1. A compound that is a chemical analog of adenosine receptor antagonist
8-Ethoxy-9-ethyl-9H-purin-6-amine with general formula of Formula II
##STR00013## where R3 is a heteromethyl, cycloalkyl, or tetrahydrofuran.
2. The compound of claim 1, wherein R3 is difluoro methyl, cyclopropyl,
cyclobutyl, or .beta.-tetrahydrofuran.
3. A method for treating a breast cancer or lung cancer patient
comprising administering to the patient an effective amount of one or
more compounds of claim 1.
Description
[0001] This Application is a continuation application of U.S. patent
application Ser. No. 16/267,106, filed Feb. 4, 2019, which is a
continuation application of U.S. patent application Ser. No. 15/544,056,
filed Jul. 17, 2017 (issued as U.S. Pat. No. 10,214,529), which is a
national phase application under 35 U.S.C. .sctn. 371 of International
Application No. PCT/US2016/013645, filed Jan. 15, 2016, which claims
priority to U.S. Provisional Application Ser. No. 62/104,705 filed Jan.
17, 2015, each of which is incorporated herein by reference in its
entirety.
BACKGROUND
[0002] The bone is the most common site of metastasis in patients with
advanced cancers including breast and prostate cancers (Jin et al. (2011)
Int. J. Cancer 128, 2545-2561; Kohno, (2008) Int. J Clin. Oncol. 13,
18-23). Bone metastases are major, potentially fatal complications in
patients with advanced cancers. Almost all patients with skeletal
metastases have significantly decreased quality of life due to intense
pain, pathological fractures, spinal cord compression, and metabolic
complications (Welch et al. (2003) J. Musculoskelet. Neuronal Interact.
3, 30-38). In fact, post-mortem studies have shown that over 70% of
breast cancer patients exhibited skeletal metastases, and only 20% of
these patients are still alive five years after the discovery of the
metastases (Roodman (2004) N. Engl. J Med 350, 1655-1664; Welch et al.
(2003) J. Musculoskelet. Neuronal Interact. 3, 30-38). The high affinity
that cancer has for bone is explained by the "seed-and-soil hypothesis",
which was proposed over a century ago (Paget (1889) Lancet 1, 571-573).
It reveals that bone tissues are preferred sites of cancer metastasis due
to their microenvironment, which provides a fertile setting in which
tumor cells can grow. Many features, such as increased blood flow as well
as the release of growth factors from cells in the bone matrix, account
for the frequency of bone metastases (van der Pluijm et al. (2001) J.
Bone Miner. Res. 16, 1077-1091). Thus far, the critical factors and
mechanisms responsible for bone metastases are largely unknown.
[0003] Bisphosphonate drugs are used to treat bone cancer metastasis and
result in decreased tumor growth, reduced bone destruction, and reduced
pain (Brown and Guise (2007) Cur. Osteopor. Rep. 5, 120-127).
Bisphosphonate therapy is associated with adverse side effects, which
include atrial fibrillation; arthralgia and osteonecrosis of the jaw; and
ophthalmic, dermatologic and renal complications; as well as
medication-induced fractures (Junquera et al. (2009) Am. J. Otolaryngol.
30, 390-395; Truong et al. (2010) J. Am. Acad. Dermatol. 62, 672-676).
Despite advances in the diagnosis and treatment of bone metastasis from
solid tumors, the mechanism of how bisphosphonate treatment inhibits bone
metastasis at the molecular level remains to be established.
[0004] Adenosine receptor antagonist analog compounds can be used for
treatment of cancer (WO2014074529). However, there still remains a need
for additional non-hydrolysable ATP analog compounds and adenosine
receptor antagonists.
