mg (23%) of pure 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid benzothiazol-6-ylamide. 1H-NMR (300 MHz, CDCl3): 9.09 (s, 1H), 8.87 (s, 1H), 8.32 (d, 1H), 8.00 (d, 1H), 7.41 (s, 1H), 7.33-7.24 (m, 2H), 6.82 (s, 1H), 4.34 (m, 2H), 4.08 (s, 6H), 3.64 (m, 1H), 3.22 (m, 2H), 2.30-1.90 (m, 4H). LC/MS (ESI): calcd mass 449.2. found 450.2 (MH)+.
This was prepared as described in Example 3b except that N-(4-amino-phenyl)-acetamide was used in place of 4-imidazol-1-yl-phenylamine. Purification by Preparative TLC (silica gel, 5% MeOH/DCM) yielded 4.2 mg (10%) of pure 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-acetylamino-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): 9.07 (s, 1H), 7.47-7.35 (m, 3H), 7.33-7.25 (m, 3H), 6.64 (s, 1H), 4.30 (m, 2H), 4.08 (s, 6H), 3.62 (m, 1H), 3.17 (m, 2H), 2.24-2.06 (m, 5H), 1.99 (m, 2H). LC/MS (ESI): calcd 449.2. found 450.4 (MH)+.
To a solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (27.5 mg, 0.1 mmol), as prepared in Example 1d, in anhydrous DMF, was added 4-dimethylamino-phenylisocyanate (25 mg, 0.15 mmol) and the mixture was stirred at rt overnight. It was then concentrated in vacuo and the residue was purified by Preparative TLC (silica gel, 5% MeOH/DCM) to yield 19 mg (44%) of pure 4-(6,7-Dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-dimethylamino-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): 9.07 (s, 1H), 7.33 (s, 1H), 7.28-7.17 (m, 3H), 6.9-6.56 (bs, 2H), 6.50-6.22 (bs, 1H), 4.26 (m, 2H), 4.06 (s, 6H), 3.57 (m, 1H), 3.14 (m, 2H), 3.02-2.76 (m, 6H), 2.22-1.90 (m, 4H). LC/MS (ESI): calcd mass 435.2. found 436.5 (MH)+.
This was prepared as described in Example 26 except that 5-isocyanato-2,3-dihydro-benzofuran was used in place of 4-dimethylamino-phenylisocyanate. Purification by Preparative TLC (silica gel, 5% MeOH/DCM) yielded 15.7 mg (36%) of pure 4-(6,7-Dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (2,3-dihydro-benzofuran-5-yl)-amide. 1H-NMR (300 MHz, CDCl3): 9.08 (s, 1H), 7.39 (s, 1H), 7.34 (s, 1H), 7.28-7.25 (s, 1H), 6.92 (d, 1H), 6.70 (d, 1H), 6.34 (s, 1H), 4.55 (t, 2H), 4.27 (m, 2H), 4.07 (s, 6H), 3.60 (m, 1H), 3.24-3.10 (m, 4H), 2.24-2.06 (m, 2H), 2.04-1.94 (m, 2H). LC/MS (ESI): calcd mass 434.2. found 435.4 (MH)+.
To a solution of 4-isopropylphenylacetic acid (36 mg, 0.2 mmol) in anhydrous DCM (1 mL) was added PS-carbodiimide (100 mg, 0.3 mmol) and the mixture was shaken at rt for 15 min. Then, a solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (27.5 mg, 0.1 mmol), as prepared in Example 1d, in anhydrous DMF (1 mL) was added to the mixture and it was shaken overnight at rt. It was then filtered and the resin was washed with THF/DCM and the combined filtrate and washings were concentrated in vacuo. The crude product was purified by flash column chromatography (silica gel, 1% MeOH/DCM) to yield 13.4 mg (31%) of pure 1-[4-(6,7-dimethoxy-quinazolin-4-yl)-piperidin-1-yl]-2-(4-isopropyl-phenyl)-ethanone. 1H-NMR (300 MHz, CDCl3): δ 9.06 (s, 1H), 7.39 (s, 1H), 7.26 (s, 1H), 7.23-7.19 (m, 4H), 4.82 (d, 1H), 4.17-4.00 (m, 7H), 3.76 (m, 2H), 3.57 (m, 1H), 3.23 (m, 1H), 2.96-2.80 (m, 2H), 2.06-1.80 (m, 4H), 1.23 (d, 6H). LC/MS (ESI): calcd mass 433.2. found 434.4 (MH)+.
To a stirred mixture of 4,7-Dichloroquinazoline (800 mg, 4 mmol) and piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (1.2 g, 5.2 mmol), as prepared in Example 1b, in a sealed vial at rt was added drop-wise a 1 M solution of LiHMDS in THF (6 mL, 6 mmol). The mixture was stirred at rt overnight. It was then quenched with aqueous NaH2PO4 and the mixture was extracted with DCM. The DCM layer was drawn off, washed with water, brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo to obtain 2.2 g (>100%) of crude 4-(7-chloro-quinazolin-4-yl)-piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (29a) as a yellow semi-solid which was used as such for the next step.
Solid KOH (224 mg, 4 mmol) was added to a suspension of 4-(7-chloro-quinazolin-4-yl)-piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (29a; 41 mg, 0.1 mmol) in a 1:1 mixture of dioxane and water (1 mL). The mixture was stirred at 100° C. for 3 h. It was then cooled to rt and concentrated in vacuo. The residue was dissolved in DCM and washed with water, brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo to obtain crude 4-(7-chloro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (29b). This was dissolved in 2 mL of 3M HCl/MeOH was stirred at rt for 1 h and then concentrated in vacuo to obtain crude 4-(7-chloro-quinazolin-4-yl)-piperidine (29c) as a di-HCl salt. To a suspension of (29c) in anhydrous MeOH, was added DIEA (45 μL, 0.25 mmol) followed by (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester (48 mg, 0.15 mmol) and the mixture was stirred at rt for 1h. It was then concentrated in vacuo and the residue was purified by flash column chromatography (silica gel, 1% MeOH/DCM) to obtain 5 mg (12% overall yield from 29a) of pure 4-(7-chloro-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): δ 9.25 (s, 1H), 8.15-8.06 (m, 2H), 7.62 (d, 1H), 7.23 (d, 2H), 6.85 (d, 2H), 6.30 (s, 1H), 4.49 (m, 1H), 4.26 (m, 2H), 3.70 (m, 1H), 3.15 (m, 2H), 2.23-2.05 (m, 2H), 2.05-1.92 (m, 2H), 1.32 (d, 6H). LC/MS (ESI): calcd mass 424.2. found 425.4 (MH)+.
This was prepared as described in Example 29 except that (4-isopropyl-phenyl)-carbamic acid 4-nitro-phenyl ester, as prepared in Example 4a, was used in place of (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester. Purification by flash column chromatography (silica gel, 1% MeOH/DCM) yielded 11 mg (27% overall yield from 29a) of pure 4-(7-chloro-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): δ 9.24 (s, 1H), 8.15-8.05 (m, 2H), 7.62 (d, 1H), 7.31-7.25 (d, 2H), 7.16 (d, 2H), 6.38 (s, 1H), 4.28 (m, 2H), 3.72 (m, 1H), 3.16 (m, 2H), 2.87 (m, 1H), 2.25-2.05 (m, 2H), 2.05-1.93 (m, 2H), 1.23 (d, 6H). LC/MS (ESI): calcd mass 408.2. found 409.4 (MH)+.
Solid KOH (224 mg, 4 mmol) was added to a solution of 4-(7-chloro-quinazolin-4-yl)-piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (29a; 41 mg, 0.1 mmol), prepared as described in Example 29, in anhydrous MeOH (1 mL). The mixture was stirred at 100° C. for 3 h. It was then cooled to rt and concentrated in vacuo. The residue was dissolved in DCM and washed with water, brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo to obtain crude 4-(7-methoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (31a). This was dissolved in 2 mL of 3M HCl/MeOH was stirred at rt for 1 h and then concentrated in vacuo to obtain crude 4-(7-methoxy-quinazolin-4-yl)-piperidine (31b) as a di-HCl salt. To a suspension of (31b) in anhydrous MeOH (2 mL), was added DIEA (45 μL, 0.25 mmol) followed by (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester (48 mg, 0.15 mmol), as prepared in Example 1a, and the mixture was stirred at rt for 1 h. It was then concentrated in vacuo and the residue was purified by flash column chromatography (silica gel, 1% MeOH/DCM) to obtain 5.4 mg (13% overall yield from 29a) of pure 4-(7-methoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): δ 9.14 (s, 1H), 8.06 (d, 1H), 7.35 (d, 1H), 7.30-7.25 (m, 3H), 6.84 (d, 2H), 6.30 (s, 1H), 4.48 (m, 1H), 4.26 (m, 2H), 3.99 (s, 3H), 3.69 (m, 1H), 3.14 (m, 2H), 2.23-2.05 (m, 2H), 2.03-1.92 (m, 2H), 1.31 (d, 6H). LC/MS (ESI): calcd mass 420.2. found 421.4 (MH)+.
This was prepared as described in Example 31 except that (4-isopropyl-phenyl)-carbamic acid 4-nitro-phenyl ester, as prepared in Example 4a, was used in place of (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester. Purification by flash column chromatography (silica gel, 1% MeOH/DCM) yielded 14.1 mg (35% overall yield from 15a) of pure 4-(7-chloro-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): δ 9.14 (s, 1H), 8.06 (d, 1H), 7.34 (d, 1H), 7.31-7.24 (m, 3H), 7.16 (d, 2H), 6.39 (s, 1H), 4.27 (m, 2H), 3.98 (s, 3H), 3.69 (m, 1H), 3.14 (m, 2H), 2.87 (m, 1H), 2.23-2.05 (m, 2H), 2.04-1.92 (m, 2H), 1.23 (d, 6H). LC/MS (ESI): calcd mass 404.2. found 405.4 (MH)+.
Solid KOH (112 mg, 2 mmol) was added to a mixture of 4-(7-chloro-quinazolin-4-yl)-piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (29a; 82 mg, 0.2 mmol), prepared as described in Example 29, and 3-hydroxypropylpiperidine (0.25 mL). The mixture was stirred at 100° C. for 3 h. It was then cooled to rt and diluted with water. The mixture was extracted with DCM and the organic layer was drawn off and washed with water thrice, with brine once, then dried over anhydrous MgSO4, filtered and concentrated in vacuo. To this was added 3 mL of 3M HCl/MeOH and the mixture was stirred at rt for 2 h and then concentrated in vacuo. This was suspended in anhydrous MeOH (3 mL), and to it DIEA (1.75 mL, 0.6 mmol) was added followed by (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester (96 mg, 0.3 mmol), as prepared in Example 1a, and the mixture was stirred at rt overnight. It was then concentrated in vacuo and the residue was dissolved in DCM and washed extensively with water thrice and brine once and then dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (silica gel, 1% MeOH/DCM followed by 90:9:1 DCM:MeOH:NH4OH) to obtain 14 mg (13% overall yield from 29a) of pure 4-(7-(3-piperidin-1-yl-propoxy)-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): δ 9.13 (s, 1H), 8.05 (d, 1H), 7.35-7.21 (m, 4H), 6.84 (d, 2H), 6.33 (s, 1H), 4.48 (m, 1H), 4.32-4.15 (m, 4H), 3.68 (m, 1H), 3.13 (m, 2H), 2.7-2.45 (m, 6H), 2.20-1.90 (m, 8H), 1.75-1.58 (m, 4H), 1.31 (d, 6H). LC/MS (ESI): calcd mass 531.3. found 532.6 (MH)+.
This was prepared as described in Example 33 except that 2-hydroxyethylpiperidine (0.5 mL) was used in place of 3-hydroxypropylpiperidine (0.25 mL). Purification by flash column chromatography (silica gel, 5% MeOH/DCM) yielded 45 mg (43% overall yield from 29a) of pure 4-(7-(2-piperindin-1-yl-ethoxy)-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): δ 9.12 (s, 1H), 8.05 (d, 1H), 7.34-7.21 (m, 4H), 6.83 (d, 2H), 6.42 (s, 1H), 4.47 (m, 1H), 4.37 (m, 2H), 4.26 (m, 2H), 3.67 (m, 1H), 3.19-3.02 (m, 2H), 2.98 (m, 2H), 2.68 (m, 4H), 2.21-2.03 (m, 2H), 1.96 (m, 2H), 1.72 (m, 4H), 1.50 (m, 2H), 1.31 (d, 6H). LC/MS (ESI): calcd mass 517.3. found 518.5 (MH)+.
This was prepared as described in Example 33 except that 2-diethylaminoethanol (0.5 mL) was used in place of 3-hydroxypropylpiperidine (0.25 mL). Purification by flash column chromatography (silica gel, 5% MeOH/DCM followed by 90:9:1 DCM:MeOH:NH4OH) yielded 30 mg (30% overall yield from 29a) of pure 4-[7-(2-diethylamino-ethoxy)-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): δ 9.13 (s, 1H), 8.05 (d, 1H), 7.35-7.21 (m, 4H), 6.84 (d, 2H), 6.30 (s, 1H), 4.48 (m, 1H), 4.31-4.20 (m, 4H), 3.68 (m, 1H), 3.14 (m, 2H), 3.00 (m, 2H), 2.70 (m, 4H), 2.22-2.04 (m, 2H), 1.97 (m, 2H), 1.31 (d, 6H), 1.12 (d, 6H). LC/MS (ESI): calcd mass 505.3. found 506.6 (MH)+.
This was prepared as described in Example 33 except that 3-diethylaminopropanol (0.5 mL) was used in place of 3-hydroxypropylpiperidine (0.25 mL). Purification by flash column chromatography (silica gel, 5% MeOH/DCM followed by 90:9:1 DCM:MeOH:NH4OH) yielded 20 mg (19% overall yield from 29a) of pure 4-[7-(3-diethylamino-propoxy)-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): δ 9.13 (s, 1H), 8.04 (d, 1H), 7.34-7.21 (m, 4H), 6.84 (d, 2H), 6.33 (s, 1H), 4.48 (m, 1H), 4.32-4.15 (m, 4H), 3.68 (m, 1H), 3.14 (m, 2H), 2.74-2.54 (m, 6H), 2.22-1.90 (m, 6H), 1.31 (d, 6H), 1.07 (t, 6H). LC/MS (ESI): calcd mass 519.3. found 520.6 (MH)+.
This was prepared as described in Example 33 except that 2-hydroxyethylmorrpholine (0.5 mL) was used in place of 3-hydroxypropylpiperidine (0.25 mL). Purification by flash column chromatography (silica gel, 5% MeOH/DCM followed by 90:9:1 DCM:MeOH:NH4OH) yielded 25 mg (24% overall yield from 29a) of pure 4-[7-(2-morpholin-4-yl-ethoxy)-quinazolin-4-yl)]-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): δ 9.14 (s, 1H), 8.05 (d, 1H), 7.33-7.20 (m, 4H), 6.84 (d, 2H), 6.32 (s, 1H), 4.48 (m, 1H), 4.33-4.20 (m, 4H), 3.79-3.61 (m, 5H), 3.13 (m, 2H), 2.90 (m, 2H), 2.26 (m, 4H), 2.22-2.03 (m, 2H), 1.96 (m, 2H), 1.31 (d, 6H). LC/MS (ESI): calcd mass 519.3. found 520.6 (MH)+.
This was prepared as described in Example 33 except that 3-hydroxypropylmorpholine (0.5 mL) was used in place of 3-hydroxypropylpiperidine (0.25 mL). Purification by flash column chromatography (silica gel, 5% MeOH/DCM followed by 90:9:1 DCM:MeOH:NH4OH) yielded 15 mg (14% overall yield from 29a) of pure 4-[7-(3-morpholin-4-yl-propoxy)-quinazolin-4-yl)]-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): δ 9.13 (s, 1H), 8.05 (d, 1H), 7.35-7.21 (m, 4H), 6.85 (d, 2H), 6.30 (s, 1H), 4.48 (m, 1H), 4.31-4.17 (m, 4H), 3.76-3.61 (m, 5H), 3.14 (m, 2H), 2.57 (m, 2H), 2.49 (m, 4H), 2.22-1.90 (m, 6H), 1.32 (d, 6H). LC/MS (ESI): calcd mass 533.3. found 534.6 (MH)+.
This was prepared as described in Example 33 except that 3-(4-methyl-piperazin-1-yl)-propan-1-ol (0.5 mL) was used in place of 3-hydroxypropylpiperidine (0.25 mL). Purification by flash column chromatography (silica gel, 5% MeOH/DCM followed by 90:9:1 DCM:MeOH:NH4OH) yielded 25 mg (23% overall yield from 29a) of pure 4-{7-[3-(4-methyl-piperazin-1-yl)-propoxy]-quinazolin-4-yl)}-piperidine-1-carboxylic acid (4-isopropoxy-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): δ 9.13 (s, 1H), 8.05 (d, 1H), 7.34-7.21 (m, 4H), 6.84 (d, 2H), 6.31 (s, 1H), 4.48 (m, 1H), 4.31-4.15 (m, 4H), 3.68 (m, 1H), 3.13 (m, 2H), 2.70-2.40 (m, 8H), 2.32 (s, 3H), 2.22-1.90 (m, 8H), 1.31 (d, 6H). LC/MS (ESI): calcd mass 546.3. found 547.6 (MH)+.
A mixture of 4-iodoaniline (219 mg, 1.0 mmol), 2-methoxyethanol (152 mg, 2.0 mmol), copper iodide (19.0 mg, 0.1 mmol), cesium carbonate (554 mg, 1.7 mmol) and 1,10-phenanthroline (36.0 mg, 0.2 mmol) was stirred in toluene (0.5 mL) at 110° C. overnight. The reaction was then cooled to RT and filtered through silica gel and washed with diethyl ether. The ether was removed in vacuo to obtain a crude solid. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a solid (8.9 mg, 5.3%). 1H NMR (300 MHz, CDCl3) δ 6.82-6.72 (m, 4H), 4.06 (t, 2H), 3.72 (t, 2H), 3.45 (s, 3H).
A mixture of 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carbonyl chloride (18 mg, 0.0536 mmol), as prepared in Example 3a, 4-(2-methoxy-ethoxy)-phenylamine (8.9 mg, 0.0533 mmol), as prepared in the previous step, and triethylamine (14 μL, 0.1 mmol) was stirred in DMSO (0.5 mL) at 50° C. overnight. The reaction was then cooled to RT, partitioned between EtOAc (10 mL) and H2O (10 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a brown solid (5.7 mg, 23%). 1H NMR (300 MHz, CDCl3) δ 9.16 (s, 1H), 7.28-7.25 (m, 4H), 6.89 (m, 2H), 6.33 (br s, NH), 4.29-4.24 (m, 2H), 4.12-4.07 (m, 8H), 3.74 (m, 2H), 3.59 (m, 1H), 3.45 (s, 3H), 3.17 (m, 2H), 2.22-2.08 (m, 2H), 2.05-1.97 (m, 2H); LC/MS (ESI): calcd mass 466.2, found 467.4 [M+1]+.
To a solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (30 mg, 0.110 mmol), as prepared in Example 1d, in DMF (1 mL) was treated with 1-isocyanato-4-methoxy-benzene (24.5 mg, 0.164 mmol) at RT overnight. The reaction was then partitioned between EtOAc (10 mL) and H2O (10 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a yellow solid (25.9 mg, 56%). 1H NMR (300 MHz, CDCl3) δ 9.10 (s, 1H), 7.29 (m, 4H), 6.88 (m, 2H), 6.30 (br s, NH), 4.30-4.26 (m, 2H), 4.08 (s, 6H), 3.80 (s, 3H), 3.61 (m, 1H), 3.17 (m, 2H), 2.19-2.14 (m, 2H), 2.03-1.97 (m, 2H); LC/MS (ESI): calcd mass 422.2. found 423.3 [M+1]+.
A solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (30 mg, 0.110 mmol), as prepared in Example 1d, in DMF (1 mL) was treated with isocyanato-cyclohexane (20.6 mg, 0.165 mmol) at RT overnight. The reaction was then partitioned between EtOAc (10 ImL) and H2O (10 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a light yellow solid (22 mg, 50%). 1H NMR (300 MHz, CDCl3) δ 9.08 (s, 1H), 7.38 (s, 1H), 7.26 (s, 1H), 4.35 (d, 1H), 4.14 (d, 1H), 4.07 (s, 6H), 3.68 (m, 1H), 3.53 (m, 1H), 3.03 (m, 2H), 2.12-1.90 (m, 4H), 1.70-1.55 (m, 5H), 1.40-1.09 (m, 5H); LC/MS (ESI): calcd mass 398.2. found 399.3 [M+1]+.
A solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (30 mg, 0.110 mmol), as prepared in Example 1d, in DMF (1 mL) was treated with 1-butyl-4-isocyanato-benzene (28.8 mg, 0.165 mmol) at RT overnight. The reaction was then partitioned between EtOAc (10 mL) and H2O (10 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a light yellow solid (20.3 mg, 41%). 1H NMR (300 MHz, CDCl3) δ 9.09 (s, 1H), 7.40 (s, 1H), 7.28 (m, 3H), 7.13-7.10 (m, 2H), 6.36 (br s, NH), 4.30-4.26 (m, 2H), 4.08 (s, 6H), 3.61 (m, 1H), 3.17 (m, 2H), 2.57 (m, 2H), 2.17 (m, 2H), 2.02-1.98 (m, 2H), 1.34 (m, 4H), 0.94-0.80 (m, 3H); LC/MS (ESI): calcd mass 448.3. found 449.3 [M+1]+.
A solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (30 mg, 0.110 mmol), as prepared in Example 1d, in DMF (1 mL) was treated with 1-ethoxy-4-isocyanato-benzene (26.8 mg, 0.164 mmol) at RT overnight. The reaction was then partitioned between EtOAc (10 mL) and H2O (10 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a light brown solid (9.7 mg, 20%). 1H NMR (300 MHz, CDCl3) δ 9.09 (s, 1H), 7.41 (m, 1H), 7.26 (m, 3H), 6.87 (m, 2H), 6.29 (br s, NH), 4.30-4.25 (m, 2H), 4.08 (s, 6H), 4.01 (q, 2H), 3.61 (m, 1H), 3.17 (m, 2H), 2.17 (m, 2H), 2.02-2.01 (m, 2H), 1.40 (t, 3H); LC/MS (ESI): calcd mass 436.2. found 437.3 [M+1]+.
A solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (30 mg, 0.110 mmol), as prepared in Example 1d, in DMF (1 mL) was treated with isocyanato-benzene (19.6 mg, 0.165 mmol) at RT overnight. The reaction was then partitioned between EtOAc (10 mL) and H2O (10 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a yellow solid (11.4 mg, 27%). 1H NMR (300 MHz, CDCl3) δ 9.09 (s, 1H), 7.37 (m, 6H), 7.06 (m, 1H), 6.42 (br s, NH), 4.31-4.27 (m, 2H), 4.08 (s, 6H), 3.62 (m, 1H), 3.19 (m, 2H), 2.17 (m, 2H), 2.04-1.98 (m, 2H); LC/MS (ESI): calcd mass 392.2. found 393.3 [M+1]+.
A solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (20 mg, 0.0733 mmol), as prepared in Example 1d, in DMF (1 mL) was treated with 1-isocyanato-4-trifluoromethyl-benzene (20 mg, 0.107 mmol) at RT overnight. The reaction was then partitioned between EtOAc (10 mL) and H2O (10 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a yellow solid (9.0 mg, 27%). 1H NMR (300 MHz, CDCl3) δ 9.10 (s, 1H), 7.54 (m, 2H), 7.39 (m, 1H), 7.28 (m, 2H), 6.69 (m, 1H), 6.63 (br s, NH), 4.33-4.29 (m, 2H), 4.09 (s, 6H), 3.65 (m, 1H), 3.22 (m, 2H), 2.17 (m, 2H), 2.06-2.01 (m, 2H); LC/MS (ESI) calcd mass 460.2. found 461.3 [M+1]+.
A solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (20 mg, 0.0733 mmol), as prepared in Example 1d, in DMF (1 mL) was treated with 4-phenoxyphenyl isocyanate (23 mg, 0.109 mmol) at RT overnight. The reaction was then partitioned between EtOAc (10 mL) and H2O (10 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a brown solid (15.7 mg, 44%). 1H NMR (300 MHz, CDCl3) δ 9.10 (s, 1H), 7.48 (m, 1H), 7.33 (m, 5H), 7.07 (m, 1H), 6.99 (m, 4H), 6.41 (br s, NH), 4.32-4.27 (m, 2H), 4.09 (s, 6H), 3.63 (m, 1H), 3.20 (m, 2H), 2.17 (m, 2H), 2.03-1.99 (m, 2H); LC/MS (ESI) calcd mass 484.2. found 485.3 [M+1]+.
A solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (20 mg, 0.0733 mmol), as prepared in Example 1d, in DMF (1 mL) was treated with 1-isocyanato-4-methyl-benzene (15 mg, 0.113 mmol) at RT overnight. The reaction was then partitioned between EtOAc (10 mL) and H2O (10 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a brown solid (25.1 mg, 84%). 1H NMR (300 MHz, CDCl3) δ 9.09 (s, 1H), 7.26-7.21 (m, 3H), 7.12 (m, 3H), 6.37 (br s, NH), 4.30-4.26 (m, 2H), 4.07 (s, 6H), 3.60 (m, 1H), 3.18 (m, 2H), 2.30 (s, 3H), 2.17 (m, 2H), 2.01-1.98 (m, 2H); LC/MS (ESI) calcd mass 406.2. found 407.3 [M+1]+.
A solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (20 mg, 0.0733 mmol), as prepared in Example 1d, in DMF (1 mL) was treated with 1-chloro-4-isocyanato-benzene (16.8 mg, 0.1 10 mmol) at RT overnight. The reaction was then partitioned between EtOAc (10 mL) and H2O (10 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a yellow solid (9.2 mg, 29%). 1H NMR (300 MHz, CDCl3) δ 9.08 (s, 1H), 7.38-7.33 (m, 4H), 7.26 (m, 2H), 6.44 (br s, NH), 4.29-4.26 (m, 2H), 4.07 (s, 6H), 3.62 (m, 1H), 3.19 (m, 2H), 2.16 (m, 2H), 2.02-1.99 (m, 2H); LC/MS (ESI) calcd mass 426.2. found 427.2 [M+1]+.
A solution of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (20 mg, 0.0733 mmol), as prepared in Example 1d, in DMF (1 mL) was treated with 1-isocyanato-4-trifluoromethoxy-benzene (22 mg, 0.108 mmol) at RT overnight. The reaction was then partitioned between EtOAc (10 mL) and H2O (10 mL). The organic phase was dried over Na2SO4 and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound as a yellow solid (18.2 mg, 52%). 1H NMR (300 MHz, CDCl3) δ 9.08 (s, 1H), 7.39 (m, 3H), 7.16 (m, 2H), 7.00 (m, 1H), 6.52 (br s, NH), 4.30-4.27 (m, 2H), 4.07 (s, 6H), 3.62 (m, 1H), 3.20 (m, 2H), 2.18-2.11 (m, 2H), 2.03-1.99 (m, 2H); LC/MS (ESI) calcd mass 476.2. found 477.3 [M+1]+.
To a solution of 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carbonyl chloride (46.9 mg, 0.14 mmol), as prepared in Example 3a, in DMSO (1 mL) was added 4-(difluoromethoxy)aniline (26.6 mg, 0.17 mmol), followed by DIEA (35.9 mg, 0.28 mmol). The mixture was heated at 100° C. with stirring. After 2 h, it was cooled to room temperature and partitioned between EtOAc and water. The combined EtOAc extracts were dried (Na2SO4) and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (EtOAc→5% MeOH/EtOAc as eluent) to afford the title compound as a white solid (20.4 mg, 32%). 1H NMR (300 MHz, CDCl3) δ 9.09 (s, 1H), 7.40 (s, 1H), 7.38 (d, J=8.99 Hz, 2H), 7.27 (s, 1H), 7.07 (d, J=8.93 Hz, 2H), 6.48 (s, 1H), 6.45 (t, J=74.22 Hz, 1H), 4.28 (m, 2H), 4.08 (s, 3H), 4.07 (s, 3H), 3.62 (m, 1H), 3.20 (td, J=13.02 and 2.64 Hz, 2H), 2.14 (m, 2H), 2.01 (m, 2H). LC-MS (ESI) calcd mass 458.2. found 459.3 (MH+).
Similar to the synthesis of Example 51, Examples 52-56 were synthesized by the reactions of 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carbonyl chloride with the corresponding aniline or amine in the presence of DIEA.
1H NMR (300 MHz, CDCl3) δ 9.10 (s, 1H), 7.51 (s, 1H), 7.29 (d, J=8.39 Hz, 2H), 7.28 (s, 1H), 7.11 (d, J=8.58 Hz, 2H), 6.41 (s, 1H), 4.29 (m, 2H), 4.09 (s, 3H), 4.08 (s, 3H), 3.63 (m, 1H), 3.18 (td, J=13.00 and 2.41 Hz, 2H), 2.55 (m, 1H), 2.18 (m, 2H), 1.99 (m, 2H), 1.57 (m, 2H), 1.21 (d, J=6.96 Hz, 3H), 0.81 (t, J=7.35 Hz, 3H). LC-MS (ESI) calcd mass 448.3. found 449.4 (MH+).
1H NMR (300 MHz, CDCl3) δ 9.09 (s, 1H), 7.40 (s, 1H), 7.31 (d, J=3.51 Hz, 2H), 7.26 (d, J=3.42 Hz, 2H), 6.39 (s, 1H), 4.28 (m, 2H), 4.08 (s, 3H), 4.07 (s, 3H), 3.61 (m, 1H), 3.18 (td, J=13.41 and 3.06 Hz, 2H), 2.17 (m, 2H), 1.99 (m, 2H), 1.30 (s, 9H). LC-MS (ESI) calcd mass 448.3. found 449.4 (MH+).
1H NMR (300 MHz, CDCl3) δ 9.07 (s, 1H), 7.70 (m, 0.5H), 7.53 (m, 0.5H), 7.35 (s, 1H), 7.25 (s, 1H), 4.28 (m, 1H), 4.12 (m, 2H), 4.05 (s, 6H), 3.53 (m, 1H), 3.02 (td, J=12.78 and 2.39 Hz, 2H), 1.64-2.12 (m, 4H), 0.86-1.32 (m, 9H), 0.85 (s, 9H). LC-MS (ESI) calcd mass 454.3. found 455.4 (MH+).
1H NMR (300 MHz, CDCl3) δ 9.08 (s, 1H), 7.34 (m, 5H), 6.47 (s, 1H), 4.87 (q, J=6.30 Hz, 1H), 4.28 (m, 2H), 4.07 (s, 3H), 4.06 (s, 3H), 3.61 (m, 1H), 3.18 (td, J=13.00 and 2.60 Hz, 2H), 2.15 (m, 2H), 1.99 (m, 2H), 1.48 (d, J=6.45 Hz, 3H). LC-MS (ESI) calcd mass 436.2. found 437.4 (MH+).
To a solution of 2-chloro-5-nitro-pyridine (450 mg, 2.84 mmol) in isopropanol (10 mL)/DMF (7 mL) was added 60% NaH (57 mg). The mixture was stirred at 80° C. for 4 h and the organic solvents were evaporated under reduced pressure. The residue was partitioned between EtOAc and water. The EtOAc extracts were dried (Na2SO4) and evaporated. The crude product was used for the next step reaction without further purification. 1H NMR (300 MHz, CDCl3) δ 9.06 (d, J=2.81 Hz, 1H), 8.32 (dd, J=8.79 and 2.53 Hz, 1H), 6.74 (d, J=8.61 Hz, 1H), 5.43 (m, 1H), 1.38 (d, J=6.20 Hz, 6H).
To a solution of 2-isopropoxy-5-nitro-pyridine, as prepared in the previous step, in MeOH (5 mL) was added 20 mg of 10% Pd/C. The mixture was degassed several times and stirred under hydrogen atmosphere for 4 h. It was filtered through a pad of celite and the filtrate was evaporated. The residue was purified by flash column chromatography on silica gel (EtOAc as eluent). 1H NMR (400 MHz, CDCl3) δ 7.65 (d, J=2.96 Hz, 1H), 7.02 (dd, J=8.71 and 2.99 Hz, 1H), 6.54 (d, J=8.67 Hz, 1H), 5.14 (m, 1H), 1.31 (d, J=6.17 Hz, 6H). LC-MS (ESI) calcd mass 152.1. found 153.2 (MH+).
1H NMR (300 MHz, CDCl3) δ 9.09 (s, 1H), 8.04 (d, J=2.69 Hz, 1H), 7.81 (dd, J=8.92 and 2.56 Hz, 1H), 7.39 (s, 1H), 7.27 (s, 1H), 6.68 (d, J=8.86 Hz, 1H), 6.49 (s, 1H), 5.21 (m, 1H), 4.30 (m, 2H), 4.08 (s, 3H), 4.07 (s, 3H), 3.62 (m, 1H), 3.19 (td, J=13.00 and 2.74 Hz, 2H), 2.17 (m, 2H), 2.00 (m, 2H), 1.34 (d, J=6.17 Hz, 6H). LC-MS (ESI) calcd mass 451.2. found 452.4 (MH+).
