HK1240937B - Small molecules for the treatment of primary cancer and cancer metastasis - Google Patents

Description

Small molecules for the treatment of primary cancer and cancer metastasis

This application claims priority to U.S. Provisional Application No. 62/104,705, filed January 17, 2015, which is incorporated herein by reference in its entirety.

Background Art

In patients with advanced cancers including breast cancer and prostate cancer, bone is the most common site of metastasis (Jin et al., (2011) Int. J. Cancer 128, 2545-2561; Kohno, (2008) Int. J. Clin. Oncol. 13, 18-23). Bone metastasis is a major, potentially fatal complication in patients with advanced cancer. Due to severe pain, pathological fractures, spinal cord compression, and metabolic complications, almost all patients with bone metastasis have a significantly reduced quality of life (Welch et al. (2003) J. Musculoskelet. Neuronal Interact. 3, 30-38). In fact, autopsy studies have shown that more than 70% of breast cancer patients exhibit bone metastasis, and only 20% of these patients are still alive 5 years after the discovery of metastasis (Roodman (2004) N. Engl. J. Med. 350, 1655-1664; Welch et al. (2003) J. Musculoskelet. Neuronal Interact. 3, 30-38). The high affinity of cancer for bone is explained by the "seed and soil hypothesis" proposed more than a century ago (Paget (1889) Lancet 1, 571-573). It reveals that bone tissue is a preferred site for cancer metastasis because its microenvironment provides a fertile environment in which tumor cells can grow. Many characteristics, such as increased blood flow and the release of growth factors by cells in the bone matrix, lead to the frequent occurrence of bone metastasis (van der Pluijm et al. (2001) J. Bone Miner. Res. 16, 1077-1091). To date, the key factors and mechanisms leading to bone metastasis are largely unknown.

Bisphosphonate drugs are used to treat bone cancer metastasis and result in reduced tumor growth, reduced bone damage, and reduced pain (Brown and Guise (2007) Cur. Osteopor. Rep. 5, 120-127). Bisphosphonate therapy is associated with adverse side effects, including atrial fibrillation, jaw joint pain and osteonecrosis, as well as ophthalmic, dermatological, and renal complications, and drug-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 solid tumor bone metastases, the molecular mechanism by which bisphosphonate therapy inhibits bone metastasis remains to be determined.

Previous studies have suggested that ATP may exhibit anticancer effects through its binding to P2 purinergic receptors (White and Burnstock (2006) Trends Pharmacol. Sci. 27, 211-217). Several studies have determined that ATP inhibits the growth of several cell lines, including prostate cancer cells, colon adenocarcinoma cells, melanoma cells, and bladder cancer cells (Rapaport et al. (1983) Cancer Res. 43, 4402-4406; Shabbir and Burnstock (2009) Int. J. Urol. 16, 143-150; White and Burnstock (2006) Trends Pharmacol. Sci. 27, 211-217). Activation of purinergic signaling has also been reported to inhibit the proliferation and migration of human acute myeloid leukemia cells in immunodeficient mice (Salvestrini et al. (2012) Blood 119, 217-226). In addition, in vivo studies have shown that daily injection of ATP significantly inhibits tumor growth, prolongs survival time, and inhibits weight loss in mice (Rapaport (1988) Eur. J. Cancer Clin. Oncol. 24, 1491-1497). However, several studies have also proposed adverse effects of ATP, including increased tumor growth and migration. We recently reported that ATP and ATP analogs such as ATPγS inhibit breast cancer cell growth, migration, and bone metastasis, while activation of adenosine and adenosine receptors has the opposite effect by promoting the growth, migration, and bone metastasis of breast cancer cells (Zhou et al., (2014) Oncogene (Epub)).

Non-hydrolyzable ATP analog compounds and adenosine receptor antagonist analog compounds can be used to treat cancer (WO2014074529). However, there is still a need for other non-hydrolyzable ATP analog compounds and adenosine receptor antagonists.