SUMMARY
[0005] Certain embodiments are directed to non-hydrolysable ATP analogs
that inhibit migration and growth of cancer cells. The term
non-hydrolysable ATP analog refers to an ATP analog that is not
effectively hydrolyzed by ATPase, i.e., the analog is hydrolyzed, if at
all, at a rate that is less than 5, 1, or 0.1% of the rate of ATP
hydrolysis by ATPase. Certain embodiments are directed to various
chemical analogs of the non-hydrolysable ATP analog adenosine
5'-[.gamma.-thio]triphosphate (ATP.gamma.S). These chemicals inhibit
cancer cell migration and growth. Certain embodiments are directed to
chemical analogs of the non-hydrolysable adenosine ATP analog
5'-[.gamma.-thio]triphosphate (ATP.gamma.S) having the general formula of
Formula: I, including compounds P1-P6 (Table 1)
##STR00001##
where R.sub.1 and R.sub.2 are selected independently from hydrogen (H),
cyano (CN), C1 to C3 alkyl, halogen (fluoro (F), chloro (Cl), bromo (Br),
or iodo (I)), or a trifluoromethyl (CF.sub.3). In certain aspects R1 is
selected from hydrogen, cyano, C1 to C3 alkyl, halogen (fluoro (F),
chloro (Cl), bromo (Br), or iodo (I)), or a trifluoromethyl, and R2 is
hydrogen or fluoro. In a further aspect R1 is cyano and R2 is H, R1 is H
and R2 is H, R1 is trifluoromethyl and R2 is H, R1 is fluoro and R2 is H,
R1 is methyl and R2 is H, and R1 is fluoro and R2 is fluoro.
[0006] Certain embodiments are directed to administration of one or more
compounds of Formula Ito treat cancer. The compounds can be administered
alone or in combination with other anti-cancer therapies.
[0007] Adenosine exposure can promote cancer cell growth and migration,
and adenosine is produced by the metabolism of ATP. Certain embodiments
are directed to a number of chemical analogs of adenosine receptor
antagonist 8-Ethoxy-9-ethyl-9H-purin-6-amine (ANR94, A2A antogonist).
These compounds are inhibitors of cancer cell migration and growth. In
certain aspects the chemical analogs of adenosine receptor antagonist
8-Ethoxy-9-ethyl-9H-purin-6-amine have a general formula of Formula II,
including compounds P7-P10 (Table 1)
##STR00002##
Certain aspects are directed to compounds of Formula II, where R.sub.3 is
selected from dihalomethyl, C3 to C5 cycloalkyl, or tetrahydrofuran. In
certain aspects R3 is difluoromethyl, cyclopropyl, cyclobutyl, or
.beta.-tetrahydrofuran.
[0008] Certain embodiments are directed to administration of one or more
compounds having a formula of Formula II to treat cancer. The compounds
can be administered alone or in combination with compounds with Formula I
and/or other anti-cancer therapies.
[0009] In certain aspects one or more compounds having a formula of
Formula I and/or Formula II are administered to a subject in need of an
anti-cancer treatment. In certain aspects the compounds of Formula I
and/or Formula II are administered within 1, 5, 10, 20, 30, or 60 minutes
or hours of each other. In a further aspect the compounds are
administered concurrently. In another aspect one or more compounds of
Formula I are administered before, during, or after administration of one
or more compounds of Formula II.
[0010] In certain aspects a subject or patient has bladder, blood, bone,
bone marrow, brain, breast, colorectal, esophagus, gastrointestinal,
head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate,
skin, stomach, testicular, tongue, or uterine cancer. In a further aspect
the cancer is a lung, breast, or prostate cancer. In particular aspects
the cancer is a metastatic cancer, such as a bone metastasis. In certain
aspects the cancer is identified as being at risk for or having a
propensity for metastasis or there is no indication that the cancer has
yet metastasized. In certain aspects identification of a cancer at risk
of metastasis is based on assessment of a tumor biopsy.
[0011] In certain embodiments bisphosphonate drugs can be explicitly
excluded from the claimed invention due to their potential in vivo
toxicity.
[0012] As used herein, an "inhibitor" can be a chemical compound that can
reduce the activity or function of a protein. An inhibitor, for example,
can inhibit directly or indirectly the activity of a protein. Direct
inhibition can be accomplished, for example, by binding to a protein and
thereby preventing the activity of the protein, or by inhibiting an
enzymatic or other activity of the protein competitively,
non-competitively, or uncompetitively. Indirect inhibition can be
accomplished, for example, by binding to a protein's intended target,
such as a receptor or binding partner, thereby blocking or reducing
activity of the protein.
[0013] The term "effective amount" means an amount effective, at dosages
and for periods of time necessary, to achieve the desired therapeutic or
prophylactic result. An "effective amount" of an anti-cancer agent in
reference to decreasing cancer cell growth or migration, means an amount
capable of decreasing, to some extent, the growth of some cancer or tumor
cells, or the inhibition of the ability of a cancer or tumor cell to
migrate or invade non-tumor tissue, such as bone. The term includes an
amount capable of invoking a growth inhibitory, cytostatic, and/or
cytotoxic effect, and/or apoptosis of the cancer or tumor cells.