To a mixture of 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-iodo-phenyl)-amide (57.9 mg, 0. 11 mmol), as prepared in Example 2b, and pyrrolidin-2-one (13.3 mg, 0.16 mmol) in toluene (3 mL) was added CuI (1.5 mg), followed by N,N-dimethylethylenediamine (1.4 mg) and K3PO4 (56.7 mg). The reaction mixture was heated at 105° C. overnight. It was concentrated under reduced pressure and the crude residue was purified by flash column chromatography on silica gel (10% MeOH/EtOAc as eluent) to afford the desired product (8.6 mg, 16.4% yield). 1H NMR (300 MHz, CD3OD) δ 8.95 (s, 1H), 7.58 (s, 1H), 7.50 (d, J=9.26 Hz, 2H), 7.41 (d, J=9.27 Hz, 2H), 7.32 (s, 1H), 4.36 (m, 2H), 4.06 (s, 3H), 4.04 (s, 3H), 3.91 (t, J=6.93 Hz, 2H), 3.39 (m, 1H), 3.19 (m, 2H), 2.59 (t, J=8.46 Hz, 2H), 1.94-2.30 (m, 6H). LC-MS (ESI) calcd mass 475.2. found 476.4 (MH+).
To a suspension of 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-iodo-phenyl)-amide (58.2 mg, 0.11 mmol), as prepared in Example 2b, in 1 mL of toluene/EtOH (4:1, v/v) were added pyrimidine-5-boronic acid (15.3 mg, 0.12 mmol), Pd(PPh3)4 (6.5 mg) and 2M K2CO3 solution (0.23 mL). The reaction mixture was heated at 100° C. overnight. It was concentrated under reduced pressure and the black residue was purified by flash column chromatography on silica gel (5% MeOH/EtOAc as eluent) to afford the desired product (18.4 mg, 35.6% yield). 1H NMR (300 MHz, CDCl3) δ 9.17 (s, 1H), 9.09 (s, 1H), 8.94 (s, 2H), 7.55 (s, 4H), 7.38 (s, 1H), 7.27 (s, 1H), 6.59 (s, 1H), 4.32 (m, 2H), 4.09 (s, 3H), 4.08 (s, 3H), 3.65 (m, 1H), 3.23 (m, 2H), 2.19 (m, 2H), 2.03 (m, 2H). LC-MS (ESI) calcd mass 470.2. found 471.3 (MH+)
Similar to the synthesis of Example 58, Examples 59-61 were prepared by the reaction of 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-iodo-phenyl)-amide with the corresponding boronic acid or borate in the presence of Pd(PPh3)4.
1H NMR (300 MHz, CDCl3) δ 9.09 (s, 1H, 7.61 (d, J=8.74 Hz, 2H), 7.44 (m, 1H), 7.42 (d, J=8.78 Hz, 2H), 7.36 (s, 1H), 7.27 (s, 1H), 6.57 (dd, J=3.34 and 0.62 Hz, 1H), 6.50 (s, 1H), 6.45 (dd, J=3.33 and 1.80 Hz, 1H), 4.28 (m, 2H), 4.08 (s, 3H), 4.07 (s, 3H), 3.62 (m, 1H), 3.20 (td, J=12.82 and 2.66 Hz, 2H), 2.16 (m, 2H), 2.01 (m, 2H). LC-MS (ESI) calcd mass 458.2. found 459.4 (MH+).
1H NMR (300 MHz, CDCl3) δ 9.09 (s, 1H), 8.58 (dd, J=2.58 and 0.63 Hz, 1H), 7.82 (dd, J=8.29 and 2.60 Hz, 1H), 7.51 (s, 4H), 7.37 (dd, J=8.26 and 0.67 Hz, 1H), 7.36 (s, 1H), 7.27 (s, 1H), 6.58 (s, 1H), 4.31 (m, 2H), 4.08 (s, 3H), 4.07 (s, 3H), 3.63 (m, 1H), 3.22 (m, 2H), 2.18 (m, 2H), 2.02 (m, 2H). LC-MS (ESI) calcd mass 503.2. found 504.3 (MH+).
4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylic acid tert-butyl ester was used as starting material. 1H NMR (300 MHz, CD3OD) δ 8.94 (s, 1H), 7.56 (s, 1H), 7.36 (m, 4H), 7.31 (s, 1H), 6.03 (m, 1H), 4.36 (m, 2H), 4.06 (s, 3H), 4.04 (m, 2H), 4.03 (s, 3H), 3.90 (m, 1H), 3.62 (m, 2H), 3.22 (td, J=12.97 and 2.74 Hz, 2H), 2.50 (m, 2H), 1.93-2.10 (m, 4H), 1.49 (s, 9H). LC-MS (ESI) calcd mass 573.3. found 574.6 (MH+).
4-(4-{[4-(6,7-Dimethoxy-quinazolin-4-yl)-piperidine-1-carbonyl]-amino}-phenyl)-3,6-dihydro-2H-pyridine-1-carboxylic acid tert-butyl ester (10 mg, 0.017 mmol), as prepared in Example 61, was dissolved in 50% TFA/DCM (5 mL). The solution was stirred at room temperature for 4 h. It was evaporated and the residue was quenched with 2N ammonium in MeOH (6 mL). The solvent was removed and the residue was washed with water, dried in vacuo to afford the title compound as a white solid (8 mg, 100%). 1H NMR (300 MHz, CD3OD) δ 8.94 (s, 1H), 7.58 (s, 1H), 7.42 (s, 4H), 7.32 (s, 1H), 6.12 (m, 1H), 4.37 (m, 2H), 4.06 (s, 3H), 4.04 (s, 3H), 3.93 (m, 1H), 3.82 (m, 2H), 3.44 (t, J=6.29 Hz, 2H), 3.22 (m, 2H), 2.78 (m, 2H), 1.93-2.10 (m, 4H). LC-MS (ESI) calcd mass 473.2. found 474.5 (MH+).
To a solution of 4-(4-{ [4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carbonyl]-amino}-phenyl)-3,6-dihydro-2H-pyridine-1-carboxylic acid tert-butyl ester (5 mg, 0.009 mmol), as prepared in Example 61, in MeOH (5 mL) was added 10% Pd/C (5 mg). The solution was degassed and was kept stirring under hydrogen atmosphere for 2 h. It was filtered through a pad of celite and the filtrate was evaporated to afford the desired product (3.7 mg, 74% yield). 1H NMR (300 MHz, CD3OD) δ 8.94 (s, 1H), 7.57 (s, 1H), 7.32 (s, 1H), 7.30 (d, J=8.29 Hz, 2H), 7.15 (d, J=8.51 Hz, 2H), 4.35 (m, 2H), 4.20 (m, 2H), 4.06 (s, 3H), 4.03 (s, 3H), 3.91 (m, 1H), 3.21 (m, 2H), 2.85 (br, 2H), 2.67 (m, 1H), 1.93-2.10 (m, 4H), 1.80 (m, 2H), 1.57 (m, 2H), 1.48 (s, 9H). LC-MS (ESI) calcd mass 575.3. found 576.6 (MH+).
4-(4-{[4-(6,7-Dimethoxy-quinazolin-4-yl)-piperidine-1-carbonyl]-amino}-phenyl)-piperidine-1-carboxylic acid tert-butyl ester, prepared as described in Example 63, was treated essentially as described for Example 62, to afford the title compound. 1H NMR (300 MHz, CD3OD) δ 8.94 (s, 1H), 7.57 (s, 1H), 7.36 (d, J=8.62 Hz, 2H), 7.32 (s, 1H), 7.19 (d, J=8.66 Hz, 2H), 4.36 (m, 2H), 4.06 (s, 3H), 4.04 (s, 3H), 3.91 (m, 1H), 3.49 (m, 2H), 3.06-3.28 (m, 4H), 2.87 (m, 1H), 1.79-2.12 (m, 8H). LC-MS (ESI) calcd mass 475.3. found 476.5 (MH+).
A mixture of 4-chloro-7-fluoro-quinazoline (2.87 g, 15.4 mmol) (WO 9609294 A1) and piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (4.15 g, 17.1 mmol), as prepared in Example 1b, was placed in a -78° C. bath for 5 min under argon before adding a 1.08 M LiHMDS/THF solution (17.8 mL, 19.2 mmol) rapidly by syringe along the sides of the flask (to allow cooling and dispersion of the hindered base before reaction with the ester). Following completion of LiHMDS/THF addition, the reaction was manually swirled in the -78° C. bath for 2-3 min before removing the cold bath and allowing the mixture to stir with gradual warming to rt. After 2.5 h stirring at rt, the dark brown homogeneous solution was quenched with 1.0 M NaH2PO4 (38 mL) and extracted with DCM (1×150 mL and 1×25 mL). The organic layers were combined, dried (Na2SO4), and concentrated under reduced pressure, and subject to high vacuum at 90° C. with toluene chasers (3×10 mL) to provide the crude title compound as an opaque thick yellow oil that was used in the next step without further purification (6.83 g, “114%” crude yield). 1H-NMR (300 MHz, CDCl3) δ 9.26 (s, 1H), 8.11 (dd, 1H), 7.70 (dd, 1H), 7.36 (ddd, 1H), 3.74-3.64 (m, 2H), 3.62-3.51 (m, 2H), 3.61 (s, 3H), 2.47-2.38 (br m, 4H), 1.46 (s, 9H). LC/MS (ESI): calcd mass 389.2. found 390.1 (MH)+.
A mixture of 4-(7-fluoro-quinazolin-4-yl)-piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (“6.83 g”), as prepared without further purification in the previous step, LiCl (1.32 g, 31.1 mmol), water (832 μL, 46.2 mmol), and DMSO (6.0 mL) was stirred under air at 150° C. (oil bath) with an efficient condenser (to retain reagent water) for 9.5 h. The dark solution was then allowed to cool to rt, shaken with 1.0 M NaHCO3, and extracted with EtOAc (1×60 mL) and 9:1 DCM/MeOH (2×30 mL). The organic layers were combined, dried (Na2SO4), and concentrated to afford a thick clear amber oil. Flash chromatography of this residue (3:2 hexanes/EtOAc) afforded the title compound as a thick clear yellow syrup that was rubbed to a beige solid (2.37 g, 46% from 4-chloro-7-fluoroquinazoline). 1H-NMR (300 MHz, CDCl3) δ 9.23 (s, 1H), 8.20 (dd, 1H), 7.67 (dd, 1H), 7.42 (ddd, 1H), 4.42-4.25 (br m, 2H), 3.65 (m, 1H), 2.96 (m, 2H), 2.14-1.83 (m, 4H), 1.49 (s, 1H). LC/MS (ESI): calcd mass 331.2. found 332.1 (MH)+(weak).
A mixture of 3-amino-propan-1-ol (37.9 mg, 505 pmol), t-BuOK (63.1 mg, 563 μmol), and DME (505 μL) was stirred for 5 min at rt until a homogeneous yellow solution resulted. Solid 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (170.7 mg, 516 pmol), as prepared in the previous step, was added in one portion under air at “rt” (vial spontaneously warmed), and the resulting homogeneous amber solution was stirred at rt 1 h. The reaction was then diluted with DCM (1.0 mL) and stirred at 0° C. for 5 min before adding MsCl (48 μL, 620 μmol) dropwise with stirring at 0° C. over 1 min. After 1 min additional stirring at 0° C., the ice bath was removed and the hazy yellow solution was stirred at “rt” for 5 min. DIEA (94 μL, 568 μmol) was then added dropwise, and the reaction was stirred rt 2 days. The crude reaction was then loaded directly onto a flash silica column (4:3 DCM/acetone eluent) to provide the title compound as an off-white foam (186 mg, 79%). 1H-NMR (400 MHz, CDCl3) δ 9.14 (s, 1H), 8.06 (d, 1H), 7.32 (d, 1H), 7.24 (m, 1H), 4.47 (br t, 1H), 4.32 (br s, 2H), 4.26 (t, 2H), 3.61 (m, 1H), 3.43 (q, 2H), 2.99-2.89 (m, 2H), 2.98 (s, 3H), 2.17 (pentet, 1H), 2.10-1.94 (m, 2H), 1.92-1.83 (m, 2H), 1.49 (s, 9H). LC/MS (ESI): calcd mass 464.2. found 465.2 (MH)+.
A premixed solution of 1:1 TFA/CHCl3 (80 μL, 539 μmol TFA) was added to 4-[7-(3-ethanesulfonylamino-propoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (38.7 mg, 83.4 μmol), prepared as described in the previous step, and the tightly capped reaction was stirred under air at 100° C. (aluminum block) for 10 min. After cooling to rt, DIEA (117 μL, 709 μmol) was added dropwise, followed by CHCl3 (0.5 mL), and the resulting homogeneous solution was stirred at rt while (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester (32.9 mg, 104 μmol), as prepared in Example 1a, was added in one portion. The resulting homogeneous dark yellow solution was stirred at rt overnight, and then directly loaded onto a flash silica column (5:3 DCM/acetone eluent) to afford impure title compound. This material was taken up in EtOAc (2 mL) and washed with 1.0 M NaHCO3 (3×2 mL), 1.0 M NaH2PO4 (2×2 mL), and again 1.0 M NaHCO3 (2×2 mL). One contaminant was removed (apparently protonated DIEA), but another substantially remained (apparently nitrophenol), so the EtOAc layer was directly loaded onto a flash silica column (5:3 DCM/acetone eluent) to afford the title compound as a white foam (16.0 mg, 35%). 1H-NMR (400 MHz, CDCl3) δ 9.13 (s, 1H), 8.06 (d, 1H), 7.32 (d, 1H), 7.27-7.21 (m, 3H), 6.84 (m, 2H), 6.34 (br s, 1H), 4.69 (br t, 1H), 4.48 (heptet, 1H), 4.26 (m, 4H), 3.67 (tt, 1H), 3.42 (q, 2H), 3.13 (td, 2H), 2.97 (s, 3H), 2.21-2.05 (m, 4H), 2.00-1.91 (m, 2H), 1.32 (d, 6H). LC/MS (ESI): calcd mass 541.2. found 542.1 (MH)+. Anal. Calcd for C27H35N505S: C, 59.87; H, 6.51; N, 12.93. Found: C, 60.03; H, 6.51; N, 12.78.
A solution of 4-nitrophenyl chloroformate (798 mg, 3.96 mmol) in THF (2.0 mL) was added rapidly by syringe over ˜10 s at rt under air to a stirred solution of 4-morpholin-4-yl-phenylamine (675 mg, 3.79 mmol) in THF (8.8 mL), with a heavy grey precipitate forming “instantly”. The reaction was immediately capped and stirred “rt” for 30 min (vial spontaneously warmed), and was then filtered. The grey filter cake was washed with dry THF (2×10 mL), and dried under high vacuum at 80° C. to afford the title compound as a grey powder (1.361 g, 95%). A portion was partitioned with CDCl3 and aqueous 0.5 M trisodium citrate to generate the CDCl3-soluble free base: 1H-NMR (300 MHz, CDCl3) δ 8.28 (m, 2H), 7.42-7.31 (m, 4H), 6.95-6.88 (m, 3H), 3.87 (m, 4H), 3.14 (m, 4H).
4-[7-(3-methanesulfonylamino-propoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (7.4 mg, 16 μmol), as prepared in Example 65c, and TFA (100 μL, 1.35 mmol) was capped tightly and stirred at 100° C. (aluminum block) for 5 min. The reaction was then concentrated, and pyridine (100 μL) was added to give a homogeneous solution. (4-Morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride (7.5 mg, 20 μmol) was then added in one portion at rt, and the solution was stirred at 80° C. for 10 min, then at rt overnight. The reaction was then concentrated and subjected to silica flash chromatography (4:3→3:5 DCM/acetone) to afford the title compound as an off-white semisolid (3.5 mg, 39%). 1H-NMR (400 MHz, 95:5 v/v CDCl3:CD3OD) δ 9.10 (s, 1H), 8.09 (d, 1H), 7.33-7.25 (m, 4H), 6.89 (m, 2H), 4.27 (m, 4H), 3.87 (m, 4H), 3.70 (m, 1H), 3.38 (t, 2H), 3.17-3.07 (m, 6H), 2.96 (s, 3H), 2.20-2.05 (m, 4H), 2.01-1.92 (m, 2H). LC/MS (ESI): calcd mass 568.2. found 569.1 (MH)+.
To a mixture of 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester 66.9 mg, 0.20 mmol), as prepared in Example 65b, and tert-BuOK (33.4 mg, 0.30 mmol) was added 1-(3-hydroxypropyl)-2-pyrrolidone (34.7 mg, 0.24 mmol) in anhydrous THF (3 mL). The mixture was stirred at 85° C. for 15 min and the solvent was evaporated under reduced pressure to give a light brown residue, which is used for the next step reaction without purification. 1H NMR (300 MHz, CDCl3) δ 9. 10 (s, 1H), 8.03 (d, J=9.13 Hz, 1H), 7.26 (m, 1H), 7.23 (dd, J=9.05 and 2.43 Hz, 1H), 4.14 (t, J=6.08 Hz, 2H), 3.58 (m, 1H), 3.50 (t, J=6.60 Hz, 4H), 3.42 (t, J=6.98 Hz, 4H), 2.37 (t, J=8.45 Hz, 2H), 1.80-2.15 (m, 8H), 1.46 (s, 9H). LC-MS (ESI) calcd for C25H35N404 (MH+) 455.3. found 455.2.
4-{7-[3-(2-Oxo-pyrrolidin-1-yl)-propoxy]-quinazolin-4-yl}-piperidine-1-carboxylic acid tert-butyl ester (as prepared in the previous step, 0.20 mmol) was treated with 50% TFA/CH2CI2 (4 mL) for 2 h and the solvents were evaporated. To the residue was added (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester (70.2 mg, 0.22 mmol), as prepared in Example 1a, followed by DIEA (130.5 mg, 1.01 mmol) in CH3CN (4 mL). The resulting mixture was heated at 95° C. for 1 h and the solvents were evaporated under reduced pressure. The residue was purified by flash column chromatography on silica gel (5% MeOH/EtOAc as eluent) to afford the product as a white solid (95.5 mg, 89% yield). 1H NMR (300 MHz, CDCl3) δ 9.14 (s, 1H), 8.06 (d, J=9.33 Hz, 1H), 7.33 (d, J=2.46 Hz, 1H), 7.28 (dd, J=9.30 and 2.65 Hz, 1H), 7.25 (m, 2H), 6.84 (d, J=8.93 Hz, 2H), 6.33 (br, 1H), 4.48 (m, 1H), 4.26 (m, 2H), 4.17 (t, J=6.10 Hz, 2H), 3.69 (m, 1H), 3.53 (t, J=6.99 Hz, 2H), 3.45 (t, J=7.02 Hz, 2H), 3.13 (td, J=12.85 and 2.83 Hz, 2H), 2.40 (t, J=7.78 Hz, 2H), 1.94-2.20 (m, 8H), 1.31 (d, J=6.06 Hz, 6H). LC-MS (ESI) calcd for C30H38N504 (MH+) 532.3, found 532.2. Anal. Calcd for C30H37N504: C, 67.77; H, 7.01; N, 13.17. Found: C, 67.81; H, 6.96; N, 13.16.
Prepared essentially as described in Example 67b, using (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, as prepared in Example 66a. 1H NMR (300 MHz, CD3OD) δ 9.03 (s, 1H), 8.35 (d, J=9.40 Hz, 1H), 7.38 (dd, J=9.34 and 2.50 Hz, 1H), 7.31 (d, J=2.48 Hz, 1H), 7.25 (d, J=9.09 Hz, 2H), 6.93 (d, J=9.14 Hz, 2H), 4.34 (m, 2H), 4.22 (t, J=6.03 Hz, 2H), 3.92 (m, 1H), 3.82 (t, J=4.65 Hz, 4H), 3.53 (t, J=6.88 Hz, 4H), 3.16 (td, J=13.05 and 2.81 Hz, 2H), 3.08 (t, J=4.82 Hz, 4H), 2.37 (t, J=7.74 Hz, 2H), 1.89-2.17 (m, 8H). LC-MS (ESI) calcd for C31H39N604 (MH+) 559.3. found 559.2. Anal. Calcd for C31H38N604: C, 66.65; H, 6.86; N, 15.04. Found: C, 66.34; H, 6.80; N, 14.97.
To a solution of 2-chloro-5-nitropyridine (7.01 g, 44.4 mmol) in THF (30 mL) and cyclopentanol (3.9 g, 45.3 mmol) was added sodium hydride (1.3 g, 54.2 mmol) portionwise with stirring over ˜30 sec with ice-bath cooling at 0° C. After stirring at 0° C. for 5 min, the ice bath was removed and the reaction was stirred at rt for 3 h. It was then concentrated in vacuo and the residue was dissolved in DCM and washed extensively with 1 M NaHCO3 and then dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The crude product was purified by flash column chromatography (silica gel, 9:1 Hexane:Ethyl Acetate) to obtain pure 2-cyclopentyloxy-5-nitro-pyridine (0.4 g, 4%). 1H-NMR (300 MHz, CDCl3): δ 9.07 (s, 1H), 8.32 (m, 1H), 6.74 (d, 1H), 5.53 (m, 1H), 2.00 (m, 2H), 1.81 (m, 4H), 1.66 (m, 2H).
To a solution of 2-cyclopentyloxy-5-nitro-pyridine (0.3099 g, 1.49 mmol), in MeOH (2 mL) was added 10% Pd/C (90 mg). The solution was degassed and was kept stirring under hydrogen atmosphere for overnight. It was filtered through a pad of celite and the filtrate was evaporated to afford the desired product as a brown oil (248 mg, 94% yield). 1H-NMR (300 MHz, CDCl3): δ 7.69 (d, 1H), 7.04 (m, 1H), 6.56 (d, 1H), 5.25 (m, 1H), 1.93 (m, 2H), 1.78 (m, 4H), 1.60 (m, 2H). LC/MS (ESI) calcd for C10H14N2O 178.23. found [M+41+1]+220.0.
To a solution of 6-cyclopentyloxy-pyridin-3-ylamine (0.248 g, 1.39 mmol) in THF (2 mL) was added 4-nitrophenyl chloroformate (0.280 g, 1.39 mmol) portionwise. After stirring at rt for 1 h, a heavy precipitate formed in the organic layer. Filtration of the organic layer provided the title compound as a light pink solid (0.368 g, 77%). 1H-NMR (400 MHz, CDCl3): δ 11.1 (s, 1H), 9.11 (s, 1H), 9.04 (d, 1H), 8.26 (d, 2H), 7.40 (d, 2H), 7.14 (d, 1H), 5.36 (m, 1H), 2.11 (m, 2H), 1.97 (m, 2H), 1.84 (m, 2H), 1.71 (m, 2H).
A mixture of 6,7-dimethoxy-4-piperidin-4-yl-quinazoline (12 mg, 0.044 mmol), prepared as described in Example 1d, (6-cyclopentyloxy-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester (20 mg, 0.058 mmol), prepared as described in the previous step, and DCM (500 uL) was treated with TEA (6 uL, 0.043 mmol) in one portion at rt. The homogeneous amber solution was stirred at rt for 2 h, diluted with DCM (2 mL), and washed with H2O (2 mL). The aqueous layer was extracted with DCM (2×2 mL), the organic layers were combined, dried (Na2SO4) and concentrated in vacuo. Purification by prep tlc (1:9 MeOH/DCM) afforded the title compound (6.0 mg, 29%). 1H-NMR (300 MHz, CDCl3): δ 9.10 (s, 1H), 8.08 (s, 1H), 7.89 (d, 1H), 7.39 (s, 1H), 7.28 (s, 1H), 6.71 (d, 1H), 6.61 (bs, 1H), 5.30 (m, 1H), 4.32 (d, 2H), 4.08 (s, 6H), 3.62 (m, 1H), 3.20 (m, 2H), 2.16 (m, 2H), 1.98 (m, 4H), 1.79 (m, 4H), 1.62 (m, 2H). LC/MS (ESI) calcd for C26H31N5O4 477.56. found [M+1]+478.1.
4-(6,7-Dimethoxy-quinazolin-4-yl)-piperidine-1-carbonyl chloride (37 mg, 0.11 mmol), prepared as described in Example 3a, was dissolved in anhydrous dioxane (2 mL) and to it was added 4-azepan-1-yl-phenylamine (19 mg, 0.1 mmol) followed by DIEA (20 uL, 0.11 mmol) and the mixture was stirred at 100° C. for 3 h. It was then concentrated in vacuo and the residue was purified by Preparative TLC (silica gel, 5% MeOH/DCM) to obtain 3 mg (6%) of pure 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-azepan-1-yl-phenyl)-amide. 1H-NMR (300 MHz, CDCl3) 9.08 (s, 1H), 7.55-7.23 (m, 4H), 7.18 (d, 1H), 6.77 (d, 1H), 6.37 (s, 1H), 4.27 (m, 2H), 4.07 (s, 6H), 3.68-3.32 (m, 3H), 3.30-2.90 (m, 2H), 2.24-1.88 (m, 4H), 1.86-1.38 (m, 10H). LC/MS (ESI): 490.3 (MH)+.
Prepared as described in Example 70, except that 3-chloro-4-piperidin-4-yl-phenylamine was used in place of 4-azepan-1-yl-phenylamine. Purification by Preparative TLC (silica gel, 5% MeOH/DCM) yielded 8.1 mg (16%) of pure 4-(6,7-dimethoxy-quinazolin-4-yl)-piperidine-1-carboxylic acid (3-chloro-4-piperidin-1-yl-phenyl)-amide. 1H-NMR (300 MHz, CDCl3): 9.07 (s, 1H), 7.43 (d, 1H), 7.34 (s, 1H), 7.24 (d, 1H), 7.21 (d, 1H), 7.00 (d, 1H), 6.45 (d, 1H), 4.26 (m, 2H), 4.07 (s, 6H), 3.66-3.52 (m, 1H), 3.23-3.10 (m, 2H), 2.93 (m, 4H), 2.23-2.06 (m, 2H), 2.04-1.93 (m, 2H), 1.74 (m, 4H), 1.57 (m, 2H). LC/MS (ESI): 510.3 (MH)+.
Prepared essentially as described in Example 67b, using (6-morpholin-4-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared by the method outlined in Example 66a. 1H NMR (300 MHz, CD3OD) δ 9.03 (s, 1H), 8.35 (d, J=9.49 Hz, 1H), 8.12 (dd, J=2.72 and 0.62 Hz, 1H), 7.65 (dd, J=9.01 and 2.70 Hz, 1H), 7.39 (dd, J=9.32 and 2.62 Hz, 1H), 7.31 (d, J=2.41 Hz, 1H), 6.82 (d, J=9.03 Hz, 1H), 4.34 (m, 2H), 4.22 (t, J=5.88 Hz, 2H), 3.94 (m, 1H), 3.80 (t, J=4.89 Hz, 4H), 3.53 (t, J=7.09 Hz, 4H), 3.40 (t, J=4.91 Hz, 4H), 3.18 (m, 2H), 2.38 (t, J=8.09 Hz, 2H), 1.90-2.17 (8H). LC-MS (ESI) calcd for C30H38N704 (MH+) 560.3. found 560.2.
The title compound was prepared essentially as described in Example 65b, except the starting material 4-(7-fluoro-quinazolin-4-yl)-piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester was purified by silica flash chromatography (3:1→2:1 hexanes/EtOAc) before subjection to LiCl/water/DMSO decarboxylative conditions.
Solid KOtBu (1.36 g, 12.1 mmol) was added in one portion under air to a homogeneous solution of 4-(7-Fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (3.33 g, 10.1 mmol), as prepared in the preceding step, and commercial 3-(4-methyl-piperazin-1-yl)-propan-1-ol (1.50 g, 9.50 mmol) in dry THF (10 mL), while stirring on an ice bath. Following KOtBu addition, the ice bath was immediately removed, and the resulting homogeneous amber solution was stirred for 6 hr. 6 M aqueous HCl (10 mL, 60 mmol) was then added in one portion, and the reaction was stirred overnight (mild bubbles were seen following HCl addition, but these subsided after 15 min). The reaction was then partitioned with 9:1 DCM/MeOH (50 mL) and 2.5 M NaOH (28 mL, 70 mmol), and the aqueous layer was extracted with 9:1 DCM/MeOH (1×50 mL). The combined organic layers were dried (Na2SO4) and concentrated by rotary evaporation at 90° C. to provide the crude title compound as a clear yellow oil (3.79 g, “102%” crude yield). LC/MS (ESI): calcd mass 369.3. found 370.2 (MH)+.
A solution of 7-[3-(4-Methyl-piperazin-1-yl)-propoxy]-4-piperidin-4-yl-quinazoline (3.654 g, 9.9 mmol), as prepared in the previous step, in 98:2 DCM/MeOH (15 mL) was added rapidly dropwise under air in 2 mL portions to an ice bath-chilled stirred mixture of (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride (4.13 g, 10.9 mmol), as prepared in Example 66a, and dimethylethylamine (DMEA) (1.4 mL, 13 mmol) in 98:2 DCM/MeOH (20 mL). Residual quinazoline derivative was then transferred to the carbamate reaction mixture with 2×7 mL additional 98:2 DCM/MeOH. The resulting homogeneous dark amber solution was stirred for another 5 min, and the ice bath was then removed and the reaction stirred at “rt” for 1.5 hr. The homogeneous reaction solution was then directly applied to a silica flash column (79 mm diameter×6″ length) pre-equilibrated with acetone. The title compound was eluted with 1.5 L acetone→2 L 9:1 acetone/MeOH→2 L 9:1 acetone/MeOH/3% DMEA. The combined fractions were concentrated to afford the title compound contaminated with nitrophenol and DMEA, and this material was partitioned with DCM (100 mL) and 2 M aqueous K2CO3 (2×20 mL). The organic layer was dried (Na2SO4) and concentrated under high vacuum at 90° C. to afford the title compound as a lavender foam that was crushed to a powder [3.89 g, 70% over three steps from 4-(7-Fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester].
1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.04 (d, 1H), 7.34-7.22 (m, 4H), 6.88 (m, 2H), 6.32 (s, 1H), 4.31-4.23 (m, 2H), 4.22-4.15 (t, 2H), 3.89-3.82 (m, 4H), 3.75-3.60 (m, 1H), 3.20-3.05 (m, 6H), 2.70-2.45 (m, 10H), 2.35 (s, 3H), 2.22-1.88 (m, 6H). LC/MS (ESI): 574.2 (MH)+. Anal. Calcd for C32H43N7O3·0.35 H2O: C, 66.26; H, 7.59; N, 16.90. Found: C, 66.05; H, 7.47; N, 16.79. Karl Fischer: 1.09% water.
To a stirred solution of 4.9 g (30.4 mmol) of 4-pyrrolidin-1-yl-phenylamine in 70 mL of anhydrous THF at room temperature, was added dropwise a solution of 6.4 g (32 mmol) of 4-nitrophenyl chloroformate in 16 mL of anhydrous THF. After the addition was complete, the mixture was stirred for 1 h and then filtered. The precipitate was washed first with anhydrous THF (2×10 mL) and then with anhydrous DCM (3×10 mL) and dried in vacuo to yield 10 g of an off-white solid. 1H-NMR (300 MHz, CD3OD): 10.39 (s, 1H), 8.32 (d, 2H), 7.73 (d, 2H), 7.60 (d, 2H), 7.48 (d, 2H), 3.86-3.68 (bs, 4H), 2.35-2.24 (bs, 4H). LC/MS (ESI): 328 (MH)+.
Prepared essentially as described in Example 67b, using 1-(2-hydroxyethyl)-2-pyrrolidone and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride. 1H NMR (400 MHz, CDCl3) δ 9.15 (s, 1H), 8.06 (d, J=9.30 Hz, 1H), 7.30 (d, J=2.48 Hz, 1H), 7.25 (dd, J=9.28 and 2.49 Hz, 1H), 7.18 (d, J=8.94 Hz, 2H), 6.52 (d, J=8.89 Hz, 2H), 6.20 (br, 1H), 4.28 (t, J=5.17 Hz, 2H), 4.24 (m, 2H), 3.79 (t, J=5.13 Hz, 2H), 3.66 (m, 1H), 3.60 (t, J=6.95 Hz, 2H), 3.26 (t, J=6.58 Hz, 4H), 3.12 (td, J=12.71 and 2.51 Hz, 2H), 2.42 (t, J=7.80 Hz, 2H), 1.93-2.18 (m, 10 H). LC-MS (ESI) calcd for C30H37N6O3 (MH+) 529.3. found 529.1. Anal. Calcd for C30H36N6O3: C, 68.16; H, 6.86; N, 15.90. Found: C, 67.97; H, 6.76; N, 15.80.
Prepared essentially as described in Example 33 using (4-Morpholino-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride as prepared by the method outlined in Example 66a. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.05 (d, 1H), 7.33-7.22 (m, 4H), 6.88 (d, 2H), 6.31 (s, 1H), 4.30-4.23 (m, 2H), 4.22-4.17 (m, 2H), 3.88-3.83 (m, 4H), 3.72-3.63 (m, 1H), 3.18-3.06 (m, 6H), 2.74-2.36 (m, 4H), 2.20-2.05 (m, 4H), 1.97 (d, 2H), 1.76-1.42 (m, 8H). LC/MS (ESI): 559.1 (MH)+.
The title compound was prepared from 4-chloro-6-fluoroquinazoline (WO 2005021500 Al, WO 2004071460 A2, WO 9609294 Al) essentially as described in Example 65, except 3-(4-Methyl-piperazin-1-yl)-propan-1-ol at 100° C. for 1 hr was used in place of 3-amino-propan-1-ol, and the use of methanesulfonyl chloride was omitted. 1H-NMR (300 MHz, CDCl3) δ 9.13 (s, 1H), 7.98 (d, 1H), 7.56 (dd, 1H), 7.32 (d, 1H), 7.25 (m, 2H), 6.85 (m, 2H), 6.33 (br s, 1H), 4.49 (heptet, 1H), 4.27 (m, 2H), 4.19 (t, 2H), 3.65 (tt, 1H), 3.18 (td, 2H), 2.65-2.38 (m, 10H), 2.31 (t, 3H), 2.21-1.95 (m, 6H), 1.32 (d, 6H). LC/MS (ESI): calcd mass 546.3. found 547.3 (MH)+.
