U.S. patent application number 17/521255 was filed with the patent office on 2022-06-30 for pyrazolopyrimidine compounds.
This patent application is currently assigned to UNIVERSITY HEALTH NETWORK. The applicant listed for this patent is UNIVERSITY HEALTH NETWORK. Invention is credited to Radoslaw Laufer, Sze-Wan Li, Yong Liu, Grace Ng, Narendra Kumar B. Patel, Heinz W. Pauls.
Application Number | 20220204511 17/521255 |
Document ID | / |
Family ID | |
Filed Date | 2022-06-30 |
United States Patent
Application |
20220204511 |
Kind Code |
A1 |
Laufer; Radoslaw ; et
al. |
June 30, 2022 |
PYRAZOLOPYRIMIDINE COMPOUNDS
Abstract
The present teachings provide a compound represented by the
following structural formula: ##STR00001## or a pharmaceutically
acceptable salt thereof. Also described are pharmaceutical
compositions and methods of use thereof.
Inventors: |
Laufer; Radoslaw; (Oakville,
CA) ; Ng; Grace; (Markham, CA) ; Li;
Sze-Wan; (Toronto, CA) ; Pauls; Heinz W.;
(Oakville, CA) ; Liu; Yong; (Oakville, CA)
; Patel; Narendra Kumar B.; (Brampton, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY HEALTH NETWORK |
Toronto |
|
CA |
|
|
Assignee: |
UNIVERSITY HEALTH NETWORK
Toronto
CA
|
Appl. No.: |
17/521255 |
Filed: |
November 8, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16880192 |
May 21, 2020 |
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17521255 |
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16225970 |
Dec 19, 2018 |
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16880192 |
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15598402 |
May 18, 2017 |
10167289 |
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16225970 |
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14926977 |
Oct 29, 2015 |
9657025 |
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15598402 |
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PCT/CA2014/051091 |
Nov 14, 2014 |
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14926977 |
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International
Class: |
C07D 487/04 20060101
C07D487/04; C07D 519/00 20060101 C07D519/00 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 15, 2013 |
CA |
PCT/CA2013/000957 |
Claims
1. A compound represented by the following structural formula:
##STR00034## or a pharmaceutically acceptable salt thereof,
wherein: R.sup.1 is --NH--CH.sub.2--Cy, Cy is C.sub.3-C.sub.4
cycloalkyl optionally substituted with one or two groups selected
from alkyl and hydroxyl; R.sup.2 is
--NH--(C.sub.2-C.sub.6)hydroxyalkyl optionally substituted with
cyclopropyl or isopropyl; or --NH--(C.sub.3-C.sub.6)cycloalkyl
optionally substituted with hydroxyl or
(C.sub.1-C.sub.2)hydroxylalkyl; R.sup.4 is selected from hydrogen,
halogen, and (C.sub.1-C.sub.3)alkyl; and R.sup.d is
cyclopropyl.
2-6. (canceled)
7. A pharmaceutical composition comprising a compound of claim 1 or
a pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier or diluent.
8. A method for treating cancer, the method comprising
administering to a subject in need thereof an effective amount of
the compound of claim 1 or a pharmaceutically acceptable salt
thereof.
9. The method of claim 8, wherein the cancer is pancreatic cancer,
prostate cancer, lung cancer, melanoma, breast cancer, colon
cancer, or ovarian cancer.
Description
RELATED APPLICATIONS
[0001] This application is continuation application of U.S.
application Ser. No. 16/880,192, filed on May 21, 2020, which is a
continuation of U.S. application Ser. No. 16/225,970, filed on Dec.
19, 2018, which is a continuation application of U.S. application
Ser. No. 15/598,402, filed on May 18, 2017 and now U.S. Pat. No.
10,167,289, which is a divisional application of U.S. application
Ser. No. 14/926,977, filed on Oct. 29, 2015 and now U.S. Pat. No.
9,657,025, which, in turn, is a continuation of International
Application No. PCT/CA2014/051091, filed Nov. 14, 2014, which
claims benefit of International Application No. PCT/CA2013/000957,
filed on Nov. 15, 2013. The entire contents of each of the
aforementioned applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Protein kinases have been the subject of extensive study in
the search for new therapeutic agents in various diseases, for
example, cancer. Protein kinases are known to mediate intracellular
signal transduction by effecting a phosphoryl transfer from a
nucleoside triphosphate to a protein acceptor that is involved in a
signaling pathway. There are a number of kinases and pathways
through which extracellular and other stimuli cause a variety of
cellular responses to occur inside the cell.
[0003] Human TTK protein kinase (TTK), also known as tyrosine
threonine kinase, dual specificity protein kinase TTK, Monopolar
Spindle 1 (Mps1) and Phosphotyrosine-Picked Threonine Kinase (PYT),
is a conserved multispecific kinase that is capable of
phosphorylating serine, threonine and tyrosine residues when
expressed in E. coli (Mills et al., J. Biol. Chem. 22(5):
16000-16006 (1992)). TTK mRNA is not expressed in the majority of
physiologically normal tissues in human (Id). TTK mRNA is expressed
in some rapidly proliferating tissues, such as testis and thymus,
as well as in some tumors (for example, TTK mRNA was not expressed
in renal cell carcinoma, was expressed in 50% of breast cancer
samples, was expressed in testicular tumors and ovarian cancer
samples) (Id). TTK is expressed in some cancer cell lines and
tumors relative to normal counterparts (Id.; see also WO 02/068444
A1).
[0004] Therefore, agents which inhibit a protein kinase, in
particular TTK, have the potential to treat cancer. There is a need
for additional agents which can act as protein kinase inhibitors,
in particular TTK inhibitors.
[0005] In addition, cancer recurrence, drug resistance or
metastasis is one of the major challenges in cancer therapies.
Cancer patients who responded favorably to the initial anti-cancer
therapy often develop drug resistance and secondary tumors that
lead to the relapse of the disease. Recent research evidences
suggest that the capability of a tumor to grow and propagate is
dependent on a small subset of cells within the tumor. These cells
are termed tumor-initiating cells (TICs) or cancer stem cells. It
is thought that the TICs are responsible for drug resistance,
cancer relapse and metastasis. Compounds that can inhibit the
growth and survival of these tumor-initiating cells can be used to
treat cancer, metastasis or prevent recurrence of cancer.
Therefore, a need exists for new compounds that can inhibit the
growth and survival of tumor-imitating cells.
SUMMARY OF THE INVENTION
[0006] Applicants have now discovered that certain
pyrazolopyrimidine compounds are potent kinase inhibitors, such as
TTK protein kinase (see Example B). Applicants have also discovered
that these compounds have potent anticancer activity against breast
cancer, colon cancer, and ovarian cancer cells in cell culture
study (see Examples C-D). Based on these discoveries,
pyrazolopyrimidine compounds, pharmaceutical compositions thereof,
and methods of treating cancer with the pyrazolopyrimidine
compounds are disclosed herein.
[0007] The present teachings are directed, at least in part, to a
compound represented by the following structural formula:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein:
[0008] R.sup.1 is --NH--CH.sub.2--Cy,
[0009] Cy is C.sub.3-C.sub.4 cycloalkyl optionally substituted with
one or two groups selected from alkyl and hydroxyl;
[0010] R.sup.2 is --O-pyridinyl;
--NH--(C.sub.2-C.sub.6)hydroxyalkyl optionally substituted with
cyclopropyl or isopropyl; or --NH--(C.sub.3-C.sub.6)cycloalkyl
optionally substituted with hydroxyl or
(C.sub.1-C.sub.2)hydroxylalkyl;
[0011] R.sup.4 is selected from hydrogen, halogen, and
(C.sub.1-C.sub.3)alkyl; and
[0012] R.sup.d is cyclopropyl. Preferably, R.sup.4 is chlorine or
methyl.
[0013] In one embodiment, the present teachings include a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier or diluent and a compound represented by structural formula
(I) described above or a pharmaceutically acceptable salt
thereof.
[0014] In another embodiment, the present teachings provide a
method of treating a subject having cancer comprising administering
to the subject an effective amount of a compound of structural
formula (I) or a pharmaceutically acceptable salt thereof.
[0015] Another embodiment of the present teachings provides a
method of inhibiting TTK activity in a subject in need of
inhibition of TTK activity, comprising administering to the subject
an effective amount of a compound represented by Structural Formula
(I) or a pharmaceutically acceptable salt thereof.
[0016] Another embodiment of the present teachings includes the use
of a compound represented by Structural Formula (I) or a
pharmaceutically acceptable salt thereof in therapy. In some
embodiments, the therapy is for treating a subject with cancer.
Alternatively, the therapy is for inhibiting TTK activity in a
subject in need of inhibition of TTK activity.
[0017] Another embodiment of the present teachings includes the use
of a compound represented by Structural Formula (I) or a
pharmaceutically acceptable salt thereof for the manufacture of a
medicament for treating a subject with cancer.
[0018] Another embodiment of the present teachings includes the use
of a compound represented by Structural Formulas (I) or a
pharmaceutically acceptable salt thereof for the manufacture of a
medicament for inhibiting TTK activity in a subject in need of
inhibition of TTK activity.
DETAILED DESCRIPTION OF THE INVENTION
[0019] In one embodiment, the present teachings are directed to a
compound represented by Structural Formula (I) or a
pharmaceutically acceptable salt thereof. The invention also
includes the compounds depicted by structure and/or described by
name in the Exemplification, and includes both the neutral forms
and as well as pharmaceutically acceptable salts thereof.
Treatments with and/or uses of these compounds (including neutral
forms and pharmaceutically acceptable salts thereof) as described
herein are also included in the invention. Specific examples of
compounds of the invention are shown below:
##STR00003##
or a pharmaceutically acceptable salt thereof.
##STR00004##
or a pharmaceutically acceptable salt thereof;
##STR00005##
or a pharmaceutically acceptable salt thereof; and
##STR00006##
or a pharmaceutically acceptable salt thereof.
[0020] The term "alkyl" used alone or as part of a larger moiety,
such as "hydroxyalkyl", and the like, means saturated aliphatic
straight-chain or branched monovalent hydrocarbon radical. Unless
otherwise specified, an alkyl group typically has 1-6 carbon atoms,
i.e., (C.sub.1-C.sub.6)alkyl. As used herein, a
"(C.sub.1-C.sub.6)alkyl" group is means a radical having from 1 to
6 carbon atoms in a linear or branched arrangement.
[0021] "Cycloalkyl" means a saturated aliphatic cyclic hydrocarbon
radical optionally containing one or more double bonds. It can be
monocyclic, bicyclic (e.g., a bridged bicyclic ring), polycyclic
(e.g., tricyclic), or fused. For example, monocyclic
(C.sub.3-C.sub.7)cycloalkyl means a radical having from 3-7 carbon
atoms arranged in a monocyclic ring. A (C.sub.3-C.sub.7)cycloalkyl
includes, but is not limited to, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and cycloheptyl.
[0022] Certain of the compounds described herein may exist in
various stereoisomeric or tautomeric forms. Stereoisomers are
compounds which differ only in their spatial arrangement. When a
disclosed compound is named or depicted by structure without
indicating stereochemistry, it is understood that the name or
structure encompasses all possible stereoisomers, tautomers,
geometric isomers or a combination thereof.
[0023] When a geometric isomer is depicted by name or structure, it
is to be understood that the geometric isomeric purity of the named
or depicted geometric isomer is at least 60%, 70%, 80%, 90%, 99% or
99.9% pure by weight. Geometric isomeric purity is determined by
dividing the weight of the named or depicted geometric isomer in
the mixture by the total weight of all of the geomeric isomers in
the mixture.
[0024] Racemic mixture means 50% of one enantiomer and 50% of is
corresponding enantiomer. The present teachings encompass all
enantiomerically-pure, enantiomerically-enriched,
diastereomerically pure, diastereomerically enriched, and racemic
mixtures, and diastereomeric mixtures of the compounds described
herein.
[0025] Enantiomeric and diastereomeric mixtures can be resolved
into their component enantiomers or stereoisomers by well known
methods, such as chiral-phase gas chromatography, chiral-phase high
performance liquid chromatography, crystallizing the compound as a
chiral salt complex, or crystallizing the compound in a chiral
solvent. Enantiomers and diastereomers can also be obtained from
diastereomerically- or enantiomerically-pure intermediates,
reagents, and catalysts by well known asymmetric synthetic
methods.
[0026] When a compound is designated by a name or structure that
indicates a single enantiomer, unless indicated otherwise, the
compound is at least 60%, 70%, 80%, 90%, 99% or 99.9% optically
pure (also referred to as "enantiomerically pure"). Optical purity
is the weight in the mixture of the named or depicted enantiomer
divided by the total weight in the mixture of both enantiomers.