[0014] A "therapeutically effective amount" in reference to the treatment
of cancer, means an amount capable of invoking one or more of the
following effects: (1) inhibition, to some extent, of cancer or tumor
growth, including slowing down growth or complete growth arrest; (2)
reduction in the number of cancer or tumor cells; (3) reduction in tumor
size; (4) inhibition (i.e., reduction, slowing down, or complete
stopping) of cancer or tumor cell infiltration into peripheral organs;
(5) inhibition (i.e., reduction, slowing down, or complete stopping) of
metastasis; (6) enhancement of anti-tumor immune response, which may, but
is not required to, result in the regression or rejection of the tumor,
or (7) relief, to some extent, of one or more symptoms associated with
the cancer or tumor. The therapeutically effective amount may vary
according to factors such as the disease state, age, sex and weight of
the individual and the ability of one or more anti-cancer agents to
elicit a desired response in the individual. A "therapeutically effective
amount" is also one in which any toxic or detrimental effects are
outweighed by the therapeutically beneficial effects.
[0015] The phrases "treating cancer" and "treatment of cancer" mean to
decrease, reduce, or inhibit the replication of cancer cells; decrease,
reduce or inhibit the spread (formation of metastases) of cancer;
decrease tumor size; decrease the number of tumors (i.e. reduce tumor
burden); lessen or reduce the number of cancerous cells in the body;
prevent recurrence of cancer after surgical removal or other anti-cancer
therapies; or ameliorate or alleviate the symptoms of the disease caused
by the cancer.
[0016] Other embodiments of the invention are discussed throughout this
application. Any embodiment discussed with respect to one aspect of the
invention applies to other aspects of the invention as well and vice
versa. Each embodiment described herein is understood to be embodiments
of the invention that are applicable to all aspects of the invention. It
is contemplated that any embodiment discussed herein can be implemented
with respect to any method or composition of the invention, and vice
versa. Furthermore, compositions and kits of the invention can be used to
achieve methods of the invention.
[0017] The use of the word "a" or "an" when used in conjunction with the
term "comprising" in the claims and/or the specification may mean "one,"
but it is also consistent with the meaning of "one or more," "at least
one," and "one or more than one."
[0018] Throughout this application, the term "about" is used to indicate
that a value includes the standard deviation of error for the device or
method being employed to determine the value.
[0019] The use of the term "or" in the claims is used to mean "and/or"
unless explicitly indicated to refer to alternatives only or the
alternatives are mutually exclusive, although the disclosure supports a
definition that refers to only alternatives and "and/or."
[0020] As used in this specification and claim(s), the words "comprising"
(and any form of comprising, such as "comprise" and "comprises"),
"having" (and any form of having, such as "have" and "has"), "including"
(and any form of including, such as "includes" and "include") or
"containing" (and any form of containing, such as "contains" and
"contain") are inclusive or open-ended and do not exclude additional,
unrecited elements or method steps.
[0021] Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It should
be understood, however, that the detailed description and the specific
examples, while indicating specific embodiments of the invention, are
given by way of illustration only, since various changes and
modifications within the spirit and scope of the invention will become
apparent to those skilled in the art from this detailed description.
DESCRIPTION OF THE DRAWINGS
[0022] The following drawings form part of the present specification and
are included to further demonstrate certain aspects of the present
invention. The invention may be better understood by reference to one or
more of these drawings in combination with the detailed description of
the specification embodiments presented herein.
[0023] FIG. 1. Transwell cell migration assay with MDA-MB-231 human breast
cancer cells.
[0026] Certain embodiments are directed to compounds having a chemical
formula of Formula I, for example P1, P2, P3, P4, P4, P5, or P6 (Table
1).
[0027] Other embodiments are directed to compounds having a chemical
formula of Formula II, for example P7, P8, P9, or P10, which are chemical
analogs of adenosine receptor antagonist
8-Ethoxy-9-ethyl-9H-purin-6-amine (ANR94, A2A antogonist) (Table 1).
Studies have shown that all 10 compounds have inhibitory effects on cell
migration. In Transwell cell migration assay of MDA-MB-231 human breast
cancer cells, all 10 compounds, especially P2, P3, P4, P5 and P9 showed
inhibitory effects on cell migration. At 50 .mu.M, none of the compounds
exerted any toxicity to the cell.