Prepared essentially as described in Example 33 using propane-1,3-diol in place of 3-hydroxypropylpiperidine and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, as prepared by the method outlined in Example 66a, in place of (4-isopropoxy-phenyl)-carbamic acid 4-nitrophenyl ester. 1H-NMR (300 MHz, CDCl3): 9.12 (s, 1H), 8.04 (d, 1H), 7.36-7.22 (m, 4H), 6.89 (d, 2H), 6.40 (s, 1H), 4.34-4.21 (m, 4H), 3.95-3.81 (m, 6H), 3.67 (m, 1H), 3.20-3.05 (m, 6H), 2.22-2.02 (m, 4H), 2.02-1.75 (m, 3H). LC/MS (ESI): 492.1 (MH)+.
Prepared essentially as described in Example 33 using 3-methoxypropanol in place of 3-hydroxypropylpiperidine and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, as prepared by the method outlined in Example 66a, in place of (4-isopropoxy-phenyl)-carbamic acid 4-nitrophenyl ester. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.05 (d, 1H), 7.36-7.23 (m, 4H), 6.90 (d, 2H), 6.36 (s, 1H), 4.31-4.20 (m, 4H), 3.87 (m, 4H), 3.75-3.55 (m, 3H), 3.37 (s, 3H), 3.20-3.05 (m, 6H), 2.22-2.04 (m, 4H), 1.97 (d, 2H). LC/MS (ESI): 506.1 (MH)+.
Prepared essentially as described in Example 67 using 3-(2-hydroxyethyl)-oxazolidin-2-one and (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester. 1H NMR (CDCl3) δ 9.16 (s, 1H), 8.09 (d, J=9.34 Hz, 1H), 7.36 (d, J=2.48 Hz, 1H), 7.28 (m, 1H), 7.25 (d, J=8.48 Hz, 2H), 6.85 (d, J=8.97 Hz, 2H), 6.33 (br, 1H), 4.48 (m, 1H), 4.38 (t, J=7.71 Hz, 2H), 4.33 (t, J=5.13 Hz, 2H), 4.26 (m, 2H), 3.76-3.82 (4H), 3.69 (m, 1H), 3.14 (m, 2H), 1.94-2.21 (4H), 1.31 (d, J=6.06 Hz, 6H). Calcd for C28H34N5O5 (MH+) 520.3. found 520.1.
Prepared essentially as described in Example 66a using 6-morpholin-4-yl-pyridin-3-ylamine in place of 4-Morpholino-4-yl-phenylamine. LC/MS (ESI): 345.1 (MH)+.
Prepared essentially as described in Example 39 using (6-morpholin-4-yl-pyridin-3-yl)-carbamic acid 4-nitrophenyl ester hydrochloride in place of (4-isopropoxy-phenyl)-carbamic acid 4-nitrophenyl ester. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.05 (m, 2H), 7.76 (m, 1H), 7.34-7.22 (m, 2H), 6.65 (d, 1H), 6.33 (s, 1H), 4.33-4.15 (m, 4H), 3.83 (m, 4H), 3.75-3.62 (m, 1H), 3.44 (m, 4H), 3.22-3.06 (m, 2H), 2.95-2.60 (m, 10H), 2.52 (s, 3H), 2.23-1.91 (m, 6H). LCAMS (ESI): 575.2 (MH)+.
Prepared essentially as described in Example 66a using 3-fluoro-4-morpholino-4-yl-phenylamine in place of 4-morpholin-4-yl-phenylamine. LC/MS (ESI): 362.1 (MH)+.
Prepared essentially as described in Example 39 using (3-Fluoro-4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride in place of (4-isopropoxy-phenyl)-carbamic acid 4-nitrophenyl ester. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.05 (d, 1H), 7.34-7.22 (m, 3H), 7.00 (d, 1H), 6.86 (t, 1H), 6.41 (s, 1H), 4.31-4.16 (m, 4H), 3.87 (m, 4H), 3.75-3.62 (m, 1H), 3.22-2.98 (m, 8H), 2.71-2.51 (m, 8H), 2.38 (s, 3H), 2.21-1.93 (m, 6H). LC/MS (ESI): 592.2 (MH)+.
Prepared essentially as described in Example 39 using (6-cyclopentoxy-pyridin-3-yl)-carbamic acid 4-nitrophenyl ester as prepared by the method outlined in Example 69c, in place of (4-isopropoxy-phenyl)-carbamic acid 4-nitrophenyl ester. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.07-7.97 (m, 2H), 7.76 (m, 1H), 7.34-7.22 (m, 2H), 6.67 (d, 1H), 6.34 (s, 1H), 5.30 (m, 1H), 4.33-4.15 (m, 3H), 3.75-3.62 (m, 1H), 3.22-3.01 (m, 3H), 2.68-2.47 (m, 8H), 2.37 (s, 3H), 2.24-1.52 (m, 16H). LC/MS (ESI): 574.2 (MH)+.
Prepared essentially as described in Example 67 using 1-(2-hydroxy-ethyl)-pyrrolidin-2-one and (6-morpholin-4-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 80a. 1H NMR (CD3OD) δ 9.04 (s, 1H), 8.36 (d, J=9.34 Hz, 1H), 8.27 (m, 1H), 8.12 (m, 1H), 7.65 (dd, J=9.04 and 2.71 Hz, 1H), 7.39 (m, 1H), 6.81 (d, J=8.88 Hz, 1H), 4.36 (t, J=5.15 Hz, 2H), 4.32 (m, 2H), 3.94 (m, 1H), 3.80 (t, J=4.67 Hz, 4H), 3.77 (t, J=4.82 Hz, 2H), 3.64 (t, J=6.81 Hz, 2H), 3.40 (t, J=4.98 Hz, 4H), 3.18 (m, 2H), 2.40 (t, J=7.77 Hz, 2H), 1.90-2.10 (6H). Calcd for C29H36N7O4 (MH+) 546.3. found 546.1.
Prepared essentially as described in Example 69a, using 2-chloro-5-nitro-pyrimidine and pyrrolidine. 1H NMR (DMSO-d6) δ 9.11 (s, 2H), 3.62 (m, 4H), 1.97 (m, 4H).
Prepared essentially as described in Example 69b, using 5-nitro-2-pyrrolidin-1-yl-pyrimidine.
1H NMR (CDCl3) δ 7.99 (s, 2H), 3.50 (m, 4H), 3.06 (br, 2H), 1.97 (m, 4H).
Prepared essentially as described in example 69c. 1H NMR (DMSO-d6) δ 10.19 (bs, 1H), 8.45 (s, 2H), 8.30 (d, J=9.23 Hz, 2H), 7.52 (d, J=9.18 Hz, 2h), 3.45 (m, 4H).
Prepared essentially as described in Example 67 using 1-(2-hydroxy-ethyl)-pyrrolidin-2-one and (2-pyrrolidin-1-yl-pyrimidin-5-yl)-carbamic acid 4-nitro-phenyl ester. 1H NMR (CD3OD) δ 9.04 (s, 1H), 8.36 (d, J=9.31 Hz, 1H), 8.31 (s, 2H), 7.39 (dd, J=9.20 and 2.57 Hz, 1H), 7.34 (d, J=2.50 Hz, 1H), 4.36 (t, J=5.23 Hz, 2H), 4.30 (m, 2H), 3.94 (m, 1H), 3.78 (t, J=5.28 Hz, 2H), 3.64 (t, J=7.00 Hz, 2H), 3.53 (t, J=6.74 Hz, 4H), 3.19 (m, 2H), 2.40 (t, J=7.87 Hz, 2H), 1.90-2.12 (1OH). Calcd for C28H35N8O3 (MH+) 531.3. found 531.1.
Prepared essentially as described in Example 67 using 1-(2-hydroxy-ethyl)-pyrrolidin-2-one and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester, which was prepared by the method described in Example 74a. 1H NMR (CD3OD) δ 9.04 (s, 1H), 8.36 (d, J=9.59 Hz, 1H), 7.39 (dd, J=9.22 and 2.60 Hz, 1H), 7.34 (d, J=2.63 Hz, 1H), 7.25 (d, J=9.01 Hz, 2H), 6.93 (d, J=9.04 Hz, 2H), 4.36 (t, J=5.36 Hz, 2H), 4.32 (m, 2H), 3.93 (m, 1H), 3.83 (t, J=4.78 Hz, 4H), 3.78 (t, J=5.22 Hz, 2H), 3.64 (t, J=7.14 Hz, 2H), 3.16 (m, 2H), 3.08 (t, J=4.83 Hz, 4H), 2.40 (t, J=7.76 Hz, 2H), 1.90-2.12 (6H). Calcd for C30H37N6O4 (MH+) 545.3. found 545.1.
The title compound was prepared essentially as described in Example 65, but using (6-Pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared from 6-Pyrrolidin-1-yl-pyridin-3-ylamine (WO 2002048152 A2) essentially as described in Example 74a. 1H-NMR (400 MHz, CDCl3) δ 9.11 (s, 1H), 8.04 (d, 1H), 7.99 (d, 1H), 7.61 (dd, 1H), 7.31 (d, 1H), 7.24 (dd, 1H), 6.41 (br s, 1H), 6.34 (d, 1H), 5.04 (br t, 1H), 4.30-4.21 (m, 4H), 3.65 (tt, 1H), 3.45-3.37 (m, 6H), 3.11 (td, 2H), 2.96 (s, 3H), 2.19-1.89 (m, 10H). LC/MS (ESI) calcd mass 553.3, found 554.1 (MH)+.
The title compound was prepared essentially as described in Example 65, but using (6-morpholin-4-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared from commercial 6-morpholin-4-yl-pyridin-3-ylamine essentially as described in Example 66a. 1H-NMR (400 MHz, CDCl3) δ 9.11 (s, 1H), 8.06 (s, 1H), 8.05 (d, 1H), 7.74 (dd, 1H), 7.32 (d, 1H), 7.25 (dd, 1H), 6.64 (d, 1H), 6.45 (br s, 1H), 4.93 (br t, 1H), 4.30-4.22 (m, 4H), 3.82 (m, 4H), 3.67 (tt, 1H), 3.42 (m, 6H), 3.13 (td, 2H), 2.97 (s, 3H), 2.20-2.05 (m, 4H), 1.99-1.91 (m, 2H). LC/MS (ESI) calcd mass 569.2. found 570.0 (MH)+.
The title compound was prepared essentially as described in Example 65, but using (6-Cyclopentyloxy-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 69c. 1H-NMR (400 MHz, CDCl3) δ9.10 (s, 1H), 8.04 (d, 1H), 8.01 (d, 1H), 7.74 (dd, 1H), 7.31 (d, 1H), 7.25 (dd, 1H), 6.65 (d, 1H), 6.55 (br s, 1H), 5.30 (m, 1H), 5.05 (br t, 1H), 4.36 (tt, 1H), 4.30-4.22 (m, 4H), 3.41 (q, 2H), 3.13 (m, 2H), 2.97 (s, 3H), 2.20-2.04 (m, 4H), 1.94 (m, 4H), 1.78 (m, 4H), 1.61 (m, 2H). LC/MS (ESI) calcd mass 568.3. found 569.0 (MH)+.
Prepared essentially as described in Example 67b, using (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester, which was prepared from 6-Pyrrolidin-1-yl-pyridin-3-ylamine (WO 2002048152 A2) essentially as described in Example 74a. 1H NMR (CD3OD) δ 9.03 (s, 1H), 8.35 (d, J=9.33 Hz, 1H), 7.99 (d, J=2.60 Hz, 1H), 7.58 (dd, J=9.05 and 2.59 Hz, 1H), 7.39 (dd, J=9.25 and 2.52 Hz, 1H), 7.31 (d, J=2.49 Hz, 1H), 6.54 (d, J=9.24 Hz, 1H), 4.33 (m, 2H), 4.22 (t, J=5.80 Hz, 2H), 3.93 (m, 1H), 3.53 (t, J=6.88 Hz, 4H), 3.43 (t, J=6.72 Hz, 4H), 3.18 (m, 2H), 2.37 (t, J=7.82 Hz, 2H), 1.90-2.17 (12 H). Calcd for C30H38N7O3 (MH+) 544.3. found 544.1.
Prepared essentially as described in Example 67b using (2-pyrrolidin-1-yl-pyrimidin-5-yl)-carbamic acid 4-nitro-phenyl ester, which was prepared as described in Example 84c.1H NMR (CD3OD) δ 9.04 (s, 1H), 8.36 (d, J=9.43 Hz, 1H), 8.32 (s, 2H), 7.39 (dd, J=9.26 and 2.52 Hz, 1H), 7.31 (d, J=2.49 Hz, 1H), 4.33 (m, 2H), 4.22 (t, J=5.96 Hz, 2H), 3.95 (m, 1H), 3.53 (t, J=6.61 Hz, 8H), 3.20 (m, 2H), 2.38 (t, J=7.66 Hz, 2H), 1.90-2.17 (12H). Calcd for C29H37N8O3 (MH+) 545.3. found 545.1.
Prepared essentially as described in Example 79 using (6-morpholin-4-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester, which was prepared as described in Example 80a. 1H NMR (CD3OD) δ 9.05 (s, 1H), 8.37 (d, J=9.14 Hz, 1H), 8.13 (dd, J=2.71 and 0.49 Hz, 1H), 7.65 (dd, J=9.04 and 2.69 Hz, 1H), 7.42 (dd, J=9.27 and 2.61 Hz, 1H), 7.37 (d, J=2.49 Hz, 1H), 6.82 (d, J=9.02 and 0.51 Hz, 1H), 4.30-4.41 (6H), 3.94 (m, 1H), 3.74-3.84 (8H), 3.40 (t, J=5.00 Hz, 4H), 3.18 (m, 2H), 1.90-2.08 (4H). Calcd for C28H34N7O5 (MH+) 548.3. found 548.0.
Prepared essentially as described in Example 79 using (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester, which was prepared from 6-Pyrrolidin-1-yl-pyridin-3-ylamine (WO 2002048152 A2) essentially as described in Example 74a. 1H NMR (CD3OD) δ 9.04 (s, 1H), 8.37 (d, J=9.26 Hz, 1H), 7.98 (dd, J=2.65 and 0.62 Hz, 1H), 7.56 (dd, J=9.03 and 2.66 Hz, 1H), 7.41 (dd, J=9.02 and 2.49Hz, 1H), 7.36 (d, J=2.63 Hz, 1H), 6.50 (d, J=9.02 Hz, 1H), 4.39 (t, J=5.20 Hz, 2H), 4.37 (t, J=8.25 Hz, 2H), 4.33 (m, 2H), 3.94 (m, 1H), 3.73-3.84 (4H), 3.42 (t, J=6.68 Hz, 4H), 3.18 (m, 2H), 1.90-2.07 (8H). Calcd for C28H34N7O4 (MH+) 532.3. found 532.1.
Prepared essentially as described in Example 67 using (1-methyl-piperidin-4-yl)-methanol. 1H NMR (CD3OD) δ 9.03 (s, 1H), 8.34 (d, J=9.44 Hz, 1H), 7.37 (dd, J=9.19 and 2.61 Hz, 1H), 7.31 (d, J=2.55 Hz, 1H), 7.23 (d, J=9.06 Hz, 2H), 6.84 (d, J=9.00 Hz, 2H), 4.53 (m, 1H), 4.34 (m, 2H), 4.07 (d, J=5.79 Hz, 2H), 3.92 (m, 1H), 3.32 (m, 2H), 3.16 (m, 2H), 2.95 (m, 2H), 2.30 (s, 3H), 1.87-2.14 (7H), 1.51 (m, 2H), 1.28 (d, J=6.04 Hz, 6H). Calcd for C30H40N5O3 (MH+) 518.3. found 518.1.
Prepared essentially as described in Example 67 using (1-methyl-piperidin-4-yl)-methanol and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester, which was prepared as described in Example 66a. 1H NMR (CDCl3) δ 9.14 (s, 1H), 8.05 (d, J=9.34 Hz, 1H), 7.23-7.30 (4H), 6.88 (d, J=9.02 Hz, 2H), 6.30 (br, 1H), 4.26 (m, 2H), 4.04 (d, J=5.65 Hz, 2H), 3.86 (t, J=4.73 Hz, 4H), 3.68 (m, 1H), 3.20 (m, 2H), 3.16 (m, 2H), 3.10 (t, J=4.78 Hz, 4H), 3.00 (m, 2H), 2.51 (s, 3H), 1.93-2.13 (7H), 1.70 (br, 2H). Calcd for C31H41N6O3 (MH+) 545.3. found 545.1.
Prepared essentially as described in Example 67 using 2-morpholin-4-yl-ethanol and (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester, which was prepared from6-Pyrrolidin-1-yl-pyridin-3-ylamine(WO 2002048152 A2) essentially as described in Example 74a. 1H NMR (CDCl3) δ 9.13 (s, 1H), 8.05 (d, J=9.27 Hz, 1H), 7.99 (d, J=2.57 Hz, 1H), 7.67 (dd, J=9.08 and 2.78 Hz, 1H), 7.30 (dd, J=5.33 and 2.39 Hz, 1H), 7.28 (d, J=9.04 Hz, 1H), 6.42 (br, 1H), 6.37 (d, J=9.16 Hz, 1H), 4.29 (t, J=5.58 Hz, 4H), 3.75 (t, J=4.55 Hz, 4H), 3.67 (m, 1H), 3.44 (t, J=6.64 Hz, 4H), 3.13 (td, J=12.96 and 2.42 Hz, 2H), 2.90 (t, J=5.51 Hz, 2H), 2.61 (t, J=4.71 Hz, 4H), 2.13 (m, 2H), 1.92-2.03 (6H). Calcd for C29H38N7O3 (MH+) 532.3. found 532.1.
Prepared essentially as described in Example 67 using 2-morpholin-4-yl-ethanol and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester, which was prepared as described in Example 66a. 1H NMR (CDCl3) δ 9.14 (s, 1H), 8.05 (d, J=9.22 Hz, 1H), 7.26-7.33 (4H), 6.87 (d, J=9.02 Hz, 2H), 6.33 (br, 1H), 4.22-4.34 (4H), 3.86 (t, J=4.63 Hz, 4H), 3.77 (m, 4H), 3.68 (m, 1H), 3.07-3.18 (6H), 2.93 (m, 2H), 2.64 (m, 4H), 2.13 (m, 2H), 1.97 (m, 2H). Calcd for C30H39N6O4 (MH+) 547.3. found 547.1.
Prepared essentially as described in Example 67 using 1-[4-(2-hydroxy-ethyl)-piperazin-1-yl]-ethanone and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester, which was prepared as described in Example 66a. 1H NMR (CD3OD) δ 9.04 (s, 1H), 8.35 (d, J=9.36 Hz, 1H), 7.40 (dd, J=9.22 and 2.45 Hz, 1H), 7.35 (d, J=2.44 Hz, 1H), 7.26 (d, J=9.10 Hz, 2H), 6.92 (d, J=9.12 Hz, 2H), 4.36 (t, J=5.15 Hz, 2H), 4.32 (m, 2H), 3.92 (m, 1H), 3.82 (t, J=4.64 Hz, 4H), 3.62 (t, J=4.71 Hz, 2H), 3.58 (t, J=5.22 Hz, 2H), 3.16 (m, 2H), 3.08 (t, J=4.82 Hz, 4H), 2.94 (t, J=5.46 Hz, 2H), 2.66 (t, J=5.16 Hz, 2H), 2.61 (t, J=5.13 Hz, 2H), 2.10 (s, 3H), 1.89-2.08 (4H). Calcd for C32H42N7O4 (MH+) 588.3. found 588.1.
Prepared essentially as described in Example 67 using 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 66a. 1H NMR (CDCl3) δ 9.23 (s, 1H), 9.21 (dd, J=9.35 Hz and 5.85 Hz, 1H), 7.69 (dd, J=9.48 and 2.52 Hz, 1H), 7.44 (m, 1H), 7.27 (d, J=8.95 Hz, 2H), 6.89 (d, J=8.95 Hz, 2H), 6.29 (s, 1H), 4.27 (m, 2H), 3.86 (t, J=4.74 Hz, 4H), 3.73 (m, 1H), 3.17 (m, 2H), 3.11 (t, J=4.78 Hz, 4H), 2.15 (m, 2H), 1.99 (m, 2H). Calcd for C24H27FN5O2 (MH+) 436.2. found 436.1.
Prepared from 4-(7-Fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid (4-morpholin-4-yl-phenyl)-amide, synthesized as described in the previous step, and 2-piperidin-2-yl-ethanol using the protocol described in Example 67a. 1H NMR (CD3OD) δ 9.03 (s, 1H), 8.34 (d, J=9.31 Hz, 1H), 7.37 (dd, J=9.19 and 2.54 Hz, 1H), 7.33 (d, J=2.47 Hz, 1H), 7.26 (d, J=9.06 Hz, 2H), 6.93 (d, J=9.10 Hz, 2H), 4.34 (m, 2H), 4.28 (m, 2H), 3.94 (m, 1H), 3.82 (t, J=4.69 Hz, 4H), 3.16 (m, 2H), 3.08 (t, J=4.78 Hz, 4H), 3.04 (m, 1H), 2.82 (m, 1H), 2.66 (m, 1H), 1.40-2.10 (12H). Calcd for C31H41N6O3 (MH+) 545.3. found 545.1.
Prepared essentially as described in Example 67 using (1-methyl-piperidin-4-yl)-methanol and (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester, which was prepared from 6-Pyrrolidin-1-yl-pyridin-3-ylamine (WO 2002048152 A2) essentially as described in Example 74a. 1H NMR (CDCl3) δ 9.14 (s, 1H), 8.05 (d, J=9.26 Hz, 1H), 7.97 (d, J=2.61 Hz, 1H), 7.63 (dd, J=8.93 and 2.72 Hz, 1H), 7.28 (dd, J=7.00 and 2.63 Hz, 1H), 7.24 (d, J=2.37 Hz, 1H), 6.36 (d, J=8.87 Hz, 1H), 6.18 (br, 1H), 4.26 (m, 2H), 4.03 (d, J=5.79 Hz, 2H), 3.67 (m, 1H), 3.44 (t, J=6.69 Hz, 4H), 3.14 (td, J=12.22 and 2.65 Hz, 4H), 2.47 (m, 2H), 2.00 (s, 3H), 1.92-2.21 (13H). Calcd for C30H40N7O2 (MH+) 530.3. found 530.1.
Prepared essentially as described in Example 102 and the title compound was obtained as a major side-product after purification. 1H-NMR (300 MHz, CDCl3): 8.98 (s, 1H), 7.97 (d, 1H), 7.30-7.15 (m, 3H), 7.03 (d, 1H), 6.83 (d, 2H), 6.41 (s, 1H), 4.58-4.40 (m, 1H), 4.26 (d, 2H), 3.68-3.55 (m, 1H), 3.18-3.04 (m, 8H), 2.20-1.85 (m, 4H), 1.3 (d, 6H). LCAMS (ESI): 434.1 (MH)+.
Prepared essentially as described in Example 76, using 1-(3-hydroxy-propyl)-pyrrolidin-2-one. 1H NMR (CDCl3) δ 9.13 (s, 1H), 7.98 (d, J=9.22 Hz, 1H), 7.54 (dd, J=9.19 and 2.63 Hz, 1H), 7.34 (d, J=2.52 Hz, 1H), 7.26 (d, J=8.91 Hz, 2H), 6.83 (d, J=8.98 Hz, 2H), 6.43 (br, 1H), 4.47 (m, 1H), 4.26 (m, 2H), 4.16 (t, J=6.11 Hz, 2H), 3.65 (m, 1H), 3.54 (t, J=7.04 Hz, 2h), 3.47 (t, J=7.10 Hz, 2H), 3.18 (m, 2H), 2.39 (t, J=7.88 Hz, 2H), 1.96-2.18 (8H), 1.30 (d, J=6.06 Hz, 6H). Calcd for C30H38N5O4 (MH+) 532.3. found 532.1.
A mixture of 4-(3-aminopropyl)-1-methylpiperazine (0.1 mmol), Et3N (0.1 mmol) and 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (0.1 mmol), prepared as described in Example 65b, in DMF (1 mL) was stirred at 130° C. for 3 h. It was then diluted with water and extracted with EtOAc. The combined extracts were washed with water, brine, dried (anhydrous MgSO4), filtered and concentrated in vacuo. The crude product was then treated with 3M HCl/MeOH (2 mL) and stirred at rt for 2 h, then concentrated in vacuo. The crude residue was dissolved in a mixture of DCM:MeOH (1: 1; 2 mL) and neutralized with excess Et3N and treated with (4-isopropoxy-phenyl)-carbamic acid 4-nitrophenyl ester (0.11 mmol), which was prepared as described in Example 1a, at rt for 1 h. It was then concentrated in vacuo and the crude product was dissolved in DCM and washed with water, brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was then purified by Preparative TLC (silica gel; DCM:MeOH, 9:1) to obtain 3.2 mg (6% overall yield over the three steps) of the title compound. 1H-NMR (300 MHz, CDCl3): 8.97 (s, 1H), 7.88 (d, 1H), 7.28-7.22 (m, 3H), 6.97-6.81 (m, 4H), 6.33 (s, 1H), 4.53-4.43 (m, 1H), 4.30-4.20 (d, 2H), 3.66-3.32 (m, 2H), 3.11 (t, 2H), 2.85-2.55 (m, 8H), 2.43 (s, 4H), 2.20-1.85 (m, 8H), 1.31 (d, 6H). LC/MS (ESI): 546.2 (MH)+.
Prepared essentially as described in Example 102 using 1-methyl-piperazine in place of 4-(3-aminopropyl)-1-methylpiperazine. 1H-NMR (300 MHz, CDCl3): 9.05 (s, 1H), 7.99 (d, 1H), 7.35-7.20 (m, 4H), 6.84 (d, 2H), 6.33 (s, 1H), 4.54-4.42 (m, 1H), 4.25 (d, 2H), 3.69-3.50 (m, 5H), 3.13 (t, 2H), 2.74 (m, 4H), 2.46 (s, 3H), 2.20-1.88 (m, 4H), 1.31 (d, 6H). LCAMS (ESI): 489.2 (MH)+.
To a solution of 3-(4-methylpiperazin-1-yl)-propan-1-ol (0.22 mmol) in anhydrous THF (2 mL) was added NaH (0.4 mmol) and the mixture was stirred at rt for 5 min. Then, 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (0.2 mmol), prepared as described in Example 65, was added to it and the mixture was stirred at 60° C. for 2 h. It was then concentrated in vacuo and partitioned between water and DCM. The DCM layer was drawn off, washed with water, brine, dried (anhydrous MgSO4), filtered and concentrated in vacuo. This crude product was then treated with 3 M HCl/MeOH (2 mL) and stirred at rt for 2 h and then concentrated in vacuo. A portion of the crude residue (0.05 mmol) was dissolved in a mixture of DCM:MeOH (1:1;2 mL) and neutralized with excess Et3N (0.3 mmol) and treated with (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitrophenyl ester hydrochloride (0.075 mmol), which was prepared from 6-Pyrrolidin-1-yl-pyridin-3-ylamine (WO 2002048152 A2) essentially as described in Example 74a, at rt for 1 h. It was then concentrated in vacuo and the crude product was dissolved in DCM and washed with water thrice, then washed with brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was then purified by Preparative TLC (silica gel; DCM:MeOH:NH4OH, 90:9:1) to obtain 10 mg (35%) of the title compound. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.08-7.96 (m, 2H), 7.66-7.60 (m, 1H), 7.34-7.22 (m, 2H), 6.39-6.27 (m, 2H), 4.32-4.14 (m, 4H), 3.74-3.59 (m, 1H), 3.46-3.38 (m, 4H), 3.13 (t, 2H), 2.65-2.50 (m, 10H), 2.37 (s, 3H), 2.22-1.86 (m, 10H). LC/MS (ESI): 559.1 (MH)+.
Prepared essentially as described in Example 104 using (2-pyrrolidin-1-yl-pyrimidin-5-yl)-carbamic acid 4-nitrophenyl ester hydrochloride, which was prepared as described in Example 84c, in place of (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.12 (s, 1H), 8.32 (m, 2H), 8.04 (d, 1H), 7.34-7.22 (m, 2H), 6.24 (s, 1H), 4.32-4.14 (m, 4H), 3.74-3.61 (m, 1H), 3.60-3.50 (m, 4H), 3.14 (t, 2H), 2.75-2.45 (m, 10H), 2.37 (s, 3H), 2.22-1.88 (m, 10H). LC/MS (ESI): 560.1 (MH)+.
Prepared essentially as described in Example 104 using 4-(5-isothiocyanato-pyridin-2-yl)-morpholine in place of (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.11 (s, 1H), 8.08-8.00 (m, 2H), 7.58-7.51 (m, 1H), 7.34-7.22 (m, 3H), 6.64 (d, 1H), 4.86 (d, 2H), 4.19 (t, 2H), 3.86-3.70 (m, 5H), 3.52-3.30 (m, 6H), 2.63-2.40 (m, 10H), 2.34 (s, 3H), 2.30-1.86 (m, 6H). LC/MS (ESI): 591.0 (MH)+.
Prepared essentially as described in Example 67 using (1-methyl-piperidin-4-yl)-methanol and (6-cyclobutoxy-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester, which was prepared as described in Example 17c. 1H NMR (CD3OD) δ 9.03 (s, 1H), 8.34 (d, J=9.41 Hz, 1H), 8.07 (dd, J=2.73 and 0.54 Hz, 1H), 7.72 (dd, J=8.87 and 2.77 Hz, 1H), 7.38 (dd, J=9.24 and 2.48 Hz, 1H), 7.31 (d, J=2.48 Hz, 1H), 6.71 (dd, J=8.87 and 0.59 Hz, 1H), 5.05 (m, 1H), 4.34 (m, 2H), 4.06 (d, J=5.77 Hz, 2H), 3.93 (m, 1H), 3.18 (m, 2H), 2.96 (m, 2H), 2.45 (m, 2H), 2.30 (s, 3H), 1.64-2.17 (13H), 1.51 (m, 2H). Calcd for C30H39N6O3 (MH+) 531.3. found 531.0.
Prepared essentially as described in Example 67 using (1-methyl-piperidin-4-yl)-methanol and (6-morpholin-4-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester, which was prepared as described in Example 80a. 1H NMR (CD3OD) δ 9.04 (s, 1H), 8.36 (d, J=9.31 Hz, 1H), 8.12 (dd, J=2.66 and 0.57 Hz, 1H), 7.65 (dd, J=9.03 and 2.73 Hz, 1H), 7.38 (dd, J=9.14 and 2.61 Hz, 1H), 7.33 (d, J=2.48 Hz, 1H), 6.82 (d, J=9.08 Hz, 1H), 4.34 (m, 2H), 4.12 (d, J=5.75 Hz, 2H), 3.94 (m, 1H), 3.80 (t, J=4.73 Hz, 4H), 3.40 (t, J=4.97 Hz, 4H), 3.31 (m, 2H), 3.18 (m, 2H), 2.70 (m, 2H), 2.65 (s, 3H), 1.90-2.13 (7H), 1.65 (m, 2H). Calcd for C30H40N7O3 (MH+) 546.3, found 546.0.
Prepared essentially as described in Example 67 using (1-methyl-piperidin-4-yl)-methanol and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CD3OD) δ 9.03 (s, 1H), 8.35 (d, J=9.41 Hz, 1H), 7.37 (dd, J=9.20 and 2.61 Hz, 1H), 7.31 (d, J=2.59 Hz, 1H), 7.13 (d, J=8.88 Hz, 2H), 6.54 (d, J=8.98 Hz, 2H), 4.33 (m, 2H), 4.07 (d, J=5.87 Hz, 2H), 3.92 (m, 1H), 3.24 (t, J=6.80 Hz, 4H), 3.16 (m, 4H), 2.97 (m, 2H), 2.33 (s, 3H), 1.88-2.19 (13H). Calcd for C31H41N6O2 (MH+) 529.3. found 529.1.
A mixture of 4-(7-fluoro-quinazolin-4-yl)-piperidin-1-carboxylic acid tert-butyl ester (31.6 mg, 95.5 μmol), as prepared in Example 65b, 3-[1,2,4]-triazol-4-yl-propan-1-ol (ChemPacific) (12.0 mg, 94.5 μmol), and KOtBu (11.7 mg, 104 μmol) in DME (100 μL) and DMSO (50 μL) was stirred at rt for 1 hr. The resulting homogeneous amber solution was partitioned with DCM (2 mL) and 0.5M sodium phosphate/pH 7 (2 mL). The organic layer was concentrated to provide the crude title compound that was used immediately for the next step. LC/MS (ESI): calcd mass 438.2. found 439.1 (MH)+.