Summary of the Invention

Some embodiments relate to non-hydrolyzable ATP analogs that inhibit the migration and growth of cancer cells. The term non-hydrolyzable ATP analog refers to an ATP analog that is not effectively hydrolyzed by ATPase, that is, if hydrolyzed, the analog is hydrolyzed by ATPase at a rate of less than 5%, 1% or 0.1% of the ATP hydrolysis rate. Some embodiments relate to various chemical analogs of the non-hydrolyzable ATP analog 5'-[γ-thio] adenosine triphosphate (ATPγS). These chemicals inhibit the migration and growth of cancer cells. Some embodiments relate to chemical analogs of the non-hydrolyzable ATP analog 5'-[γ-thio] adenosine triphosphate (ATPγS), which have the general formula I, including compounds P1 to P6 (Table 1)

wherein R ₁ and R ₂ are independently selected from hydrogen (H), cyano (CN), C1 to C3 alkyl, halogen (fluorine (F), chlorine (Cl), bromine (Br), or iodine (I)), or trifluoromethyl (CF ₃ ). In some aspects, R 1 is selected from hydrogen, cyano, C1 to C3 alkyl, halogen (fluorine (F), chlorine (Cl), bromine (Br), or iodine (I)), or trifluoromethyl, and R 2 is hydrogen or fluorine. In another aspect, R 1 is cyano, and R 2 is H; R 1 is H, and R 2 is H; R 1 is trifluoromethyl, and R 2 is H; R 1 is fluorine, and R 2 is H; R 1 is methyl, and R 2 is H; R 1 is fluorine, and R 2 is fluorine.

Some embodiments involve administering one or more compounds of Formula I to treat cancer. The compounds can be administered alone or in combination with other anti-cancer therapies.

Exposure to adenosine, produced through ATP metabolism, can promote cancer cell growth and migration. Some embodiments relate to chemical analogs of the adenosine receptor antagonist 8-ethoxy-9-ethyl-9H-purin-6-amine (ANR94, an A2A antagonist). These compounds are inhibitors of cancer cell migration and growth. In some embodiments, chemical analogs of the adenosine receptor antagonist 8-ethoxy-9-ethyl-9H-purin-6-amine have the general formula of Formula II, which includes compounds P7 to P10 (Table 1).

Some aspects relate to compounds of formula II, wherein R ₃ is selected from dihalomethyl, C3 to C5 cycloalkyl, or tetrahydrofuran. In some aspects, R 3 is difluoromethyl, cyclopropyl, cyclobutyl, or β-tetrahydrofuran.

Some embodiments involve administering one or more compounds of Formula II to treat cancer. The compounds can be administered alone or in combination with a compound of Formula I and/or other anti-cancer therapies.

In some aspects, one or more compounds of Formula I and/or Formula II are administered to a subject in need of anticancer treatment. In some aspects, the compounds of Formula I and/or Formula II are administered within 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, or 60 minutes. In another aspect, the compounds are administered simultaneously. In another aspect, one or more compounds of Formula I are administered before, during, or after the administration of one or more compounds of Formula II.

Table 1. List of representative compounds

In some aspects, object or patient suffer from bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, cervical cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, tongue cancer or uterine cancer. On the other hand, cancer is lung cancer, breast cancer or prostate cancer. In concrete aspect, cancer is metastatic cancer, for example bone metastasis. In some aspects, cancer is accredited as having a risk of metastasis or having a tendency to metastasize, or does not have the indication of cancer metastasis. In some aspects, the evaluation of the cancer with a risk of metastasis is based on the evaluation of tumor biopsy.

In some embodiments, bisphosphonate drugs may be specifically excluded from the claimed invention due to their potential in vivo toxicity.

As used herein, an "inhibitor" can be a compound that reduces the activity or function of a protein. For example, an inhibitor can inhibit protein activity directly or indirectly. For example, direct inhibition can occur by binding to the protein and thereby preventing protein activity, or by competitively, non-competitively, or non-competitively inhibiting the enzymatic or other activity of the protein. For example, indirect inhibition can occur by binding to a predetermined target of the protein, such as a receptor or binding partner, thereby blocking or reducing protein activity.

The term "effective amount" refers to an amount effective to achieve the desired therapeutic or preventive outcome at the dosage and for the required period of time. An "effective amount" with respect to an anticancer agent that reduces cancer cell growth or migration refers to an amount that is capable of reducing the growth of some cancer cells or tumor cells to a certain extent, or inhibiting the ability of cancer cells or tumor cells to migrate or invade non-tumor tissues, such as bone. The term includes amounts that are capable of causing a growth inhibitory effect, a cytostatic effect, and/or a cytotoxic effect, and/or causing apoptosis in cancer cells or tumor cells.