[0028] Soft agar assays have been performed to determine anchorage
independent cell growth of MDA-MB-231 cells with compounds P1-P10 and
compounds P2 and P3 were found to be most effective. There was 30% and
65% decrease in cell colonies as compared to control for P2 and P3
respectively (FIG. 2).
[0029] In addition, mammary fat pad xenograft assays with MDA-MB-231 cells
were performed. MDA-MB-231 cells were xenografted in mammary fat pad of
null mice. After tumor nodules appeared, test compounds (e.g., P3) were
injected into these mice (500 .mu.l of a 400 .mu.M solution). The tumor
size for mice receiving a test compound were compared with control mice
(i.e., mice administered vehicle without test compound). After 15 days
there was more than 50% reduction in the tumor size for mice received P3
compared to those that did not (FIG. 3).
[0030] In certain aspect compounds having Formula I and/or Formula II
(e.g., compounds P1-P10) can be used to inhibit proliferation and/or
migration of cancer cells. In certain aspects the cancer is a bladder,
blood, bone, bone marrow, brain, breast, colorectal, esophagus,
gastrointestine, head, kidney, liver, lung, nasopharynx, neck, ovary,
pancreas, prostate, skin, stomach, testicular, tongue, or uterine cancer.
In a further aspect the cancer is breast cancer. In still a further
aspect the cancer is prostate cancer. In particular embodiments the
cancer is metastatic cancer, e.g., cancer that has or is at risk of
metastasizing or migrating to the bone.
[0031] In certain embodiments, the invention also provides compositions
comprising one or more compound having the chemical formula of Formula I
and/or Formula II (e.g., P1-P2) in a pharmaceutically acceptable
formulation. Thus, the use of one or more compound described herein in
the preparation of a medicament is also included. Such compositions can
be used in the treatment of a variety of cancers. In certain embodiments
the treatment is for a metastatic cancer, e.g., lung, breast, or prostate
cancer.
[0032] The compounds described herein may be formulated into therapeutic
compositions in a variety of dosage forms such as, but not limited to,
liquid solutions or suspensions, tablets, pills, powders, suppositories,
polymeric microcapsules or microvesicles, liposomes, and injectable or
infusible solutions. The preferred form depends upon the mode of
administration and the particular disease targeted. The compositions also
preferably include pharmaceutically acceptable vehicles, carriers, or
adjuvants, well known in the art.
[0033] Acceptable formulation components for pharmaceutical preparations
are nontoxic to recipients at the dosages and concentrations employed. In
addition to the compounds described herein, compositions may contain
components for modifying, maintaining, or preserving, for example, the
pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility,
stability, rate of dissolution or release, adsorption, or penetration of
the composition. Suitable materials for formulating pharmaceutical
compositions include, but are not limited to, amino acids (such as
glycine, glutamine, asparagine, arginine or lysine); antimicrobials;
antioxidants (such as ascorbic acid, sodium sulfite or sodium
hydrogen-sulfite); buffers (such as acetate, borate, bicarbonate,
Tris-HCl, citrates, phosphates or other organic acids); bulking agents
(such as mannitol or glycine); chelating agents (such as ethylenediamine
tetraacetic acid (EDTA)); complexing agents (such as caffeine,
polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;
disaccharides; and other carbohydrates (such as glucose, mannose or
dextrins); proteins (such as serum albumin, gelatin or immunoglobulins);
coloring, flavoring and diluting agents; emulsifying agents; hydrophilic
polymers (such as polyvinylpyrrolidone); low molecular weight
polypeptides; salt-forming counter ions (such as sodium); preservatives
(such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic
acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol
or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol);
suspending agents; surfactants or wetting agents (such as pluronics, PEG,
sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80,
triton, tromethamine, lecithin, cholesterol, tyloxapal); stability
enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents
(such as alkali metal halides, preferably sodium or potassium chloride,
mannitol sorbitol); delivery vehicles; diluents; excipients and/or
pharmaceutical adjuvants. (see Remington's Pharmaceutical Sciences, 18 th
Ed., (A. R. Gennaro, ed.), 1990, Mack Publishing Company), hereby
incorporated by reference.