The crude 4-[7-(3-[1,2,4]Triazol-4-yl-propoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester, as prepared in the previous step, was treated with TFA (70 μL) at 100° C. in a sealed vial for 10 min (aluminum block). CHCl3 (450 μL) and DMEA (140 μL, 1.3 mmol) were added, and one-half of the resulting homogeneous amber solution was treated with (6-morpholin-4-yl-pyridin-3-yl)-carbamic acid-4-nitrophenyl ester hydrochloride (22 mg, 58 μmol), as prepared in Example 80a, and stirred at 40° C. for 1.5 hr. (The other one-half of the homogenous solution was diverted to the synthesis given in Example 114. ) The reaction was then partitioned with 2M K2CO3 (2 mL) and DCM (2 mL), and the aqueous layer was extracted with 9:1 DCM/MeOH (1×2 mL). The combined organic layers were concentrated and the residue was partially purified with a 5 g silica flash cartridge (97:3 acetone/MeOH eluent with 2% DMEA), and further purified with HPLC (C 18 column) to provide the title compound as a powder after lyophilization [2.1 mg, 8.1% overall from 4-(7-fluoro-quinazolin-4-yl)-piperidin-1-carboxylic acid tert-butyl ester.] 1H-NMR (400 MHz, 95:5 CDCl3/CD3OD) δ 9.12 (s, 1H), 8.43 (dd, 1H), 8.30 (br s, 2H), 8.13 (m, 2H), 7.32 (m, 1H), 7.26 (dd, 1H), 6.96 (d, 1H), 4.37 (m, 4H), 4.21 (t, 2H), 3.88 (m, 4H), 3.75 (m, 1H), 3.59 (m, 4H), 3.14 (m, 2H), 2.42 (pentet, 2H), 2.16-1.95 (m, 4H). LC/MS (ESI) calcd mass 543.3. found 544.1 (MH)+.
The title compound was prepared essentially as described for Example 65, except 3-Dimethylamino-4-methoxy-cyclobut-3-ene-1,2-dione [Inorganic Chemistry (1997), 36(14), 3096-3101] at 80° C. for 1 hr replaced methanesulfonyl chloride at rt, and (6-Pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared from 6-Pyrrolidin-1-yl-pyridin-3-ylamine (WO 2002048152 A2) essentially as described in Example 74a, was used. Work-up of the crude reaction and HPLC purification was essentially as described in Example 110. 1H-NMR (400 MHz, 95:5 CDCl3/CD3OD) δ 9.08 (s, 1H), 8.35 (dd, 1H), 8.10 (d, 1H), 8.04 (d, 1H), 7.31-7.26 (m, 2H), 6.75 (d, 1H), 4.38 (m, 2H), 4.27 (t, 2H), 3.93 (t, 2H), 3.72 (m, 1H), 3.58 (m, 4H), 3.23 (s, 6H), 3.12 (m, 2H), 2.25-1.92 (m, 10H). LC/MS (ESI) calcd mass 598.3. found 599.0 (MH)+.
The title compound was prepared essentially as described in Example 111, except commercial 4-morpholinecarbonyl chloride replaced 3-Dimethylamino-4-methoxy-cyclobut-3-ene-1,2-dione. 1H-NMR (400 MHz, 95:5 CDCl3/CD3OD) δ 9.10 (s, 1H), 8.35 (dd, 1H), 8.11 (d, 1H), 8.03 (d, 1H), 7.36 (d, 1H), 7.28 (dd, 1H), 6.75 (d, 1H), 4.39 (m, 2H), 4.24 (t, 2H), 3.80-3.66 (m, 5H), 3.58 (m, 4H), 3.47 (t, 2H), 3.12 (m, 2H), 3.36 (m, 4H), 2.19-1.92 (m, 10H). LC/MS (ESI) calcd mass 588.3. found 589.1 (MH)+.
The title compound was prepared essentially as described in Example 112, using (6-morpholin-4-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester hydrochloride (prepared as described in Example 80a). 1H-NMR (400 MHz, 95:5 CDCl3/CD3OD) δ9.12 (s, 1H), 8.38 (dd, 1H), 8.16 (d, 1H), 8.09 (d, 1H), 7.36 (d, 1H), 7.30-7.25 (m, 1H), 6.90 (d, 1H), 4.37 (m, 2H), 4.24 (m, 2H), 3.87 (m, 4H), 3.70 (m, 4H), 3.58 (m, 4H), 3.48 (m, 2H), 3.36 (m, 4H), 3.13 (m, 2H), 2.20-1.95 (m, 6H). LC/MS (ESI): calcd mass 604.3. found 605.1 (MH)+.
The title compound was prepared essentially as described in Example 110 using (6-Pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared from 6-Pyrrolidin-1-yl-pyridin-3-ylamine (WO 2002048152 A2) essentially as described in Example 74a. 1H-NMR (400 MHz, ˜9:1 CDC13/CD3OD) δ9.14 (s, 1H), 8.43 (dd, 1H), 8.26 (s, 2H), 8.12 (d, 1H), 8.09 (d, 1H), 7.33 (d, 1H), 7.27-7.23 (m, 1H), 6.73 (d, 1H), 4.43-4.32 (m, 4H), 4.20 (t, 2H), 3.72 (tt, 1H), 3.59 (m, 4H), 3.13 (td, 2H), 2.41 (pentet, 2H), 2.18-2.05 (m, 6H), 2.02-1.94 (m, 2H). LC/MS (ESI): calcd mass 527.3. found 528.1 (MH)+.
4-[7-(-Hydroxy-propoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester was prepared as described in Example 33 using propane-1,3-diol in place of 3-hydroxypropylpiperidine. To a solution of 4-[7-(-hydroxy-propoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (0.3 mmol) in anhydrous DCM, was added Et3N (0.6 mmol) and methanesulfonyl chloride (0.6 mmol) and the mixture was stirred at rt for 2 h. It was then washed with water (3×), dried over anhydrous MgSO4, filtered and concentrated in vacuo to obtain 4-[7-(3-methanesulfonyloxy-propoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester. This (0.05 mmol) was dissolved in anhydrous dioxane together with 1-ethyl-piperazine (0.1 mmol) and the mixture was stirred at 100° C. overnight and then concentrated in vacuo, then diluted with water and extracted with DCM. The DCM extract was washed with water (3×), dried over anhydrous MgSO4, filtered and concentrated in vacuo. To this was added 3M HCI/MeOH (1 mL) and the mixture was stirred at rt for 2 h and then concentrated in vacuo and the residue was dissolved in a 1:1 mixture of DCM:MeOH, neutralized with excess Et3N and treated with (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride (0.06 mmol), which was prepared as described in Example 66a. The mixture was stirred at rt overnight and then concentrated in vacuo and partitioned between water and DCM. DCM layer was drawn off, washed with water thrice, then dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by Preparative TLC (silica gel; DCM:MeOH:NH4OH; 90:9:1) to obtain 9.4 mg (32%) of the title compound. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.04 (d, 1H), 7.35-7.22 (m, 4H), 6.88 (d, 2H), 6.33 (s, 1H), 4.32-4.15 (m, 4H), 3.89-3.81 (m, 4H), 3.74-3.60 (m, 1H), 3.20-3.04 (m, 7H), 2.66-2.35 (m, 12H), 2.22-1.88 (m, 5H), 1.10 (t, 3H). LC/MS (ESI): 588.1 (MH)+.
Prepared essentially as described in Example 115 using 2-piperazin-1-yl-ethanol in place of 1-ethyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.12 (s, 1H), 8.05 (d, 1H), 7.36-7.22 (m, 4H), 6.88 (d, 2H), 6.36 (s, 1H), 4.32-4.16 (m, 4H), 3.90-3.81 (m, 4H), 3.74-3.6 (m, 1H), 3.31-3.21 (m, 4H), 3.15-3.05 (m, 7H), 2.79 (s, 3H), 2.67-2.53 (m, 6H), 2.22-1.90 (m, 6H). LC/MS (ESI): 604.1 (MH)+.
Prepared essentially as described in Example 115 using 1-acetyl-piperazine in place of 1-ethyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.05 (d, 1H), 7.35-7.23 (m, 4H), 6.88 (d, 2H), 6.29 (s, 1H), 4.31-4.18 (m, 4H), 3.89-3.83 (m, 4H), 3.70-3.43 (m, 5H), 3.20-3.07 (m, 6H), 2.64-2.39 (m, 6H), 2.22-1.90 (m, 9H). LC/MS (ESI): 602.1 (MH)+.
4-[7-(3-Methanesulfonyloxy-propoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (0.1 mmol), prepared as described in Example 115, was dissolved in anhydrous dioxane together with piperazine (0.5 mmol) and the mixture was stirred at 100° C. overnight and then concentrated in vacuo, then diluted with water and extracted with DCM. The DCM extract was washed with water thrice, dried over anhydrous MgSO4, filtered and concentrated in vacuo to obtain 4-[7-(3-piperazin-1-yl-propoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester. This (0.05 mmol) was dissolved in anhydrous DCM (1 mL) and treated with Et3N (0.1 mmol) followed by methanesulfonyl chloride (0.1 mmol) and the mixture was stirred at rt overnight and then washed with water thrice, then dried over anhydrous MgSO4, filtered and concentrated in vacuo. To this was added 3M HCl/MeOH (1 mL) and the mixture was stirred at rt for 2 h and then concentrated in vacuo and the residue was dissolved in a 1:1 mixture of DCM:MeOH, neutralized with excess Et3N and treated with (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride (0.06 mmol), which was prepared as described in Example 66a. The mixture was stirred at rt overnight and then concentrated in vacuo and partitioned between water and DCM. DCM layer was drawn off, washed with water thrice, then dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by Preparative TLC (silica gel; DCM:MeOH:NH4OH; 90:9:1) to obtain 14.3 mg (45%) of the title compound. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.04 (d, 1H), 7.35-7.22 (m, 4H), 6.88 (d, 2H), 6.33 (s, IH), 4.31-4.13 (m, 4H), 3.89-3.80 (m, 4H), 3.74-3.56 (m, 3H), 3.20-3.03 (m, 6H), 2.61-2.38 (m, 11H), 2.22-1.88 (m, 6H). LC/MS (LST): 638.1 (MH)+.
Prepared essentially as described in Example 115 using (S)-prolinol in place of 1-ethyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.05 (d, 1H), 7.35-7.23 (m, 4H), 6.88 (d, 2H), 6.31 (s, 1H), 4.31-4.18 (m, 4H), 3.89-3.81 (m, 4H), 3.72-3.62 (m, 2H), 3.50-3.00 (m, 9H), 2.78-2.26 (m, 4H), 2.22-1.66 (m, 10H). LC/MS (ESI): 575.1 (MH)+.
Prepared essentially as described in Example 118 using N,N-dimethylcarbamyl chloride in place of methanesulfonyl chloride. 1H-NMR (300 MHz, CDCl3): 9.12 (s, 1H), 8.04 (d, 1H), 7.35-7.21 (m, 4H), 6.87 (d, 2H), 6.38 (s, 1H), 4.32-4.15 (m, 4H), 3.90-3.80 (m, 4H), 3.75-3.60 (m, 1H), 3.32-3.23 (m, 4H), 3.15-3.06 (m, 6H), 2.82 (s, 6H), 2.63-2.43 (m, 6H), 2.22-1.90 (m, 6H). LC/MS (ESI): 631.1 (MH)+.
To 4-[7-(3-methanesulfonyloxy-propoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (0.1 mmol), prepared as described in Example 115, was added 3M HCl/MeOH (2 mL) and the mixture was stirred at rt for 2 h and then concentrated in vacuo and the residue was dissolved in a 1:1 mixture of DCM:MeOH, neutralized with excess Et3N and treated with (4-isopropoxy-phenyl)-carbamic acid 4-nitrophenyl ester (0.11 mmol), which was prepared as described in Example 1a. The mixture was stirred at rt overnight and then concentrated in vacuo and partitioned between water and DCM. DCM layer was drawn off, washed with water thrice, then dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by Preparative TLC (silica gel; DCM:MeOH; 9.5:0.5) to obtain 40 mg (75%) of the title compound. 1H-NMR (300 MHz, CDCl3): 9.12 (s, 1H), 8.05 (d, 1H), 7.32-7.20 (m, 4H), 6.81 (d, 2H), 6.53 (s, 1H), 4.50-4.40 (m, 3H), 4.25 (t, 4H), 3.72-3.59 (m, 1H), 3.16-3.03 (m, 2H), 3.01 (s, 3H), 2.36-2.26 (m, 2H), 2.18-2.00 (m, 2H), 1.99-1.87 (m, 2H), 1.29 (d, 6H). LCAMS (ESI): 543.1 (MH)+.
Prepared essentially as described in Example 121 using (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, as prepared by the method outlined in Example 66a, in place of (4-isopropoxy-phenyl)-carbamic acid 4-nitrophenyl ester. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.06 (d, 1H), 7.35-7.23 (m, 4H), 6.87 (d, 2H), 6.40 (s, 1H), 4.48 (t, 2H), 4.31-4.20 (m, 4H), 3.88-3.80 (m, 4H), 3.73-3.61 (m, 1H), 3.18-3.05 (m, 6H), 3.02 (s, 3H), 2.38-2.27 (m, 2H), 2.20-1.85 (m, 4H). LC/MS (ESI): 570.1 (MH)+.
4-[7-(3-Piperazin-1-yl-propoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (0.05 mmol), prepared as described in Example 118, was dissolved in anhydrous DCM (1 mL) and treated with Et3N (0.05 mmol) followed by FMOC-Cl (0.1 mmol) and the mixture was stirred at rt overnight and then washed with water thrice, then dried over anhydrous MgSO4, filtered and concentrated in vacuo. To this was added 3M HCl/MeOH (1 mL) and the mixture was stirred at rt for 2 h and then concentrated in vacuo and the residue was dissolved in a 1:1 mixture of DCM:MeOH, neutralized with excess Et3N and treated with (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride (0.06 mmol), as prepared by the method outlined in Example 66a. The mixture was stirred at rt overnight and then concentrated in vacuo and partitioned between water and DCM. DCM layer was drawn off, washed with water thrice, then dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by Preparative TLC (silica gel; DCM:MeOH:NH4OH; 90:9:1) to obtain the pure product. This was dissolved in anhydrous DCM (1 mL) and diethylamine (0.25 mL) was added and the mixture was stirred at rt overnight. It was then concentrated in vacuo and purified by Preparative TLC (silica gel; DCM:MeOH:NH4OH; 90:9:1) to obtain 2.8 mg (10%) of the title compound. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.05 (d, 1H), 7.35-7.23 (m, 4H), 6.88 (d, 2H), 6.35 (s, 1H), 4.32-4.14 (m, 4H), 3.90-3.80 (m, 4H), 3.75-3.60 (m, 1H), 3.20-2.91 (m, 10H), 2.75-2.55 (m, 6H), 2.18-1.85 (m, 7H). LC/MS (ESI): 560.0 (MH)+.
Prepared essentially as described in Example 115 using pyrrolidine in place of 1-ethyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.05 (d, 1H), 7.34-7.23 (m, 4H), 6.88 (d, 2H), 6.30 (s, 1H), 4.32-4.18 (m, 4H), 3.90-3.83 (m, 4H), 3.75-3.60 (m, 2H), 3.20-3.05 (m, 5H), 2.80-2.55 (m, 6H), 2.22-1.76 (m, 10H). LC/MS (ESI): 545.0 (MH)+.
Prepared essentially as described in Example 115 using 1-methyl-[1,4]diazepane in place of 1-ethyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.05 (d, 1H), 7.34-7.23 (m, 4H), 6.88 (d, 2H), 6.30 (s, 1H), 4.31-4.16 (m, 4H), 3.89-3.83 (m, 4H), 3.74-3.63 (m, 2H), 3.20-3.07 (m, 5H), 2.83-2.67 (m, 9H), 2.43 (s, 3H), 2.22-1.84 (m, 9H). LC/MS (ESI): 588.2 (MH)+.
A mixture of 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (34.9 mg, 0.105 mmol), which was prepared as described in Example 65b, and (R)-(+)-3-pyrrolidinol (32 mg, 0.368 mmol) in DMSO (0.4 mL) was heated at 120° C. with stirring for 40 min. It was partitioned between ethyl acetate and water, the combined organic extracts were washed with brine, dried over Na2SO4 and evaporated to afford almost pure product (40 mg, 95.7%). 1H NMR (CDCl3) δ 8.97 (s, 1H), 7.96 (d, J=9.39 Hz, 1H), 7.01 (dd, J=9.33 and 2.45 Hz, 1H), 6.88 (d, J=2.19 Hz, 1H), 4.71 (m, 1H), 4.32 (m, 2H), 3.67 (m, 2H), 3.58 (m, 1H), 3.51 (m, 2H), 2.93 (m, 2H), 1.80-2.28 (6H), 1.49 (s, 9H). Calcd for C22H31N4O3 (MH+) 399.2. found 399.0.
4-[7-(R)-3-Hydroxy-pyrrolidin-1-yl)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (21 mg, 0.053 mmol) was treated with 2.5 mL of 50% TFA/CH2Cl2 for 2 h, it was evaporated and the dry residue was re-dissolved in CH3CN (1.5 mL). To the CH3CN solution was added DIPEA (64 μL), followed by (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride (27.9 mg, 0.074 mmol), which was prepared as described in Example 66a. The resulting mixture was stirred at room temperature for 1 h and the solvents were removed under reduced pressure. The residue was washed with water and purified by flash column chromatography on silica gel (EtOAc→15% MeOH/EtOAc as eluent). 1H NMR (CD3OD) δ 8.95 (s, 1H), 7.96 (d, J=9.47 Hz, 1H), 7.29 (d, J=8.96 Hz, 2H), 7.03 (dd, J=9.35 and 2.53 Hz, 1H), 6.92 (d, J=1.94 Hz, 1H), 6.87 (d, J=8.87 Hz, 2H), 4.69 (m, 1H), 4.25 (m, 2H), 3.86 (t, J=4.78 Hz, 4H), 3.46-3.72 (5H), 3.07-3.14 (6H), 2.04-2.24 (4H), 1.92 (m, 2H). Calcd for C28H35N6O3 (MH+) 503.3. found 503.1.
Prepared essentially as described in Example 67 using 1-methyl-piperidin-4-ol and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester, which was prepared as described in Example 66a. 1H NMR (CD3OD) δ 9.02 (s, 1H), 8.35 (d, J=9.25 Hz, 1H), 7.37 (dd, J=9.12 and 2.44 Hz, 1H), 7.34 (d, J=2.48 (Hz, 1H), 7.26 (d, J=8.87 Hz, 2H), 6.93 (d, J=8.96 Hz, 2H), 4.72 (m, 1H), 4.34 (m, 2H), 3.92 (m, 1H), 3.82 (t, J=4.66 Hz, 4H), 3.17 (m, 2H), 3.08 (t, J=4.83 Hz, 4H), 2.77 (m, 2H), 2.47 (m, 2H), 2.34 (s, 3H), 1.87-2.18 (8H). Calcd for C30H39N6O3 (MH+) 531.3. found 531.1.
Prepared essentially as described in Example 126b, using (S)-(+)-3-pyrrolidinol. 1H NMR (CDCl3) δ 8.95 (s, 1H), 7.96 (d, J=9.48 Hz, 1H), 7.29 (m, 3H), 7.03 (dd, J=9.19 and 2.29 Hz, 1H), 6.91 (d, J=1.78 Hz, 1H), 6.88 (m, 2H), 6.46 (br, 1H), 4.69 (m, 1H), 4.25 (m, 2H), 3.86 (t, J=4.48 Hz, 4H), 3.55-3.72 (4H), 3.48 (m, 1H), 3.09 (m, 6H), 2.04-2.26 (4H), 1.91 (m, 2H). Calcd for C28H35N6O3 (MH+) 503.3. found 503.1.
Prepared essentially as described in Example 126 using (S)-(+)-3-pyrrolidinol and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CD3OD) δ 8.83 (s, 1H), 8.29 (d, J=9.70 Hz, 1H), 7.29 (dd, J=9.34 and 2.62 Hz, 1H), 7.16 (d, J=8.91 Hz, 2H), 6.77 (d, J=2.31 Hz, 1H), 6.59 (m, 2H), 4.62 (m, 1H), 4.34 (m, 2H), 3.89 (m, 1H), 3.63-3.73 (4H), 3.47 (m, 1H), 3.08-3.34 (4H), 1.86-2.26 (10H). Calcd for C27H34N7O2 (MH+) 487.3. found 487.1.
Prepared essentially as described in Example 126 using (R)-2-(methoxymethyl)pyrrolidine. 1H NMR (CDCl3) δ 8.98 (s, 1H), 7.95 (d, J=9.47 Hz, 1H), 7.27 (d, J=6.95 Hz, 2H), 7.13 (dd, J=9.42 and 2.52 Hz, 1H), 6.95 (d, J=2.41 Hz, 1H), 6.87 (d, J=9.00 Hz, 2H), 6.31 (br, 1H), 4.25 (m, 2H), 4.11 (m, 1H), 3.86 (t, J=4.65 Hz, 4H), 3.61 (m, 1H), 3.54 (dd, J=9.34 and 3.54 Hz, 2H), 3.38 (s, 3H), 3.32 (m, 2H), 3.08-3.17 (6H), 1.91-2.19 (8H). Calcd for C30H39N6O3 (MH+) 531.3. found 530.1.
Prepared essentially as described in Example 76, using 1-methyl-piperazine. 1H NMR (CDCl3) δ 9.08 (s, 1H), 7.93 (d, J=9.31 Hz, 1H), 7.65 (dd, J=9.32 and 2.57 Hz, 1H), 7.25 (d, J=8.92 Hz, 2H), 7.24 (d, J=4.74 Hz, 1H), 6.84 (d, J=8.93 Hz, 2H), 6.37 (br, 1H), 4.48 (m, 1H), 4.25 (m, 2H), 3.66 (m, 1H), 3.40 (t, J=4.89 Hz, 4H), 3.17 (td, J=12.74 and 3.04 Hz, 2H), 2.73 (m, 4H), 2.45 (s, 3H), 1.96-2.19 (4H), 1.31 (d, J=6.06 Hz, 6H). Calcd for C28H37N6O2 (MH+) 489.3. found 489.1.
Prepared essentially as described in Example 126 using (R)-2-pyrrolidinemethanol. 1H NMR (CDCl3) δ 8.96 (s, 1H), 7.93 (d, J=9.45 Hz, 1H), 7.27 (d, J=9.08 Hz, 2H), 7.14 (dd, J=9.21 and 2.22 Hz, 1H), 6.95 (d, J=2.26 Hz, 1H), 6.86 (d, J=8.99 Hz, 2H), 6.40 (br, 1H), 4.24 (m, 2H), 4.08 (m, 1H), 3.85 (t, J=4.70 Hz, 4H), 3.77 (dd, J=10.77 and 3.67 Hz, 1H), 3.68 (dd, J=10.73 and 7.22 Hz, 1H), 3.60 (m, 2H), 3.50 (m, 1H), 3.06-3.14 (6H), 2.03-2.17 (6H), 1.92 (m, 2H). Calcd for C29H37N6O3 (MH+) 517.3. found 517.1.
Prepared essentially as described in Example 33 using 3-morpholin-4-yl-propan-1-ol in place of 3-hydroxypropylpiperidine and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, as prepared by the method outlined in Example 66a, in place of (4-isopropoxy-phenyl)-carbamic acid 4-nitrophenyl ester. 1H-NMR (300 MHz, CDCl3): 9.13 (s, IH), 8.04 (d, IH), 7.35-7.22 (m, 4H), 6.87 (d, 2H), 6.37 (s, 1H), 4.32-4.16 (m, 4H), 3.90-3.60 (m, 9H), 3.20-3.04 (m, 6H), 2.43-2.62 (m, 6H), 2.21-1.90 (m, 6H). LCAMS (ESI): 561.1 (MH)+.
Prepared essentially as described in Example 33 using 3-diethylamino-propan-1-ol in place of 3-hydroxypropylpiperidine and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, as prepared by the method outlined in Example 66a, in place of (4-isopropoxy-phenyl)-carbamic acid 4-nitrophenyl ester. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.04 (d, 1H), 7.35-7.23 (m, 4H), 6.88 (d, 2H), 6.33 (s, 1H), 4.32-4.15 (m, 4H), 3.90-3.81 (m, 4H), 3.74-3.60 (m, 1H), 3.20-3.04 (m, 6H), 2.72-2.51 (m, 6H), 2.22-1.89 (m, 6H), 1.06 (t, 6H). LC/MS (ESI): 547.2 (MH)+.
A mixture of 1-methylpiperazine (0.11 mmol) and 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (0.05 mmol), prepared as described in Example 65, in DMSO (1 mL) was stirred at 120° C. for 1 h. It was then diluted with water and extracted with DCM. The combined extracts were washed with water, brine, dried (anhydrous MgSO4), filtered and concentrated in vacuo. The crude product was then treated with 3M HCl/MeOH (2 mL) and stirred at rt for 2 h and then concentrated in vacuo. The crude residue was dissolved in a mixture of DCM:MeOH (1:1; 2 mL) and neutralized with excess Et3N and treated with (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride (0.06 mmol), as prepared by the method outlined in Example 66a, at rt overnight. It was then concentrated in vacuo and the crude product was dissolved in DCM and washed with water thrice, then washed with brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was then purified by Preparative TLC (silica gel; DCM:MeOH:NH4OH, 90:9:1) to obtain 5.5 mg (21%) of the title compound. 1H-NMR (300 MHz, CDCl3): 9.04 (s, 1H), 7.98 (d, 1H), 7.36-7.18 (m, 4H), 6.88 (d, 2H), 6.32 (s, 1H), 4.25 (m, 2H), 3.89-3.83 (m, 4H), 3.69-3.55 (m, 1H), 3.53-3.43 (m, 4H), 3.19-3.04 (m, 6H), 2.66-2.58 (m, 4H), 2.39 (s, 3H), 2.20-1.90 (m, 4H). LC/MS (ESI): 516.1 (MH)+.
Prepared essentially as described in Example 135 using 1-ethyl-piperazine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.04 (s, 1H), 7.98 (d, 1H), 7.38-7.17 (m, 4H), 6.87 (d, 2H), 6.35 (s, 1H), 4.25 (m, 2H), 3.89-3.82 (m, 4H), 3.69-3.56 (m, 1H), 3.53-3.44 (m, 4H), 3.18-3.05 (m, 6H), 2.69-2.60 (m, 4H), 2.55-2.45 (q, 2H), 2.20-1.90 (m, 4H), 1.15 (t, 3H). LC/MS (ESI): 530.1 (MH)+.
Prepared essentially as described in Example 135 using 1-(2-hydroxyethyl)-piperazine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.05 (s, 1H), 7.99 (d, 1H), 7.38-7.19 (m, 4H), 6.88 (d, 2H), 6.34 (s, 1H), 4.25 (m, 2H), 3.89-3.81 (m, 4H), 3.74-3.57 (m, 3H), 3.51-3.43 (m, 4H), 3.19-3.04 (m, 6H), 2.75-2.60 (m, 6H), 2.20-1.90 (m, 5H). LC/MS (ESI): 546.1 (MH)+.
Prepared essentially as described in Example 135 using 1-methyl-[1,4]diazepane in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 8.98 (s, 1H), 7.95 (d, 1H), 7.31-7.24 (m, 2H), 7.20-7.10 (m, 1H), 7.01 (d, 1H), 6.88 (d, 2H), 6.33 (s, 1H), 4.25 (m, 2H), 3.90-3.51 (m, 9H), 3.18-3.05 (m, 6H), 2.83-2.75 (m, 2H), 2.65-2.55 (m, 2H), 2.41 (s, 3H), 2.20-1.90 (m, 6H). LC/MS (ESI): 530.1 (MH)+.
Prepared essentially as described in Example 126 using (S)-2-pyrrolidinemethanol.1H NMR (CDCl3) δ 8.96 (s, 1H), 7.92 (d, J=9.37 Hz, 1H), 7.27 (d, J=9.14 Hz, 2H), 7.14 (dd, J=9.35 and 2.44 Hz, 1H), 6.95 (d, J=2.30 Hz, 1H), 6.86 (d, J=8.97 Hz, 2H), 6.41 (br, 1H), 4.24 (m, 2H), 4.08 (m, 1H), 3.85 (t, J=4.67 Hz, 4H), 3.77 (dd, J=11.02 and 4.00 Hz, 1H), 3.68 (dd, J=10.88 and 6.80 Hz, 1H), 3.54-3.63 (2H), 3.35 (m, 1H), 3.06-3.14 (6H), 2.04-2.18 (6H), 1.92 (m, 2H). Calcd for C29H37N6O3 (MH+) 517.3. found 517.1.
A mixture of piperazine (5 mmol) and 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (1 mmol) in DMSO (1 mL) was stirred at 120° C. for 1 h. It was then diluted with water and extracted with DCM. The combined extracts were washed with water, brine, dried (anhydrous MgSO4), filtered and concentrated in vacuo to obtain 4-(7-piperazin-1-yl-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester. This (0.1 mmol) was dissolved in anhydrous DCM (1 mL) and treated with Et3N (0.2 mmol) followed by 9-fluorenylmethyl chloroformate (FMOC-Cl, 0.2 mmol) and the mixture was stirred at rt overnight and then washed with water thrice, then dried over anhydrous MgSO4, filtered and concentrated in vacuo. To this was then added 3M HCl/MeOH (2 mL) and stirred at rt for 2 h and then concentrated in vacuo. The crude residue was dissolved in a mixture of DCM:MeOH (1:1; 2 mL) and neutralized with excess Et3N and treated with (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride (0.11 mmol), as prepared by the method outlined in Example 66a, at rt overnight. It was then concentrated in vacuo and the crude product was dissolved in DCM and washed with water thrice, then washed with brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was then purified by Preparative TLC (silica gel; DCM:MeOH:NH4OH, 90:9:1) to obtain 5.6 mg (11%) of the title compound. 1H-NMR (300 MHz, CDCl3): 9.04 (s, 1H), 7.97 (d, 1H), 7.36-7.18 (m, 4H), 6.88 (d, 2H), 6.31 (s, 1H), 4.25 (m, 2H), 3.89-3.82 (m, 4H), 3.70-3.56 (m, 1H), 3.46-3.38 (m, 4H), 3.19-3.04 (m, 10H), 2.21-1.90 (m, 5H). LCAMS (ESI): 502.1 (MH)+.
Prepared essentially as described in Example 140 using acetyl chloride in place of FMOC-Cl. 1H-NMR (300 MHz, CDCl3): 9.05 (s, 1H), 8.01 (d, 1H), 7.36-7.20 (m, 3H), 6.86 (d, 3H), 6.48 (s, 1H), 4.25 (m, 2H), 3.90-3.76 (m, 6H), 3.74-3.56 (m, 3H), 3.53-3.40 (m, 4H), 3.19-3.00 (m, 6H), 2.20-2.01 (m, 5H), 2.00-1.85 (m, 2H). LC/MS (ESI): 544.1 (MH)+.
Prepared essentially as described in Example 140 using methanesulfonyl chloride in place of FMOC-Cl. 1H-NMR (300 MHz, CDCl3+CD3OD): 8.92 (s, 1H), 7.99 (d, 1H), 7.33-7.12 (m, 4H), 6.81 (d, 2H), 4.21 (m, 2H), 3.82-3.75 (m, 4H), 3.67-3.48 (m, 5H), 3.40-3.32 (m, 4H), 3.09-2.96 (m, 6H), 2.79 (s, 3H), 2.08-1.81 (m, 4H). LC/MS (ESI): 580.1 (MH)+.
Prepared essentially as described in Example 140 using N,N-dimethylcarbamoyl chloride in place of FMOC-Cl. 1H-NMR (300 MHz, CDCl3): 9.04 (s, 1H), 7.99 (d, 1H), 7.35-7.17 (m, 4H), 6.86 (d, 2H), 6.47 (s, 1H), 4.25 (m, 2H), 3.88-3.81 (m, 4H), 3.65-3.56 (m, 1H), 3.49-3.39 (m, 8H), 3.17-3.04 (m, 6H), 2.89 (s, 6H), 2.20-1.85 (m, 4H). LC/MS (ESI): 573.1 (MH)+.
Prepared essentially as described in Example 140 using N,N-dimethylaminoacetyl chloride in place of FMOC-Cl. 1H-NMR (300 MHz, CDCl3): 9.05 (s, 1H), 8.01 (d, 1H), 7.35-7.17 (m, 4H), 6.86 (d, 2H), 6.46 (s, 1H), 4.25 (m, 2H), 3.88-3.76 (m, 8H), 3.70-3.55 (m, 1H), 3.50-3.40 (m, 4H), 3.20-3.03 (m, 8H), 2.30 (s, 6H), 2.19-1.87 (m, 4H). LC/MS (ESI): 587.1 (MH)+.
Prepared essentially as described in Example 135 using morpholine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.07 (s, 1H), 8.01 (d, 1H), 7.36-7.19 (m, 4H), 6.88 (d, 2H), 6.32 (s, 1H), 4.25 (m, 2H), 3.94-3.81 (m, 8H), 3.70-3.57 (m, 1H), 3.44-3.37 (m, 4H), 3.19-3.05 (m, 6H), 2.20-1.87 (m, 4H). LC/MS (ESI): 503.1 (MH)+.
Prepared essentially as described in Example 126 using 2-methanesulfonyl-ethylamine. 1H NMR (CDCl3) δ 9.03 (s, 1H), 7.94 (d, J=8.91 Hz, 1H), 7.27 (d, J=9.01 Hz, 2H), 6.96-7.01 (2H), 6.87 (d, J=8.98 Hz, 2H), 5.35 (br, 1H), 5.23 (t, J=5.65 Hz, 1H), 4.26 (m, 2H), 3.83-3.92 (6H), 3.61 (m, 1H), 3.39 (m, 2H), 3.07-3.17 (6H), 2.99 (s, 3H), 2.11 (m, 2H), 1.93 (m, 2H). Calcd for C27H35N6O2S (MH+) 539.2. found 539.0.