[0027] When the stereochemistry of a disclosed compound is named or
depicted by structure, and the named or depicted structure
encompasses more than one stereoisomer (e.g., as in a
diastereomeric pair), it is to be understood that one of the
encompassed stereoisomers or any mixture of the encompassed
stereoisomers are included. It is to be further understood that the
stereoisomeric purity of the named or depicted stereoisomers at
least 60%, 70%, 80%, 90%, 99% or 99.9% by weight. The
stereoisomeric purity in this case is determined by dividing the
total weight in the mixture of the stereoisomers encompassed by the
name or structure by the total weight in the mixture of all of the
stereoisomers.
[0028] Included in the present teachings are pharmaceutically
acceptable salts of the compounds disclosed herein. The disclosed
compounds have basic amine groups and therefore can form
pharmaceutically acceptable salts with pharmaceutically acceptable
acid(s). Suitable pharmaceutically acceptable acid addition salts
of the compounds described herein include salts of inorganic acids
(such as hydrochloric acid, hydrobromic, phosphoric, nitric, and
sulfuric acids) and of organic acids (such as, acetic acid,
benzenesulfonic, benzoic, methanesulfonic, and p-toluenesulfonic
acids). Compounds of the present teachings with acidic groups such
as carboxylic acids can form pharmaceutically acceptable salts with
pharmaceutically acceptable base(s). Suitable pharmaceutically
acceptable basic salts include ammonium salts, alkali metal salts
(such as sodium and potassium salts) and alkaline earth metal salts
(such as magnesium and calcium salts). Compounds with a quaternary
ammonium group also contain a counteranion such as chloride,
bromide, iodide, acetate, perchlorate and the like. Other examples
of such salts include hydrochlorides, hydrobromides, sulfates,
methanesulfonates, nitrates, benzoates and salts with amino acids
such as glutamic acid.
[0029] Compounds described herein can inhibit various kinases,
including the TTK. Thus, generally, compounds described herein are
useful in the treatment of diseases or conditions associated with
such kinases. In some embodiments, compounds described herein can
inhibit TTK.
[0030] In one embodiment, the compounds described herein are TTK
inhibitors, and are useful for treating diseases, such as cancer,
associated with such kinase(s).
[0031] Another aspect of the present teachings relates to a method
of treating a subject with cancer comprising administering to the
subject an effective amount of a compound described herein. In one
embodiment, the compounds described herein inhibit the growth of a
tumor. For example, the compounds described herein inhibit the
growth of a tumor that overexpresses TTK.
[0032] Cancers that can be treated (including reduction in the
likelihood of recurrence) by the methods of the present teachings
include lung cancer, breast cancer, colon cancer, brain cancer,
neuroblastoma, prostate cancer, melanoma, glioblastoma multiform,
ovarian cancer, lymphoma, leukemia, melanoma, sarcoma,
paraneoplasia, osteosarcoma, germinoma, glioma and mesothelioma. In
one embodiment, the cancer is selected from leukemia, acute myeloid
leukemia, chronic myelogenous leukemia, breast cancer, brain
cancer, colon cancer, colorectal cancer, head and neck cancer,
hepatocellular carcinoma, lung adenocarcinoma, metastatic melanoma,
pancreatic cancer, prostate cancer, ovarian cancer and renal
cancer. In one embodiment, the cancer is lung cancer, colon cancer,
brain cancer, neuroblastoma, prostate cancer, melanoma,
glioblastoma mutiform or ovarian cancer. In another embodiment, the
cancer is pancreatic cancer, prostate cancer, lung cancer,
melanoma, breast cancer, colon cancer, or ovarian cancer. In yet
another embodiment, the cancer is breast cancer, colon cancer and
ovarian cancer. In yet another embodiment, the cancer is a breast
cancer. In yet another embodiment, the cancer is a basal sub-type
breast cancer or a luminal B sub-type breast cancer. In yet another
embodiment, the cancer is a basal sub-type breast cancer that
overexpresses TTK. In yet another embodiment, the basal sub-type
breast cancer is ER (estrogen receptor), HER2 and PR (progesterone
receptor) negative breast cancer. In yet another embodiment, the
cancer is a soft tissue cancer. A "soft tissue cancer" is an
art-recognized term that encompasses tumors derived from any soft
tissue of the body. Such soft tissue connects, supports, or
surrounds various structures and organs of the body, including, but
not limited to, smooth muscle, skeletal muscle, tendons, fibrous
tissues, fatty tissue, blood and lymph vessels, perivascular
tissue, nerves, mesenchymal cells and synovial tissues. Thus, soft
tissue cancers can be of fat tissue, muscle tissue, nerve tissue,
joint tissue, blood vessels, lymph vessels, and fibrous tissues.
Soft tissue cancers can be benign or malignant. Generally,
malignant soft tissue cancers are referred to as sarcomas, or soft
tissue sarcomas. There are many types of soft tissue tumors,
including lipoma, lipoblastoma, hibernoma, liposarcoma, leiomyoma,
leiomyosarcoma, rhabdomyoma, rhabdomyosarcoma, neurofibroma,
schwannoma (neurilemoma), neuroma, malignant schwannoma,
neurofibrosarcoma, neurogenic sarcoma, nodular tenosynovitis,
synovial sarcoma, hemangioma, glomus tumor, hemangiopericytoma,
hemangioendothelioma, angiosarcoma, Kaposi sarcoma, lymphangioma,
fibroma, elastofibroma, superficial fibromatosis, fibrous
histiocytoma, fibrosarcoma, fibromatosis, dermatofibrosarcoma
protuberans (DFSP), malignant fibrous histiocytoma (MFH), myxoma,
granular cell tumor, malignant mesenchymomas, alveolar soft-part
sarcoma, epithelioid sarcoma, clear cell sarcoma, and desmoplastic
small cell tumor. In a particular embodiment, the soft tissue
cancer is a sarcoma selected from the group consisting of a
fibrosarcoma, a gastrointestinal sarcoma, a leiomyosarcoma, a
dedifferentiated liposarcoma, a pleomorphic liposarcoma, a
malignant fibrous histiocytoma, a round cell sarcoma, and a
synovial sarcoma.
[0033] In some embodiments, the present teachings provide methods
of inhibiting the growth of tumor-initiating cells or reducing the
likelihood of recurrence of a cancer in a subject who is undergoing
an anti-cancer therapy. The method comprises the steps of:
[0034] a) assessing the subject to determine whether the cancer is
in remission; and
[0035] b) if the cancer is in remission; then administering to the
subject an effective amount of a TTK inhibitor (e.g., a compound
represented by Structural Formula (I). If the cancer is not in
remission, the method optionally further comprises the step of
continuing the anti-cancer therapy until the cancer goes into
remission and then the step b) of administering an effective amount
of a TTK inhibitor (e.g., a compound represented by Structural
Formula (I).
[0036] As used herein, the term "tumor-initiating cells" or "TICs"
refer to cells present within some tumors that possess the ability
to self-renew and proliferate. These cells are sometimes called
cancer stem cells (CSCs) and may be observed to share certain
characteristics with normal stem cells, including a stem cell-like
phenotype and function. In some embodiments, TICs are characterized
by their ability to form tumors after xenotransplantation in
immunodeficient mice.
[0037] In some embodiments, the present teachings provide methods
of inhibiting the growth of tumor-initiating cells or reducing the
likelihood of recurrence of a cancer in a subject whose cancer is
in remission comprising administering to the subject an effective
amount of a TTK inhibitor (e.g, a compound represented by
Structural Formula (I)).
[0038] In some embodiments, e.g., where the subject is being
treated to reduce the likelihood of recurrence of a cancer, the
subject has already been treated with an anti-cancer therapy.
Alternatively, the subject has already been treated with an
anti-cancer therapy and the subject is in remission.
[0039] In some embodiments, the present teachings provide methods
of treating a subject with a cancer comprising administering to the
subject an effective amount of a compound represented by Structural
Formula (I) in combination with an effective anti-cancer therapy.
In one embodiment, the cancer is a metastatic cancer. A "metastatic
cancer" is a cancer that has spread from its primary site to other
parts of the body.
[0040] In another embodiment, the present teachings are directed to
a method of treating a subject with a drug-resistant cancer. A
"drug-resistant cancer" is a cancer that is not responsive to one,
two, three, four, five or more drugs that are typically used for
the treatment of the cancer. In one embodiment, the drug-resistant
cancer is mediated by the growth of tumor-initiating cells.
[0041] Suitable methods known in the art can be used for assessing
a subject to determine whether the cancer is in remission. For
example, the size of the tumor and/or tumor markers, usually
proteins associated with tumors, can be monitored to determine the
state of the cancer. Size of the tumor can be monitored with
imaging devices, such as X-ray, MRI, CAT scans, ultrasound,
mammography, PET and the like or via biopsy.
[0042] For methods described herein, e.g., coadministration
methods, the anti-cancer therapy are selected from the group
consisting of surgery, radiation therapy, immunotherapy, endocrine
therapy, gene therapy and administration of an anti-cancer agent.
Alternatively, the anti-cancer therapy is radiation therapy. In
another alternative, the anti-cancer therapy is immunotherapy. In
another alternative, the anti-cancer therapy is administration of
an anti-cancer agent. In yet another alternative, the anti-cancer
therapy is surgery.
[0043] Radiation therapy is the use of radiation to kill, destroy
or treat the cancers. Exemplary radiation therapy includes, but is
not limited to, gamma-radiation, neutron beam radiotherapy,
electron beam radiotherapy, proton therapy, brachytherapy, and
radioiosotope therapy (i.e., systemic radioactive isotopes
therapy),
[0044] An endocrine therapy is a treatment that adds, blocks or
removes hormones. For example, chemotherapeutic agents that can
block the production or activity of estrogen have been used for
treating breast cancer. In addition, hormonal stimulation of the
immune system has been used to treat specific cancers, such as
renal cell carcinoma and melanoma. In one embodiment, the endocrine
therapy comprises administration of natural hormones, synthetic
hormones or other synthetic molecules that may block or increase
the production of the body's natural hormones. In another
embodiment, the endocrine therapy includes removal of a gland that
makes a certain hormone.
[0045] As use herein, a gene therapy is the insertion of genes into
a subject's cell and biological tissues to treat diseases, such as
cancer. Exemplary gene therapy includes, but is not limited to, a
germ line gene therapy and a somatic gene therapy.
[0046] Immunotherapy (also called biological response modifier
therapy, biologic therapy, biotherapy, immune therapy, or
biological therapy) is treatment that uses parts of the immune
system to fight disease. Immunotherapy can help the immune system
recognize cancer cells, or enhance a response against cancer cells.
Immunotherapies include active and passive immunotherapies. Active
immunotherapies stimulate the body's own immune system while
passive immunotherapies generally use immune system components
created outside of the body.
[0047] Examples of active immunotherapies include, but are not
limited to vaccines including cancer vaccines, tumor cell vaccines
(autologous or allogeneic), dendritic cell vaccines, antigen
vaccines, anti-idiotype vaccines, DNA vaccines, viral vaccines, or
Tumor-Infiltrating Lymphocyte (TIL) Vaccine with Interleukin-2
(IL-2) or Lymphokine-Activated Killer (LAK) Cell Therapy.
[0048] Examples of passive immunotherapies include but are not
limited to monoclonal antibodies and targeted therapies containing
toxins. Monoclonal antibodies include naked antibodies and
conjugated monoclonal antibodies (also called tagged, labeled, or
loaded antibodies). Naked monoclonal antibodies do not have a drug
or radioactive material attached whereas conjugated monoclonal
antibodies are joined to, for example, a chemotherapy drug
(chemolabeled), a radioactive particle (radiolabeled), or a toxin
(immunotoxin). Examples of these naked monoclonal antibody drugs
include, but are not limited to Rituximab (Rituxan), an antibody
against the CD20 antigen used to treat, for example, B cell
non-Hodgkin lymphoma; Trastuzumab (Herceptin), an antibody against
the HER2 protein used to treat, for example, advanced breast
cancer; Alemtuzumab (Campath), an antibody against the CD52 antigen
used to treat, for example, B cell chronic lymphocytic leukemia
(B-CLL); Cetuximab (Erbitux), an antibody against the EGFR protein
used, for example, in combination with irinotecan to treat, for
example, advanced colorectal cancer and head and neck cancers; and
Bevacizumab (Avastin) which is an antiangiogenesis therapy that
works against the VEGF protein and is used, for example, in
combination with chemotherapy to treat, for example, metastatic
colorectal cancer. Examples of the conjugated monoclonal antibodies
include, but are not limited to Radiolabeled antibody Ibritumomab
tiuxetan (Zevalin) which delivers radioactivity directly to
cancerous B lymphocytes and is used to treat, for example, B cell
non-Hodgkin lymphoma; radiolabeled antibody Tositumomab (Bexxar)
which is used to treat, for example, certain types of non-Hodgkin
lymphoma; and immunotoxin Gemtuzumab ozogamicin (Mylotarg) which
contains calicheamicin and is used to treat, for example, acute
myelogenous leukemia (AML). BL22 is a conjugated monoclonal
antibody for treating, for example, hairy cell leukemia,
immunotoxins for treating, for example, leukemias, lymphomas, and
brain tumors, and radiolabeled antibodies such as OncoScint for
example, for colorectal and ovarian cancers and ProstaScint for
example, for prostate cancers.