[0034] Formulation components are present in concentrations that are
acceptable to the site of administration. Buffers are advantageously used
to maintain the composition at physiological pH or at a slightly lower
pH, typically within a pH range of from about 4.0 to about 8.5, or
alternatively, between about 5.0 to 8.0. Pharmaceutical compositions can
comprise TRIS buffer of about pH 6.5-8.5, or acetate buffer of about pH
4.0-5.5, which may further include sorbitol or a suitable substitute
therefor.
[0035] The pharmaceutical composition to be used for in vivo
administration is typically sterile. Sterilization may be accomplished by
filtration through sterile filtration membranes. If the composition is
lyophilized, sterilization may be conducted either prior to or following
lyophilization and reconstitution. The composition for parenteral
administration may be stored in lyophilized form or in a solution. In
certain embodiments, parenteral compositions are placed into a container
having a sterile access port, for example, an intravenous solution bag or
vial having a stopper pierceable by a hypodermic injection needle, or a
sterile pre-filled syringe ready to use for injection.
[0036] The above compositions can be administered using conventional modes
of delivery including, but not limited to, intravenous, intraperitoneal,
oral, intralymphatic, subcutaneous administration, intraarterial,
intramuscular, intrapleural, intrathecal, and by perfusion through a
regional catheter. Local administration to a tumor or a metastasis in
question is also contemplated by the present invention. When
administering the compositions by injection, the administration may be by
continuous infusion or by single or multiple boluses. For parenteral
administration, the agents may be administered in a pyrogen-free,
parenterally acceptable aqueous solution comprising the desired compound
in a pharmaceutically acceptable vehicle. A particularly suitable vehicle
for parenteral injection is sterile distilled water in which one or more
anti-cancer agents are formulated as a sterile, isotonic solution,
properly preserved.
[0037] Once the pharmaceutical composition of the invention has been
formulated, it may be stored in sterile vials as a solution, suspension,
gel, emulsion, solid, or as a dehydrated or lyophilized powder. Such
formulations may be stored either in a ready-to-use form or in a form
(e.g., lyophilized) that is reconstituted prior to administration.
[0038] If desired, stabilizers that are conventionally employed in
pharmaceutical compositions, such as sucrose, trehalose, or glycine, may
be used. Typically, such stabilizers will be added in minor amounts
ranging from, for example, about 0.1% to about 0.5% (w/v). Surfactant
stabilizers, such as TWEEN.RTM.-20 or TWEEN.RTM.-80 (ICI Americas, Inc.,
Bridgewater, N.J., USA), may also be added in conventional amounts.
[0039] The components used to formulate the pharmaceutical compositions
are preferably of high purity and are substantially free of potentially
harmful contaminants (e.g., at least National Food (NF) grade, generally
at least analytical grade, and more typically at least pharmaceutical
grade). Moreover, compositions intended for in vivo use are usually
sterile. To the extent that a given compound must be synthesized prior to
use, the resulting product is typically substantially free of any
potentially toxic agents. Compositions for parental administration are
also sterile, substantially isotonic and made under GMP conditions.
[0040] For the compounds described herein, alone or as part of a
pharmaceutical composition, such doses are between about 0.001 mg/kg and
1 mg/kg body weight, preferably between about 1 and 100 .mu.g/kg body
weight, most preferably between 1 and 10 .mu.g/kg body weight. In certain
aspects, compounds described herein can be administered by infusion to
patients in daily dosages at rates ranging from 20, 25, 30, 35, 40 to 30,
35, 40, 45, 50 .mu.g/kg/min (including all values and ranges there
between) for up to 8 hours, including 1, 2, 3, 4, 5, 6, 7, or 8 hours.
Compounds described herein can be administered orally at about 1, 10, 20,
30, 40, 50, 60 to 50, 60, 70, 80 90, 100 .mu.g/kg or mg/kg of body weight
per day. In certain aspects the compounds described herein can be
administered at about 0.01 to 10 mg/kg of body weight per day.
[0041] Therapeutically effective doses will be easily determined by one of
skill in the art and will depend on the severity and course of the
disease, the patient's health and response to treatment, the patient's
age, weight, height, sex, previous medical history and the judgment of
the treating physician.
[0042] In some methods of the invention, the cancer cell is a tumor cell.
The cancer cell may be in a patient. The patient may have a solid tumor.