Prepared essentially as described in Example 67 using 3-(2-hydroxy-ethyl)-oxazolidin-2-one and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CDCl3) δ9.16 (s, 1H), 8.07 (d, J=9.22 Hz, 1H), 7.31 (d, J=2.61 Hz, 1H), 7.25 (m, 1H), 7.18 (d, J=8.92 Hz, 2H), 6.52 (d, J=8.93 Hz, 2H), 6.19 (br, 1H), 4.38 (t, J=7.90 Hz, 2H), 4.33 (t, J=4.79 Hz, 2H), 4.26 (m, 2H), 3.80 (t, J=8.21 Hz, 2H), 3.78 (t, J=4.75 Hz, 2H), 3.67 (m, 1H), 3.26 (t, J=6.65 Hz, 4H), 3.12 (td, J=12.53 and 2.63 Hz, 2H), 2.13 (m, 2H), 1.93-2.01 (6H). Calcd for C29H35N6O4 (MH+) 531.3. found 531.1.
Prepared essentially as described in Example 67 using 3-(2-hydroxy-ethyl)-oxazolidin-2-one and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 66a. 1H NMR (CDCl3) δ9.15 (s, 1H), 8.07 (d, J=9.34 Hz, 1H), 7.32 (d, J=2.44 Hz, 1H), 7.24-7.29 (3H), 6.88 (d, J=8.97 Hz, 2H), 6.29 (br, 1H), 4.38 (t, J=8.04 Hz, 2H), 4.33 (t, J=4.89 Hz, 2H), 4.26 (m, 2H), 3.86 (t, J=4.67 Hz, 4H), 3.80 (t, J=8.04 Hz, 2H), 3.79 (t, J=5.05 Hz, 2H), 3.68 (m, 1H), 3.14 (td, J=13.86 and 3.07 Hz, 2H), 3.10 (t, J=4.80 Hz, 4H), 2.13 (m, 2H), 1.97 (m, 2H). Calcd for C29H35N6O5 (MH+) 547.3. found 547.0.
Prepared essentially as described in Example 126 using (3R)-(+)-3-(dimethylaminopyrrolidine) and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CDCl3) δ 8.98 (s, 1H), 7.95 (d, J=9.34 Hz, 1H), 7.18 (d, J=8.87 Hz, 2H), 6.99 (dd, J=9.29 and 2.46 Hz, 1H), 6.84 (d, J=2.38 Hz, 1H), 6.51 (d, J=8.92 Hz, 2H), 6.20 (br, 1H), 4.24 (m, 2H), 3.65 (m, 2H), 3.58 (m, 1H), 3.47 (m, 1H), 3.30 (t, J =8.68 Hz, 1H), 3.25 (t, J=6.61 Hz, 4H), 3.09 (td, J=12.94 and 2.28 Hz, 2H), 2.90 (m, 1H), 2.34 (s, 6H), 2.28 (m, 1H), 2.11 (m, 2H), 1.90-2.02 (7H). Calcd for C30H40N7O (MH+) 514.3. found 514.1.
Prepared essentially as described in Example 126 using (3R)-(+)-3-(dimethylaminopyrrolidine). 1H NMR (CDCl3) δ 8.98 (s, 1H), 7.95 (d, J=9.32 Hz, 1H), 7.27 (d, J=9.00 Hz, 2H), 7.00 (dd, J=9.19 and 2.38 Hz, 1H), 6.87 (d, J=8.96 Hz, 2H), 6.84 (d, J=2.31 Hz, 1H), 6.31 (br, 1H), 4.24 (m, 2H), 3.86 (t, J=4.65 Hz, 4H), 3.65 (m, 2H), 3.60 (m, 1H), 3.48 (m, 1H), 3.31 (t, J=8.68 HZ, 1H), 3.13 (m, 2H), 3.10 (t, J=4.85 Hz, 4H), 2.92 (m, 1H), 2.35 (s, 6H), 2.29 (m, 1H), 2.11 (m, 2H), 1.97 (m, 3H). Calcd for C30H40N7O2 (MH+) 530.3. found 530.1.
Prepared essentially as described in Example 67 using (S)-(-)-1-methyl-2-pyrrolidinemethanol and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CDCl3) δ 9.13 (s, 1H), 8.04 (d, J=9.34 Hz, 1H), 7.26-7.34 (2H), 7.18 (d, J=8.47 Hz, 2H), 6.53 (d, J=8.63 Hz, 2H), 6.18 (br, 1H), 4.25 (m, 2H), 4.12 (m, 2H), 3.68 (m, 1H), 3.25 (m, 4H), 3.12 (m, 3H), 2.73 (m, 1H), 2.51 (s, 3H), 2.33 (m, 1h), 1.78-2.18 (12H). Calcd for C30H49N6O2 (MH+) 515.3. found 515.3.
4-(7-Fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (97.4 mg, 0.294 mmol), which was prepared as described in Example 65b, was added to ethane-1,2-diol (2.98 g, 48.01 mmol) and the suspension was heated to 90° C. to allow the starting material totally dissolved in ethane-1,2-diol. KOH (130.7 mg) was added and the mixture was stirred at 120° C. for 2 h. It was partitioned between ethyl acetate and water and the combined organic extracts were washed with brine, dried over Na2SO4 and evaporated to afford the product as a white solid (90 mg, 82%). 1H NMR (CDCl3) δ 9.12 (s, 1H), 8.05 (d, J=9.27 Hz, 1H), 7.32 (d, J=2.46 Hz, 1H), 7.28 (dd, J=9.21 and 2.54 Hz, 1H), 4.31 (br, 1H), 4.26 (t, J=4.01 Hz, 2H), 4.20 (m, 1H), 4.06 (t, J=4.67 Hz, 2H), 3.83 (m, 1H), 3.60 (m, 1H), 2.93 (m, 2H), 1.80-2.11 (4H), 1.47 (s, 9H). Calcd for C20H28N3O4 (MH+) 374.2. found 374.2.
To a mixture of 4-[7-(2-hydroxy-ethoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (90 mg, 0.24 mmol) and DIPEA (167.2 μL) in CH2Cl2 (5 mL) was added MsCl (37.2 μL). The reaction mixture was stirred for 4 h and the solvents were evaporated. The residue was purified by flash column chromatography on silica gel (EtOAc as eluent) to afford almost pure product. 1H NMR (CDCl3) δ 9.15 (s, 1H), 8.09 (d, J=9.33 Hz, 1H), 7.33 (d, J=2.44 Hz, 1H), 7.29 (dd, J=9.18 and 2.59 Hz, 1H), 4.66 (t, J=4.29 Hz, 2H), 4.42 (t, J=4.39 Hz, 2H), 4.33 (m, 2H), 3.61 (m, 1H), 3.11 (s, 3H), 2.94 (m, 2H), 1.83-2.10 (4H), 1.48 (s, 9H). Calcd for C21H30N3O6S (MH+) 452.2. found 452.2.
To a solution of 4-[7-(2-methanesulfonyloxy-ethoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (40.6 mg, 0.09 mmol) in DMSO (0.4 mL) was added (S)-(+)-2-pyrrolidinemethanol (90.9 mg, 0.9 mmol). The mixture was stirred at 120° C. overnight and subsequently partitioned between EtOAc and water. The combined organic extracts were washed with brine, dried over Na2SO4 and evaporated. The residue was treated with 50% TFA/CH2Cl2 (8 mL) for 2 h, the solvents (TFA/CH2Cl2) were removed under reduced pressure and half of the residue was re-dissolved in CH2Cl2. DIPEA (55 μL) was added to the above solution, followed by (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride (19.6 mg, 0.054 mmol), which was prepared as described in Example 74a. The reaction mixture was stirred for 1 h, diluted with water. The organic phase was collected and the solvents were evaporated. The crude residue was purified by flash column chromatography on silica gel (15% MeOH/EtOAc as eluent) to afford a white solid (10 mg, 40.8% overall yield). 1H NMR (CD3OD) δ 9.03 (s, 1H), 8.35 (d, J=9.44 Hz, 1H), 7.40 (dd, J=9.25 and 2.54 Hz, 1H), 7.34 (d, J=2.48 Hz, 1H), 7.13 (d, J=8.87 Hz, 2H), 6.54 (d, J=8.96 Hz, 2H), 4.29-4.36 (4H), 3.92 (m, 1H), 3.60 (dd, J=10.98 and 4.81 Hz, 1H), 3.52 (dd, J=11.00 and 5.90 Hz, 1H), 3.40 (m, 1H), 3.27 (m, 1H), 3.24 (t, J=6.61 Hz, 4H), 3.15 (td, J=12.57 and 2.55 Hz, 2H), 2.88 (dt, J=13.67 and 5.50 Hz, 1H), 2.71 (m, 1H), 2.47 (m, 1H), 1.90-2.09 (9H), 1.79 (m, 2H), 1.66 (m, 1H). Calcd for C31H41N6O3 (MH+) 545.3. found 545.3.
Prepared essentially as described in Example 152 using (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 66a. 1H NMR (CD3OD) δ 9.08 (s, 1H), 8.41 (d, J=9.42 Hz, 1H), 7.48 (dd, J=9.22 Hz and 2.54 Hz, 1H), 7.41 (d, J=2.49 Hz, 1H), 7.26 (d, J=9.09 Hz, 2H), 6.93 (d, J=9.08 Hz, 2H), 4.56 (m, 2H), 4.34 (m, 2H), 3.87-3.98 (2H), 3.83 (t, J=4.60 Hz, 4H), 3.68-3.76 (4H), 3.60 (m, 1H), 3.24 (m, 1H), 3.17 (m, 2H), 3.08 (t, J=4.77 Hz, 4H), 1.92-2.28 (8H). Calcd for C31H41N6O4 (MH+) 561.3. found 561.2.
To a solution of KOt-Bu (55.1 mg, 0.47 mmol) in THF (1 mL) was added (R)-hydroxypyrrolidine (37.7 mg, 0.43 mmol), followed by 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (110.3 mg, 0.33 mmol), which was prepared as described in Example 65b, in THF (1 mL). The mixture was stirred for 1 h at room temperature, quenched with (CH3CO)2O. The mixture was then partitioned between EtOAc and water. The organic extracts were washed with brine and evaporated and the residue was used for the next step reaction without further purification. LC/MS for C24H33N4O4 (MH+) 440.2. found 440.5.
Prepared essentially as described in Example 67b, using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CDCl3) δ 9.14 (s, 1H), 8.07 (d, J=9.67 Hz, 1H), 7.27 (m, 1H), 7.23 (m, 1H), 7.18 (d, J=8.88 Hz, 2H), 6.52 (d, J=8.87 Hz, 2H), 6.20 (br, 1H), 5.14 (m, 1H), 4.24 (m, 2H), 3.58-3.88 (5H), 3.26 (t, J=6.57 Hz, 4H), 3.12 (m, 2H), 2.11 (s, 3H), 1.92-2.12 (IOH). Calcd for C30H37N6O3 (MH+) 529.3. found 529.1.
Prepared essentially as described in Example 126 using piperidine-4-carboxylic acid methylamide and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CDCl3) δ9.03 (s, 1H), 7.96 (d, J=9.52 Hz, 1H), 7.31 (dd, J=9.48 and 2.47 Hz, 1H), 7.18 (d, J =8.88 Hz, 2H), 7.17 (m, 1H), 6.52 (d, J=8.90 Hz, 2H), 6.19 (br, 1H), 5.54 (m, 1H), 4.25 (m, 2H), 4.03 (m, 2H), 3.60 (m, 1H), 3.26 (t, J=6.64 Hz, 4H), 3.10 (td, J=12.63 and 2.98 Hz, 2H), 3.00 (td, J=12.41 and 2.91 Hz, 2H), 2.83 (d, J=4.82 Hz, 3H), 2.35 (m, 1H), 2.11 (m, 2H), 1.84-2.02 (10H). Calcd for C31H40N7O2 (MH+) 542.3. found 542.2.
Prepared essentially as described in Example 152 using 1-methyl-piperazine and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 66a. 1H NMR (CDCl3) δ 9.12 (s, 1H), 8.02 (d, J=9.23 Hz, 1H), 7.23-7.31 (3H), 6.86 (d, J=9.07 Hz, 2H), 6.28 (br, 1H), 4.27 (t, J=5.84 Hz, 2H), 4.22 (m, 2H), 3.84 (t, J=4.65 Hz, 4H), 3.66 (m,lH), 3.06-3.18 (6H), 2.90 (t, J=5.54 Hz, 2H), 2.63 (m, 4H), 2.47 (m, 4H), 2.29 (s, 3H), 2.12 (m, 2H), 1.95 (m, 2H). Calcd for C31H42N7O3 (MH+) 560.3. found 560.1.
Prepared essentially as described in Example 152 using 1-methyl-piperazine 1H NMR (CD3OD) δ 9.04 (s, 1H), 8.36 (d, J=9.34 Hz, 1H), 7.40 (dd, J=9.30 and 2.64 Hz, 1H), 7.35 (d, J=2.48 Hz, 1H), 7.13 (d, J=8.99 Hz, 2H), 6.54 (d, J=9.01 Hz, 2H), 4.35 (t, J=5.32 Hz, 4H), 3.93 (m, 1H), 3.24 (m, 6H), 3.16 (m, 2H), 2.93 (t, J=5.23 Hz, 2H), 2.58 (4H), 2.32 (s, 3H), 1.91-2.05 (1OH). Calcd for C31H42N7O2 (MH+) 544.3. found 544.3.
Prepared essentially as described in Example 104 using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, prepared by the method as outlined in Example 74a, in place of (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.12 (s, 1H), 8.04 (d, 1H), 7.35-7.12 (m, 4H), 6.52 (d, 2H), 6.26 (s, 1H), 4.31-4.11 (m, 4H), 3.71-3.57 (m, 1H), 3.31-3.00 (m, 6H), 2.74-2.46 (m, 8H), 2.39 (s, 3H), 2.20-1.82 (m, 12H). LC/MS (ESI): 558.1 (MH)+.
Prepared essentially as described in Example 135 using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, prepared by the method as outlined in Example 74a, in place of (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.03 (s, 1H), 7.97 (d, 1H), 7.36-7.13 (m, 4H), 6.51 (d, 2H), 6.29 (s, 1H), 4.24 (m, 2H), 3.66-3.54 (m, 1H), 3.51-3.41 (m, 4H), 3.30-3.16 (m, 4H), 3.14-3.01 (m, 2H), 2.65-2.55 (m, 4H), 2.37 (s, 3H), 2.18-1.85 (m, 8H). LC/MS (ESI): 500.1 (MH)+.
Prepared essentially as described in Example 135 using (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitrophenyl ester hydrochloride, which was prepared from 6-Pyrrolidin-1-yl-pyridin-3-ylamine (WO 2002048152 A2) essentially as described in Example 74a, in place of (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.03 (s, 1H), 8.06-7.96 (m, 2H), 7.75 (d, 1H), 7.35-7.18 (m, 2H), 6.76-6.60 (s, 1H), 6.40 (s, 1H), 4.30 (m, 2H), 3.68-3.40 (m, llH), 2.70-2.51 (m, 4H), 2.41 (s, 3H), 2.18-1.87 (m, 8H). LC/MS (ESI): 501.1 (MH)+.
Prepared essentially as described in Example 115 using (S)-prolinol in place of 1-ethyl-piperazine and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, prepared by the method as outlined in Example 74a, in place of (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.04 (s, 1H), 8.06 (d, 1H), 7.43-6.95 (m, 4H), 6.49 (d, 2H), 6.28 (s, 1H), 4.33-4.15 (m, 4H), 3.72-3.57 (m, 4H), 3.36-2.97 (m, 15H), 2.22-1.70 (m, 10H). LC/MS (ESI): 559.2 (MH)+.
Prepared essentially as described in Example 154, using (S)-(+)-2-pyrrolidinemethanol. 1H NMR (CDCl3) δ 9.12 (s, 1H), 8.04 (d, J=9.35 Hz, 1H), 7.37 (d, J=2.54 Hz, 1H), 7.27 (dd, J=9.16 and 2.56 Hz, 1H), 7.18 (d, J=8.91 Hz, 2H), 6.52 (d, J=8.95 Hz, 2H), 6.23 (br, 1H), 4.52 (m, 1H), 4.34 (dd, J=9.38 and 3.09 Hz, 1H), 4.23 (m, 2H), 4.15 (dd, J=9.38 and 7.00 Hz, 1H), 3.64 (m, 1H), 3.43-3.60 (2H), 3.25 (t, J=6.63 Hz, 4H), 3.10 (m, 2H), 2.10-2.18 (2H), 2.09 (s, 3H), 1.92-2.08 (10H). Calcd for C31H39N6O3 (MH+) 543.3. found 543.2.
Prepared essentially as described in Example 154, using piperidin-4-yl-methanol. 1H NMR (CD3OD) δ 9.03 (s, 1H), 8.34 (d, J=9.42 Hz, 1H), 7.38 (dd, J=9.28 and 2.56 Hz, 1H), 7.32 (d, J=2.51 Hz, 1H), 7.13 (d, J=8.99 Hz, 2H), 6.54 (d, J=9.01 Hz, 2H), 4.59 (m, 1H), 4.33 (m, 2H), 4.08 (d, J=6.21 Hz, 2H), 4.00 (m, 1H), 3.91 (m, 1H), 3.24 (t, J=6.59 Hz, 4H), 3.11-3.20 (3H), 2.70 (td, J=12.73 and 2.58 Hz, 1H), 2.19 (m, 1H), 2.12 (s, 3H), 1.89-2.08 (1OH), 1.28-1.48 (2H). Calcd for C32H41N6O3 (MH+) 557.3. found 557.3.
Prepared essentially as described in Example 118 using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, prepared by the method as outlined in Example 74a, in place of (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.13 (s, 1H), 8.05 (d, 1H), 7.35-7.13 (m, 4H), 6.53 (d, 2H), 6.22 (s, 1H), 4.30-4.17 (m, 4H), 3.71-3.61 (m, 1H), 3.38-3.00 (m, 10H), 2.79 (s, 3H), 2.73-2.55 (m, 6H), 2.19-1.90 (m, 10H). LC/MS (ESI): 622.3 (MH)+.
Prepared essentially as described in Example 120 using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, prepared by the method as outlined in Example 74a, in place of (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.12 (s, 1H), 8.05 (d, 1H), 7.37-7.13 (m, 4H), 6.52 (d, 2H), 6.25 (s, 1H), 4.34-4.16 (m, 4H), 3.71-3.59 (m, 1H), 3.40-3.04 (m, 9H), 2.89-2.79 (m, 5H), 2.68-2.41 (m, 6H), 2.21-1.88 (m, 12H). LC/MS (ESI): 615.3 (MH)+.
Prepared essentially as described in Example 141 using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, prepared by the method as outlined in Example 74a, in place of (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.05 (s, 1H), 8.02 (d, 1H), 7.35-7.11 (m, 4H), 6.90-6.00 (bm, 3H), 4.25 (d, 2H), 3.86-3.78 (m, 2H), 3.72-3.56 (m, 3H), 3.5-3.40 (m, 4H), 3.20-3.00 (m, 4H), 2.20-2.04 (m, 5H), 2.03-1.77 (m, 8H). LC/MS (ESI): 528.2 (MH)+.
Prepared essentially as described in Example 142 using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, prepared by the method as outlined in Example 74a, in place of (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.08 (s, 1H), 8.02 (d, 1H), 7.37-7.11 (m, 4H), 6.90-6.00 (bm, 3H), 4.25 (d, 2H), 3.68-2.92 (bm, 13H), 2.84 (s, 3H), 2.18-1.87 (m, 10H). LC/MS (ESI): 564.2 (MH)+.
Prepared essentially as described in Example 143 using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride, prepared by the method as outlined in Example 74a, in place of (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.04 (s, 1H), 7.99 (d, 1H), 7.35-7.12 (m, 4H), 6.60-6.25 (bm, 3H), 4.24 (d, 2H), 3.65-3.55 (m, 1H), 3.50-3.37 (m, 8H), 3.32-3.01 (m, 4H), 2.88 (s, 6H), 2.16-1.80 (m, 10H). LC/MS (ESI): 557.2 (MH)+.
KOtBu (1.17 g, 10.4 mmol) was added to ethylene glycol (10 mL, 179 mmol) to provide a homogeneous solution. 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (2.61 g, 7.89 mmol), as prepared in Example 65b, was added, and the opaque white slurry was stirred at rt for 3.5 hr. DMSO (5 mL) was then added, and the mixture stirred at 110° C. for 20 min at which point it became a homogeneous solution. The reaction was then stirred at rt overnight, at which point it became a translucent white slurry. The mixture was then diluted with 0.1M NaHCO3 and extracted with EtOAc (2×50 mL). The combined organic layers were washed with 0.1M NaHCO3 (1×100 mL), dried (Na2SO4), concentrated, and dissolved in ˜15 mL toluene. The title compound crystallized upon standing at rt, was filtered, and the crystalline filter cake washed with toluene (1×10 mL). The filter cake was dried under high vacuum at 100° C. to afford the title compound as a white powder (2.38 g, 81%). LC/MS (ESI): calc mass 373.2. found 374.2.
The title compound was prepared essentially as described for Example 10b, using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, prepared as described in Example 74a. The title compound was purified by filtration of the crude reaction slurry. The resulting filter cake was taken up in 95:5 DCM/MeOH and washed sequentially with 2M K2CO3 and water. The hazy organic layer was then diluted with DCM and MeOH until a clear solution resulted, and was then dried (Na2SO4) and concentrated to afford the title compound (6.3 mg, 12%). 1H-NMR (400 MHz, 95:5 CDCl3/CD3OD) δ 9.10 (s, 1H), 8.11 (d, 1H), 7.36-7.30 (m, 2H), 7.17 (m, 2H), 6.53 (m, 2H), 4.28 (m, 4H), 4.03 (t, 2H), 3.70 (tt, 1H), 3.26 (m, 4H), 3.11 (td, 2H), 2.16-1.92 (m, 8H). LC/MS (ESI) calcd mass 461.2. found 462.3 (MH)+.
A mixture of Azetidin-3-ol hydrochloride (Oakwood) (461 mg, 4.21 mmol), KOtBu (1.02 g, 9.11 mmol), and dry DMSO (4.2 mL) was stirred at rt for 30 min until a translucent solution resulted. Then 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (1.46 g, 4.41 mmol), as prepared in Example 65b, was added, and the resulting opaque orange mixture (no visible precipitate) was stirred at rt for 3.5 hr. The reaction was then shaken with water (40 mL) and extracted with DCM (1×20 mL) and 9:1 DCM/MeOH (1×20 mL). The combined organic layers were washed with 0.2 M K2CO3 (3×20 mL), dried (Na2SO4), and concentrated to give 1.715 g of the title compound as an off-white solid (“106%”crude yield). LC/MS (ESI): calcd mass 384.2. found 385.3 (MH)+.
Acetic anhydride (66 μL, 703 μmol) was added dropwise with stirring at rt to a mixture of 4-[7-(Azetidin-3-yloxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (180 mg, 469 μmol), as prepared in the previous step, in DCM (1.0 mL). The resulting homogeneous yellow solution was stirred overnight, and was then partitioned with DCM (3 mL) and IM NaHCO3 (1×4 mL). The organic layer was dried (Na2SO4), concentrated, and purified by silica flash chromatography (8:2 DCM/acetone/3% DMEA eluent) to afford the title compound as a white crystalline film (88.3 mg, 44% over two steps). LC/MS (ESI): calcd mass 426.2. found 426.9 (MH)+.
The title compound was prepared from 4-[7-(1-Acetyl-azetidin-3-yloxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (44.1 mg, 103 μmol), as synthesized in the previous step, using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, prepared in Example 74a, and using essentially the reaction and work-up procedure described in Example 110b. The title compound was purified by silica flash cartridge chromatography (9:1 DCM/acetone/3% DMEA eluent). The combined fractions (10 mL) were washed with 1M NaHCO3 (1×5 mL) to remove a DMEA+impurity, and were dried (Na2SO4) and concentrated to provide the title compound (13.8 mg, 26%). 1H-NMR (400 MHz, 95:5 CDCl3/CD3OD) δ 9.13 (s, 1H), 8.16 (d, 1H), 7.31 (dd, 1H), 7.17 (m, 2H), 7.03 (d, 1H), 6.54 (m, 2H), 5.16 (m, 1H), 4.67 (ddd, 1H), 4.51 (dd, 1H), 4.32-4.23 (m, 3H), 4.15 (dd, 1H), 3.70 (tt, 1H), 3.26 (m, 4H), 3.11 (tt, 2H), 2.17-1.97 (m, 8H), 1.94 (s, 3H). LC/MS (ESI): calcd mass 514.3. found 515.3 (MH)+.
The title compound was prepared essentially as described for Example 170b-c, using methanesulfonyl chloride and 1.5 equivalents of TEA in place of acetic anhydride. 1H-NMR (400 MHz, 95:5 CDCl3/CD3OD) δ 9.13 (s, 1H), 8.15 (d, 1H), 7.31 (dd, 1H), 7.17 (m, 2H), 7.04 (d, 1H), 6.53 (m, 2H), 5.15 (m, 1H), 4.43 (m, 2H), 4.27 (m, 2H), 4.15 (m, 2H), 3.70 (tt, 1H), 3.26 (m, 4H), 3.11 (td, 2H), 2.97 (s, 3H), 2.16-2.04 (m, 2H), 2.03-1.93 (m, 6H). LC/MS (ESI): calcd mass 550.2. found 551.2 (MH)+.
A mixture of morpholine (107.4 mg, 1.23 mmol) and methyl glycolate (77.5 mg, 860 μmol) was stirred at 150° C. for 3 hr. The resulting homogeneous clear amber oil was taken up in toluene (2×2 mL) with repeated rotary evaporation to remove methanol. The residue was taken up in dry THF (860 μL) and KOtBu was added (113 mg, 1.01 mmol). The mixture was stirred at 100° C. for 5-10 min until a brown slurry formed with no visible chunks. The mixture was then allowed to cool to rt, 4-(7-fluoro-quinazolin -4-yl)-piperidine-1-carboxylic acid tert-butyl ester (302 mg, 912 μmol), as prepared in Example 65b, was added, and the resulting nearly homogeneous reddish-brown solution was stirred at rt for 1 hr, at which point the reaction solidified into a paste. The reaction was taken up in DCM (4 mL) and washed with 1M NaHCO3 (1×2 mL) and 1M NaH2PO4 (1×2 mL), and the organic layer was dried (Na2SO4) and concentrated. The residue was purified by silica flash chromatography (9:1 DCM/acetone→8:2→8:2 DCM/acetone/3% DMEA eluent) to provide the title compound as a pale yellow oil (94.8 mg, 24% over two steps). LC/MS (ESI): calcd mass 456.2. found 457.3 (MH)+.
The title compound was prepared from 4-[7-(2-Morpholin-4-yl-2-oxo-ethoxy)-quinazolin -4-yl]-piperidine-1-carboxylic acid tert-butyl ester as synthesized in the previous step, using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride (preparation given in Example 74a), essentially as described in Example 170c. 1H-NMR (400 MHz, CDCl3) δ 9.14 (s, 1H), 8.10 (d, 1H), 7.38 (dd, 1H), 7.29 (d, 1H), 7.18 (m, 2H), 6.51 (m, 2H), 6.34 (s, 1H), 4.88 (s, 2H), 4.26 (m, 2H), 3.75-3.61 (m, 7H), 3.55 (m, 2H), 3.25 (m, 4H), 3.10 (td, 2H), 2.16-2.04 (m, 2H), 2.02-1.90 (m, 6H). LC/MS (ESI): calcd mass 544.3. found 545.3 (MH)+.
Prepared essentially as Example 126 using azetidine and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CD3OD) δ 8.80 (s, 1H), 8.20 (d, J=9.30 Hz, 1H), 7.13 (d, J=8.98 Hz, 2H), 6.95 (dd, J=9.20 and 2.36 Hz, 1H), 6.56 (d, J=2.34 Hz, 1H), 6.54 (d, J=9.00 Hz, 2H), 4.32 (m, 2H), 4.13 (t, J=7.41 Hz, 4H), 3.82 (m, 1H), 3.24 (t, J=6.69 Hz, 4H), 3.12 (td, J=13.10 and 2.99 Hz, 2H), 2.50 (m, 2H), 1.96-2.07 (6H), 1.88 (m, 2H). Calcd for C27H33N6O (MH+) 457.3. found 457.3.
Prepared essentially as Example 67 using pyridin-3-ol and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CDCl3) δ 9.14 (s, 1H), 8.53-8.54 (2H), 8.19 (d, J=9.22 Hz, 1H), 7.50 (ddd, J=8.34, 2.78, and 1.44 Hz, 1H), 7.44 (dd, J=9.19 and 2.56 Hz, 1H), 7.40 (ddd, J=8.34, 4.73 and 0.64 Hz, 1H), 7.31 (d, J=2.53 Hz, 1H), 7.17 (d, J=8.91 Hz, 2H), 6.51 (d, J=8.95 Hz, 2H), 6.25 (br, 1H), 4.23 (m, 2H), 3.70 (m, 1H), 3.25 (t, J=6.61 Hz, 4H), 3.12 (td, J=13.18 and 2.66 Hz, 2H), 2.13 (m, 2H), 1.94-2.01 (6H). Calcd for C29H31N6O2 (MH+) 495.2. found 495.2.
Prepared essentially as Example 126 using 2-amino-ethanol and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CD3OD) δ 8.78 (s, 1H), 8.10 (d, J=9.42 Hz, 1H), 7.15 (dd, J=9.29 and 2.38 Hz, 1H), 7.13 (d, J=8.97 Hz, 2H), 6.77 (d, J=2.36 Hz, 1H), 4.32 (m, 2H), 3.81 (m, 1H), 3.79 (t, J=5.77 Hz, 2H), 3.40 (t, J=5.77 Hz, 2H), 3.24 (t, J=6.62 Hz, 4H), 3.12 (td, J=13.22 and 2.52 Hz, 2H), 1.95-2.06 (6H), 1.87 9m, 2H). Calcd for C26H33N6O2 (MH+) 461.3. found 461.3.
To a solution of 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (139.6 mg, 0.42 mmol), which was prepared as described in Example 65b, in DMSO (0.8 mL) was added ethanolamine (256.2 mg, 4.2 mmol). The mixture was stirred at 120° C. overnight and subsequently partitioned between EtOAc and water.
The combined organic extracts were washed with brine, dried over Na2SO4 and evaporated. The residue was re-dissolved in CH2Cl2 (4 mL), treated with COCl2 (1 mL of 1M solution in toluene) and TEA (200 mg). The mixture was partitioned between CH2Cl2 and water. The CH2Cl2 extracts were evaporated and the residue was purified by flash column chromatography on silica gel (hexanes/EtOAc 1:1, v/v) to afford the desired product. LC/MS for C21H27N4O4 (MH+) 399.2. found 399.2.
Prepared essentially as Example 67b using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CDCl3) δ 9.19 (s, 1H), 8.60 (dd, J=9.38 and 2.38 Hz, 1H), 8.17 (d, J=9.45 Hz, 1H), 7.54 (d, J=2.35 Hz, 1H), 7.18 (m, 2H), 6.53 (m, 2H), 6.22 (br, 1H), 4.59 (t, J=7.99 Hz, 2H), 4.26 (m, 2H), 4.21 (t, J=8.01 Hz, 2H), 3.72 (m, 1H), 3.26 (m, 4H), 3.13 (t, J=12.39 Hz, 2H), 2.10 (td, J=12.16 and 3.85 Hz, 2H), 1.99 (m, 6H). Calcd for C27H31N6O3 (MH+) 487.3. found 487.3.
Prepared essentially as Example 154 with the sole exception that the intermediate generated was quenched with MsCl. 1H NMR (CDCl3) δ 9.16 (s, 1H), 8.10 (d, J=9.33 Hz, 1H), 7.26 (m, 1H), 7.21 (dd, J=9.15 and 2.60 Hz, 1H), 7.18 (m, 2H), 6.52 (m, 2H), 6.20 (br, 1H), 5.14 (m, 1H), 4.25 (m, 2H), 3.72-3.78 (3H), 3.61-3.72 (2H), 3.52 (td, J=10.45 and 7.09 Hz, 2H), 3.10-3.30 (4H), 2.87 (s, 3H), 2.28-2.46 (2H), 2.13 (m, 2H), 1.98 (m, 6H). Calcd for C29H37N6O4S (MH+) 565.3. found 565.3.
To a mixture of 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (458 mg, 1.38 mmol), which was prepared as described in Example 65b, and (2-amino -ethyl)-carbamic acid benzyl ester hydrochloride (446 mg, 1.93 mmol) in DMSO (1.0 mL) was added K2CO3 (1.52 g, 11.04 mmol). The mixture was stirred at 115° C. overnight and subsequently partitioned between EtOAc and water. The combined organic extracts were washed with brine, dried over Na2SO4 and evaporated. The residue was purified by flash column chromatography on silica gel (EtOAc as eluent) to afford the desired product as a white solid (400 mg, 73%). 1H NMR (CDCl3) δ 9.13 (s, 1H), 8.69 (dd, J=9.40 and 2.35 Hz, 1H), 8.08 (d, J=9.53 Hz, 1H), 7.42 (d, J=2.33 Hz, 1H), 5.25 (br, 1H), 4.31 (m, 2H), 4.09 (t, J=8.21 Hz, 2H), 3.69 (t, J=8.14 Hz, 2H), 3.63 (m, 1H), 2.95 (m, 2H), 1.77-2.04 (4H), 1.48 (s, 9H). Calcd for C21H28N5O3 (MH+) 398.3. found 398.3.