[0049] Further examples of therapeutic antibodies that can be used
include, but are not limited to, HERCEPTIN.RTM. (Trastuzumab)
(Genentech, CA) which is a humanized anti-HER2 monoclonal antibody
for the treatment of patients with metastatic breast cancer;
REOPRO.RTM. (abciximab) (Centocor) which is an anti-glycoprotein
IIb/IIIa receptor on the platelets for the prevention of clot
formation; ZENAPAX.RTM. (daclizumab) (Roche Pharmaceuticals,
Switzerland) which is an immunosuppressive, humanized anti-CD25
monoclonal antibody for the prevention of acute renal allograft
rejection; PANOREX.TM. which is a murine anti-17-IA cell surface
antigen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2 which is a
murine anti-idiotype (GD3 epitope) IgG antibody (ImClone System);
IMC-C225 which is a chimeric anti-EGFR IgG antibody (ImClone
System); VITAXIN.TM. which is a humanized anti-aVD3 integrin
antibody (Applied Molecular Evolution/MedImmune); Campath 1H/LDP-03
which is a humanized anti CD52 IgGI antibody (Leukosite); Smart
M195 which is a humanized anti-CD33 IgG antibody (Protein Design
Lab/Kanebo); RITUXAN.TM. which is a chimeric anti-CD20 IgGI
antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE.TM.
which is a humanized anti-CD22 IgG antibody (Immunomedics);
LYMPHOCIDE.TM. Y-90 (Immunomedics); Lymphoscan (Tc-99m-labeled;
radioimaging; Immunomedics); Nuvion (against CD3; Protein Design
Labs); CM3 is a humanized anti-ICAM3 antibody (ICOS Pharm);
IDEC-114 is a primatied anti-CD80 antibody (IDEC Pharm/Mitsubishi);
ZEVALIN.TM. is a radiolabelled murine anti-CD20 antibody
(IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody
(IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC);
IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMART
anti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is
a humanized anti-complement factor 5 (C5) antibody (Alexion Pharm);
D2E7 is a humanized anti-TNF-.alpha. antibody (CAT/BASF); CDP870 is
a humanized anti-TNF-.alpha. Fab fragment (Celltech); IDEC-151 is a
primatized anti-CD4 IgGI antibody (IDEC Pharm/SmithKline Beecham);
MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab);
CD20-sreptdavidin (+biotin-yttrium 90; NeoRx); CDP571 is a
humanized anti-TNF-.alpha. IgG4 antibody (Celltech); LDP-02 is a
humanized anti-.alpha.4.beta.7 antibody (LeukoSite/Genentech);
OrthoClone OKT4A is a humanized anti-CD4 IgG antibody (Ortho
Biotech); ANTOVA.TM. is a humanized anti-CD40L IgG antibody
(Biogen); ANTEGREN.TM. is a humanized anti-VLA-4 IgG antibody
(Elan); and CAT-152 is a human anti-TGF-.beta..sub.2 antibody
(Cambridge Ab Tech).
[0050] Immunotherapies that can be used in the present teachings
include adjuvant immunotherapies. Examples include cytokines, such
as granulocyte-macrophage colony-stimulating factor (GM-CSF),
granulocyte-colony stimulating factor (G-CSF), macrophage
inflammatory protein (MIP)-1-alpha, interleukins (including IL-1,
IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, and IL-27),
tumor necrosis factors (including TNF-alpha), and interferons
(including IFN-alpha, IFN-beta, and IFN-gamma); aluminum hydroxide
(alum); Bacille Calmette-Gudrin (BCG); Keyhole limpet hemocyanin
(KLH); Incomplete Freund's adjuvant (IFA); QS-21; DETOX;
Levamisole; and Dinitrophenyl (DNP), and combinations thereof, such
as, for example, combinations of, interleukins, for example, IL-2
with other cytokines, such as IFN-alpha.
[0051] Alternatively, the anti-cancer therapy described herein
includes administration of an anti-cancer agent. An "anti-cancer
agent" is a compound, which when administered in an effective
amount to a subject with cancer, can achieve, partially or
substantially, one or more of the following: arresting the growth,
reducing the extent of a cancer (e.g., reducing size of a tumor),
inhibiting the growth rate of a cancer, and ameliorating or
improving a clinical symptom or indicator associated with a cancer
(such as tissue or serum components) or increasing longevity of the
subject.
[0052] The anti-cancer agent suitable for use in the methods
described herein includes any anti-cancer agents that have been
approved for the treatment of cancer. In one embodiment, the
anti-cancer agent includes, but is not limited to, a targeted
antibody, an angiogenesis inhibitor, an alkylating agent, an
antimetabolite, a vinca alkaloid, a taxane, a podophyllotoxin, a
topoisomerase inhibitor, a hormonal antineoplastic agent and other
antineoplastic agents.
[0053] Examples of alkylating agents useful in the methods of the
present teachings include but are not limited to, nitrogen mustards
(e.g., mechloroethamine, cyclophosphamide, chlorambucil, melphalan,
etc.), ethylenimine and methylmelamines (e.g., hexamethlymelamine,
thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,
carmustine, lomusitne, semustine, streptozocin, etc.), or triazenes
(decarbazine, etc.). Examples of antimetabolites useful in the
methods of the present teachings include but are not limited to
folic acid analog (e.g., methotrexate), or pyrimidine analogs
(e.g., fluorouracil, floxouridine, Cytarabine), purine analogs
(e.g., mercaptopurine, thioguanine, pentostatin). Examples of plant
alkaloids and terpenoids or derivatives thereof include, but are
not limited to, vinca alkaloids (e.g., vincristine, vinblastine,
vinorelbine, vindesine), podophyllotoxin, and taxanes (e.g.,
paclitaxel, docetaxel). Examples of a topoisomerase inhibitor
include, but are not limited to, irinotecan, topotecan, amsacrine,
etoposide, etoposide phosphate and teniposide. Examples of
antineoplastic agents include, but are not limited to, actinomycin,
anthracyclines (e.g., doxorubicin, daunorubicin, valrubicin,
idarubicin, epirubicin), bleomycin, plicamycin and mitomycin.
[0054] In one embodiment, the anti-cancer agents that can be used
in the present teachings include Adriamycin, Dactinomycin,
Bleomycin, Vinblastine, Cisplatin, acivicin; aclarubicin; acodazole
hydrochloride; acronine; adozelesin; aldesleukin; altretamine;
ambomycin; ametantrone acetate; aminoglutethimide; amsacrine;
anastrozole; anthramycin; asparaginase; asperlin; azacitidine;
azetepa; azotomycin; batimastat; benzodepa; bicalutamide;
bisantrene hydrochloride; bisnafide dimesylate; bizelesin;
bleomycin sulfate; brequinar sodium; bropirimine; busulfan;
cactinomycin; calusterone; caracemide; carbetimer; carboplatin;
carmustine; carubicin hydrochloride; carzelesin; cedefingol;
chlorambucil; cirolemycin; cladribine; crisnatol mesylate;
cyclophosphamide; cytarabine; dacarbazine; daunorubicin
hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine
mesylate; diaziquone; doxorubicin; doxorubicin hydrochloride;
droloxifene; droloxifene citrate; dromostanolone propionate;
duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin;
enloplatin; enpromate; epipropidine; epirubicin hydrochloride;
erbulozole; esorubicin hydrochloride; estramustine; estramustine
phosphate sodium; etanidazole; etoposide; etoposide phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide;
floxuridine; fludarabine phosphate; fluorouracil; flurocitabine;
fosquidone; fostriecin sodium; gemcitabine; gemcitabine
hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;
ilmofosine; interleukin II (including recombinant interleukin II,
or rIL2), interferon alfa-2a; interferon alfa-2b; interferon
alfa-n1; interferon alfa-n3; interferon beta-I a; interferon
gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide
acetate; letrozole; leuprolide acetate; liarozole hydrochloride;
lometrexol sodium; lomustine; losoxantrone hydrochloride;
masoprocol; maytansine; mechlorethamine hydrochloride; megestrol
acetate; melengestrol acetate; melphalan; menogaril;
mercaptopurine; methotrexate; methotrexate sodium; metoprine;
meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;
mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazole; nogalamycin;
ormaplatin; oxisuran; pegaspargase; peliomycin; pentamustine;
peplomycin sulfate; perfosfamide; pipobroman; piposulfan;
piroxantrone hydrochloride; plicamycin; plomestane; porfimer
sodium; porfiromycin; prednimustine; procarbazine hydrochloride;
puromycin; puromycin hydrochloride; pyrazofurin; riboprine;
rogletimide; safingol; safingol hydrochloride; semustine;
simtrazene; sparfosate sodium; sparsomycin; spirogermanium
hydrochloride; spiromustine; spiroplatin; streptonigrin;
streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur;
teloxantrone hydrochloride; temoporfin; teniposide; teroxirone;
testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin;
tirapazamine; toremifene citrate; trestolone acetate; triciribine
phosphate; trimetrexate; trimetrexate glucuronate; triptorelin;
tubulozole hydrochloride; uracil mustard; uredepa; vapreotide;
verteporfin; vinblastine sulfate; vincristine sulfate; vindesine;
vindesine sulfate; vinepidine sulfate; vinglycinate sulfate;
vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate;
vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin
hydrochloride.
[0055] Yet other anti-cancer agents/drugs that can be used in the
present teachings include, but are not limited to: 20-epi-1,25
dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;
acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK
antagonists; altretamine; ambamustine; amidox; amifostine;
aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist
G; antarelix; anti-dorsalizing morphogenetic protein-1;
antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;
antisense oligonucleotides; aphidicolin glycinate; apoptosis gene
modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;
arginine deaminase; asulacrine; atamestane; atrimustine;
axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin;
azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL
antagonists; benzochlorins; benzoylstaurosporine; beta lactam
derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF
inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;
bisnafide; bistratene A; bizelesin; breflate; bropirimine;
budotitane; buthionine sulfoximine; calcipotriol; calphostin C;
camptothecin derivatives; canarypox IL-2; capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN
700; cartilage derived inhibitor; carzelesin; casein kinase
inhibitors (ICOS); castanospermine; cecropin B; cetrorelix;
chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; 9-dioxamycin; diphenyl spiromustine;
docosanol; dolasetron; doxifluridine; droloxifene; dronabinol;
duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab;
eflornithine; elemene; emitefur; epirubicin; epristeride;
estramustine analogue; estrogen agonists; estrogen antagonists;
etanidazole; etoposide phosphate; exemestane; fadrozole;
fazarabine; fenretinide; filgrastim; finasteride; flavopiridol;
flezelastine; fluasterone; fludarabine; fluorodaunorunicin
hydrochloride; forfenimex; formestane; fostriecin; fotemustine;
gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix;
gelatinase inhibitors; gemcitabine; glutathione inhibitors;
hepsulfam; heregulin; hexamethylene bisacetamide; hypericin;
ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine;
ilomastat; imidazoacridones; imiquimod; immunostimulant peptides;
insulin-like growth factor-1 receptor inhibitor; interferon
agonists; interferons; interleukins; iobenguane; iododoxorubicin;
ipomeanol, 4-; iroplact; irsogladine; isobengazole;
isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F;
lamellarin-N triacetate; lanreotide; leinamycin; lenograstim;
lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting
factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;
lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;
masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase
inhibitors; menogaril; merbarone; meterelin; methioninase;
metoclopramide; MIF inhibitor; mifepristone; miltefosine;
mirimostim; mismatched double stranded RNA; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal antibody, human chorionic gonadotrophin; monophosphoryl
lipid A+myobacterium cell wall sk; mopidamol; multiple drug
resistance gene inhibitor; multiple tumor suppressor 1-based
therapy; mustard anticancer agent; mycaperoxide B; mycobacterial
cell wall extract; myriaporone; N-acetyldinaline; N-substituted
benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin;
naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid;
neutral endopeptidase; nilutamide; nisamycin; nitric oxide
modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine;
octreotide; okicenone; oligonucleotides; onapristone; ondansetron;
ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;
oxaliplatin; oxaunomycin; palauamine; palmitoylrhizoxin; pamidronic
acid; panaxytriol; panomifene; parabactin; pazelliptine;
pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;
pentrozole; perflubron; perfosfamide; perillyl alcohol;
phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil;
pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A;
placetin B; plasminogen activator inhibitor; platinum complex;
platinum compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU;
sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence
derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal transduction modulators; single chain
antigen-binding protein; sizofiran; sobuzoxane; sodium borocaptate;
sodium phenylacetate; solverol; somatomedin binding protein;
sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stem cell inhibitor; stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;
superactive vasoactive intestinal peptide antagonist; suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;
tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;
tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;
temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine; thiocoraline; thrombopoietin; thrombopoietin
mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;
thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;
titanocene bichloride; topsentin; toremifene; totipotent stem cell
factor; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride;
tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor; urokinase
receptor antagonists; vapreotide; variolin B; vector system,
erythrocyte gene therapy; velaresol; veramine; verdins;
verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole;
zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.