In such cases, embodiments may further involve performing surgery on the
patient, such as by resecting all or part of the tumor. Compositions may
be administered to the patient before, after, or at the same time as
surgery. In additional embodiments, patients may also be administered
directly, endoscopically, intratracheally, intratumorally, intravenously,
intralesionally, intramuscularly, intraperitoneally, regionally,
percutaneously, topically, intrarterially, intravesically, or
subcutaneously. Therapeutic compositions may be administered 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more times,
and they may be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, or 1, 2, 3, 4, 5,
6, 7 days, or 1, 2, 3, 4, 5 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12 months.
[0043] Methods of treating cancer may further include administering to the
patient chemotherapy or radiotherapy, which may be administered more than
one time. Chemotherapy includes, but is not limited to, cisplatin (CDDP),
carboplatin, procarbazine, mechlorethamine, cyclophosphamide,
camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea,
dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,
mitomycin, etoposide (VP16), tamoxifen, taxotere, taxol, transplatinum,
5-fluorouracil, vincristin, vinblastin, methotrexate, gemcitabine,
oxaliplatin, irinotecan, topotecan, or any analog or derivative variant
thereof. Radiation therapy includes, but is not limited to, X-ray
irradiation, UV-irradiation, .gamma.-irradiation, electron-beam
radiation, or microwaves. Moreover, a cell or a patient may be
administered a microtubule stabilizing agent, including, but not limited
to, taxane, as part of methods of the invention. It is specifically
contemplated that any of the compounds or derivatives or analogs, can be
used with these combination therapies.
[0044] Various chemical definitions related to such compounds are provided
as follows.
[0045] As used herein, the term the term "fluro" designates --F; the term
"cyano" means --CN; the term "methyl" means --CH.sub.3; the term
"difluromethyl" means --CF.sub.2H; the term "triflurometyl" means
--CF.sub.3; the term "cyclopropyl" means a three membered saturated
cycloalkyl ring; the term "cyclobutyl" means four membered saturated
cycloalkyl ring; and the term ".beta.-tetrahydrofuran" means a five
membered saturated heterocyclyl ring with O as heteroatom and is
substituted at the .beta. carbon from the heteroatom.
[0046] As used herein, the term "halo" designates --F, --Cl, --Br or --I;
the term "mercapto" means --SH; the term "cyano" means --CN; the term
"azido" means --N.sub.3; and the term "hydroxy" means --OH.
[0047] The term "alkyl," by itself or as part of another substituent,
means, unless otherwise stated, a linear (i.e. unbranched) or branched
carbon chain, which may be fully saturated, mono- or polyunsaturated. An
unsaturated alkyl group is one having one or more double bonds or triple
bonds. Saturated alkyl groups include those having one or more
carbon-carbon double bonds (alkenyl) and those having one or more
carbon-carbon triple bonds (alkynyl). The groups, --CH.sub.3 (Me),
--CH.sub.2CH.sub.3 (Et), --CH.sub.2CH.sub.2CH.sub.3 (n-Pr),
--CH(CH.sub.3).sub.2 (iso-Pr), --CH.sub.2CH.sub.2CH.sub.2CH.sub.3 (n-Bu),
--CH(CH.sub.3)CH.sub.2CH.sub.3 (sec-butyl), --CH.sub.2CH(CH.sub.3).sub.2
(iso-butyl), --C(CH.sub.3).sub.3 (tert-butyl),
--CH.sub.2C(CH.sub.3).sub.3 (neo-pentyl), are all non-limiting examples
of alkyl groups.
[0048] The term "heteroalkyl," by itself or in combination with another
term, means, unless otherwise stated, a linear or branched chain having
at least one carbon atom and at least one heteroatom selected from the
group consisting of O, N, S, P, and Si. In certain embodiments, the
heteroatoms are selected from the group consisting of O and N. The
heteroatom(s) may be placed at any interior position of the heteroalkyl
group or at the position at which the alkyl group is attached to the
remainder of the molecule. Up to two heteroatoms may be consecutive. The
following groups are all non-limiting examples of heteroalkyl groups:
trifluoromethyl, --CH.sub.2F, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2OH,
--CH.sub.2OCH.sub.3, --CH.sub.2OCH.sub.2CF.sub.3,
--CH.sub.2OC(O)CH.sub.3, --CH.sub.2NH.sub.2, --CH.sub.2NHCH.sub.3,
--CH.sub.2N(CH.sub.3).sub.2, --CH.sub.2CH.sub.2Cl, --CH.sub.2CH.sub.2OH,
CH.sub.2CH.sub.2OC(O)CH.sub.3,
--CH.sub.2CH.sub.2NHCO.sub.2C(CH.sub.3).sub.3, and
--CH.sub.2Si(CH.sub.3).sub.3.