Prepared essentially as Example 67b using (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CDCl3) δ 9.13 (s, 1H), 8.71 (dd, J=9.40 and 2.33 Hz, 1H), 8.09 (d, J=9.53 Hz, 1H), 7.41 (d, J=2.32 Hz, 1H), 7.17 (d, J=8.83 Hz, 2H), 6.51 (d, J=8.47 Hz, 2H), 6.28 (br, 1H), 5.10 (br, 1H), 4.25 (m, 2H), 4.07 (t, J=6.17 Hz, 2H), 3.71 (m, 1H), 3.67 (m, 2H), 3.24 (m, 4H), 3.11 (td, J=12.75 and 2.13 Hz, 2H), 1.93-2.13 (8H). Calcd for C27H32N7O2 (MH+) 486.3. found 486.3.
Prepared essentially as described in Example 159 using pyrrolidine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 8.95 (s, 1H), 7.94 (d, 1H), 7.17 (d, 2H), 7.01 (m, 1H), 6.83 (d, 1H), 6.51 (d, 2H), 6.28 (s, 1H), 4.24 (d, 2H), 3.65-3.52 (m, 1H), 3.49-3.39 (m, 4H), 3.28-3.20 (m, 4H), 3.13-3.02 (m, 2H), 2.16-1.78 (m, 12H). LC/MS (ESI): 471.3 (MH)+.
Prepared essentially as described in Example 159 using imidazole in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.28 (s, 1H), 8.32 (d, 1H), 8.11-8.04 (m, 2H), 7.74 (m, 1H), 7.49 (m, 1H), 7.30 (m, 1H), 7.18 (d, 2H), 6.52 (d, 2H), 6.26 (s, 1H), 4.28 (d, 2H), 3.80-3.69 (m, 1H), 3.29-3.10 (m, 6H), 2.22-1.90 (m, 8H). LC/MS (ESI): 468.3 (MH)+.
Prepared essentially as described in Example 159 using morpholine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.05 (s, 1H), 8.00 (d, 1H), 7.34-7.14 (m, 4H), 6.51 (d, 2H), 6.29 (s, 1H), 4.24 (d, 2H), 3.93-3.87 (m, 4H), 3.66-3.56 (m, 1H), 3.43-3.36 (m, 4H), 3.28-3.19 (m, 4H), 3.14-3.04 (m, 2H), 2.17-1.89 (m, 8H). LC/MS (ESI): 487.3 (MH)+.
Prepared essentially as described in Example 159 using thiomorpholine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.03 (s, 1H), 7.99 (d, 1H), 7.28-7.14 (m, 4H), 6.52 (d, 2H), 6.22 (s, 1H), 4.25 (d, 2H), 3.92-3.85 (m, 4H), 3.65-3.55 (m, 1H), 3.30-3.04 (m, 6H), 2.77-2.72 (m, 4H), 2.18-1.88 (m, 8H). LC/MS (ESI): 503.3 (MH)+.
Prepared essentially as described in Example 159 using piperazin-2-one in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.07 (s, 1H), 8.05 (d, 1H), 7.30-7.15 (m, 4H), 6.55-6.46 (m, 3H), 6.25 (s, 1H), 4.29-4.10 (m, 4H), 3.78-3.55 (m, 5H), 3.29-3.05 (m, 6H), 2.18-1.89 (m, 8H). LC/MS (ESI): 500.2 (MH)+.
Prepared essentially as described in Example 159 using 1-methyl-piperazin-2-one in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.07 (s, 1H), 8.05 (d, 1H), 7.27-7.04 (m, 4H), 6.52 (d, 2H), 6.22 (s, 1H), 4.25 (d, 2H), 4.12 (s, 2H), 3.76-3.70 (m, 2H), 3.68-3.53 (m, 4H), 3.31-3.19 (m, 4H), 3.17-3.04 (m, 4H), 2.18-1.89 (m, 8H). LC/MS (ESI): 514.3 (MH)+.
Prepared essentially as described in Example 159 using 1-(2-hydroxyethyl)-piperazine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.03 (s, 1H), 7.98 (d, 1H), 7.32 (m, 1H), 7.20-7.14 (m, 3H), 6.51 (d, 2H), 6.31 (s, 1H), 4.24 (d, 2H), 3.71-3.65 (m, 2H), 3.65-3.55 (m, 1H), 3.49-3.42 (m, 4H), 3.28-3.20 (m, 4H), 3.13-3.03 (m, 2H), 2.74-2.65 (m, 4H), 2.65-2.59 (m, 2H), 2.16-1.81 (m, 9H). LC/MS (ESI): 530.3 (MH)+.
Prepared essentially as described in Example 159 using 1-(2-methoxyethyl)-piperazine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.03 (s, 1H), 7.97 (d, 1H), 7.31 (m, 1H), 7.20-7.14 (m, 3H), 6.51 (d, 2H), 6.24 (s, 1H), 4.24 (d, 2H), 3.65-3.53 (m, 3H), 3.51-3.45 (m, 4H), 3.38 (s, 3H), 3.28-3.21 (m, 4H), 3.14-3.04 (m, 2H), 2.72-2.62 (m, 6H), 2.16-1.88 (m, 8H). LC/MS (ESI): 544.3 (MH)+.
Prepared essentially as described in Example 159 using 1-ethyl-piperazine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.03 (s, 1H), 7.97 (d, 1H), 7.32 (m, 1H), 7.21-7.14 (m, 3H), 6.51 (d, 2H), 6.27 (s, 1H), 4.24 (d, 2H), 3.65-3.55 (m, 1H), 3.51-3.44 (m, 4H), 3.28-3.20 (m, 4H), 3.14-3.03 (m, 2H), 2.68-2.58 (m, 4H), 2.53-2.44 (m, 2H), 2.16-1.84 (m, 8H), 1.14 (t, 3H). LC/MS (ESI): 514.3 (MH)+.
A mixture of (tetrahydro-pyran-4-yl)-methanol (0.2 mmol), KOtBu (0.2 mmol) and 4-(7-fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (0.1 mmol), prepared as described in Example 65b, in DMSO (1 mL), was stirred at 80° C for 1 h. It was then diluted with water and extracted with DCM. The combined extracts were washed with water, brine, dried with MgSO4, filtered, and concentrated in vacuo. The crude product was then treated with 3M HCl/MeOH (2 mL) and stirred at rt for 2 h and then concentrated in vacuo. The crude deprotected intermediate was dissolved in a mixture of DCM:MeOH (1:1; 2 mL) and neutralized with excess Et3N and treated with (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride (0.11 mmol), prepared by the method as outlined in Example 74a, at rt overnight. It was then concentrated in vacuo and the crude product was dissolved in DCM and washed with water thrice, then washed with brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was then purified by Preparative TLC (silica gel; DCM:MeOH, 9.5:0.5) followed by a further purification by Preparative HPLC to obtain 1.5 mg (3%) of the title compound. 1H-NMR (300 MHz, CDCl3+CD3OD): 9.05 (s, 1H), 8.09 (m, 1H), 7.52-7.23 (m, 6H), 4.30 (m, 2H), 4.03-3.94 (m, 4H), 3.75-3.50 (m, 8H), 3.48-3.38 (m, 2H), 2.30-2.18 (m, 4H), 2.17-2.00 (m, 2H), 1.97-1.85 (m, 2H), 1.75 (m, 2H), 1.55-1.41 (m, 2H). LC/MS (ESI): 516.2 (MH)+.
Prepared essentially as described in Example 188 using tetrahydro-pyran-4-ol in place of (tetrahydro-pyran-4-yl)-methanol. 1H-NMR (300 MHz, CDCl3): 9.12 (s, 1H), 8.07 (d, 1H), 7.34-7.15 (m, 4H), 6.52 (d, 2H), 6.21 (s, 1H), 4.76-4.68 (m, 1H), 4.26 (d, 2H), 4.06-3.98 (m, 2H), 3.71-3.58 (m, 3H), 3.30-3.19 (m, 4H), 3.16-3.06 (m, 2H), 2.19-2.05 (m, 4H), 2.03-1.82 (m, 8H). LC/MS (ESI): 502.2 (MH)+.
Prepared essentially as described in Example 188 using (S)-tetrahydro-furan-3-ol in place of (tetrahydro-pyran-4-yl)-methanol. 1H-NMR (300 MHz, CDCl3): 9.14 (s, 1H), 8.07 (d, 1H), 7.28-7.15 (m, 4H), 6.52 (d, 2H), 6.22 (s, 1H), 5.11 (m, 1H), 4.26 (d, 2H), 4.12-3.99 (m, 3H), 3.98-3.90 (m, 1H), 3.72-3.61 (m, 1H), 3.31-3.18 (m, 4H), 3.16-3.05 (m, 2H), 2.41-2.29 (m, 1H), 2.28-2.04 (m, 3H), 2.03-1.90 (m, 6H). LC/MS (ESI): 488.2 (MH)+.
Prepared essentially as described in Example 188 using (R)-tetrahydro-furan-3-ol in place of (tetrahydro-pyran-4-yl)-methanol. 1H-NMR (300 MHz, CDCl3): 9.14 (s, 1H), 8.07 (d, 1H), 7.28-7.15 (m, 4H), 6.52 (d, 2H), 6.22 (s, 1H), 5.11 (m, 1H), 4.26 (d, 2H), 4.12-3.99 (m, 3H), 3.98-3.90 (m, 1H), 3.71-3.61 (m, 1H), 3.31-3.18 (m, 4H), 3.16-3.05 (m, 2H), 2.41-2.29 (m, 1H), 2.28-2.05 (m, 3H), 2.03-1.91 (m, 6H). LC/MS (ESI): 488.3 (MH)+.
Prepared essentially as described in Example 159 using 1-pyridin-2-yl-piperazine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.05 (s, 1H), 8.22 (d, 1H), 8.01 (d, 1H), 7.53 (m, 1H), 7.36 (m, 1H), 7.24-7.15 (m, 3H), 6.74-6.66 (m, 2H), 6.51 (d, 2H), 6.27 (s, 1H), 4.25 (d, 2H), 3.80-3.72 (m, 4H), 3.67-3.54 (m, 5H), 3.30-3.19 (m, 4H), 3.15-3.04 (m, 2H), 2.17-1.88 (m, 8H). LC/MS (ESI): 563.3 (MH)+.
Prepared essentially as described in Example 159 using 1-pyrimidin-2-yl-piperazine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.05 (s, 1H), 8.35 (d, 2H), 8.01 (d, 1H), 7.39-7.14 (m, 4H), 6.58-6.47 (m, 3H), 6.28 (s, 1H), 4.25 (d, 2H), 4.07-3.98 (m, 4H), 3.67-3.48 (m, 5H), 3.28-3.18 (m, 4H), 3.15-3.03 (m, 2H), 2.17-1.88 (m, 8H). LC/MS (ESI): 564.3 (MH)+.
Prepared essentially as described in Example 159 using 1-pyridin-4-yl-piperazine in place of 1-methyl-piperazine. 1H-NMR (300 MHz, CDCl3): 9.00 (s, 1H), 8.27 (d, 2H), 7.97 (d, 1H), 7.30-7.09 (m, 4H), 6.65 (d, 2H), 6.46 (d, 2H), 6.14 (s, 1H), 4.19 (d, 2H), 3.61-3.47 (m, 9H), 3.22-3.15 (m, 4H), 3.10-3.00 (m, 2H), 2.12-1.99 (m, 2H), 1.95-1.84 (m, 6H). LC/MS (ESI): 563.3 (MH)+.
Prepared essentially as Example 126 using 4-fluoro-piperidine and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H NMR (CDCl3) δ 8.97 (s, 1H), 7.91 (d, J=9.49 Hz, 1H), 7.25 (dd, J=9.40 and 2.62 Hz, 1H), 7.15 (d, J=2.58 Hz, 1H), 7.11 (d, J=8.88 Hz, 2H), 6.45 (d, J=8.92 Hz, 2H), 4.84 (m, 1H), 4.18 (m, 2H), 3.43-3.60 (5H), 3.19 (t, J=6.60 Hz, 4H), 3.30 (td, J=12.63 and 2.62 Hz, 2H), 1.84-2.10 (12 H). Calcd for C29H36FN6O (MH+) 503.3. found 503.3.
Prepared essentially as Example 126 using 4-fluoro-piperidine. 1H NMR (CDCl3) δ 9.04 (s, 1H), 7.98 (d, J=9.49 Hz, 1H), 7.32 (dd, J=9.42 and 2.61 Hz, 1H), 7.27 (d, J=8.91 Hz, 2H), 7.22 (d, J=2.57 Hz, 1H), 6.87 (d, J=9.04 Hz, 2H), 6.31 (br, 1H), 4.90 (m, 1H), 4.25 (m, 2H), 3.86 (t, J=4.71 Hz, 4H), 3.50-3.67 (5H), 3.14 (dd, J=13.15 and 2.72 Hz, 2H), 3.10 (t, J=4.83 Hz, 4H), 1.92-2.17 (8H). Calcd for C29H36FN6O2 (MH+) 519.3. found 519.3.
Prepared essentially as Example 178b using (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 66a. 1H NMR (DMSO-d6) δ 9.04 (s, 1H), 8.39 (dd, J=9.33 and 2.04 Hz, 1H), 8.36 (s, 1H), 8.34 (s, 1H), 7.65 (d, J=2.23 Hz, 1H), 7.38 (s, 1H), 7.30 (d, J=9.10 Hz, 2H), 6.82 (d, J=9.18 Hz, 2H), 4.24 (m, 2H), 3.87 (m, 1H), 3.71 (m, 2H), 3.32-3.45 (8H), 2.99 (m, 4H), 1.78-1.85 (4H). Calcd for C27H32N7O3 (MH+) 502.3. found 502.3.
Prepared essentially as described in Example 131 using (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 66a. 1H NMR (CDCl3) δ 9.08 (s, 1H), 7.94 (d, J=9.27 Hz, 1H), 7.62 (dd, J=9.31 and 2.57 Hz, 1H), 7.30 (d, J=9.00 Hz, 2H), 7.27 (d, J=3.23 Hz, 1H), 6.86 (d, J=9.02 Hz, 2H), 6.53 (br, 1H), 4.28 (m, 2H), 3.84 (t, J=4.66 Hz, 4H), 3.58-3.68 (3H), 3.54 (m, 4H), 3.17 (m, 2H), 3.04-3.10 (4H), 2.96 (m, 2H), 2.61 (s, 3H), 1.93-2.18 (4H). Calcd for C29H38N7O2(MH+) 516.3. found 516.1.
Prepared essentially as described in Example 140 using ethyl isocyanate in place of FMOC-Cl and (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitrophenyl ester hydrochloride, as prepared by the method outlined in Example 74a, in place of (4-morpholin -4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.04 (s, 1H), 8.00 (d, 1H), 7.32-7.14 (m, 4H), 6.51 (d, 2H), 6.30 (s, 1H), 4.58 (m, 1H), 4.25 (m, 2H), 3.66-3.54 (m, 5H), 3.51-3.43 (m, 4H), 3.35-3.17 (m, 6H), 3.15-3.04 (m, 3H), 2.17-2.03 (m, 2H), 2.02-1.88 (m, 5H), 1.16 (t, 3H). LC/MS (ESI): 557.3 (MH)+.
Prepared essentially as described in Example 140 using methoxyacetyl chloride in place of FMOC-Cl and (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitrophenyl ester hydrochloride, as prepared by the method outlined in Example 74a, in place of (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride. 1H-NMR (300 MHz, CDCl3): 9.07 (s, 1H), 8.02 (d, 1H), 7.35-7.14 (m, 4H), 6.52 (d, 2H), 6.25 (s, 1H), 4.25 (m, 2H), 4.17 (s, 2H), 3.86-3.56 (m, 6H), 3.49-3.42 (m, 6H), 3.30-3.18 (m, 4H), 3.10 (t, 2H), 2.18-2.04 (m, 2H), 2.02-1.87 (m, 6H). LC/MS (ESI): 558.3 (MH)+.
4-(7-Piperazin-1-yl-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (0.1 mmol), prepared as described in Example 140, was added to a mixture of t-butoxyacetic acid (0.15 mmol) and PS-carbodiimide (0.2 mmol) in anhydrous DCM (2 mL). The mixture was shaken at rt overnight. It was then filtered and the resin washed with DCM. The combined filtrate and washings were concentrated in vacuo. To this was then added 3M HCl/MeOH (2 mL) and stirred at rt for 2 h and then concentrated in vacuo. The crude residue was dissolved in a mixture of DCM:MeOH (1:1; 2 mL), neutralized with excess Et3N and treated with (6-pyrrolidin-1-yl-pyridin-3-yl)-carbamic acid 4-nitrophenyl ester hydrochloride (0.11 mmol), as prepared by the method outlined in Example 74a, at rt overnight. It was then concentrated in vacuo and the crude product was dissolved in DCM and washed with water thrice, then washed with brine, dried over anhydrous MgSO4, filtered and concentrated in vacuo. The crude product was then purified by Preparative TLC (silica gel; DCM:MeOH, 95:5) followed by a further purification by Preparative HPLC to obtain 1 mg (1%) of the title compound. 1H-NMR (300 MHz, CDCl3): 9.08 (s, 1H), 8.04 (d, 1H), 7.34-7.15 (m, 4H), 6.52 (d, 2H), 6.19 (s, 1H), 4.30-4.19 (m, 4H), 3.92-3.32 (m, 12H), 3.29-3.20 (m, 4H), 3.11 (t, 2H), 2.18-1.87 (m, 6H). LC/MS (ESI): 544.3 (MH)+.
To a solution of 4-[7-(-hydroxy-ethoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (0.5 mmol), prepared as described in Example 169a, in anhydrous DCM, was added Et3N (1 mmol) and methanesulfonyl chloride (1 mmol) and the mixture was stirred at rt for 2 h. It was then washed with water (3×), dried over anhydrous MgSO4, filtered and concentrated in vacuo to obtain crude 4-[7-(3-methanesulfonyloxy-ethoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester. This (0.1 mmol) was dissolved in anhydrous DMSO together with 1-methyl-piperazin-2-one (0.2 mmol) and the mixture was stirred at 100° C. for 2 h and then diluted with water and extracted with DCM. The DCM extract was washed with water (3×), dried over anhydrous MgSO4, filtered and concentrated in vacuo. To this was added 3M HCl/MeOH (1 mL) and the mixture was stirred at rt for 2 h and then concentrated in vacuo and the residue was dissolved in a 1:1 mixture of DCM:MeOH, neutralized with excess Et3N and treated with (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitrophenyl ester hydrochloride (0.11 mmol), as prepared by the method outlined in Example 74a. The mixture was stirred at rt overnight and then concentrated in vacuo and partitioned between water and DCM. DCM layer was drawn off, washed with water thrice, then dried over anhydrous MgSO4, filtered and concentrated in vacuo. The residue was purified by Preparative TLC (silica gel; DCM:MeOH, 95:5) followed by a further purification by Preparative HPLC to obtain 5.6 mg (6%) of the title compound. 1H-NMR (300 MHz, CD3OD): 9.10 (s, 1H), 8.44 (d, 1H), 7.59-7.31 (m, 6H), 4.62 (t, 2H), 4.37 (m, 2H), 4.04-3.93 (m, 4H), 3.78-3.54 (m, 8H), 3.21 (m, 2H), 3.03 (s, 3H), 2.30-2.18 (m, 5H), 2.11-1.91 (m, 4H). LC/MS (ESI): 558.3 (MH)+.
The title compound was prepared from 4-chloro-6-methoxyquinazoline (WO 2001032632 A2, WO 9609294 A1) essentially as described for Example 1, except the methyl ester intermediate was stirred in KOH/MeOH at 100° C. for 3 hr instead of 1 hr. 1H-NMR (300 MHz, CDCl3) δ 9.15 (s, 1H), 7.99 (d, 1H), 7.56 (dd, 1H), 7.33 (d, 1H), 7.25 (m, 2H), 6.85 (m, 2H), 6.31 (br s, 1H), 4.49 (heptet, 1H), 4.27 (m, 2H), 4.00 (s, 3H), 3.66 (tt, 1H), 3.17 (td, 2H), 2.22-1.97 (m, 4H), 1.32 (d, 6H). LC/MS (ESI): calcd mass 420.2. found 421.2 (MH)+.
A mixture of 4-hydroxybutyronitrile (24.2 mg, 285 μmol) [Organometallics (1996), 15(4), 1236-41], KOtBu (34.8 mg, 311 μmol), and DME was stirred at rt, followed by the addition of 4-(7-Fluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (48.8 mg, 147 μmol) (prepared as described in Example 65b). The resulting homogeneous solution was stirred at rt for 2 hr, and was then directly loaded onto a 5 g Jones silica cartridge pre-equilibrated with 9:1 DCM/acetone, and eluted with 9:1→8:2 DCM/acetone to afford the title intermediate (24.5 mg, 42%) as a colorless oil. LC/MS (ESI) calcd mass 396.2. found 397.1 (MH)+.
A mixture of 4-[7-(3-Cyano-propoxy)-quinazolin-4-yl]-piperidine-1-carboxylic acid tert-butyl ester (24.5 mg, 62 μmol), as prepared in the preceding step, NaN3 (13.4 mg, 206 μmol), TEA.HCl (25.5 mg, 185 μmol), and toluene (100 μL) was tightly capped and stirred at 100° C. for 6.5 hr. The reaction was then allowed to cool to rt, partitioned with EtOAc (1 mL) and 0.1 M HCl (1 mL). The aqueous layer was then extracted with EtOAc (2×1 mL), the organic layers were combined, dried (Na2SO4), and concentrated. The residue was purified via flash silica chromatography (3:2 EtOAc/acetone) to yield the title intermediate as an off-white solid (12.2 mg, 44%). LC/MS (ESI) calcd mass 439.2. found 440.1 (MH)+.
A solution of 4-{7-[3-(1H-Tetrazol-5-yl)-propoxy]-quinazolin-4-yl}-piperidine-1-carboxylic acid tert-butyl ester (6.1 mg, 14 μmol), as prepared in the previous step, in 9:1 TFA/anisole (100 μL) was stirred at 100° C. for 10 min. The solution was then concentrated. Pyridine (100 μL) and (4-isopropoxy-phenyl)-carbamic acid 4-nitro-phenyl ester (5.8 mg, 18 μmol), as prepared in Example 1a, were added, and the solution was stirred at 80° C. for 15 min. The reaction was concentrated, taken up in 1M NaH2PO4 (2 mL), and extracted with 95:5 DCM/MeOH (2×2 mL). The combined organic layers were dried (Na2SO4), concentrated, and purified by flash silica cartridge chromatography (EtOAc→acetone eluent) to provide the title compound (1.0 mg, 14%). 1H-NMR (400 MHz, 95:5 CDCl3/CD3OD) δ 9.09 (s, 1H), 8.08 (s, 1H), 7.30-7.21 (m, 4H), 6.85 (m, 2H), 4.49 (septet, 1H), 4.27 (m, 2H), 4.24 (t, 2H), 3.70 (tt, 1H), 3.19 (t, 2H), 3.12 (td, 2H), 2.40 (m, 2H), 2.17-1.92 (m, 4H), 1.32 (d, 6H). LC/MS (ESI) calcd mass 516.3. found 517.2 (MH)+.
A mixture of 4,5-difluoroanthranilic acid (20.43 g, 118 mmol) and formamidine acetate (13.55 g, 130 mmol) in reagent EtOH was stirred at 120° C. (oil bath) for 3 hr. The reaction was briefly a homogeneous brown solution, and then became an opaque mixture. The reaction was allowed to cool to rt, and the resulting solid was filtered, washed with denatured EtOH (1×10 mL), and allowed to air dry. Powdering with a mortar and pestle provided 4-hydroxy-6,7-difluoroquinazoline as a beige powder (16.9 g, 79%). 16.6 g of this material (91.1 mmol) was taken up in SOCl2 (66 mL), DCE (66 mL), and DMF (7.05 mL, 91 mmol), and was stirred at 110 ° C. (oil bath) for 1 hr. The resulting homogeneous amber solution was then concentrated under rotary evaporation, and taken up in toluene (2×100 mL) with repeated rotary evaporation to provide the crude title compound as a beige solid. A portion of this material (8.4 g of 17.7 g total) was taken up in DCM (80 mL) and gently shaken with 2M trisodium citrate (1×40 mL) until a homogeneous clear organic layer resulted. This organic layer was immediately applied (without drying) directly onto a silica flash column (79 mm×6″) pre-equilibrated with 1:1 hexanes/EtOAc. Trivial elution with 1:1 hexanes/EtOAc, followed by repeated rotary evaporation from toluene (2×50 mL) of the combined fractions afforded the title compound as a light yellow solid (6.79 g, 78%). 1H-NMR (400 MHz, CDCl3) δ 9.05 (s, 1H), 8.05 (dd, 1H), 7.86 (dd, 1H).
A solution of piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (1.27 g, 5.23 mmol) in dry THF (2 mL) was added dropwise over 2 minutes with stirring to 1.01M LiHMDS/THF (5.75 mL, 5.81 mmol) at −78° C. under argon. After 5 min at −78° C., the cold bath was removed and the reaction was allowed to stir at “rt” for 30 min. A portion of this enolate solution (5.1 mL, ˜3 mmol enolate) was added dropwise over 2-3 min to a stirred homogeneous solution of 4-chloro-6,7-difluoroquinazoline (600 mg, 2.99 mmol) in dry THF (3 mL) at 0° C. under argon. The reaction was stirred for 30 min at 0° C., and was then quenched with 1M NaH2PO4 (50 mL) and extracted with EtOAc (1×50 mL). The organic layer was washed with 4M NaCl (1×50 mL), dried (Na2SO4), and concentrated. The residue was purified with silica flash chromatography (3:1 hexanes/EtOAc) to afford the title compound as a yellow oil (451 mg, 37%). LC/MS (ESI): calcd mass 407.2. found 408.2 (MH)+.
A mixture of 4-(6,7-Difluoro-quinazolin-4-yl)-piperidine-1,4-dicarboxylic acid 1-tert-butyl ester 4-methyl ester (451 mg, 1.11 mmol), as prepared in the previous step, LiCl (89 mg, 2.12 mmol), water (60 μL, 3.3 mmol), and DMSO (430 μL) was stirred at 150° C. for 7.5 hrs with a reflux condenser. The reaction was then allowed to cool to rt, shaken with 1M NaCl (5 mL), and extracted with DCM (1×3 mL) and 9:1 DCM/MeOH (1×3 mL). The organic layers were combined, dried (Na2SO4), and concentrated. The residue was purified by silica flash chromatography (3:1 hex/EtOAc→2:1 eluent) to provide the title compound (151.8 mg, 39%). 1H-NMR (300 MHz, CDCl3) δ 9.22 (s, 1H), 7.90 (dd, 1H), 7.81 (dd, 1H), 4.33 (br m, 2H), 3.50 (tt, 1H), 2.96 (br t, 2H), 2.11-1.82 (m, 4H), 1.49 (s, 9H). LC/MS (ESI): calcd mass 349.2. found 368.3 (MH.H2O)+.
A solution of 1.19M KOtBu in THF (128 μL, 152 μmol) was added dropwise with stirring over 2.5 min to a 0° C. homogeneous solution of 4-(6,7-Difluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (38. 1 mg, 109 μmol), as prepared in the previous step, and 3-(4-Methyl-piperazin-1-yl)-propan-1-ol (22.4 mg, 142 μmol) in THF (170 μL). The reaction was stirred at 0° C. for 1.5 hr, and was then partitioned with DCM (2 mL) and 1M NaCl (2 mL). The aq layer was back-extracted with DCM (1×2 mL), and the combined cloudy white organic layers were dried (Na2SO4) and concentrated. The residue was purified by silica flash chromatography (1:2 hex/EtOAc/3% DMEA eluent) to yield the title compound as an off-white foam (32.6 mg, 61%). NOe experiments support the assigned regioisomer. Select 1H-NMR resonances and nOes (300 MHz, CDCl3) δ 7.73 (d, J=11.4 Hz, 1H), 7.43 (d, J=8.1 Hz, 1H), 3.46 (tt, 1H). Irradiation of the diagnostic methine proton at δ 3.46 generates an nOe to the quinazoline C5 proton at δ 7.73, but not to the quinazoline C8 proton at δ 7.43. The C5 proton has a larger coupling constant than the C8 proton, indicating fluorine substitution at C6 of the quinazoline. LC/MS (ESI): calcd mass 487.3. found 488.3 (MH)+.
The title compound was prepared from 4-{6-Fluoro-7-[3-(4-methyl-piperazin-1-yl)-propoxy]-quinazolin-4-yl}-piperidine-1-carboxylic acid tert-butyl ester, prepared in the previous step, and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, prepared as described in Example 66a, using essentially the protocol given for Example 170c. 1H-NMR (400 MHz, CDCl3) δ 9.14 (s, 1H), 7.74 (d, 1H), 7.44 (d, 1H), 7.27 (m, 2H), 6.88 (m, 2H), 6.32 (s, 1H), 4.27 (m, 4H), 3.86 (m, 4H), 3.54 (tt, 1H), 3.18-3.08 (m, 6H), 2.58 (t, 2H), 2.64-2.35 (br, 8H), 2.30 (s, 3H), 2.12 (m, 4H), 1.96 (m, 2H). LC/MS (ESI): calcd mass 591.3. found 592.4 (MH)+.
Prepared as for Example 205d-e using 1-(2-Hydroxy-ethyl)-pyrrolidin-2-one and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, which was prepared as described in Example 74a. 1H-NMR (400 MHz, CDCl3) δ 9.15 (s, 1H), 7.76 (d, 1H), 7.40 (d, 1H), 7.19 (m, 2H), 6.53 (m, 2H), 6.25 (s, 1H), 4.35 (t, 2H), 4.26 (m, 2H), 3.82 (t, 2H), 3.66 (m, 2H), 3.52 (tt, 1H), 3.26 (m, 4H), 3.11 (td, 2H), 2.42 (m, 2H), 2.17-2.02 (m, 4H), 2.02-1.90 (m, 6H). LC/MS (ESI): calcd mass 546.3. found 547.4 (MH)+.
A mixture of 4-{6-Fluoro-7-[3-(4-methyl-piperazin-1-yl)-propoxy]-quinazolin-4-yl}-piperidine-1-carboxylic acid tert-butyl ester (32.6 mg, 66.9 μmol), as prepared in Example 205d, DMSO (50 μL), and 0.31M KOMe/MeOH (270 μL, 83.9 μmol KOMe in 6.4 mmol MeOH) was stirred at 100° C. for 9 hr, and then 110° C. for 2 hr. The resulting pale yellow homogeneous solution was allowed to cool to rt, diluted with DCM (2 mL), and washed with 4M NaCl (1×2 mL). The aq layer was back-extracted with DCM (1×2 mL), and the combined organic layers were dried (Na2SO4) and concentrated. Purification of the residue by silica flash chromatography (1:2 hex/EtOAc→1:2 hex/EtOAc/3% DMEA→9:1 EtOAc/acetone/3% DMEA eluent) afforded the title compound (18.4 mg, 55%). NOe experiments support the assigned regioisomer. Select 1H-NMR resonances and nOes (300 MHz, CDCl3) δ 7.34 (s, 1H), 7.24 (s, 1H), 4.04 (s, 3H), 3.51 (m, 1H). Irradiation of the diagnostic methine proton at 67 3.51 generates an nOe to the quinazoline C5 proton at δ 7.24, but not to the quinazoline C8 proton at δ 7.34. Irradiation of the methoxy protons at δ 4.04 generates an nOe to the C5 proton at δ 7.24, but not to the C8 proton at δ 7.34. This indicates methoxy substitution at C6 of the quinazoline. LC/MS (ESI): calcd mass 499.3. found 500.4 (MH)+.
The title compound was prepared from 4-{6-Methoxy-7-[3-(4-methyl-piperazin-1-yl)-propoxy]-quinazolin-4-yl}-piperidine-1-carboxylic acid tert-butyl ester, prepared in the previous step, and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, prepared as described in Example 66a, using essentially the protocol given for Example 170c. 1H-NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 7.34 (s, 1H), 7.28 (m, 2H), 7.25 (s, 1H), 6.88 (m, 2H), 6.34 (s, 1H), 4.27 (m, 4H), 4.05 (s, 3H), 3.86 (m, 4H), 3.59 (tt, 1H), 3.16 (td, 2H), 3.11 (m, 4H), 2.57 (m, 2H), 2.65-2.34 (br, 8H), 2.30 (s, 3H), 2.21-2.08 (m, 4H), 2.03-1.95 (m, 2H). LC/MS (ESI): calcd mass 603.4. found 604.4 (MH)+.
The title compound was prepared from 4-{6-Methoxy-7-[3-(4-methyl-piperazin-1-yl) -propoxy]-quinazolin-4-yl}-piperidine-1-carboxylic acid tert-butyl ester, prepared as described in Example 207a, and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, prepared as described in Example 74a, using essentially the protocol given for Example 170c. 1H-NMR (400 MHz, CDCl3) δ 9.07 (s, 1H), 7.34 (s, 1H), 7.25 (s, 1H), 7.19 (m, 2H), 6.52 (m, 2H), 6.24 (s, 1H), 4.27 (m, 4H), 4.04 (s, 3H), 3.57 (tt, 1H), 3.26 (m, 4H), 3.14 (td, 2H), 2.58 (m, 2H), 2.64-2.35 (br, 8H), 2.30 (s, 3H), 2.20-2.08 (m, 4H), 2.04-1.93 (m, 6H). LC/MS (ESI): calcd mass 587.4. found 588.4 (MH)+.