Preferred additional anti-cancer drugs are 5-fluorouracil and
leucovorin.
[0056] In one embodiment, the anti-cancer agents that can be used
in methods described herein are selected from the group consisting
of paclitaxel, docetaxel, 5-fluorouracil, trastuzumab, lapatinib,
bevacizumab, letrozole, goserelin, tamoxifen, cetuximab,
panitumumab, gemcitabine, capecitabine, irinotecan, oxaliplatin,
carboplatin, cisplatin, doxorubicin, epirubicin, cyclophosphamide,
methotrexate, vinblastine, vincristine, melphalan and a combination
thereof.
[0057] In one embodiment, the anti-cancer agent and the compound
represented by Structural Formula (I) are administered
contemporaneously. When administered contemporaneously, the
anti-cancer agent and the compound can be administered in the same
formulation or in different formulations. Alternatively, the
compound and the additional anti-cancer agent are administered
separately. Alternatively, the compound and the additional
anti-cancer agent can be administered sequentially, as separate
compositions, within an appropriate time frame (e.g., a cancer
treatment session/interval (e.g., about 1.5 to about 5 hours to
about 10 hours to about 15 hours to about 20 hours; about 1 day to
about 2 days to about 5 days to about 10 days to about 14 days)) as
determined by the skilled clinician (e.g., a time sufficient to
allow an overlap of the pharmaceutical effects of the therapies).
The compound and the additional anti-cancer agent can be
administered in a single dose or multiple doses in an order and on
a schedule suitable to achieve a desired therapeutic effect (e.g.,
inhibition of tumor growth).
[0058] In one embodiment, the subject in the methods described
herein has not been previously treated with a TTK inhibitor (e.g.,
the compound represented by Structural Formula (I).
[0059] The term "inhibiting the growth of tumor-initiating cells"
refers to decreasing the rate of the proliferation and/or survival
of the tumor-initiating cells.
[0060] As used herein, the term "reducing the likelihood of
recurrence of a cancer" means partially or totally inhibiting,
delaying the return of a cancer at or near a primary site and/or at
a secondary site after a period of remission. It also means that
the cancer is less likely to return with treatment described herein
than in its absence.
[0061] As used herein, the term "remission" refers to a state of
cancer, wherein the clinical symptoms or indicators associated with
a cancer have disappeared or cannot be detected, typically after
the subject has been successfully treated with an anti-cancer
therapy.
[0062] As used herein, "treatment" is an approach for obtaining
beneficial or desired results, including clinical results.
Beneficial or desired clinical results can include, but are not
limited to, alleviation or amelioration of one or more symptoms or
conditions, diminishment of extent of disease, stabilized (i.e.,
not worsening) state of disease, reducing the likelihood of the
spread of the disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, whether detectable
or undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment.
"Treatment" also includes reducing the likelihood of reoccurrence
of the disease.
[0063] As used herein, "treating a subject with a cancer" includes
achieving, partially or substantially, one or more of the
following: arresting the growth, reducing the extent of the cancer
(e.g., reducing size of a tumor), inhibiting the growth rate of the
cancer, ameliorating or improving a clinical symptom or indicator
associated with the cancer (such as tissue or serum components) or
increasing longevity of the subject; and reducing the likelihood of
recurrence of the cancer.
[0064] Generally, an effective amount of a compound taught herein
varies depending upon various factors, such as the given drug or
compound, the pharmaceutical formulation, the route of
administration, the type of disease or disorder, the identity of
the subject or host being treated, and the like, but can
nevertheless be routinely determined by one skilled in the art. An
effective amount of a compound of the present teachings may be
readily determined by one of ordinary skill by routine methods
known in the art.
[0065] The term an "effective amount" means an amount when
administered to the subject which results in beneficial or desired
results, including clinical results, e.g., inhibits, suppresses or
reduces the cancer (e.g., as determined by clinical symptoms or the
amount of cancer cells) in a subject as compared to a control.
[0066] In an embodiment, an effective amount of a compound taught
herein ranges from about 0.1 to about 1000 mg/kg body weight,
alternatively about 1 to about 500 mg/kg body weight, and in
another alternative, from about 20 to about 300 mg/kg body weight.
In another embodiment, an effective amount of a compound taught
herein ranges from about 0.5 to about 5000 mg/m.sup.2,
alternatively about from 5 to about 2500 mg/m.sup.2, and in another
alternative from about 50 to about 1000 mg/m.sup.2. The skilled
artisan will appreciate that certain factors may influence the
dosage required to effectively treat a subject suffering from
cancer or reduce the likelihood of recurrence of a cancer. These
factors include, but are not limited to, the severity of the
disease or disorder, previous treatments, the general health and/or
age of the subject and other diseases present.
[0067] Moreover, for methods described herein (including treating a
subject with a cancer or reducing the likelihood of recurrence of a
cancer), a "treatment" or dosing regimen of a subject with an
effective amount of the compound of the present teachings may
consist of a single administration, or alternatively comprise a
series of applications. For example, the compound of the present
teachings may be administered at least once a week. However, in
another embodiment, the compound may be administered to the subject
from about one time per week to once daily for a given treatment.
The length of the treatment period depends on a variety of factors,
such as the severity of the disease, the age of the patient, the
concentration and the activity of the compounds of the present
teachings, or a combination thereof. It will also be appreciated
that the effective dosage of the compound used for the treatment
may increase or decrease over the course of a particular treatment
regime. Changes in dosage may result and become apparent by
standard diagnostic assays known in the art. In some instances,
chronic administration may be required.
[0068] A "subject" is a mammal, preferably a human, but can also be
an animal in need of veterinary treatment, e.g., companion animals
(e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep,
pigs, horses, and the like) and laboratory animals (e.g., rats,
mice, guinea pigs, and the like).
[0069] The compounds taught herein can be administered to a patient
in a variety of forms depending on the selected route of
administration, as will be understood by those skilled in the art.
The compounds of the present teachings may be administered, for
example, by oral, parenteral, buccal, sublingual, nasal, rectal,
patch, pump or transdermal administration and the pharmaceutical
compositions formulated accordingly. Parenteral administration
includes intravenous, intraperitoneal, subcutaneous, intramuscular,
transepithelial, nasal, intrapulmonary, intrathecal, rectal and
topical modes of administration. Parenteral administration can be
by continuous infusion over a selected period of time.
[0070] The compounds taught herein can be suitably formulated into
pharmaceutical compositions for administration to a subject. The
pharmaceutical compositions of the present teachings optionally
include one or more pharmaceutically acceptable carriers and/or
diluents therefor, such as lactose, starch, cellulose and dextrose.
Other excipients, such as flavoring agents; sweeteners; and
preservatives, such as methyl, ethyl, propyl and butyl parabens,
can also be included. More complete listings of suitable excipients
can be found in the Handbook of Pharmaceutical Excipients (5.sup.th
Ed., Pharmaceutical Press (2005)). A person skilled in the art
would know how to prepare formulations suitable for various types
of administration routes. Conventional procedures and ingredients
for the selection and preparation of suitable formulations are
described, for example, in Remington's Pharmaceutical Sciences
(2003-20th edition) and in The United States Pharmacopeia: The
National Formulary (USP 24 NF19) published in 1999. The carriers,
diluents and/or excipients are "acceptable" in the sense of being
compatible with the other ingredients of the pharmaceutical
composition and not deleterious to the recipient thereof.
[0071] Typically, for oral therapeutic administration, a compound
of the present teachings may be incorporated with excipient and
used in the form of ingestible tablets, buccal tablets, troches,
capsules, elixirs, suspensions, syrups, wafers, and the like.
[0072] Typically for parenteral administration, solutions of a
compound of the present teachings can generally be prepared in
water suitably mixed with a surfactant such as
hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, DMSO and mixtures thereof
with or without alcohol, and in oils. Under ordinary conditions of
storage and use, these preparations contain a preservative to
prevent the growth of microorganisms.
[0073] Typically, for injectable use, sterile aqueous solutions or
dispersion of, and sterile powders of, a compound described herein
for the extemporaneous preparation of sterile injectable solutions
or dispersions are appropriate.
[0074] For nasal administration, the compounds of the present
teachings can be formulated as aerosols, drops, gels and powders.
Aerosol formulations typically comprise a solution or fine
suspension of the active substance in a physiologically acceptable
aqueous or non-aqueous solvent and are usually presented in single
or multidose quantities in sterile form in a sealed container,
which can take the form of a cartridge or refill for use with an
atomizing device. Alternatively, the sealed container may be a
unitary dispensing device such as a single dose nasal inhaler or an
aerosol dispenser fitted with a metering valve which is intended
for disposal after use. Where the dosage form comprises an aerosol
dispenser, it will contain a propellant which can be a compressed
gas such as compressed air or an organic propellant such as
fluorochlorohydrocarbon. The aerosol dosage forms can also take the
form of a pump-atomizer.
[0075] For buccal or sublingual administration, the compounds of
the present teachings can be formulated with a carrier such as
sugar, acacia, tragacanth, or gelatin and glycerine, as tablets,
lozenges or pastilles.
[0076] For rectal administration, the compounds described herein
can be formulated in the form of suppositories containing a
conventional suppository base such as cocoa butter.
[0077] The compounds of invention may be prepared by methods known
to those skilled in the art, as illustrated by the general schemes
and procedures below and by the preparative examples that follow.
All starting materials are either commercially available or
prepared by methods known to those skilled in the art and the
procedures described below.
[0078] In accordance with another aspect of the present invention,
the compounds of the invention can be prepared by processes
analogues to those established in the art. General methods to
synthesize the claimed compounds are elaborated below in Example
A.
EXEMPLIFICATION
Example A: Synthesis
[0079] General Methods
[0080] Commercially available starting materials, reagents, and
solvents were used as received. In general, anhydrous reactions
were performed under an inert atmosphere such as nitrogen or Argon.
PoraPak.RTM. Rxn CX refers to a commercial cation-exchange resin
available from Waters. Microwave reactions were performed with a
Biotage Initiator microwave reactor. Reaction progress was
generally monitored by TLC using Merck silica gel plates with
visualization by UV at 254 nm, by analytical HPLC or by LCMS
(Bruker Exquire 4000 or Waters Acquity UPLC system). Flash column
chromatographic purification of intermediates or final products was
performed using 230-400 mesh silica gel 60 from EMD chemicals or
Silicycle, or purified using a Biotage Isolera with KP-SIL or
HP-SIL silica cartridges, or KP-NH basic modified silica and
corresponding samplets. Reverse-phase HPLC purification was
performed on a Varian PrepStar model SD-1 HPLC system with a Varian
Monochrom 10p C-18 reverse-phase column using a of about 5-30% MeCN
or MeOH/0.05% TFA-H.sub.2O to 70-90% MeCN or MeOH/0.05% TFA in
H.sub.2O over a 20-40-min period at a flow rate of 30-80 mL/min.
Reverse phase purification was also performed using a Biotage
Isolera equipped with a KP-C18-H column using a between 10-95% MeOH
or CH.sub.3CN/0.1% TFA in H.sub.2O. Proton NMRs were recorded on a
Bruker 400 MHz spectrometer, and mass spectra were obtained using a
Bruker Esquire 4000 spectrometer or Waters Acquity UPLC system.
[0081] Compound names were generated using the software built into
CambridgeSoft-PerkinElmer's ChemBioDraw Ultra version 11.0 or
12.0.