[0049] The terms "cycloalkyl" and "heterocyclyl," by themselves or in
combination with other terms, means cyclic versions of "alkyl" and
"heteroalkyl", respectively. Additionally, for heterocyclyl, a heteroatom
can occupy the position at which the heterocycle is attached to the
remainder of the molecule.
[0050] The term "aryl" means a polyunsaturated, aromatic, hydrocarbon
substituent. Aryl groups can be monocyclic or polycyclic (e.g., 2 to 3
rings that are fused together or linked covalently). The term
"heteroaryl" refers to an aryl group that contains one to four
heteroatoms selected from N, O, and S. A heteroaryl group can be attached
to the remainder of the molecule through a carbon or heteroatom.
Non-limiting examples of aryl and heteroaryl groups include phenyl,
1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,
3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,
4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,
5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,
2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,
4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,
1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl,
and 6-quinolyl. Substituents for each of the above noted aryl and
heteroaryl ring systems are selected from the group of acceptable
substituents described below.
[0051] Various groups are described herein as substituted or unsubstituted
(i.e., optionally substituted). Optionally substituted groups may include
one or more substituents independently selected from: halogen, nitro,
cyano, hydroxy, amino, mercapto, formyl, carboxy, oxo, carbamoyl,
substituted or unsubstituted alkyl, substituted or unsubstituted
heteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl).sub.2amino,
alkylsulfinyl, alkyl sulfonyl, arylsulfonyl, substituted or unsubstituted
cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or
unsubstituted aryl, and substituted or unsubstituted heteroaryl. In
certain aspects the optional substituents may be further substituted with
one or more substituents independently selected from: halogen, nitro,
cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl,
unsubstituted alkyl, unsubstituted heteroalkyl, alkoxy, alkylthio,
alkylamino, (alkyl).sub.2amino, alkylsulfinyl, alkyl sulfonyl, aryl
sulfonyl, unsubstituted cycloalkyl, unsubstituted heterocyclyl,
unsubstituted aryl, or unsubstituted heteroaryl. Exemplary optional
substituents include, but are not limited to: --OH, oxo(.dbd.O), --Cl,
--F, Br, C.sub.1-4alkyl, phenyl, benzyl, --NH.sub.2,
--NH(C.sub.1-4alkyl), --N(C.sub.1-4alkyl).sub.2, --NO.sub.2,
--S(C.sub.1-4alkyl), --SO.sub.2(C.sub.1-4alkyl),
--CO.sub.2(C.sub.1-4alkyl), and --O(C.sub.1-4alkyl).
[0052] The term "pharmaceutically acceptable salts," as used herein,
refers to salts of compounds of this invention that are substantially
non-toxic to living organisms. Typical pharmaceutically acceptable salts
include those salts prepared by reaction of a compound of this invention
with an inorganic or organic acid, or an organic base, depending on the
substituents present on the compounds of the invention.
[0053] Non-limiting examples of inorganic acids which may be used to
prepare pharmaceutically acceptable salts include: hydrochloric acid,
phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid,
phosphorous acid and the like. Examples of organic acids which may be
used to prepare pharmaceutically acceptable salts include: aliphatic
mono- and dicarboxylic acids, such as oxalic acid, carbonic acid, citric
acid, succinic acid, phenyl-heteroatom-substituted alkanoic acids,
aliphatic and aromatic sulfuric acids and the like. Pharmaceutically
acceptable salts prepared from inorganic or organic acids thus include
hydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate, bisulfate,
sulfite, bisulfate, phosphate, monohydrogenphosphate,
dihydrogenphosphate, metaphosphate, pyrophosphate, hydroiodide, hydro
fluoride, acetate, propionate, formate, oxalate, citrate, lactate,
p-toluenesulfonate, methanesulfonate, maleate, and the like.
[0054] Suitable pharmaceutically acceptable salts may also be formed by
reacting the agents of the invention with an organic base such as
methylamine, ethylamine, ethanolamine, lysine, ornithine and the like.
Pharmaceutically acceptable salts include the salts formed between
carboxylate or sulfonate groups found on some of the compounds of this
invention and inorganic cations, such as sodium, potassium, ammonium, or
calcium, or such organic cations as isopropylammonium, trimethylammonium,
tetramethylammonium, and imidazolium.