Prepared as for Example 208 using 1-(2-Hydroxy-ethyl)-pyrrolidin-2-one instead of 3-(4-methyl-piperazin-1-yl)-propan-1-ol 1H-NMR (400 MHz, CDCl3) δ 9.07 (s, 1H), 7.30 (s, 1H), 7.26 (s, 1H), 7.19 (m, 2H), 6.53 (m, 2H), 6.23 (s, 1H), 4.32 (t, 2H), 4.26 (m, 2H), 4.04 (s, 3H), 3.82 (t, 2H), 3.66 (m, 2H), 3.57 (tt, 1H), 3.26 (m, 4H), 3.14 (td, 2H), 2.41 (m, 2H), 2.22-1.94 (m, 10H). LC/MS (ESI): calcd mass 558.3. found 559.4 (MH)+.
A solution of 4-(6,7-Difluoro-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (37.8 mg, 108 μmol) (preparation in Example 205c) and morpholine (19.8 μL, 227 μmol) in THF (100 μL) and DMSO (50 μL) was heated at 100° C. for 1 hr. The crude reaction was loaded onto a flash silica cartridge (1:1 hexanes/EtOAc eluent) to provide the title compound (40.2 mg, 89%). NOe experiments support the assigned regioisomer. Select 1H-NMR resonances and nOes (300 MHz, CDCl3) δ 7.68 (d, J=13.7 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 3.45 (tt, 1H), 3.31 (m, 4H). Irradiation of the diagnostic methine proton at δ 3.45 generates an nOe to the quinazoline C5 proton at δ 7.68, but not to the quinazoline C8 proton at δ 7.37. The C5 proton has a larger coupling constant than the C8 proton, indicating fluorine substitution at C6 of the quinazoline. Furthermore, irradiation of the C8 proton at δ 7.37 generates an nOe only to the morpholine C3 protons at δ 3.31, while irradiation of the C5 proton generates an nOe only to the methine proton at δ 3.45. These data indicate morpholine substitution at the quinazoline C7 carbon. LC/MS (ESI): calcd mass 416.2. found 417.3 (MH)+.
The title compound was prepared from 4-(6-Fluoro-7-morpholin-4-yl-quinazolin-4-yl) -piperidine-1-carboxylic acid tert-butyl ester, prepared as described in Example 210a, and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, prepared as described in Example 74a, using essentially the protocol given for Example 170c. 1H-NMR (400 MHz, CDCl3) δ 9.12 (s, 1H), 7.70 (d, 1H), 7.38 (d, 1H), 7.18 (m, 2H), 6.52 (m, 2H), 6.24 (s, 1H), 4.26 (m, 2H), 3.93 (m, 4H), 3.51 (tt, 1H), 3.31 (m, 4H), 3.26 (m, 4H), 3.11 (td, 2H), 2.17-2.05 (m, 2H), 2.03-1.90 (m, 6H). LC/MS (ESI): calcd mass 504.3. found 505.3.
A mixture of 4-(6-Fluoro-7-morpholin-4-yl-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester (28.9 mg, 69.5 μmol), as prepared in Example 210a, DMSO (50 μL), and 1.0M KOMe/MeOH (140 μL, 140 μmol) was stirred in a sealed vial at 100° C. (aluminum block) for 13 hr. The crude reaction was then diluted with toluene and directly loaded onto a silica flash column (1:2 hexanes/EtOAc eluent) to provide the title compound (20.0 mg, 67%). NOe experiments support the assigned regioisomer. Select 1H-NMR resonances and nOes (300 MHz, CDCl3) δ 7.36 (s, 1H), 7.25 (s, 1H), 4.05 (s, 3H), 3.51 (m, 1H). Irradiation of the diagnostic methine proton at δ 3.51 generates an nOe to the quinazoline C5 proton at δ 7.25, but not to the quinazoline C8 proton at δ 7.36. Irradiation of the methoxy protons at δ 4.05 generates an nOe to the C5 proton at δ 7.25, but not to the C8 proton at δ 7.36. This indicates methoxy substitution at C6 of the quinazoline. LC/MS (ESI): calcd mass 428.2. found 429.3 (MH)+.
The title compound was prepared from 4-(6-methoxy-7-morpholin-4-yl-quinazolin-4-yl) -piperidine-1-carboxylic acid tert-butyl ester, prepared as described in Example 211 a, and (4-pyrrolidin-1-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, prepared as described in Example 74a, using essentially the protocol given for Example 170c. 1H-NMR (400 MHz, CDCl3) δ 9.07 (s, 1H), 7.37 (s, 1H), 7.25 (s, 1H), 7.19 (m, 2H), 6.53 (m, 2H), 6.21 (s, 1H), 4.26 (m, 2H), 4.05 (s, 3H), 3.94 (m, 4H), 3.57 (tt, 1H), 3.31-3.24 (m, 8H), 3.15 (td, 2H), 2.20-2.08 (m, 2H), 2.03-1.94 (m, 6H). LC/MS (ESI): calcd mass 516.3. found 517.3 (MH)+.
The title compound was prepared from 4-(6-methoxy-7-morpholin-4-yl-quinazolin-4-yl)-piperidine-1-carboxylic acid tert-butyl ester, prepared in Example 211a, and (4-morpholin-4-yl-phenyl)-carbamic acid 4-nitro-phenyl ester hydrochloride, prepared as described in Example 66a, using essentially the protocol given for Example 170c. 1H-NMR (400 MHz, CDCl3) δ 9.06 (s, 1H), 7.37 (s, 1H), 7.28 (m, 2H), 7.25 (s, 1H), 6.89 (m, 2H), 6.35 (s, 1H), 4.27 (m, 2H), 4.06 (s, 3H), 3.94 (m, 4H), 3.86 (m, 4H), 3.59 (tt, 1H), 3.29 (m, 4H), 3.16 (td, 2H), 3.11 (m, 4H), 2.21-2.08 (m, 2H), 2.03-1.95 (m, 2H). LC/MS (ESI): calcd mass, 532.3. found 533.3 (MH)+.
Biological Activity of FLT3 Inhibitors of Formula I′
The following representative assays were performed in determining the biological activities of the FLT3 inhibitors of Formula I′. They are given to illustrate the invention in a non-limiting fashion.
In Vitro Assays
The following representative in vitro assays were performed in determining the biological activities of the FLT3 inhibitors of Formula I′ within the scope of the invention. They are given to illustrate the invention in a non-limiting fashion.
Inhibition of FLT3 enzyme activity, MV4-11 proliferation and Baf3-FLT3 phosphorylation exemplify the specific inhibition of the FLT3 enzyme and cellular processes that are dependent on FLT3 activity. Inhibition of Baf3 cell proliferation is used as a test of FLT3, c-Kit and TrkB independent cytotoxicity of compounds within the scope of the invention. All of the examples herein show significant and specific inhibition of the FLT3 kinase and FLT3-dependent cellular responses. Examples herein also show specific inhibition of the TrkB and c-kit kinase in an enzyme activity assay. The FLT3 inhibitors of Formula I′ are also cell permeable.
FLT3 Fluorescence Polarization Kinase Assay
To determine the activity of the FLT3 inhibitors of Formula I′ in an in vitro kinase assay, inhibition of the isolated kinase domain of the human FLT3 receptor (a.a. 571-993) was performed using the following fluorescence polarization (FP) protocol. The FLT3 FP assay utilizes the fluorescein-labeled phosphopeptide and the anti-phosphotyrosine antibody included in the Panvera Phospho-Tyrosine Kinase Kit (Green) supplied by Invitrogen. When FLT3 phosphorylates polyGlu4Tyr, the fluorescein-labeled phosphopeptide is displaced from the anti-phosphotyrosine antibody by the phosphorylated poly Glu4Tyr, thus decreasing the FP value. The FLT3 kinase reaction is incubated at room temperature for 30 minutes under the following conditions: 10 nM FLT3 571-993, 20 ug/mL poly Glu4Tyr, 150 uM ATP, 5 mM MgCl2, 1% compound in DMSO. The kinase reaction is stopped with the addition of EDTA. The fluorescein-labeled phosphopeptide and the anti-phosphotyrosine antibody are added and incubated for 30 minutes at room temperature.
All data points are an average of triplicate samples. Inhibition and IC50 data analysis was done with GraphPad Prism using a non-linear regression fit with a multiparamater, sigmoidal dose-response (variable slope) equation. The IC50 for kinase inhibition represents the dose of a compound that results in a 50% inhibition of kinase activity compared to DMSO vehicle control.
Inhibition Of MV4-11 and Baf3 Cell Proliferation
To assess the cellular potency of the FLT3 inhibitors of Formula I′, FLT3 specific growth inhibition was measured in the leukemic cell line MV4-11 (ATCC Number: CRL-9591). MV4-11 cells are derived from a patient with childhood acute myelomonocytic leukemia with an 11q23 translocation resulting in a MLL gene rearrangement and containing an FLT3-ITD mutation (AML subtype M4)(see Drexler H G. The Leukemia-Lymphoma Cell Line Factsbook. Academic Pres: San Diego, Calif., 2000 and Quentmeier H, Reinhardt J, Zaborski M, Drexler H G. FLT3 mutations in acute myeloid leukemia cell lines. Leukemia. 2003 Jan; 17:120-124.). MV4-11 cells cannot grow and survive without active FLT3ITD.
The IL-3 dependent, murine b-cell lymphoma cell line, Baf3, were used as a control to confirm the selectivity of the FLT3 inhibitors of Formula I′ by measuring non-specific growth inhibition by the FLT3 inhibitors of Formula I′.
To measure proliferation inhibition by test compounds, the luciferase based CellTiterGlo reagent (Promega), which quantifies total cell number based on total cellular ATP concentration, was used. Cells are plated at 10,000 cells per well in 100 ul of in RPMI media containing penn/strep, 10% FBS and 1 ng/ml GM-CSF or 1 ng/ml IL-3 for MV4-11 and Baf3 cells respectively.
Compound dilutions or 0.1% DMSO (vehicle control) are added to cells and the cells are allowed to grow for 72 hours at standard cell growth conditions (37° C., 5% CO2). For activity measurements in MV4-11 cells grown in 50% plasma, cells were plated at 10,000 cells per well in a 1:1 mixture of growth media and human plasma (final volume of 100 μL). To measure total cell growth an equal volume of CellTiterGlo reagent was added to each well, according to the manufacturer's instructions, and luminescence was quantified. Total cell growth was quantified as the difference in luminescent counts (relative light units, RLU) of cell number at Day 0 compared to total cell number at Day 3 (72 hours of growth and/or compound treatment). One hundred percent inhibition of growth is defined as an RLU equivalent to the Day 0 reading. Zero percent inhibition was defined as the RLU signal for the DMSO vehicle control at Day 3 of growth. All data points are an average of triplicate samples. The IC50 for growth inhibition represents the dose of a compound that results in a 50% inhibition of total cell growth at day 3 of the DMSO vehicle control. Inhibition and IC50 data analysis was done with GraphPad Prism using a non-linear regression fit with a multiparamater, sigmoidal dose-response (variable slope) equation.
MV4-11 cells express the FLT3 internal tandem duplication mutation, and thus are entirely dependent upon FLT3 activity for growth. Strong activity against the MV4-11 cells is anticipated to be a desirable quality of the invention. In contrast, the Baf3 cell proliferation is driven by the cytokine IL-3 and thus are used as a non-specific toxicity control for test compounds. All compound examples in the present invention showed <50% inhibition at a 3 uM dose (data is not included), suggesting that the compounds are not cytotoxic and have good selectivity for FLT3.
Cell-Based FLT3 Receptor Elisa
Specific cellular inhibition of FLT ligand-induced wild-type FLT3 phosphorylation was measured in the following manner: Baf3 FLT3 cells overexpressing the FLT3 receptor were obtained from Dr. Michael Heinrich (Oregon Health and Sciences University). The Baf3 FLT3 cell lines were created by stable transfection of parental Baf3 cells (a murine B cell lymphoma line dependent on the cytokine IL-3 for growth) with wild-type FLT3. Cells were selected for their ability to grow in the absence of IL-3 and in the presence of FLT3 ligand. Baf3 cells were maintained in RPMI 1640 with 10% FBS, penn/strep and 10 ng/ml FLT ligand at 37° C., 5% CO2. To measure direct inhibition of the wild-type FLT3 receptor activity and phosphorylation a sandwich ELISA method was developed similar to those developed for other RTKs (see Sadick, M D, Sliwkowski, M X, Nuijens, A, Bald, L, Chiang, N, Lofgren, J A, Wong W L T. Analysis of Heregulin-Induced ErbB2 Phosphorylation with a High-Throughput Kinase Receptor Activation Enzyme-Linked Immunsorbent Assay, Analytical Biochemistry. 1996; 235:207-214 and Baumann C A, Zeng L, Donatelli R R, Maroney A C. Development of a quantitative, high-throughput cell-based enzyme-linked immunosorbent assay for detection of colony-stimulating factor-1 receptor tyrosine kinase inhibitors. J Biochem Biophys Methods. 2004; 60:69-79.). 200 μL of Baf3FLT3 cells (1×106 /mL) were plated in 96 well dishes in RPMI 1640 with 0.5% serum and 0.01 ng/mL IL-3 for 16 hours prior to 1 hour compound or DMSO vehicle incubation. Cells were treated with 100 ng/mL Flt ligand (R&D Systems Cat# 308-FK) for 10 min. at 37° C. Cells were pelleted, washed and lysed in 100 ul lysis buffer (50 mM Hepes, 150 mM NaCl, 10% Glycerol, 1% Triton-X-100, 10 mM NaF, 1 mM EDTA, 1.5 mM MgCl2, 10 mM NaPyrophosphate) supplemented with phosphatase (Sigma Cat# P2850) and protease inhibitors (Sigma Cat #P8340). Lysates were cleared by centrifugation at 1000×g for 5 minutes at 4° C. Cell lysates were transferred to white wall 96 well microtiter (Costar #9018) plates coated with 50 ng/well anti-FLT3 antibody (Santa Cruz Cat# sc-480) and blocked with SeaBlock reagent (Pierce Cat#37527). Lysates were incubated at 4° C. for 2 hours. Plates were washed 3× with 200 ul/well PBS/0.1% Triton-X-100. Plates were then incubated with 1:8000 dilution of HRP-conjugated anti-phosphotyrosine antibody (Clone 4G10, Upstate Biotechnology Cat#16-105) for 1 hour at room temperature. Plates were washed 3× with 200 ul/well PBS/0.1% Triton-X-100. Signal detection with Super Signal Pico reagent (Pierce Cat#37070) was done according to manufacturer's instruction with a Berthold microplate luminometer. All data points are an average of triplicate samples. The total relative light units (RLU) of Flt ligand stimulated FLT3 phosphorylation in the presence of 0.1% DMSO control was defined as 0% inhibition and 100% inhibition was the total RLU of lysate in the basal state. Inhibition and IC50 data analysis was done with GraphPad Prism using a non-linear regression fit with a multiparamater, sigmoidal dose-response (variable slope) equation.
Biological Data for FLT3
activity of representative FLT3 inhibitors of Formula I′ is presented in the charts hereafter. All activities are in μM and have the following uncertainties: FLT3 kinase: ±10%; MV4-11 and Baf3-FLT3: ±20%.
* Except where indicated, compound names were derived using nomenclature rules well known to those skilled in the art, by either standard IUPAC nomenclature references, such as Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F and H, (Pergamon Press, Oxford, 1979, Copyright 1979 IUPAC) and A Guide to IUPAC
1Not determined.
In vivo Evaluation of Oral Anti-tumorEfficacy
The following representative in vivo assay was performed in determining the biological activities of the FLT3 inhibitors of Formula I′ within the scope of the invention. They are given to illustrate the invention in a non-limiting fashion.
The oral anti-tumor efficacy of a subset of the compounds of the invention was evaluated in vivo using a nude mouse MV4-11 human tumor xenograft regression model.
Female athymic nude mice (CD-1, nu/nu, 9-10 weeks old) were obtained from Charles River Laboratories (Wilmington, Mass.) and were maintained according to NIH standards. All mice were group housed (5 mice/cage) under clean-room conditions in sterile micro-isolator cages on a 12-hour light/dark cycle in a room maintained at 21-22° C. and 40-50% humidity. Mice were fed irradiated standard rodent diet and water ad libitum. All animals were housed in a Laboratory Animal Medicine facility that is fully accredited by the American Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). All procedures involving animals were conducted in compliance with the NIH Guide for the Care and Use of Laboratory Animals and all protocols were approved by an Internal Animal Care and Use Committee (IACUC).
The human leukemic MV4-11 cell line was obtained from the American Type Culture Collection (ATCC Number: CRL-9591) and propagated in RPMI medium containing 10% FBS (fetal bovine serum) and 5 ng/mL GM-CSF (R&D Systems). MV4-11 cells are derived from a patient with childhood acute myelomonocytic leukemia with an 11q23 translocation resulting in a MLL gene rearrangement and containing an FLT3-ITD mutation (AML subtype M4)(1,2). MV4-11 cells express constitutively active phosphorylated FLT3 receptor as a result of a naturally occurring FLT3/ITD mutation. Strong anti-tumor activity against MV4-11 tumor growth in the nude mouse tumor xenograft model is anticipated to be a desirable quality of the invention.
In pilot growth studies, the following conditions were identified as permitting MV4-11 cell growth in nude mice as subcutaneous solid tumor xenografts: Immediately prior to injection, cells were washed in PBS and counted, suspended 1:1 in a mixture of PBS:Matrigel (BD Biosciences) and then loaded into pre-chilled 1 cc syringes equipped with 25 gauge needles. Female athymic nude mice weighing no less than 20-21 grams were inoculated subcutaneously in the left inguinal region of the thigh with 5×106 tumor cells in a delivery volume of 0.2 mL. For regression studies, the tumors were allowed to grow to a pre-determined size prior to initiation of dosing. Approximately 3 weeks after tumor cell inoculation, mice bearing subcutaneous tumors ranging in size from 106 to 439 mm3 (60 mice in this range) were randomly assigned to treatment groups such that all treatment groups had similar starting mean tumor volumes of ˜200 mm3. Mice were dosed orally by gavage with vehicle (control group) or compound at various doses twice-daily (b.i.d.) during the week and once-daily (q.d.) on weekends. Dosing was continued for 11 consecutive days, depending on the kinetics of tumor growth and size of tumors in vehicle-treated control mice. If tumors in the control mice reached ˜10% of body weight (˜2.0 grams), the study was to be terminated. The FLT3 inhibitors of Formula I′ were prepared fresh daily as a clear solution (@ 1, 3 and 10 mg/mL) in 20% HPβCD/2% NMP/10 mM Na Phosphate, pH 3-4 (NMP=Pharmasolve, ISP Technologies, Inc.) or other suitable vehicle and administered orally as described above. During the study, tumor growth was measured three times-a-week (M, W, F) using electronic Vernier calipers. Tumor volume (mm3) was calculated using the formula (L×W)2/2, where L=length (mm) and W=width (shortest distance in mm) of the tumor. Body weight was measured three times-a-week and a loss of body weight >10% was used as an indication of lack of compound tolerability. Unacceptable toxicity was defined as body weight loss >20% during the study. Mice were closely examined daily at each dose for overt clinical signs of adverse, drug-related side effects.
On the day of study termination, a final tumor volume and final body weight were obtained on each animal. Mice were euthanized using 100% CO2 and tumors were immediately excised intact and weighed, with final tumor wet weight (grams) serving as a primary efficacy endpoint.
The time course of the inhibitory effects of the FLT3 inhibitors of Formula I′ on the growth of MV4-11 tumors is illustrated in
A similar reduction of final tumor weight was noted at study termination. (See
a: FLT3 inhibitor Compound B (Compound 73 of Formula I′) administered orally by gavage at doses of 10, 30 and 100 mg/kg b.i.d. for 11 consecutive days, produced statistically significant, dose-dependent inhibition of growth of MV4-11 tumors grown subcutaneously in nude mice. On the last day of treatment (Day 11), mean tumor volume was dose-dependently decreased by 44%, 84% (p<0.01) and 94% (p<0.01) at doses of 10, 30 and 100 mg/kg, respectively, compared to the mean tumor volume of the vehicle-treated group. Tumor regression was observed at doses of 30 mg/kg and 100 mg/kg, with statistically significant decreases of 42% and 77%, respectively, versus the starting mean tumor volumes on Day 1. At the lowest dose tested of 10 mg/kg, modest growth delay was observed (44% I vs Control), however this effect did not achieve statistical significance.
a: Following eleven consecutive days of oral dosing, FLT3 inhibitor Compound B (Compound 73 of Formula I′) produced statistically significant, dose-dependent reductions of final tumor weight compared to the mean tumor weight of the vehicle-treated group, with 48%, 85% (p<0.01) and 99% (p<0.01) decreases at 10, 30 and 100 mg/kg doses, respectively. In some mice, at the high dose of Compound B, final tumors had regressed to non-palpable, non-detectable tumors.
b: FLT3 inhibitor Compound C (Compound 74 of Formula I′) administered orally by gavage at doses of 10, 30 and 100 mg/kg b.i.d. for 11 consecutive days, also produced statistically significant, dose-dependent inhibition of growth of MV4-11 tumors grown subcutaneously in nude mice. On the last day of treatment (Day 11), mean tumor volume was dose-dependently decreased by 22%, 54% (p<0.01) and 96% (p<0.01) at doses of 10, 30 and 100 mg/kg, respectively, compared to the mean tumor volume of the vehicle-treated group. Tumor regression was observed at a dose of 100 mg/kg, with a statistically significant decrease of 79% versus the starting mean tumor volume on Day 1. Significant growth delay was observed at a dose of 30 mg/kg (54% I vs Control) and, at the lowest dose tested of 10 mg/kg, some growth delay was observed (22% I vs Control); however this effect did not achieve statistical significance.
b: Following eleven consecutive days of oral dosing, FLT3 inhibitor Compound C (Compound 74 of Formula I′) produced statistically significant, dose-dependent reductions of final tumor weight compared to the mean tumor weight of the vehicle-treated group, with 12%, 43% (p<0.01) and 91% (p<0.01) decreases at 10, 30 and 100 mg/kg doses, respectively. In some mice, at the high dose of Compound C, final tumors had regressed to non-palpable, non-detectable tumors.
Mice were weighed three times each week (M, W, F) during the study and were examined daily at the time of dosing for overt clinical signs of any adverse, drug-related side effects. No overt toxicity was noted for either Compound B or C and no significant adverse effects on body weight were observed during the 11-day treatment period with either Compound B or C at doses up to 200 mg/kg/day. Overall, across all dose groups for both Compound B and C the mean loss of body weight was <3% of initial body weight, indicating that the FLT3 inhibitors of Formula I′ were well-tolerated.
To establish further that FLT3 inhibitors of Formula I′ reached the expected target in tumor tissue, the level of FLT3 phosphorylation in tumor tissue obtained from vehicle- and compound-treated mice was measured. Results for FLT3 inhibitor Compound B (Compound 73 of Formula I′) and FLT3 inhibitor Compound C (Compound 74 of Formula I′) are shown in
Harvested tumors were processed for immunoblot analysis of FLT3 phosphorylation in the following manner: 100 mg of tumor tissue was dounce homogenized in lysis buffer (50 mM Hepes, 150 mM NaCl, 10% Glycerol, 1% Triton-X-100, 10 mM NaF, 1 mM EDTA, 1.5 mM MgCl2, 10 mM NaPyrophosphate) supplemented with phosphatase (Sigma Cat# P2850) and protease inhibitors (Sigma Cat #P8340). Insoluble debris was removed by centrifugation at 1000×g for 5 minutes at 4° C. Cleared lysates (15 mg of total potein at 10 mg/ml in lysis buffer) were incubated with 10 μg of agarose conjugated anti-FLT3 antibody, clone C-20 (Santa Cruz cat # sc-479ac), for 2 hours at 4° C. with gentle agitation. Immunoprecipitated FLT3 from tumor lysates were then washed four times with lysis buffer and separated by SDS-PAGE. The SDS-PAGE gel was transfered to nitrocellulose and immunoblotted with anti-phosphotyrosine antibody (clone-4G10, UBI cat. #05-777), followed by alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Novagen cat. # 401212). Detection of protein was done by measuring the fluorescent product of the alkaline phosphatase reaction with the substrate 9H-(1,3-dichloro-9,9-dimethylacridin -2-one-7-yl) phosphate, diammonium salt (DDAO phosphate) (Molecular Probes cat. # D 6487) using a Molecular Dynamics Typhoon Imaging system (Molecular Dynamics, Sunnyvale, Calif.). Blots were then stripped and reprobed with anti-FLT3 antibody for normalization of phosphorylation signals.
As illustrated in
Other FLT3 Inhibitors
Other FLT3 kinase inhibitors which can be employed in accordance with the present include: AG1295 and AG1296; Lestaurtinib (also known as CEP 701, formerly KT-5555, Kyowa Hakko, licensed to Cephalon); CEP-5214 and CEP-7055 (Cephalon); CHIR-258 (Chiron Corp.); EB-10 and IMC-EB10 (ImClone Systems Inc.); GTP 14564 (Merk Biosciences UK). Midostaurin (also known as PKC 412 Novartis AG); MLN 608 (Millennium USA); MLN-518 (formerly CT53518, COR Therapeutics Inc., licensed to Millennium Pharmaceuticals Inc.); MLN-608 (Millennium Pharmaceuticals Inc.); SU-11248 (Pfizer USA); SU-11657 (Pfizer USA); SU-5416 and SU 5614; THRX-165724 (Theravance Inc.); AMI-10706 (Theravance Inc.); VX-528 and VX-680 (Vertex Pharmaceuticals USA, licensed to Novartis (Switzerland), Merck & Co USA); and XL 999 (Exelixis USA).
Formulation
The FLT3 kinase inhibitors and the farnesyl transferase inhibitors of the present invention can be prepared and formulated by methods known in the art, and as described herein. In addition to the preparation and formulations described herein, the farnesyltransferase inhibitors of the present invention can be prepared and formulated into pharmaceutical compositions by methods described in the art, such as the publications cited herein. For example, for the farnesyltransferase inhibitors of formulae (I), (II) and (III) suitable examples can be found in WO-97/21701. The farnesyltransferase inhibitors of formulae (IV), (V), and (VI) can be prepared and formulated using methods described in WO 97/16443, farnesyltransferase inhibitors of formulae (VII) and (VIII) according to methods described in WO 98/40383 and WO 98/49157 and farnesyltransferase inhibitors of formula (IX) according to methods described in WO 00/39082 respectively. Tipifarnib (Zarnestra™, also known as R115777) and its less active enantiomer can be synthesized by methods described in WO 97/21701. Tipifarnib is expected to be available commercially as ZARNESTRA™ in the near future, and is currently available upon request (by contract) from Johnson & Johnson Pharmaceutical Research & Development, L.L.C. (Titusville, N.J.).
Where separate pharmaceutical compositions are utilized, the FLT3 kinase inhibitor or farnesyl transferase inhibitor, as the active ingredient, is intimately admixed with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques, which carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g., oral or parenteral such as intramuscular. A unitary pharmaceutical composition having both the FLT3 kinase inhibitor and farnesyl transferase inhibitor as active ingredients can be similarly prepared.
In preparing either of the individual compositions, or the unitary composition, in oral dosage form, any of the usual pharmaceutical media may be employed. Thus, for liquid oral preparations, such as for example, suspensions, elixirs and solutions, suitable carriers and additives include water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like; for solid oral preparations such as, for example, powders, capsules, caplets, gelcaps and tablets, suitable carriers and additives include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar coated or enteric coated by standard techniques. For parenterals, the carrier will usually comprise sterile water, though other ingredients, for example, for purposes such as aiding solubility or for preservation, may be included. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents and the like may be employed. In preparation for slow release, a slow release carrier, typically a polymeric carrier, and a compound of the present invention are first dissolved or dispersed in an organic solvent. The obtained organic solution is then added into an aqueous solution to obtain an oil-in-water-type emulsion. Preferably, the aqueous solution includes surface-active agent(s). Subsequently, the organic solvent is evaporated from the oil-in-water-type emulsion to obtain a colloidal suspension of particles containing the slow release carrier and the compound of the present invention.
The pharmaceutical compositions herein will contain, per dosage unit, e.g., tablet, capsule, powder, injection, teaspoonful and the like, an amount of the active ingredient necessary to deliver an effective dose as described above. The pharmaceutical compositions herein will contain, per unit dosage unit, e.g., tablet, capsule, powder, injection, suppository, teaspoonful and the like, from about 0.01 mg to 200 mg/kg of body weight per day. Preferably, the range is from about 0.03 to about 100 mg/kg of body weight per day, most preferably, from about 0.05 to about 10 mg/kg of body weight per day. The compounds may be administered on a regimen of 1 to 5 times per day. The dosages, however, may be varied depending upon the requirement of the patients, the severity of the condition being treated and the compound being employed. The use of either daily administration or post-periodic dosing may be employed.
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 composition 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. 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 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. 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 material can be used for such enteric layers or coatings, such materials including a number of polymeric acids with such materials as shellac, acetyl alcohol and cellulose acetate.
The liquid forms in which the FLT3 kinase inhibitor and the farnesyl transferase inhibitor individually (or both in the case of a unitary composition) 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 liquid forms in suitably flavored suspending or dispersing agents may also include the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.
Advantageously, the FLT3 kinase inhibitor and the farnesyl transferase inhibitor may be administered in a single daily dose (individually or in a unitary composition), or the total daily dosage may be administered in divided doses of two, three or four times daily. Furthermore, compounds for the present invention (individually or in a unitary composition) can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
For instance, for oral administration in the form of a tablet or capsule, the active drug component (the FLT3 kinase inhibitor and the farnesyl transferase inhibitor individually, or together in the case of a unitary composition) can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders; lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture.
Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
The daily dosage of the products of the present invention may be varied over a wide range from 1 to 5000 mg per adult human per day. For oral administration, the compositions are preferably provided in the form of tablets containing, 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250 and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. An effective amount of the drug is ordinarily supplied at a dosage level of from about 0.01 mg/kg to about 200 mg/kg of body weight per day. Particularly, the range is from about 0.03 to about 15 mg/kg of body weight per day, and more particularly, from about 0.05 to about 10 mg/kg of body weight per day. The FLT3 kinase inhibitor and the farnesyl transferase inhibitor individually, or together in the case of a unitary composition, may be administered on a regimen up to four or more times per day, preferably of 1 to 2 times per day.
Optimal dosages to be administered may be readily determined by those skilled in the art, and will vary with the particular compound used, the mode of administration, the strength of the preparation, the mode of administration, and the advancement of the disease condition. In addition, factors associated with the particular patient being treated, including patient age, weight, diet and time of administration, will result in the need to adjust dosages.
The FLT3 kinase inhibitor and the farnesyl transferase inhibitor of the present invention can also be administered (individually or in a unitary composition) in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of lipids, including but not limited to amphipathic lipids such as phosphatidylcholines, sphingomyelins, phosphatidylethanolamines, phophatidylcholines, cardiolipins, phosphatidylserines, phosphatidylglycerols, phosphatidic acids, phosphatidylinositols, diacyl trimethylammonium propanes, diacyl dimethylammonium propanes, and stearylamine, neutral lipids such as triglycerides, and combinations thereof. They may either contain cholesterol or may be cholesterol-free.
The FLT3 kinase inhibitor and the farnesyl transferase inhibitor of the present invention can also be administered (individually or in a unitary composition) locally. Any delivery device, such as intravascular drug delivery catheters, wires, pharmacological stents and endoluminal paving, may be utilized. The delivery system for such a device may comprise a local infusion catheter that delivers the compound at a rate controlled by the administor.
The present invention provides a drug delivery device comprising an intraluminal medical device, preferably a stent, and a therapeutic dosage of the FLT3 kinase inhibitor and the farnesyl transferase inhibitor of the invention. Alternatively, the present invention provides for individual administration of a therapeutic dosage of one or both of the FLT3 kinase inhibitor and the farnesyl transferase inhibitor of the invention by means of a drug delivery device comprising an intraluminal medical device, preferably a stent
The term “stent” refers to any device capable of being delivered by a catheter. A stent is routinely used to prevent vascular closure due to physical anomalies such as unwanted inward growth of vascular tissue due to surgical trauma. It often has a tubular, expanding lattice-type structure appropriate to be left inside the lumen of a duct to relieve an obstruction. The stent has a lumen wall-contacting surface and a lumen-exposed surface. The lumen-wall contacting surface is the outside surface of the tube and the lumen-exposed surface is the inner surface of the tube. The stent can be polymeric, metallic or polymeric and metallic, and it can optionally be biodegradable.
The FLT3 kinase inhibitor and farnesyl transferase inhibitor of the present invention (individually or in a unitary composition) can be incorporated into or affixed to the stent in a number of ways and in utilizing any number of biocompatible materials. In one exemplary embodiment, the compound is directly incorporated into a polymeric matrix, such as the polymer polypyrrole, and subsequently coated onto the outer surface of the stent. The compound elutes from the matrix by diffusion through the polymer. Stents and methods for coating drugs on stents are discussed in detail in the art. In another exemplary embodiment, the stent is first coated with as a base layer comprising a solution of the compound, ethylene-co-vinylacetate, and polybutylmethacrylate. Then, the stent is further coated with an outer layer comprising only polybutylmethacrylate. The outlayer acts as a diffusion barrier to prevent the compound from eluting too quickly and entering the surrounding tissues. The thickness of the outer layer or topcoat determines the rate at which the compound elutes from the matrix. Stents and methods for coating are discussed in detail in WIPO publication WO9632907, U.S. Publication No. 2002/0016625 and references disclosed therein.
To better understand and illustrate the invention and its exemplary embodiments and advantages, reference is made to the following experimental section.