[0082] Abbreviations: [0083] aq aqueous [0084] Ar argon [0085] Boc
tert-butoxycarbonyl [0086] br. broad [0087] calcd calculated [0088]
d doublet [0089] DCM dichloromethane [0090] DIPEA
diisopropylethylamine [0091] DMF N,N-dimethylformamide [0092] DMSO
dimethylsulfoxide [0093] dppf 1,1'-bis(diphenylphosphino) ferrocene
[0094] h hour [0095] HPLC high performance liquid chromatography
[0096] LC-MS liquid chromatography coupled to mass spectrometry
[0097] min minute [0098] m multiplet [0099] MS ESI mass spectra,
electrospray ionization [0100] NMR nuclear magnetic resonance
[0101] O/N overnight [0102] PE petroleum ether [0103] PMB
para-methoxybenzyl [0104] prep preparative [0105] rt room
temperature [0106] s singlet [0107] t triplet [0108] TFA
trifluoroacetic acid [0109] THF tetrahydrofuran
##STR00007##
[0109] Intermediates
4-bromo-N-cyclopropyl-2-methylbenzamide
##STR00008##
[0111] To a suspension of 4-bromo-2-methylbenzoic acid (43.0 g, 200
mmol) and oxalyl dichloride (30.5 g, 240 mmol) in DCM (300 mL) was
added DMF (0.1 mL). The resulting reaction mixture was stirred at
rt for 16 h. The reaction turned into a clear yellow solution
slowly over 16 h. Solvent was then removed in vacuo, and the crude
product was used in the next step without further purification. The
crude product was redissolved in DCM (300 mL) and cooled to
0.degree. C. A mixture of TEA (42 mL, 300 mmol) and
cyclopropylamine (12.6 g, 220 mmol) in DCM (100 mL) was added
slowly over 15 min, and the resulting mixture was stirred at rt for
2 h. The reaction was diluted with DCM (200 mL) and water was
added. The resulting mixture was extracted with DCM and the
combined organic extracts were dried over MgSO.sub.4 and
concentrated to give the desired product as a pale pink solid (50.1
g, 99%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 7.37 (s,
1H), 7.31 (d, J=8.0 Hz, 1H), 7.17 (d, J=8.1 Hz, 1H), 5.93 (br. s,
1H), 2.90-2.85 (m, 1H), 2.40 (s, 3H), 0.90-0.85 (m, 2H), 0.62-0.58
(m, 2H); MS ESI [M+H].sup.+ 253.9, calcd for
[C.sub.11H.sub.12BrNO+H].sup.+254.0.
N-(4-bromo-2-methylphenyl)cyclopropanecarboxamide
##STR00009##
[0113] In a 100 mL RBF, 4-bromo-2-methylaniline (3.7 g, 20 mmol)
and DIPEA (6.95 mL, 40 mmol) were combined with DMF (40 mL). The
reaction was cooled to 0.degree. C. in an ice bath and
cyclopropanecarbonyl chloride (2.1 g, 20 mmol) was added. The
mixture was stirred at 0.degree. C. for 1 h. Water and EtOAc were
added to separate the phases and the aqueous phase was extracted
with EtOAc. The organic layers were combined, dried over
Na.sub.2SO.sub.4, filtered, and concentrated under reduced pressure
to give the title compound as a white solid (4.76 g, 94%). .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. ppm 7.85-7.72 (m, 1H), 7.38-7.28
(m, 2H), 7.15-7.02 (m, 1H), 2.27 (s, 3H), 1.57-1.48 (m, 1H), 1.10
(quint, J=3.9 Hz, 2H), 0.92-0.79 (m, 2H); MS ESI [M+H].sup.+ 253.9,
calcd for [C.sub.11H.sub.12BrNO+H].sup.+254.0.
N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benz-
amide
##STR00010##
[0115] To a mixture of 4-bromo-N-cyclopropyl-2-methylbenzamide
(3.73 g, 14 mmol), Bis(pinacolato)diboron (5.59 g, 22 mmol), anh
KOAc (4.29 g, 43 mmol) in DMF (37 mL) was purged with Ar for 10 min
at rt. Then PdCl.sub.2(dppf) DCM (0.59 g, 5 mol %) was added and
the reaction was heated at 100.degree. C. in oil bath for 4 h.
After reaction completion the reaction mass was diluted with EtOAc
(200 mL) and H.sub.2O (100 mL). The combined layer filtered through
celite pad and washed it with little EtOAc. The aq. layer further
extracted with EtOAc (50 mL) and the combined organic layer washed
with brine, dried over Na.sub.2SO.sub.4, filtered, and concentrated
to give crude oily residue. The crude product was purified by flash
chromatography (gradient: EtOAc/hex 0-100%) to give the title
compound as a creamy solid (4.15 g, 94%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. ppm 7.66 (s, 1H), 7.63-7.61 (d, J=7.6 Hz, 1H),
7.32-7.30 (d, J=7.6 Hz, 1H), 5.85 (s, 1H), 2.93-2.87 (m, 1H), 2.46
(s, 3H), 1.35 (s, 12H), 0.90-0.86 (m, 2H), 0.63-0.59 (m, 2H); MS
ESI [M+H].sup.+ 302.2, calcd for
[C.sub.17H.sub.24BNO.sub.3+H].sup.+302.2.
3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine
##STR00011##
[0117] To a stirred solution of sodium ethoxide in EtOH, which was
prepared from sodium (281.3 g, 12.0 mol) and EtOH (10 L) by the
conventional method, were added diethyl malonate (963.7 g, 6.02
mol) at ambient temperature and then compound 1H-pyrazol-3-amine
(500 g, 6.02 mol). The reaction mixture was refluxed for 12 hours.
After cooled to room temperature, the precipitates were collected
by filtration and dissolved in water. The aqueous solution was
acidified with 2 M HCl (pH=2). The resulting precipitates were
collected by filtration and dried under reduced pressure to afford
pyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione (649 g, 71%) as a yellow
solid, which was used for the next reaction without further
purification.
[0118] A stirred suspension of
pyrazolo[1,5-a]pyrimidine-5,7(4H,6H)-dione (265 g, 1.75 mol) and
N,N-dimethylaniline (335.6 mL) in POCl.sub.3 (2.00 kg, 13.2 mol)
was refluxed for 4 hours. After cooled to room temperature, the
reaction mixture was poured into ice-water, and stirred for 30 min,
neutralized with saturated aqueous sodium carbonate and extracted
with EtOAc. The combined organic layers were washed with water,
brine and dried over MgSO.sub.4, filtered and evaporated. The
residue was purified by column chromatography on silica gel
(gradient: EtOAc/PE 1:10) to give
5,7-dichloropyrazolo[1,5-a]pyrimidine (287 g, 87%) as a yellow
solid.
[0119] To a solution 5,7-dichloropyrazolo[1,5-a]pyrimidine (246.6
g, 1.31 mol) in CH.sub.3CN (1.8 L) was added NBS (245 g, 1.38 mol).
The resulting mixture was stirred at room temperature for 2 hours.
After removal of the solution, the reaction mixture was purified by
column chromatography on silica gel (gradient: EtOAc/PE 1:5) to
give the title compound (313.5 g, 89%) as light yellow solid.
.sup.1H NMR (300 MHz, CDCl.sub.3): .delta. ppm 7.04 (s, 1H), 8.21
(s, 1H); MS ESI [M+H].sup.+ 265.9, calcd for
[C.sub.6H.sub.2BrCl.sub.2N.sub.3+H].sup.+265.9.
3-bromo-5-chloro-N-(cyclopropylmethyl)pyrazolo[1,5-a]pyrimidin-7-amine
##STR00012##
[0121] To a solution of
3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine (5.0 g, 19 mmol) in
DCM (50 mL) was added cyclopropylmethanamine (1.48 g, 21 mmol), and
DIPEA (6.6 mL, 38 mmol). The reaction was stirred at rt for 2 h.
Water and DCM were added to separate the phases and the aqueous
phase was extracted with DCM. The combined organic extracts were
dried over NaSO.sub.4, filtered and concentrated. The crude product
was purified by flash chromatography (gradient: EtOAc/hex 0-50%) to
give the title compound as a yellow solid (5.25 g, 92%). .sup.1H
NMR (400 MHz, CDCl.sub.3) .delta. ppm 7.97 (s, 1H), 6.50 (br. s,
1H), 5.97 (s, 1H), 3.25 (dd, J=7.3, 5.5 Hz, 2H), 1.26-1.13 (m, 1H),
0.74-0.65 (m, 2H), 0.37 (q, J=5.0 Hz, 2H); MS ESI [M+H].sup.+
301.0, calcd for [C.sub.10H.sub.10BrClN.sub.4+H].sup.+301.0.
(1s,3s)-3-(((3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)amino)methyl)-1-
-methylcyclobutanol
##STR00013##
[0123] To a solution of
3-bromo-5,7-dichloropyrazolo[1,5-a]pyrimidine (14.6 g, 55.3 mmol)
in DCM (200 mL) was added
cis-hydroxy-3-methylcyclobutane-1-methylamine (7.0 g, 60.9 mmol),
and DIPEA (19.2 mL, 110.6 mmol). The reaction was stirred at rt for
16 h. Water and DCM were added to separate the phases and the
aqueous phase was extracted with DCM. The combined organic extracts
were dried over Na.sub.2SO.sub.4, filtered and concentrated to give
the title compound as a yellow solid (18.1 g, 95%). .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. ppm 7.96 (s, 1H), 6.60-6.49 (m, 1H),
5.99 (s, 1H), 3.47 (t, J=6.0 Hz, 2H), 2.38-2.27 (m, 3H), 1.96-1.85
(m, 2H), 1.43 (s, 3H); MS ESI [M+H].sup.+ 345.1, calcd for
[C.sub.12H.sub.14BrClN.sub.4O+H].sup.+345.0.
Tert-butyl
(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(cyclopropylmeth-
yl)carbamate
##STR00014##
[0125] To a solution of
3-bromo-5-chloro-N-(cyclopropylmethyl)pyrazolo[1,5-a]pyrimidin-7-amine
(5.25 g, 17.5 mmol) in DCM (150 mL) was added Boc.sub.2O (5.70 g,
26.2 mmol), DMAP (0.21 g, 1.75 mmol), and TEA (7.3 mL, 52.5 mmol).
The reaction was stirred at rt for 4 h. Water and DCM were added to
separate the phases and the aqueous phase was extracted with DCM.
The combined organic extracts were dried over MgSO.sub.4, filtered
and concentrated. The crude product was purified by flash
chromatography (gradient: EtOAc/hex 0-40%) to give the title
compound as a yellow solid (6.24 g, 89%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. ppm 8.12 (s, 1H), 6.85 (s, 1H), 3.72 (d, J=7.3
Hz, 2H), 1.39 (s, 9H), 1.05-0.94 (m, 1H), 0.46-0.37 (m, 2H),
0.13-0.03 (m, 2H); MS ESI [M-C.sub.4H.sub.8].sup.+ 345.0, calcd for
[C.sub.15H.sub.18BrClN.sub.4O.sub.2--C.sub.4H.sub.8].sup.+345.0.
Tert-butyl
(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(((1s,3s)-3-((te-
rt-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate
##STR00015##
[0127] To a solution of
(1s,3s)-3-(((3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)amino)methyl)--
1-methylcyclobutanol (18.1 g, 52.6 mmol), Boc.sub.2O (34.3 g, 158
mmol), and), and TEA (22 mL, 157.8 mmol) in DCM (200 mL) was added
DMAP (1.28 g, 10.5 mmol). The reaction was stirred at 40.degree. C.
for 16 h. The solvent was removed in vacuo and water and DCM were
added to separate the phases and the aqueous phase was extracted
with DCM. The combined organic extracts were dried over NaSO.sub.4,
filtered and concentrated. The crude product was purified by flash
chromatography (3 Biotage 100 g SiO.sub.2 columns in parallel,
gradient: EtOAc/hex 0-30%) to give the title compound as a beige
solid (12.7 g, 44%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm
8.11 (s, 1H), 6.74 (s, 1H), 3.88 (d, J=6.5 Hz, 2H), 2.17 (s, 3H),
1.99-1.88 (m, 2H), 1.47 (s, 3H), 1.45 (s, 9H), 1.35 (s, 9H); MS ESI
[M+H].sup.+ 545.1, calcd for
[C.sub.22H.sub.30BrClN.sub.4O.sub.5+H].sup.+545.1.
(1s,3s)-3-(((3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(4-methoxybenzy-
l)amino)methyl)-1-methylcyclobutanol
##STR00016##
[0129] To a solution of
(1s,3s)-3-(((3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)amino)methyl)--
1-methylcyclobutanol (12.0 g, 34.7 mmol) and 4-methoxybenzyl
chloride (5.2 mL, 38.2 mmol) in DMF (50 mL) was added
K.sub.2CO.sub.3 (9.6 g, 69.4 mmol). The resulting mixture was
stirred at 60.degree. C. for 3 h. Water and EtOAc were added to
separate the phases and the aqueous phase was extracted with EtOAc.
The combined organic extracts were washed with water, dried over
MgSO.sub.4, filtered and concentrated. The crude product was
purified by flash chromatography (gradient: EtOAc/hex 10-100%)
using three columns in parallel. Impure fractions were collected
and purified by flash chromatography using the above conditions.