[0055] It should be recognized that the particular anion or cation forming
a part of any salt of this invention is not critical, so long as the
salt, as a whole, is pharmacologically acceptable.
[0056] Additional examples of pharmaceutically acceptable salts and their
methods of preparation and use are presented in Handbook of
Pharmaceutical Salts: Properties, Selection and Use (2002), which is
incorporated herein by reference.
[0057] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method or
composition of the invention, and vice versa. Furthermore, compositions
of the invention can be used to achieve methods of the invention.
I. EXAMPLES
[0058] The following examples as well as the figures are included to
demonstrate preferred embodiments of the invention. It should be
appreciated by those of skill in the art that the techniques disclosed in
the examples or figures represent techniques discovered by the inventor
to function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However, those
of skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific embodiments
which are disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention.
[0059] A. Materials & Methods
[0060] Cell lines and cell cultures. MDA-MB-231 cells were grown in
McCoy's 5A Modified Media (Gibco) supplemented with 10% FBS (Hyclone).
Py8119 cells were grown in F12K nutrient media (Gibco) supplemented with
5% Fetal Clone II (Fisher Scientific). All cell lines were incubated in a
5% CO.sub.2 incubator at 37.degree. C.
[0061] Cell migration assay. Migration assays were performed in transwell
membrane filter inserts in 24-well tissue culture plates (BD Biosciences
San Jose, Calif., USA). The transwell membrane filter inserts contained
6.5-mm diameter, 8-.mu.m pore size, 10-nm thick polycarbonate membranes.
Five-hundred microliter breast cancer cell suspensions were added to the
upper side of the inserts at a density of 10.times.10.sup.4 cells/insert
and 750 .mu.l CM with or without other compounds was added to the lower
wells. Cells were incubated at 37.degree. C. for 18-20 hr. Cells that did
not migrate through the filters were removed using cotton swabs, and
cells that migrated through the inserts were fixed and stained with Hema
3 Stat Pack (Fisher Scientific). The number of migrated cells in 5 fields
of view per insert was counted under a light microscope at magnification
10.times..
[0062] Soft agar colony formation assay. For anchorage-independent cell
growth, MDA-MB-231 cells were plated in 0.4% agarose with complete medium
supplemented with 50 .mu.M compound (P1 to P10) on top of a 0.8% agarose
base supplemented with complete medium. Cells were maintained for about 2
weeks before staining with p-iodonitrotetrazolium violet (Sigma-Aldrich,
St. Louis, Mo.). Images were captured by using a scanner and the numbers
of colonies were counted.
[0063] Animals. Four-week-old female athymic nude mice (Harlan
Sprague-Dawley, Indianapolis, Ind., USA) were used for the mammary fat
pad injections. Four- to five-week old female C57bl/6 mice were used for
the intratibial injections. Animals were maintained under the care and
supervision of the Laboratory Animal Research facility at the University
of Texas Health Science Center, San Antonio, Tex. The animal protocol was
approved and monitored by the Institutional Animal Care and Use
Committee.
[0064] In vivo xenograft experiment. MDA-MB-231 cells were injected
subcutaneously in the mammary fat pad of 4-week-old female nu/nu athymic
nude mice. Each mouse received bilateral subcutaneous inoculation in both
the left and right inguinal mammary fat pad areas with 100 .mu.l of cell
suspension containing .about.1.times.10.sup.7 cells/ml in serum-free
media. Animals were randomly assigned to 3 different groups, and solid
tumors were allowed to form up to about 5 mm.sup.3 volume before
treatments began. Compound P3 400 .mu.mol/500 .mu.l saline, or saline as
a control was administered intraperitoneally (IP) three times a week for
3 weeks. The growth of xenograft tumors was monitored twice a week and
tumor size was measured with a caliper in two dimensions. Tumor volumes
were calculated with the equation V=(L.times.W.sup.2).times.0.5
(mm.sup.3), where L is length and W is width of a tumor.
[0065] Statistical analysis. Unless otherwise specified in the Figure
Legends, the data are presented as the mean.+-.S.E.M. of at least three
determinations. Asterisks indicate the degree of significant differences
compared with the controls (*, P<0.05; **, P<0.01; ***,
P<0.001). One-way analysis of variance (ANOVA) and Student
Newman-Keuls test were used to compare groups using GraphPad Prism 5.04
software (GraphPad).
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