Inhibition of AML cell growth with the combination of an FTI and a FLT3 inhibitor was tested. Two FTIs, Tipifarnib and FTI Compound 176 (“FTI-176), and eight novel FLT3 inhibitors: Compounds A, B, C, D, E, F G and H were used to inhibit the growth of FLT3-dependent cell types in vitro (see
The cell lines that were tested included those that are dependent on FLT3ITD mutant activity for growth (MV4-11 and Baf3-FLT3ITD), FLT3wt activity for growth (Baf3FLT3) and those that grow independent of FLT3 activity (THP-1). MV4-11 (ATCC Number: CRL-9591) cells are derived from a patient with childhood acute myelomonocytic leukemia with an 11q23 translocation resulting in a MLL gene rearrangement and containing an FLT3-ITD mutation (AML subtype M4) (see Drexler H G. The Leukemia-Lymphoma Cell Line Factsbook. Academic Pres: San Diego, Calif., 2000 and Quentmeier H, Reinhardt J, Zaborski M, Drexler H G. FLT3 mutations in acute myeloid leukemia cell lines. Leukemia. 2003 January; 17:120-124. ). Baf3-FLT3 and Baf3-FLT3ITD cell lines were obtained from Dr. Michael Henrich and the Oregon Health Sciences University. The Baf3 FLT3 cell lines were created by stable transfection of parental Baf3 cells (a murine B cell lymphoma line dependent on the cytokine IL-3 for growth) with either wild-type FLT3 or FLT3 containing the ITD insertion in the juxatamembrane domain of the receptor resulting in its constitutive activation. Cells were selected for their ability to grow in the absence of IL-3 and in either the presence of FLT3 ligand (Baf3-FLT3) or independent of any growth factor (Baf3-ITD). THP-1 (ATCC Number: TIB-202) cells were isolated from a childhood AML patient with an N-Ras mutation and no FLT3 abnormality. Although the cells express a functional FLT3 receptor, THP-1 cells are not dependent on FLT3 activity for viability and growth (data not shown).
Dose responses for the individual compounds alone were determined for each cell line using a standard 72-hour cell proliferation assay (see
The effect of a single (sub- IC50) dose of the FLT3 inhibitor Compound A on Tipifarnibpotency was then examined. Each cell line was simultaneously treated with one dose of the FLT3 inhibitor Compound A and varying doses of Tipifarnib and the proliferation of the cells was evaluated in the standard 72-hour cell proliferation protocol. The IC50 for Tipifarnib was then calculated according to the procedure described in the Biological Activity section hereafter (see
The FLT3 inhibitor Compound A significantly increased the potency of the FTI Tipifarnib for the inhibition of AML (MV4-11) and FLT3 dependent (Baf3-ITD and Baf3-FLT3) cell proliferation. With a single sub-IC50 dose of FLT3 inhibitor Compound A in (a) MV4-11 (50 nM); (b) Baf3-ITD (50 nM) and (c) Baf3-FLT3 (100 nM) cells, Tipifarnib increased in potency by more than 3-fold in each cell line tested. This is indicative of significant synergy.
Next, single dose combinations of the FTI Tipifarnib and the FLT3 inhbitor Compound A were evaluated in the MV4-11, Baf3-ITD and Baf3-FLT3 cell lines. This single dose combination scenario more closely represents dosing strategies for chemotherapeutic combinations that are used in the clinic. With this method cells are simultaneously treated with a single sub- IC50 of dose of each compound or a combination of compounds and inhibition of proliferation was monitored. Using this method it is observed that combinations of a sub- IC50 dose of the FTI Tipifarnib and the FLT3 inhibitor Compound A are beyond additive in inhibiting the growth of the AML cell line MV4-11 and other FLT3-dependent cells (see
Additionally, single dose combinations of a FLT3 inhibitor and a FTI were examined to determine if this activity was compound specific or mechanism based. A single sub- IC50 of dose of either FLT3 inhibitor Compound B or D with Tipifarnib was tested for its inhibition of MV4-11 proliferation. It is observed, similar to combinations of Tipifarnib and FLT3 inhibitor Compound A, that the combinations of either FLT3 inhibitor Compound B or D with Tipifarnib inhibits the proliferation of FLT3-dependent MV4-11 cells with greater that additive efficacy. This suggests that the combination of any FLT3 inhibitor and FTI will synergistically inhibit the proliferation of FLT3-dependent AML cells. This observation is novel and non-obvious to those skilled in the art. Synergy was also observed with the combination of either FLT3 inhihbitor Compound B or D and cytarabine.
To statistically evaluate the synergy of a FLT3 inhibitor and an FTI in FLT3 dependent cell lines, dosing combinations were evaluated by the method of Chou and Talalay. See Chou T C, Talalay P. (1984) “Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors.” Adv Enzyme Regul. 22:27-55. Using this method inhibitors are added simultaneously to cells in a ratio of the IC50 dose of each compound alone. The data is collected and subject to isobolar analysis of fixed ratio dose combinations as described by Chou and Talalay. This analysis is used to generate a combination index or CI. The CI value of 1 corresponds to compounds that behave additively; CI values <0.9 are considered synergistic and CI values of >1.1 are considered antagonistic. Using this method, multiple FTI and FLT3 combinations were evaluated. For each experimental combination IC50s were calculated for each individual compound (see
Data points for combinations that are antagonistic fall to the right, or over, the isobolar line for a given dose effect (CI>1.1).
Additionally,
To further expand the combination studies, each of the FLT3 inhibitors shown to demonstrate synergy with Tipifarnib were also tested in combination with another farnesyl transferase inhibitor, FTI-176. Tables 1-3 summarize the results of all the combinations tested in the three FLT3-dependent cell lines described above. The combination indecies for each combination are contained within Tables 1-3.
Synergy of combination dosing is observed with all FTI and FLT3 combinations tested in all FLT3 dependent cell lines used. The combination of an FTI and FLT3 inhibitor reduces the individual compounds antiproliferative effect by an average of 3-4fold. It can be concluded that the synergy observed for combinations of a FLT3 inhibitor and an FTI is a mechanism based phenomena and not related to the specific chemical structures of individual FTIs or FLT3 inhibitors. Accordingly, synergistic growth inhibition would be observed with any combination of a FLT3 inhibitor and Tipifarnib or any other FTI.
The ultimate goal of treatment for FLT3 related disorders is to kill the disease causative cells and to cause regression of disease. To examine if the FTI/FLT3 inhibitor combination is synergistic for cell death of FLT3 dependent disease causative cells, particularly AML, ALL and MDS cells, the combination of Tipifarnib and the FLT3 inhibitor Compound A was tested for its ability to induce an increase in fluorescent labeled Annexin V staining in MV4-11 cells. Annexin V binding to phosphotidyl serine that has translocated from the inner leaflet of the plasma membrane to the outer leaflet of the plasma membrane and is a well established way to measure apoptosis of cells. See van Engeland M., L. J. Nieland ,et al. (1998) “Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure.” Cytometry. 31(1):1-9.
Tipifarnib and FLT3 inhibitor Compound A were incubated with MV4-11 cells alone or in a fixed ratio (4:1 based on the calculated EC50 for each agent alone) for 48 hours in standard cell culture conditions. After the compound incubations, treated cells were harvested and stained with Annexin V-PE and 7-AAD using the Guava Nexin apoptosis kit according to the protocol in the Biological Activity Measurements section hereafter. Annexin V staining peaks at 60% because cells late in apoptosis begin to fall apart and are considered debris. However, EC50s can be calculated from this data because of its consistent sigmoidal kinetics. From the data summarized in
To confirm that the combination of a FLT3 inhibitor and an FTI synergistically activates apoptosis of FLT3 dependent cells, the combination of several FLT3 inhibitors and the FTI Tipifarnib was tested for its ability to induce the activity of caspase 3/7 in MV4-11 cells. Caspase activation, a critical step in the final execution of the apoptotic cellular death process, can be induced by a variety of cellular stimuli including growth factor withdrawal or growth factor receptor inhibition See Hengartner, M O. (2000) “The biochemistry of apoptosis.” Nature 407:770-76 and Nunez G, Benedict M A, Hu Y, Inohara N. (1998) “Caspases: the proteases of the apoptotic pathway.” Oncogene 17:3237-45. Cellular caspase activation can be monitored using a synthetic caspase 3/7 substrate that is cleaved to release a substrate for the enzyme luciferase, that may convert the substrate to a luminescent product. See Lovborg H, Gullbo J, Larsson R. (2005) “Screening for apoptosis-classical and emerging techniques.” Anticancer Drugs 16:593-9. Caspase activation was monitored using the Caspase Glo technology from Promega (Madison, Wis.) according to the protocol in the Biological Activity Measurement section hereafter. Individual EC50 determinations were done to establish dose ratios for combination analysis of synergy.
It is well established that phosphorylation of the FLT3 receptor and downstream kinases such as MAP kinase are required for proliferative effects of FLT3 receptor. See Scheijen, B. and J. D. Griffin (2002) “Tyrosine kinase oncogenes in normal hematopoiesis and hematological disease.” Oncogene 21(21): 3314-33. We postulate that the molecular mechanism of the synergy observed with a FLT3 inhibitor and an FTI is related to the compound induced decrease of FLT3 receptor signaling required for AML cell proliferation and survival. To test this we looked at phosphorylation state of both the FLT3-ITD receptor and a downstream target of FLT3 receptor activity, MAP kinase (erk1/2) phosphorylation in MV4-11 cells, using commercially available reagents according to the protocol detailed in the Biological Activity Measurements section hereafter. MV4-11 cells were treated with indicated concentrations of FLT3 inhibitor Compound A alone or in combination with Tipifarnib for 48 hours under standard cell growth conditions. For analysis of FLT3 phosphorylation, cells were harvested and FLT3 was immunoprecipitated and separated by SDS-PAGE. For analysis of MAP kinase (erk1/2) phosphorylation, cells were harvested, subjected to lysis, separated by SDS-Page and transferred to nitrocellulose for immunoblot analysis. For quantitative analysis of FLT3 phosphorylation, immunoblots were probed with phosphotyrosine antibody and the phophoFLT3 signal was quantified using Molecular Dynamics Typhoon Image Analysis. The immunoblots were then stripped and reprobed to quantify the total FLT3 protein signal. This ratio of phosphorylation to total protein signal was used to calculate the approximate IC50 of the compound dose responses. For quantitative analysis of MAP kinase (ERK1/2) phosphorylation, immunoblots were probed with a phosphospecific ERK1/2 antibody and the phophoERK1/2 signal was quantified using Molecular Dynamics Typhoon Image Analysis. The immunoblots were then stripped and reprobed to quantify the total ERK1/2 protein signal. This ratio of phosphorylation to total protein signal was used to calculate the approximate IC50 of the compound dose responses. IC50 values were calculated using GraphPad Prism software. The result of this work is summarized in
It is observed that the combination of Tipifarnib and FLT3 inhibitor Compound A increases the potency of FLT3 inhibitor Compound A two to three fold for both inhibition of FLT3 phosphorylation and MAP kinase phosphorylation. This is consistent with the increase in potency of the compounds anti-proliferative effects. The effect of FLT3 phosphorylation that was observed with the FTI/FLT3 inhihbitor combination has not been reported previously. The mechanism for this effect on FLT3 phosphorylation is unknown but would be predicted to occur for any FTI/FLT3 inhibitor combination based on the experimental data collected for proliferation inhibition described above.
In Vitro Biological Activity Measurements
Reagents and Antibodies. Cell Titerglo proliferation reagent was obtained from Promega Corporation. Proteases inhibitor cocktails and phosphatase inhibitor cocktails II were purchased from Sigma (St. Louis, Mo.). The GuavaNexin apoptosis reagent was purchased from Guava technologies (Hayward, Calif.). Superblock buffer and SuperSignal Pico reagent were purchased from Pierce Biotechnology (Rockford, Ill.). Fluorescence polarization tyrosine kinase kit (Green) was obtained from Invitrogen. Mouse anti-phosphotyrosine (4G10) antibody was purchased from Upstate Biotechnology, Inc (Charlottesville, Va.). Anti-human FLT3 (rabbit IgG) was purchased from Santa Cruz biotechnology (Santa Cruz, Calif.). Anti-phospho Map kinase and total p42/44 Map kinase antibodies were purchased form Cell Signaling Technologies (Beverly, Mass.) Alkaline phosphatase-conjugated goat-anti-rabbit IgG, and goat-anti-mouse IgG antibody purchased from Novagen (San Diego, Calif.). DDAO phosphate was purchased from Molecular Probes (Eugene, Oreg.). All tissue culture reagents were purchase from BioWhitaker (Walkersville, Md.).
Cell lines. THP-1 (Ras mutated, FLT3 wild type) and human MV4-11 (expressing constitutively FLT3-Internal tandem duplication or ITD mutant isolated from an AML patient with a t15;17 translocation) AML cells)(see Drexler H G. The Leukemia-Lymphoma Cell Line Factsbook. Academic Pres: San Diego, Calif., 2000 and Quentmeier H, Reinhardt J, Zaborski M, Drexler H G. FLT3 mutations in acute myeloid leukemia cell lines. Leukemia. 2003 January;17:120-124.) were obtained from ATCC (Rockville, Md.). The IL-3 dependent murine B-cell progenitor cell line Baf3 expressing human wild-type FLT3 (Baf3-FLT3) and ITD-mutated FLT3 (Baf3-ITD) were obtained from Dr. Michael Heinrich (Oregon Health Sciences University). Cells were maintained in RPMI media containing penn/strep, 10% FBS alone (THP-1, Baf3-ITD) and 2 ng/ml GM-CSF (MV4-11) or 1 ng/ml FLT ligand (Baf3-FLT3). MV4-11, Baf3-ITD and Baf3-FLT3 cells are all absolutely dependent on FLT3 activity for growth. GM-CSF enhances the activity of the FLT3-ITD receptor in the MV4-11 cells.
Cell proliferation assay for MV4-11, Baf3-ITD, Baf3-FLT3 and THP-1 cells. To measure proliferation inhibition by test compounds the luciferase based CellTiterGlo reagent (Promega) was used. Cells are plated at 10,000 cells per well in 100 ul of in RPMI media containing penn/strep, 10% FBS alone (THP-1, Baf3-ITD) and 0.2 ng/ml GM-CSF (MV4-11) or 10 ng/ml FLT ligand (Baf3-FLT3). Compound dilutions or 0.1% DMSO (vehicle control) are added to cells and the cells are allowed to grow for 72 hours at standard cell growth conditions (37° C., 5% CO2). In combination experiments test agents were added simultaneously to the cells. Total cell growth is quantified as the difference in luminescent counts (relative light units, RLU) of cell number at Day 0 compared to total cell number at Day 3 (72 hours of growth and/or compound treatment). One hundred percent inhibition of growth is defined as an RLU equivalent to the Day 0 reading. Zero percent inhibition is defined as the RLU signal for the DMSO vehicle control at Day 3 of growth. All data points are an average of triplicate samples. The IC50 for growth inhibition represents the dose of a compound that results in a 50% inhibition of total cell growth at Day 3 of the DMSO vehicle control. IC50 data analysis was done with GraphPad Prism using a non-linear regression fit with a multiparameter, sigmoidal dose-response (variable slope) equation.
Immunoprecipitation and Quantitative Immunoblot Analysis. MV4-11 cells were grown in DMEM supplemented with 10% fetal bovine serum, 2 ng/ml GM-CSF and kept between 1×105 and 1×106 cells/ml. For western blot analysis of Map Kinase phosphorylation 1×106 MV4-11 cells per condition were used. For immunoprecipitation experiments examining FLT3-ITD phosphorylation, 1×107 cells were used for each experimental condition. After compound treatment, MV4-11 cells were washed once with cold 1×PBS and lysed with HNTG lysis buffer (50 mM Hepes, 150 mM NaCl, 10% Glycerol, 1% Triton-X-100, 10 mM NaF, 1 mM EDTA, 1.5 mM MgCl2, 10 mM NaPyrophosphate)+4 ul/ml Protease Inhibitor Cocktail (Sigma cat.#P8340)+4ul/ml Phosphatase Inhibitor Cocktail (Sigma Cat#P2850). Nuclei and debris were removed from cell lysates by centrifugation (5000 rpm for 5 min. at 4° C.). Cell lysates for immunoprecipitation were cleared with agarose-Protein A/G for 30 minutes at 4° C. and immunoprecipitated using the 3 ug of FLT3 antibody for 1 hours at 4° C. Immune complexes were then incubated with agarose-Protein A/G for 1 hour at 4° C. Protein A/G immunoprecipitates were washed three times in 1.0 ml of HNTG lysis buffer. Immunoprecipitates and cell lysates (40 ug total protein) were resolved on a 10% SDS-PAGE gel, and the proteins were transferred to nitrocellulose membrane. For anti-phosphotyrosine immunoblot analysis, membranes were blocked with SuperBlock (Pierce) and blotted for 2 hours with anti-phosphotyrosine (clone 4G10, Upstate Biotechnologies) followed by alkaline phosphatase-conjugated goat anti-mouse antibody. For anti-phosphoMAP kinase western blotting, membranes were blocked Super block for 1 hour and blotted overnight in primary antibody, followed by an incubation with an AP conjugated goat-anti rabbit secondary antibody. Detection of protein was done by measuring the fluorescent product of the alkaline phosphatase reaction with the substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate, diammonium salt (DDAO phosphate) (Molecular Probes) using a Molecular Dynamics Typhoon Imaging system (Molecular Dynamics, Sunyvale, Calif.). Blots were stripped and reprobed with anti-FLT3 antibody for normalization of phosphorylation signals. Quantitation of DDAO phosphate signal and IC50 determinations were done with Molecular Dynamics ImageQuant and GraphPad Prism software.
Annexin V Staining. To examine the apoptosis of the leukemic MV4-11 cell line, cells were treated with Tipifarnib and/or FLT3 inhibitor Compound A, and Annexin V binding to phosphotidylserine on the outer leaflet of the plasma membrane of apoptotic cells was monitored using the GuavaNexin assay reagent and the Guava personal flow cytometry system (Guava Technologies; Hayward, Calif.). MV4-11 cells were plated at 200,000 cells per ml in tissue culture media containing varying concentrations of Tipifarnib and/or FLT3 inhibitor Compound A and incubated for 48 hours at 37° C., 5% CO2. Cells were harvested by centrifugation at 400×g for 10 minutes at 4° C. Cells were then washed with 1×PBS and resuspended in 1× Nexin buffer at 1×106 cells/ml. 5 μl of Annexin V-PE ad 5 μl of 7-AAD was added to 40 μl of cell suspension and incubated on ice for 20 minutes protected from light. 450 ml of cold 1× Nexin buffer was added to each sample and the cells were then acquired on the Guava cytometer according to the manufacturer's instructions. All annexin positive cells were considered apoptotic and percent Annexin positive cells was calculated.
Caspase 3/7 Activation Assay. MV4-11 cells were grown in RPMI media containing pen/strep, 10% FBS and 1 ng/mL GM-CSF. Cells were maintained between 2×105 cells/mL and 8×105 cells/mL feeding/splitting every 2-3 days. Cells were centrifuged and resuspend at 2×105 cells/mL RPMI media containing Penn/Strep, 10% FBS and 0.1 ng/mL GM-CSF. MV4-11 cells were plated at 20,000 cells per well in 100 μL of in RPMI media containing penn/strep, 10% FBS alone and 0.1 ng/mL GM-CSF (Corning Costar Cat # 3610) in the presence of various concentrations of test compounds or DMSO. In combination experiments test agents were added simultaneously to the cells. Cells were incubated for 24 hours at 37° C., 5% CO2. After 24-hour incubation, caspase activity was measured with the Promega CaspaseGlo reagent (Cat# G8090) according to the manufacture's instructions. Briefly, CaspaseGlo substrate is diluted with 10 mL Caspase Glo buffer. One volume of diluted Caspase Glo reagent was added to one volume of tissue culture media and mixed for two minutes on rotating orbital shaker. Following incubation at room temperature for 60 minutes, light emission was measured on a Berthold luminometer with the 1 second program. Baseline caspase activity was defined as an RLU equivalent to DMSO vehicle (0.1% DMSO) treated cells. EC50 data analysis was completed with GraphPad Prism using a non-linear regression fit with a multiparameter, sigmoidal dose-response (variable slope) equation.
Combination Index Analysis. To determine growth inhibition synergy of a FTI and FLT3 inhibitor combination based on the method of Chou and Talalay (Chou and Talalay. See Chou T C, Talalay P. (1984) “Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors.” Adv Enzyme Regul. 22:27-55. ), fixed ratio combination dosing with isobolar statistical analysis was performed. Test agents were combined at a fixed ratio of the individual IC50 for proliferation for each cell line and dosed at varying concentrations including 9, 3, 1, 1/3, 1/9 times the determined IC50 dose. To measure proliferation inhibition by test combinations the luciferase based CellTiterGlo reagent (Promega) was used. Cells are plated at 10,000 cells per well in 100 ul of in RPMI media containing penn/strep, 10% FBS alone (THP-1, Baf3-ITD) and 0.1ng/ml GM-CSF (MV4-11) or 100 ng/ml FLT ligand (Baf3-FLT3). Total cell growth is quantified as the difference in luminescent counts (relative light units, RLU) of cell number at Day 0 compared to total cell number at Day 3 (72 hours of growth and/or compound treatment). All data points are an average of triplicate samples. One hundred percent inhibition of growth is defined as an RLU equivalent to the Day 0 reading. Zero percent inhibition is defined as the RLU signal for the DMSO vehicle control at Day 3 of growth. Inhibition data was analyzed using Calcsyn (BioSoft, Ferguson, Mo.) and the combination index (C.I.) calculated. C.I. values <0.9 are considered synergistic.
In vivo Combination Studies
The effect of combination treatment of the FLT3 Inhibitor FLT3 inhibitors of Formula I′ and Tipifarnib (Zarnestra™) on the growth of MV-4-11 human AML tumor xenografts in nude mice was tested using FLT3 inhibitor Compound B. The in vivo study was designed to extend the in vitro observations to evaluate the potential for a synergistic anti-tumor effect of FLT3 inhibitor Compound B administered orally together with Tipifarnib to nude mice bearing established MV-4-11 tumor xenografts.
MV-4-11 tumor-bearing nude mice were prepared as described above, in the aforementioned in vivo evaluation of the oral anti-tumor efficacy of FLT3 inhibitor Compound B.
Nude mice with MV-4-11 tumors were randomized to five treatment groups of 15 mice each with mean tumor size was equivalent in each treatment group. Tumor volume (mm3) was calculated using the formula (L×W)2/2, where L=length (mm) and W=width (shortest distance in mm) of the tumor. The starting mean tumor volume for each treatment group was approximately 250 mm3.
Mice were dosed orally twice-daily (bid) during the week and once-daily (qd) on weekends with either Vehicle (20% HPβCD/2% NMP10 mM Na Phosphate, pH 3-4 (NMP=Pharmasolve, ISP Technologies, Inc.), a sub-efficacious dose of FLT3 inhibitor Compound B (10 mg/kg), an-efficacious dose of FLT3 inhibitor Compound B (20 mg/kg) and Tipifarnib (50 mg/kg) alone or in combination with each dose of FLT3 inhibitor Compound B. Dosing was continued for nine consecutive days. Tumor growth was measured three times during the study using electronic Vernier calipers. Body weight was measured three times during the study and a loss of body weight >10% was used as an indication of lack of compound tolerability.
The time course of the effect of treatment with FLT3 inhibitor Compound B and Tipifarnib alone and in combination on the growth of MV-4-11 tumors is illustrated in
Again as shown in
No overt toxicity was noted and no significant adverse effects on body weight were observed during the 9-day treatment period with either agent alone or in combination. In summary, combination treatment with FLT3 inhibitor Compound B and Tipifarnib produced significantly greater inhibition of tumor growth compared to either FLT3 inhibitor Compound B or Tipifarnib administered alone.
To further the observation that any FLT3 inhibitor and FTI combination will act synergistically both in vitro and in vivo to kill FLT3-dependent AML cell growth, the effect of combination treatment of the FLT3 Inhibitor FLT3 inhibitors of Formula I′ and Tipifarnib (Zarnestra™) on the growth of MV-4-11 human AML tumor xenografts in nude mice was tested using FLT3 inhibitor Compound D. The in vivo study was designed to extend the in vitro observations to evaluate the potential for a synergistic anti-tumor effect of FLT3 inhibitor Compound D administered orally together with Tipifarnib to nude mice bearing established MV-4-11 tumor xenografts.
The oral anti-tumor efficacy of FLT3 inhibitor Compound D of the present invention was evaluated in vivo using a nude mouse MV4-11 human tumor xenograft regression model in athymic nude mice Using the method described in the above section “In vivo Evaluation of Oral Anti-tumor Efficacy.”
Female athymic nude mice weighing no less than 20-21 grams were inoculated subcutaneously in the left inguinal region of the thigh with 5×106 tumor cells in a delivery volume of 0.2 mL. For regression studies, the tumors were allowed to grow to a pre-determined size prior to initiation of dosing. Approximately 3 weeks after tumor cell inoculation, mice bearing subcutaneous tumors ranging in size from 100 to 586 mm3 (60 mice in this range; mean of 288±133 mm3 (SD) were randomly assigned to treatment groups such that all treatment groups had statistically similar starting mean tumor volumes (mm3). Mice were dosed orally by gavage with vehicle (control group) or compound at various doses twice-daily (b.i.d.) during the week and once-daily (qd) on weekends. Dosing was continued for 11 consecutive days, depending on the kinetics of tumor growth and size of tumors in vehicle-treated control mice. If tumors in the control mice reached ˜10% of body weight (˜2.0 grams), the study was to be terminated. FLT3 inhibitor Compound D was prepared fresh daily as a clear solution (@ 1, 5 and 10 mg/mL) in 20% HPβCD/D5W, pH 3-4 or other suitable vehicle and administered orally as described above. During the study, tumor growth was measured three times-a-week (M, W, F) using electronic Vernier calipers. Tumor volume (mm3) was calculated using the formula (L×W)2/2, where L=length (mm) and W=width (shortest distance in mm) of the tumor. Body weight was measured three times-a-week and a loss of body weight >10% was used as an indication of lack of compound tolerability. Unacceptable toxicity was defined as body weight loss >20% during the study. Mice were closely examined daily at each dose for overt clinical signs of adverse, drug-related side effects.
On the day of study termination (Day 12), a final tumor volume and final body weight were obtained on each animal. Mice were euthanized using 100% CO2 and tumors were immediately excised intact and weighed, with final tumor wet weight (grams) serving as a primary efficacy endpoint.
The time course of the inhibitory effects of FLT3 inhibitor Compound D of the present invention on the growth of MV4-11 tumors is illustrated in
As seen in
FLT3 inhibitor Compound D of the present invention produced virtually complete regression of tumor mass as indicated by no measurable remant tumor at study termination. (See
Following eleven consecutive days of oral dosing, FLT3 inhibitor Compound D of the present invention produced dose-dependent reductions of final tumor weight compared to the mean tumor weight of the vehicle-treated group, with complete regression of tumor mass noted at the 50 mg/kg dose. (See
Mice were weighed three times each week (M, W, F) during the study and were examined daily at the time of dosing for overt clinical signs of any adverse, drug-related side effects. No overt toxicity was noted for FLT3 inhibitor Compound D of the present invention and no significant adverse effects on body weight were observed during the 11-day treatment period at doses up to 200 mg/kg/day (See
To establish further that FLT3 inhibitor Compound D of the present invention reached the expected target in tumor tissue, the level of FLT3 phosphorylation in tumor tissue obtained from vehicle- and compound-treated mice was measured. Results for FLT3 inhibitor Compound D of the present invention are shown in
Harvested tumors were frozen and processed for immunoblot analysis of FLT3 phosphorylation in the following manner: 200 mg of tumor tissue was dounce homogenized in lysis buffer (50 mM Hepes, 150 mM NaCl, 10% Glycerol, 1% Triton -X-100, 10 mM NaF, 1 mM EDTA, 1.5 mM MgCl2, 10 mM NaPyrophosphate) supplemented with phosphatase (Sigma Cat# P2850) and protease inhibitors (Sigma Cat #P8340). Insoluble debris was removed by centrifugation at 1000×g for 5 minutes at 4° C. Cleared lysates (15mg of total potein at 10 mg/ml in lysis buffer) were incubated with 10 μg of agarose conjugated anti-FLT3 antibody, clone C-20 (Santa Cruz cat # sc-479ac), for 2 hours at 4° C. with gentle agitation.
Immunoprecipitated FLT3 from tumor lysates were then washed four times with lysis buffer and separated by SDS-PAGE. The SDS-PAGE gel was transfered to nitrocellulose and immunoblotted with anti-phosphotyrosine antibody (clone-4G 10, UBI cat. #05-777), followed by alkaline phosphatase-conjugated goat anti-mouse secondary antibody (Novagen cat. # 401212). Detection of protein was done by measuring the fluorescent product of the alkaline phosphatase reaction with the substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate, diammonium salt (DDAO phosphate) (Molecular Probes cat. # D 6487) using a Molecular Dynamics Typhoon Imaging system (Molecular Dynamics, Sunyvale, Calif.). Blots were then stripped and reprobed with anti-FLT3 antibody for normalization of phosphorylation signals.
As illustrated in
To demonstrate in vivo synergy of the combination of FLT3 inhibitor Compound D and Tipifarnib in MV-4-11 xenograft model, tumor-bearing nude mice were prepared as described above, in the aforementioned In vivo Evaluation of Oral Anti-tumorEfficacy section.
Nude mice with MV-4-11 tumors were randomized to four treatment groups of 10 mice each with mean tumor size was equivalent in each treatment group. Tumor volume (mm3) was calculated using the formula (L×W)2/2, where L=length (mm) and W=width (shortest distance in mm) of the tumor. The starting mean tumor volume for each treatment group was approximately 250 mm3.
Mice were dosed orally twice-daily (bid) during the week and once-daily (qd) on weekends with either Vehicle (20% HPβ-CD, pH 3-4) or sub-efficacious doses of FLT3 inhibitor Compound D (25 mg/kg) or Tipifarnib (50 mg/kg) alone or in combination. Dosing was continued for sixteen consecutive days. Tumor growth was measured three times-a-week (Monday, Wednesday, Friday) using electronic Vernier calipers. Body weight was measured three times-a-week and a loss of body weight >10% was used as an indication of lack of compound tolerability.
The time course of the effect of treatment with FLT3 inhibitor Compound D and Tipifarnib alone and in combination on the growth of MV-4-11 tumors is illustrated in
As shown in
No overt toxicity was noted and no significant adverse effects on body weight were observed during the 16-day treatment period with either agent alone or in combination. Plasma and tumor samples were collected two hours after the last dose of compounds for determination of drug levels. In summary, combination treatment with FLT3 inhibitor Compound D and Tipifarnib produced significantly greater inhibition of tumor growth compared to either FLT3 inhibitor Compound D or Tipifarnib administered alone.
Herein we provide significant evidence that the combination of an FTI and a FLT3 inhibitor synergistically inhibits the growth of and induces the death of FLT3-dependent cells in vitro and in vivo (such as AML cells derived from patients with FLT3-ITD mutations). In vitro studies, in multiple FLT3-dependent cell lines, demonstrated synergistic inhibition of AML cell proliferation with the FTI/FLT3 inhibitor combination by both the combination index method of Chou and Talalay and the median effect method using a combination of single sub-optimal doses of each compound. Additionally, the combination of an FTI and a FLT3 inhibitor induced dramatic cell death in FLT3-dependent AML cells. This effect on apoptotsis induction was significantly greater than either agent alone. This synergistic effect of an FTI/FLT3 inhibitor combination was observed for multiple, structurally distinct FLT3 inhibitors and two different FTIs. Accordingly, this synergistic inhibition of proliferation and induction of apoptosis would occur for any FLT3 inhibitor/FTI combination. Interestingly, the combination of the FTI Tipifarnib with a FLT3 inhibitor significantly increases the potency of FLT3 inhibitor mediated decrease in FLT3 receptor signaling. Furthermore, the synergy observed using in vitro methods was recapitulated in an in vivo tumor model using FLT3-dependent AML cells (MV4-11) with the combination of the FTI Tipifarnib and two chemically distinct FLT3 inhibitors (FLT3 inhibitor Compounds B and D). Accordingly, this effect would be seen for any FLT3 inhibitor/FTI combination. To our knowledge, this is the first time that synergistic AML cell killing has been observed with the combination of an FTI and a FLT3 inhibitor. Additionally, the synergies observed in the combination were not obvious to those skilled in the art based on previous data. The observed synergy is likely related to FTIs known inhibition small GTPase (Ras and Rho) and NfkB driven proliferation and survival and the FLT3 inhibitors' ability to decrease proliferation and survival signaling by the FLT3 receptor. Additionally, the FTI/FLT3 inhibitor combination had significant effects on the activity of the FLT3 receptor itself. Although the mechanism for this is currently unknown, it is likely to have a significant role in both the inhibition of cell proliferation and activation of cell death observed with the FLT3 inhibitor/FTI combination. In sum, these studies represent a novel treatment paradigm for FLT3 disorders, particularly hematological malignancies expressing wild-type or mutant FLT3 and the basis for the design of clinical trials to test FTI and FLT3 inhibitor combinations for the treatment of FLT3 disorders, particularly AML, ALL and MDS.
While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations and/or modifications as come within the scope of the following claims and their equivalents.
This application claims priority to U.S. Provisional Application for Patent No. 60/690,070, filed Jun. 10, 2005, the entire disclosure of which is hereby incorporated in its entirely.
| Number | Date | Country | |
|---|---|---|---|
| 60690070 | Jun 2005 | US |