Pure fractions were combined and concentrated to give the desired
product as a pale yellow solid (13.1 g, 81%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. ppm 8.01 (s, 1H), 7.17 (d, J=8.7 Hz, 2H), 6.67
(d, J=8.7 Hz, 2H), 6.02 (s, 1H), 4.98 (s, 2H), 3.83 (d, J=6.4 Hz,
2H), 3.81 (s, 3H), 2.25-2.16 (m, 3H), 1.76-1.72 (m, 2H), 1.36 (s,
3H); MS ESI [M+H].sup.+ 467.1, calcd for
[C.sub.20H.sub.22BrClN.sub.4O.sub.2+H].sup.+467.1.
Tert-butyl
(3-bromo-5-(((1S,2R)-2-hydroxycyclohexyl)amino)pyrazolo[1,5-a]p-
yrimidin-7-yl)(cyclopropylmethyl)carbamate
##STR00017##
[0131] To a solution of using tert-butyl
(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(cyclopropylmethyl)carbama-
te (9.0 g, 22.5 mmol) in NMP (90 mL) was added
(1R,2S)-2-aminocyclohexanol HCl (4.08 g, 27.0 mmol), and DIPEA
(3.47 g, 25.1 mmol). The reaction was divided and sealed into six
microwave vials. Each vial was microwaved for 3 h at 130.degree. C.
Water and EtOAc were added to separate the phases and the aqueous
phase was extracted with EtOAc. The combined organic extracts were
dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude
product was purified by flash chromatography (gradient: EtOAc/hex
0-50%) using three columns in parallel to give the title compound
as a beige solid (8.51 g, 79%). .sup.1H NMR (400 MHz, CDCl.sub.3)
.delta. ppm 7.80 (s, 1H), 6.08 (s, 1H), 5.33-5.19 (m, 1H),
4.26-4.14 (m, 1H), 4.13-4.06 (m, 1H), 3.61 (d, J=7.3 Hz, 2H), 2.51
(br. s, 1H), 1.89-1.62 (m, 8H), 1.55-1.45 (m, 2H), 1.40 (s, 9H),
1.06-0.95 (m, 1H), 0.47-0.38 (m, 2H), 0.17-0.07 (m, 2H); MS ESI
[M+H].sup.+ 408.3, calcd for
[C.sub.21H.sub.30BrN.sub.5O.sub.3+H].sup.+480.2.
Tert-butyl
(3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(cycl-
opropylmethyl)carbamate
##STR00018##
[0133] A mixture of tert-butyl
(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(cyclopropylmethyl)carbama-
te (400 mg, 1.0 mmol), 3-hydroxypyridine (475 mg, 5.0 mmol), DBU
(0.75 mL, 5.0 mmol) in DME (5 mL) was combined and sealed into a
microwave vial. The mixture was microwaved for 1 h at 85.degree. C.
Water and EtOAc were added to separate the phases and the aqueous
phase was extracted with EtOAc. The combined organic extracts were
dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude
product was purified by flash chromatography (gradient: EtOAc/hex
0-50%) to give the title compound as a brown oil (367 mg, 80%).
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 8.64 (d, J=2.5 Hz,
1H) 8.54 (dd, J=4.5, 1.5 Hz, 1H) 7.97 (s, 1H), 7.78-7.72 (m, 1H),
7.44-7.38 (m, 1H) 6.61 (s, 1H) 3.71 (d, J=7.3 Hz, 2H) 1.39 (s, 9H),
1.06-1.01 (m, 1H) 0.45 (s, 2H), 0.13-0.09 (m, 2H); MS ESI
[M+H].sup.+ 460.0, calcd for
[C.sub.20H.sub.22BrN.sub.5O.sub.3+H].sup.+460.1.
Tert-butyl
(3-bromo-5-(cyclopentylamino)pyrazolo[1,5-a]pyrimidin-7-vi)(((1-
s,3s)-3-((tert-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate
##STR00019##
[0135] To a solution of tert-butyl
(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(((1s,3s)-3-((tert-butoxyc-
arbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate (0.45 g, 0.83
mmol) in NMP (4 mL) was added cyclopentylamine (0.085 g, 1.0 mmol),
and DIPEA (0.21 g, 1.66 mmol). The reaction was microwaved for 3 h
at 130.degree. C. Water and EtOAc were added to separate the phases
and the aqueous phase was extracted with EtOAc. The combined
organic extracts were dried over Na.sub.2SO.sub.4, filtered and
concentrated. The crude product was purified by flash
chromatography (gradient: EtOAc/hex 0-60%) to give the title
compound as a white solid (0.40 g, 67%). .sup.1H NMR (400 MHz,
CDCl.sub.3) .delta. ppm 7.79 (s, 1H), 5.94 (s, 1H), 5.08 (br. s,
1H), 4.23 (br. s, 1H), 3.85-3.72 (m, 2H), 2.28-2.06 (m, 5H),
2.02-1.89 (m, 2H), 1.81-1.61 (m, 5H), 1.48 (s, 4H), 1.44 (s, 9H),
1.36 (s, 9H); MS ESI [M+H].sup.+ 594.3, calcd for
[C27H.sub.40BrN.sub.5O.sub.5+H].sup.+594.2.
Tert-butyl
(3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(((1s-
,3s)-3-((tert-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate
##STR00020##
[0137] A mixture of tert-butyl
(3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(((1s,3s)-3-((tert-butoxyc-
arbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate (12.7 g, 23.3
mmol), 3-hydroxypyridine (4.43 g, 46.6 mmol), and K.sub.2CO.sub.3
(9.65 g, 69.9 mmol) was combined in DMF (100 mL) and stirred at rt
overnight. The reaction mixture was diluted with EtOAc and washed
with water, dried over Na.sub.2SO.sub.4, filtered and concentrated
in vacuo. The crude product was triturated with Et.sub.2O and
filtered to give the title compound as a beige solid (8.43 g, 14.0
mmol). The mother liquor was concentrated in vacuo and purified by
flash chromatography (gradient: EtOAc/hex 0-40%) to give the title
compound as a light orange solid (4.07 g, 6.74 mmol). The solids
obtained through trituration and flash chromatography were combined
to give the title compound as a light orange solid (12.50 g, 89%)
.sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 8.65 (d, J=2.8 Hz,
1H), 8.55 (dd, J=4.6, 1.4 Hz, 1H), 7.98 (s, 1H), 7.78-7.73 (m, 1H),
7.46-7.40 (m, 1H), 6.50 (s, 1H), 3.89 (d, J=6.8 Hz, 2H), 2.30-2.15
(m, 3H), 2.02-1.93 (m, 2H), 1.50 (s, 3H), 1.45 (s, 9H), 1.38 (s,
9H); MS ESI [M+H].sup.+ 604.3, calcd for
[C.sub.27H.sub.34BrN.sub.5O.sub.6+H].sup.+604.2.
(1s,3s)-3-(((3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(4-m-
ethoxybenzyl)amino)methyl)-1-methylcyclobutanol
##STR00021##
[0139] To a solution of
(1s,3s)-3-(((3-bromo-5-chloropyrazolo[1,5-a]pyrimidin-7-yl)(4-methoxybenz-
yl)amino)methyl)-1-methylcyclobutanol (13.1 g, 28.2 mmol) and
3-hydroxypyridine (4.0 g, 42.3 mmol) in DMF (70 mL) was added
K.sub.2CO.sub.3 (7.8 g, 56.3 mmol). The resulting mixture was
stirred at 80.degree. C. for 22 h. A mixture of 60% NaH (0.6 g, 15
mmol) and 3-hydroxypyridine (1.4 g, 14.7 mmol) in DMF was stirred
at rt for 30 min before adding to the reaction mixture. The
resulting solution was stirred at 100.degree. C. for 20 h. Water
and EtOAc were added to separate the phases and the aqueous phase
was extracted with EtOAc. The combined organic extracts were washed
with water, dried over MgSO.sub.4, filtered and concentrated. The
crude product was purified by flash chromatography (gradient:
EtOAc/hexanes 10-100%) using three columns in parallel. Impure
fractions were collected and purified by flash chromatography
(gradient: EtOAc/hexanes 50-100%). Pure fractions were combined and
concentrated to give the desired product as a pale yellow solid
(9.5 g, 64%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 8.56
(d, J=4 Hz, 1H), 8.45 (d, J=8 Hz, 1H), 8.00 (s, 1H), 7.69-7.65 (m,
1H), 7.36 (dd, J=4.8, 0.4 Hz, 1H), 7.19 (d, J=8.7 Hz, 2H), 6.85 (d,
J=8.7 Hz, 2H), 5.71 (s, 1H), 4.91 (s, 2H), 3.87 (d, J=6.7 Hz, 2H),
3.87 (s, 3H), 2.30-2.15 (m, 3H), 1.77 (t, J=8.1 Hz, 2H), 1.37 (s,
3H); MS ESI [M+H].sup.+ 524.2, calcd for
[C.sub.25H.sub.26BrN.sub.5O.sub.3+H].sup.+524.1.
N-cyclopropyl-4-(7-((((s,
3s)-3-hydroxy-3-methylcyclobutyl)methyl)(4-methoxybenzyl)amino)-5-(pyridi-
n-3-yloxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-methylbenzamide
##STR00022##
[0141] A mixture of
(1s,3s)-3-(((3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(4--
methoxybenzyl)amino)methyl)-1-methylcyclobutanol (9.5 g, 18.1
mmol),
N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ben-
zamide (7.6 g, 25.4 mmol), 2M K.sub.3PO.sub.4 (23 mL, 45.3 mmol) in
THF (100 mL) was purged with Ar for 10 min before the addition of
PdCl.sub.2dppfDCM (1.5 g, 1.8 mmol). The resulting mixture was
heated under reflux in an oil bath at 84.degree. C. for 16 h. The
reaction mixture was diluted with DCM and sat. NaHCO.sub.3. The
aqueous phase was extracted with DCM and the combined organic
extracts were dried over MgSO.sub.4, filtered and concentrated. The
crude product was purified by flash chromatography (gradient:
EtOAc/hex 50-100%) using three columns in parallel. Impure
fractions were collected and purified by flash chromatography using
the above conditions. Pure fractions were combined and concentrated
to give the desired product as an orange solid (9.6 g, 85%). 1H NMR
(400 MHz, CDCl.sub.3) .delta. ppm 8.90-8.84 (m, 1H), 8.55 (d, J=4.5
Hz, 1H), 8.29 (s, 1H). 7.88-7.83 (m, 1H), 7.63 (s, 1H), 7.58-7.53
(m, 2H), 7.35-7.31 (m, 1H), 7.23 (d, J=8.6 Hz, 2H), 6.89 (d, J=8.6
Hz, 2H), 6.19 (br. s, 1H), 5.82 (s, 1H), 4.99 (s, 2H), 3.90 (d,
J=10.0 Hz, 2H), 3.80 (s, 3H), 2.93-2.90 (m, 1H), 2.40 (s, 3H),
2.36-2.28 (m, 1H), 2.22-2.20 (m, 2H), 1.89-1.88 (m, 1H), 1.80 (t,
J=8.7 Hz, 2H), 1.38 (s, 3H), 0.88-0.85 (m, 2H), 0.68-0.64 (m, 2H);
MS ESI 619.3 [M+H].sup.+, calcd for
[C.sub.36H.sub.38N.sub.6O.sub.4+H].sup.+619.3.
Preparation of Exemplary Compounds of the Invention
A1:
N-cyclopropyl-4-(7-((cyclopropylmethyl)amino)-5-(((1S,2R)-2-hydroxycyc-
lohexyl)amino)pyrazolo[1,5-a]pyrimidin-3-yl)-2-methylbenzamide
Hydrochloride
##STR00023##
[0143] A mixture of tert-butyl
(3-bromo-5-(((1S,2R)-2-hydroxycyclohexyl)amino)pyrazolo[1,5-a]pyrimidin-7-
-yl)(cyclopropylmethyl)carbamate (8.48 g, 17.7 mmol),
N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ben-
zamide (6.92 g, 23.0 mmol), PdCl.sub.2dppfDCM (1.44 g, 1.76 mmol),
and 2M K.sub.3PO.sub.4 (26.6 mL, 106.2 mmol) in THF (60 mL) was
divided and sealed into four microwave vials. Each vial was charged
with Ar and heated in the microwave at 125.degree. C. for 3 h.
Water and EtOAc were added to separate the phases and the aqueous
phase was extracted with EtOAc. The combined organic extracts were
dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude
product was purified by flash chromatography (gradient: EtOAc/hex
20-100%) using two columns in parallel to give a yellow solid.
[0144] The above compound was dissolved in DCM (20 mL) and treated
with TFA (20 mL) at rt for 1 h. After reaction completion, solvent
was removed in vacuo and the crude was redissolved in DCM (20 mL),
neutralized with saturated aqueous sodium bicarbonate and extracted
with EtOAc. The combined organic extracts were dried over
NaSO.sub.4, filtered and concentrated. The crude product was
purified by flash chromatography (gradient: 50-100% EtOAc/hex then
MeOH/DCM 0-10%) and triturated with Et.sub.2O to give the title
compound as a free base (white solid). The free base was dissolved
in a mixture of DCM (25 mL) and MeOH (50 mL), HCl (1M Et.sub.2O, 2
equiv) was then added slowly. Solvent was removed in vacuo to give
the title compound as a pale yellow solid in HCl salt (2.09 g, 25%
over 2 steps). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 8.21
(s, 1H), 7.51-7.37 (m, 3H), 5.67 (br. s, 1H), 4.04-3.82 (m, 2H),
3.46-3.37 (m, 2H), 2.93-2.82 (m, 1H), 2.47 (s, 3H), 1.89-1.64 (m,
6H), 1.61-1.42 (m, 2H), 1.31-1.21 (m, 1H), 0.88-0.78 (m, 2H),
0.70-0.58 (m, 4H), 0.46-0.36 (m, 2H); MS ESI [M+H].sup.+ 475.3,
calcd for [C.sub.27H.sub.34N.sub.6O.sub.2+H].sup.+475.3. HPLC
purity: 98% at 254 nm.
A2:
N-cyclopropyl-4-(7-((cyclopropylmethyl)amino)-5-(pyridin-3-yloxy)pyraz-
olo[1,5-a]pyrimidin-3-yl)-2-methylbenzamide Hydrochloride
##STR00024##
[0146] A mixture of tert-butyl
(3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(cyclopropylmet-
hyl)carbamate (367 mg, 0.80 mmol)),
N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ben-
zamide (289 mg, 0.96 mmol), PdCl.sub.2dppfDCM (65 mg, 0.080 mmol),
and 2M K.sub.3PO.sub.4 (1.4 mL, 2.8 mmol) in THF (4 mL) was sealed
into a microwave vial. The vial was charged with Ar and heated in
the microwave at 130.degree. C. for 4 h. Water and EtOAc were added
to separate the phases and the aqueous phase was extracted with
EtOAc. The combined organic extracts were dried over
Na.sub.2SO.sub.4, filtered and concentrated. The crude product was
purified by flash chromatography (gradient: EtOAc/hex 20-100%) to
give a yellow oil.
[0147] The above compound was dissolved in DCM (6 mL) and treated
with TFA (2 mL) at rt for 1 h. After reaction completion, solvent
was removed in vacuo and the crude was redissolved in MeOH (4 mL),
filtered, and purified by prep HPLC. The fractions were passed
through a PoraPak column and triturated with Et.sub.2O to give the
title compound as a free base (white solid, 119 mg). A portion of
the free base (67 mg, 0.15 mmol) was then dissolved in MeOH (10
mL), and HCl (1M Et.sub.2O, 2 equiv) was then added slowly. The
mixture was concentrated and triturated with Et.sub.2O to give the
title compound as a yellow solid in HCl salt (55 mg, 25% yield over
2 steps). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm 9.10 (s,
1H), 8.82 (d, J=6.0 Hz, 1H), 8.73-8.66 (m, 1H), 8.41 (s, 1H),
8.24-8.15 (m, 1H), 7.69 (s, 1H), 7.60 (d, J=8.3 Hz, 1H), 7.23 (d,
J=8.0 Hz, 1H), 6.07 (s, 1H), 3.39 (d, J=7.0 Hz, 2H), 2.89-2.78 (m,
1H), 2.29 (s, 3H), 1.37-1.23 (m, 1H), 0.86-0.75 (m, 2H), 0.70-0.56
(m, 4H), 0.45-0.38 (m, 2H); MS ESI [M+H].sup.+ 455.2, calcd for
[C.sub.26H.sub.26N.sub.6O.sub.2+H]+455.2. HPLC purity: 95.9% at 254
nm.
A3:
4-(5-(cyclopentylamino)-7-((((1s,3s)-3-hydroxy-3-methylcyclobutyl)meth-
yl)amino)pyrazolo[1,5-a]pyrimidin-3-yl)-N-cyclopropyl-2-methylbenzamide
Hydrochloride
##STR00025##
[0149] A mixture of tert-butyl
(3-bromo-5-(cyclopentylamino)pyrazolo[1,5-a]pyrimidin-7-yl)(((1s,3s)-3-((-
tert-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate (0.40
g, 0.67 mmol),
N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
-yl)benzamide (0.26 g, 0.88 mmol), PdCl.sub.2dppfDCM (0.055 g,
0.067 mmol), and 2M K.sub.3PO.sub.4 (1 mL, 2.01 mmol) in THF (4 mL)
was charged with Ar and heated in the microwave at 130.degree. C.
for 3 h. Water and EtOAc were added to separate the phases and the
aqueous phase was extracted with EtOAc. The combined organic
extracts were dried over Na.sub.2SO.sub.4, filtered and
concentrated. The crude product was purified by flash
chromatography (gradient: EtOAc/hex 20-100%) to give a yellow
oil.
[0150] The above compound was dissolved in DCM (10 mL) and treated
with TFA (3 mL) at rt for 3 h. After reaction completion, solvent
was removed in vacuo and the crude product was dissolved in MeOH (5
mL). The mixture was filtered and purified by prep-HPLC. The
compound was passed through a PoraPak cartridge and triturated with
Et.sub.2O to give the title compound as a free base (white solid).
The free base was dissolved in MeOH (5 mL), and HCl (1M Et.sub.2O,
2 equiv) was then added slowly. Solvent was removed in vacuo to
give the title compound as a light orange solid in HCl salt (75 mg,
21% over 2 steps). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm
8.18 (s, 1H), 7.42 (s, 3H), 5.52 (br. s, 1H), 4.23-4.11 (m, 1H),
3.68-3.54 (m, 2H), 2.93-2.81 (m, 1H), 2.46 (s, 3H), 2.38-2.27 (m,
1H), 2.26-2.07 (m, 4H), 1.99-1.89 (m, 2H), 1.88-1.59 (m, 6H), 1.35
(s, 3H), 0.86-0.79 (m, 2H), 0.66-0.58 (m, 2H); MS ESI [M+H].sup.+
489.4, calcd for [C.sub.28H.sub.36N.sub.6O.sub.2+H]+489.3. HPLC
purity: 99% at 254 nm.
A4:
N-cyclopropyl-4-(7-((((s,3s)-3-hydroxy-3-methylcyclobutyl)methyl)amino-
)-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-methylbenzamide
Hydrochloride and its Free Base
##STR00026##
[0152] A). Through Boc deprotection: A mixture of tert-butyl
(3-bromo-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-7-yl)(((1s,3s)-3-((t-
ert-butoxycarbonyl)oxy)-3-methylcyclobutyl)methyl)carbamate (0.23
g, 0.38 mmol),
N-cyclopropyl-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
-yl)benzamide (0.15 g, 0.49 mmol), PdCl.sub.2dppfDCM (0.15 g, 0.49
mmol), and 2M K.sub.3PO.sub.4 (0.57 mL, 1.14 mmol) in THF (4 mL)
was charged with Ar and heated in the microwave at 130.degree. C.
for 3 h. Water and EtOAc were added to separate the phases and the
aqueous phase was extracted with EtOAc. The combined organic
extracts were dried over NaSO.sub.4, filtered and concentrated. The
crude product was purified by flash chromatography (gradient:
EtOAc/hex 20-60%) to give a yellow oil.
[0153] The above intermediate was dissolved in DCM (10 mL) and
treated with TFA (3 mL) at rt for 3 h. After reaction completion,
solvent was removed in vacuo and the crude product was dissolved in
MeOH (5 mL). The mixture was filtered and purified by prep-HPLC.
The compound was passed through a PoraPak cartridge and triturated
with Et.sub.2O to give the title compound as a free base (white
solid). The free base was dissolved in MeOH (5 mL), and HCl (1 M
Et.sub.2O, 2 equiv) was then added slowly. Solvent was removed in
vacuo to give the title compound as a beige solid in HCl salt (96
mg, 47% over 2 steps). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta.
ppm 9.14 (br. s, 1H), 8.89-8.82 (m, 1H), 8.79-8.71 (m, 1H), 8.40
(s, 1H), 8.31-8.21 (m, 1H), 7.68 (s, 1H), 7.59 (d, J=9.5 Hz, 1H),
7.23 (d, J=8.0 Hz, 1H), 6.06 (s, 1H), 3.56 (d, J=6.5 Hz, 2H),
2.88-2.79 (m, 1H), 2.40-2.31 (m, 1H), 2.29 (s, 3H), 2.26-2.18 (m,
2H), 1.99-1.89 (m, 2H), 1.37 (s, 3H), 0.85-0.76 (m, 2H), 0.63-0.53
(m, 2H); MS ESI [M+H].sup.+ 499.3, calcd for
[C.sub.28H.sub.30N.sub.6O.sub.3+H].sup.+499.2. HPLC purity: 99.5%
at 254 nm.
##STR00027##
[0154] B). Through PMB deprotection: A mixture of
N-cyclopropyl-4-(7-((((1s,3s)-3-hydroxy-3-methylcyclobutyl)methyl)(4-meth-
oxybenzyl)amino)-5-(pyridin-3-yloxy)pyrazolo[1,5-a]pyrimidin-3-yl)-2-methy-
lbenzamide (9.6 g, 15.5 mmol), TFA (50 mL) in DCE (70 mL) was
heated in an oil bath at 50.degree. C. for 4 h. After reaction
completion, solvent was removed in vacuo and the crude product was
dissolved in a mixture of MeOH/DCM (100 mL/25 mL). 2M
Na.sub.2CO.sub.3 (150 mL) was then added and the resulting mixture
was stirred at rt for 30 min. The reaction mixture was diluted with
DCM and the phases were separated. The aqueous phase was extracted
with DCM and the combined organic extracts were washed with water,
dried over MgSO.sub.4, filtered and concentrated. The crude product
was triturated and sonicated in a mixture of DCM/Et.sub.2O (10
mL/70 mL) to give the title compound as a off white solid in free
base (5.9 g, 77%). .sup.1H NMR (400 MHz, CD.sub.3OD) .delta. ppm
8.58-8.53 (m, 1H), 8.50-8.46 (m, 1H), 8.36 (s, 1H), 7.86-7.80 (m,
1H), 7.76-7.72 (m, 1H), 7.61-7.55 (m, 2H), 7.18 (d, J=8.0 Hz, 1H),
5.92 (s, 1H), 3.52 (d, J=6.8 Hz, 2H), 2.86-2.77 (m, 1H), 2.38-2.28
(m, 1H), 2.25 (s, 3H), 2.24-2.18 (m, 2H), 1.99-1.88 (m, 2H), 1.37
(s, 3H), 0.84-0.75 (m, 2H), 0.64-0.54 (in, 2H); MS ESI [M+H].sup.+
499.2, calcd for [C.sub.28H.sub.30N.sub.6O.sub.3+H]+499.2. HPLC
purity: 96.1% at 235 nm.
[0155] The following final compounds were synthesized similarly to
the syntheses of A1-4.
TABLE-US-00001 Example number MS calcd; MS ESI [M + H].sup.+ Salt
Form; Structure HPLC purity .sup.1H NMR ##STR00028## A5
[C.sub.28H.sub.30N.sub.6O.sub.3 + H].sup.+ 499.2; 499.3; free base;
98.9% at 254 nM (400 MHz, CD.sub.3OD) .delta. ppm 8.55 (dd, J =
2.8, 0.4 Hz, 1H), 8.47 (dd, J = 4.8, 1.6 Hz, 1H), 8.33 (s, 1 H),
7.83-7.80 (m, 1H), 7.72 (s, 1H), 7.58-7.55 (m, 2H), 7.18 (d, J =
8.0 Hz, 1H), 5.89 (s, 1H), 3.49 (d, J = 7.6 Hz, 2H), 2.87- 2.78 (m,
2H), 2.28-2.23 (m, 5H), 1.97-1.92 (m, 2H), 1.36 (s, 3H), 0.82-0.77
(m, 2H), 0.62-0.58 (m, 2H). ##STR00029## A6
[C.sub.27H.sub.28N.sub.6O.sub.3 + H].sup.+ 485.2; 485.3; HCl salt;
98.8% at 254 nm (400 MHz, CD.sub.3OD) .delta. ppm 9.13 (d, J = 2.3
Hz, 1H), 8.86 (d, J = 5.8 Hz, 1H), 8.77-8.69 (m, 1H), 8.40 (s, 1H),
8.26 (dd, J = 8.8, 5.8 Hz, 1H), 7.67 (s, 1H), 7.59 (d, J = 8.0 Hz,
1H), 7.24 (d, J = 8.0 Hz, 1H), 6.07 (s, 1H), 4.53- 4.39 (m, 1H),
3.57 (d, J = 8.0 Hz, 2H), 2.89- 2.79 (m, 1H), 2.76-2.61 (m, 1H),
2.30 (s, 3H), 2.29-2.21 (m, 2H), 2.21-2.08 (m, 2H), 0.85- 0.78 (m,
2H), 0.65-0.58 (m, 2H). ##STR00030## A8
[C.sub.27H.sub.34N.sub.6O.sub.2 + H].sup.+ 475.3; 475.4; HCl salt;
99.3% at 254 nm (400 MHz, CD.sub.3OD) .delta. ppm 8.22 (s, 1H),
7.51- 7.36 (m, 3H), 5.68 (s, 1H), 3.77 (s, 2H), 3.37- 3.33 (m, 2H),
2.93-2.84 (m, 1H), 2.47 (s, 3H), 2.07-1.95 (m, 4H), 1.93-1.72 (m,
4H), 1.34- 1.19 (m, 1H), 0.88-0.78 (m, 2H), 0.69-0.59 (m, 4H),
0.45-0.37 (m, 2H). ##STR00031## A11 [C.sub.27H.sub.28N.sub.6O.sub.3
+ H].sup.+ 485.2 485.2; HCl salt; 98.6% at 254 nm (400 MHz,
CD.sub.3OD) .delta. ppm 9.14 (s, 1H), 8.85 (d, J = 5.5 Hz, 1H),
8.79-8.72 (m, 1H), 8.40 (s, 1H), 8.30-8.21 (m, 1H), 7.68 (s, 1H),
7.59 (d, J = 7.8 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 6.06 (s, 1H),
4.17-4.06 (m, 1H), 3.54 (d, J = 6.5 Hz, 2H), 2.88-2.78 (m, 1H),
2.53-2.43 (m, 2H), 2.29 (s, 4H), 1.81-1.68 (m, 2H), 0.84-0.77 (m,
2H), 0.62-0.56 (m, 2H). ##STR00032## A12
[C.sub.27H.sub.34N.sub.6O.sub.2 + H].sup.+ 475.3; 475.4; HCl salt;
97.5% at 254 nm (400 MHz, CD.sub.3OD) .delta. ppm 8.18 (s, 1H),
7.55- 7.29 (m, 3H), 5.54 (br, s, 1H), 4.50-4.37 (m, 1H), 4.26-4.07
(m, 1H), 3.62 (d, J = 6.8 Hz, 2H), 2.92-2.83 (m, 1H), 2.75-2.61 (m,
1H), 2.46 (s, 3H), 2.31-2.02 (m, 6H), 1.92-1.56 (m, 6H), 0.91-0.75
(m, 2H), 0.71-0.52 (m, 2H). ##STR00033## A13
[C.sub.28H.sub.36N.sub.6O.sub.2 + H].sup.+ 489.3; 489.4 TFA salt;
98.2% at 254 nm (400 MHz, CD.sub.3OD) .delta. ppm 8.18 (s, 1H),
7.48- 7.36 (m, 3H), 5.52 (s, 1H), 4.23-4.10 (m, 1H), 3.65-3.57 (m,
2H), 2.93-2.75 (m, 2H), 2.46 (s, 3H), 2.31-2.21 (m, 2H), 2.20-2.11
(m, 3H), 2.06-1.89 (m, 2H), 1.89-1.50 (m, 6H), 1.37 (s, .3H),
0.87-0.79 (m, 2H), 0.68-0.56 (m, 2H).
Example B: TTK Inhibition Assay
[0156] Active TTK was purchased from Invitrogen as an amino
terminal GST fusion of full length human TTK. Amino terminal 6
histidine, sumo tagged human TTK (residues 1-275) was expressed in
E. coli, and purified to >95% homogeneity by Ni.sup.2+ agarose,
gel filtration, and ion exchange chromatography.
[0157] TTK activity was measured using an indirect ELISA detection
system. GST-TTK (0.68 nM) was incubated in the presence of either
16 .mu.M ATP (Sigma cat #A7699) or 100 .mu.M ATP, 50 mM Hepes pH
7.2, 1 mM EGTA, 10 mM MgCl.sub.2, and 0.1% Pluronic in a 96 well
microtitre plate pre-coated with amino terminal 6 histidine, sumo
tagged TTK (amino acid residues 1-275).
[0158] The reaction was allowed to proceed for 30 minutes, followed
by 5 washes of the plate with Wash Buffer (phosphate buffered
saline supplemented with 0.2% Tween 20), and incubation for 30
minutes with a 1:3000 dilution of primary antibody (Cell Signaling
cat #9381). The plate was washed 5 times with Wash Buffer,
incubated for 30 minutes in the presence of secondary antibody
coupled to horse radish peroxidase (BioRad cat #1721019, 1:3000
concentration), washed an additional 5 times with Wash Buffer, and
incubated in the presence of TMB substrate (Sigma cat #T0440). The
colourimetric reaction was allowed to continue for 5 minutes,
followed by addition of stop solution (0.5 N sulphuric acid), and
quantified by detection at 450 nm with either a monochromatic or
filter based plate reader (Molecular Devices M5 or Beckman DTX880,
respectively).
[0159] Compound inhibition was determined at either a fixed
concentration (10 .mu.M) or at a variable inhibitor concentration
(typically 0.5 .mu.M to 0.001 .mu.M in a 10 point dose response
titration). Compounds were pre-incubated in the presence of enzyme
for 5 minutes prior to addition of ATP and the activity remaining
quantified using the above described activity assay. The %
Inhibition of a compound was determined using the following
formula; % Inhibition=100.times.(1-(experimental value-background
value)/(high activity control-background value)). The IC.sub.50
value was determined using a non-linear 4 point logistic curve fit
(XLfit4, IDBS) with the formula; (A+(B/(1+((x/C){circumflex over (
)}D)))), where A=background value, B=range, C=inflection point,
D=curve fit parameter.
[0160] In Table 1 below, IC.sub.50 value ranges for exemplary
compounds are given using 100 uM ATP. The IC.sub.50 ranges are
indicated as "A," "B," and "C," for values less than or equal to
0.1 .mu.M; those greater than 0.1 .mu.M and less than or equal to
0.5 .mu.M; and those greater than 0.5 .mu.M, respectively.
IC.sub.50 ranges denoted with an asterisk indicated that 16 .mu.M
ATP (Sigma cat #A7699) was used in the assay.
Example C: Cancer Cell Line Data on Exemplary Compounds of the
Invention
[0161] Breast cancer cells (MDA-MB-468), colon cancer cells
(HCT116) and ovarian cancer cells (OVCAR-3) were seeded (1000 to
4000 in 80 .mu.l per well depending on the cell growth rate) into
96 well plates 24 hours before compound overlay. Compounds were
prepared as 10 mM stock solutions in 100% DMSO which were diluted
with DMEM (Dulbecco's Modified Eagle's Medium) cell growth Medium
(Invitrogen, Burlington, ON, Canada) containing 10% FBS (Fetal
Bovine Serum) to concentrations ranging from 50 nM to 250 .mu.M.
Aliquots (20 .mu.l) from each concentration were overlaid to 80
.mu.l of the pre-seeded cells in the 96 well plates to make final
concentrations of 10 nM to 50 .mu.M. The cells were cultured for 5
days before the Sulforhodamine B assay (SRB) was performed to
determine the compound's cell growth inhibition activity.
[0162] Sulforhodamine B (purchased from Sigma, Oakville, ON,
Canada) is a water-soluble dye that binds to the basic amino acids
of the cellular proteins. Thus, colorimetric measurement of the
bound dye provides an estimate of the total protein mass that is
related to the cell number. the cells are fixed in situ by gently
aspirating off the culture media and adding 50 .mu.l ice cold 10%
Trichloroacetic Acid (TCA) per well and incubate at 4.degree. C.
for 30-60 min, The plates are washed with H.sub.2O five times and
allowed to air dry for 5 min. Addition of 50 .mu.l 0.4% (w/v) SRB
solution in 1% (v/v) acetic acid to each well and incubation for 30
min at RT completes the staining reaction. Following staining,
plates are washed four times with 1% acetic acid to remove unbound
dye and then allowed to air dry for 5 min. The stain is solubilized
with 100 .mu.l of 10 mM Tris pH 10.5 per well. Absorbance is read
at 570 nm.
[0163] The percentage (%) of relative growth inhibition was
calculated by comparing to DMSO treated only cells (100%).
GI.sub.50's were determined for compounds with cytotoxic activity.
The GI.sub.50 was calculated using GraphPad PRISM software
(GraphPad Software, Inc., San Diego, Calif., USA). GI.sub.50
(growth inhibition) is the compound concentration that causes 50%
inhibition of cell growth.
[0164] In Table 1 below, GI.sub.50 value ranges for compound
examples against breast cancer cell lines (MDA-MB-468), colon
cancer cell lines (HCT116) and ovarian cancer cell lines (OVCAR-3)
are given. The example compounds demonstrated varying growth
inhibition/cell killing activity against cells of breast cancer,
colon cancer, and ovarian cancer. The GI.sub.50 ranges are
indicated as "A," "B," and "C," for values less than or equal to
0.1 .mu.M; those greater than 0.1 .mu.M and less than or equal to
0.5 .mu.M; and those greater than 0.5 .mu.M, respectively.
Example D: Colon and Ovarian Cancer Tumor-Initiating Cell Data of
Exemplary Compounds
[0165] Materials and Methods: Non-tissue or tissue cultured treated
T-75 flask and 96-well plates were purchased from VWR. Vitamin B-27
supplement, MEM NEAA (minimum essential medium non-essential amino
acids), sodium pyruvate, L-glutamine, N2 supplement,
penicillin-streptomycin and fungizone/amphotericin B were obtained
from Invitrogen. Lipid mixture, heparin and EGF were purchased from
Sigma; bFGF from BD Biosciences. Tumor Initiating Cells (TICs) from
colon were routinely maintained using non-tissue cultured treated
T-75 flasks in DMEM:F12 medium containing 0.2.times.B-27
supplement, 4 ug/ml heparin, 1.times.MEM NEAA, 1.lamda.sodium
pyruvate, 1 mM glutamine, 10 pg/ul bFGF, 20 pg/ul EGF, 1.times. N2
supplement, lipid mixture, penicillin-streptomycin and
fungizone/amphotericin B. Ovarian TICs were routinely maintained
using tissue cultured treated T-75 flasks in DMEM:F12 medium
containing 1.times.B-27 supplement, 4 ug/ml heparin, 20 pg/ul bFGF,
20 pg/ul EGF and penicillin-streptomycin.
[0166] Assay Protocol: Compounds described herein were dissolved in
DMSO and further diluted in cell culture medium for GI.sub.50
determination. Colon TICs were trypsinized and seeded into
non-tissue cultured treated 96-well plates with 4,000 cells/well.
After 24 h, compound was added into the cell culture at different
concentrations, and the final concentration of DMSO was adjusted to
0.1%. Cells were then cultured at 37.degree. C. for 9 days. Ovarian
TICs were trypsinized and seeded into tissue cultured treated
96-well plates with 1,000 cells/well. After 24 h, compound was
added into the cell culture at different concentrations, and the
final concentration of DMSO was adjusted to 0.1%. Cells were then
cultured at 37.degree. C. for 6 days. Cell viability was assessed
by Alamar Blue assay: 10 ul of Alamar Blue was added into each
well. After 4 hours incubation at 37.degree. C., fluorescence was
recorded at excitation 544 and emission 590. GI.sub.50 (Growth
inhibition) was calculated using GraphPad Prism 4.0 software. Cell
growth inhibition data for compounds described herein is tabulated
below.
[0167] In Table 1 below, GI.sub.50 value ranges for compound
examples against TICs (Colon 12 and Ovarian 2393A) are given. The
GI.sub.50 ranges are indicated as "A," "B," and "C," for values
less than or equal to 0.1 .mu.M; those greater than 0.1 .mu.M and
less than or equal to 0.5 .mu.M; and those greater than 0.5 .mu.M,
respectively.
TABLE-US-00002 TABLE 1 In vitro activity of Compound Examples
Cancer Cell Line Tumor Initiating TTK GI.sub.50 Range Cell
GI.sub.50 Range Example IC.sub.50 MDA- Ovarian # Range MB-468
HCT116 OVCAR-3 2393A Colon 12 A1 A A A A A A A2 A A A A ND ND A3 A
A A A ND ND A4 A A A A A ND A5 A A A B ND ND A6 A A A A A A A8 A B
B C ND ND A11 A A A A ND ND A12 A A A A ND ND A13 A B A B ND ND
ND--not determined
* * * * *