A "therapeutically effective amount" with respect to the treatment of cancer is an amount that produces one or more of the following effects: (1) inhibiting cancer or tumor growth to some extent, including slowing growth or halting growth altogether; (2) reducing the number of cancer cells or tumor cells; (3) reducing tumor size; (4) inhibiting (i.e., reducing, slowing, or completely stopping) cancer cells or tumor cells from penetrating peripheral organs; (5) inhibiting (i.e., reducing, slowing, or completely stopping) metastasis; (6) enhancing the anti-tumor immune response, which may, but need not, result in the regression or rejection of the tumor, or (7) alleviating to some extent one or more symptoms associated with the cancer or tumor. A therapeutically effective amount may vary depending on factors such as the individual's condition, age, sex, and weight, and the ability of one or more anticancer agents to elicit the desired response in the individual. A "therapeutically effective amount" is also an amount in which any toxic or deleterious effects are outweighed by the therapeutically beneficial effects.

The phrases "treating cancer" and "treatment of cancer" refer to reducing, decreasing, or inhibiting the replication of cancer cells; reducing, decreasing, or inhibiting the spread of cancer (the formation of metastases); decreasing the size of a tumor; decreasing the number of tumors (i.e., reducing the tumor burden); decreasing or reducing the number of cancer cells in the body; preventing the recurrence of cancer after surgical resection or other anti-cancer therapy; or ameliorating or relieving the symptoms of disease caused by cancer.

Other embodiments of the present invention are discussed throughout this application. Any embodiment discussed with respect to one aspect of the present invention may also be applied to other aspects of the present invention, and vice versa. Each embodiment described herein should be understood as an embodiment of the present invention applicable to all aspects of the present invention. It is contemplated that any embodiment discussed herein may be implemented for any method or composition of the present invention, and vice versa. In addition, the compositions and kits of the present invention may be used to implement the methods of the present invention.

When used with the term "comprising" in the claims and/or description, an element without a quantifier preceding it may mean "a", but it also complies with the meaning of "one or more", "at least one" and "one or more than one".

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of deviation for the device or method being employed to determine the value.

The use of the term "or" in the claims is intended to mean "and/or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, although this disclosure supports a definition referring to only alternatives and "and/or."

As used in this specification and claims, the words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating specific embodiments of the present invention, are given by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of this specification and are included to further demonstrate some 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 specific embodiments provided herein.

Figure 1. Transwell cell migration assay using MDA-MB-231 human breast cancer cells.

FIG2 . Soft-agar Anchorage Independent Growth assay of MDA-MB-231 cells.

Figure 3. Mammary Fat Pad Xenograft Assay.

Specific implementation plan

Some embodiments relate to compounds of Formula I, such as P1, P2, P3, P4, P5, or P6 (Table 1). These compounds are chemical analogs of the non-hydrolyzable ATP analog 5'-[γ-thio] adenosine triphosphate (ATPγS).

Other embodiments relate to compounds of formula II, such as P7, P8, P9, or P10, which are chemical analogs of the adenosine receptor antagonist 8-ethoxy-9-ethyl-9H-purin-6-amine (ANR94, an A2A antagonist) (Table 1). Studies have shown that all 10 compounds have an inhibitory effect on cell migration. In a Transwell cell migration assay of MDA-MB-231 human breast cancer cells, all 10 compounds, especially P2, P3, P4, P5, and P9, showed an inhibitory effect on cell migration. At 50 μM, no compound showed any toxicity to the cells.

A soft agar assay was performed to determine the anchorage-independent growth of MDA-MB-231 cells using compounds P1 to P10, with P2 and P3 being the most effective. P2 and P3 resulted in a 30% and 65% reduction in cell colonies, respectively, compared to the control group ( FIG2 ).

In addition, mammary fat pad xenograft analysis of MDA-MB-231 cells was performed. MDA-MB-231 cells were xenografted in the mammary fat pad of deficient mice. After tumor nodules appeared, a test compound (e.g., P3) was injected into these mice (500 μl of a 400 μM solution). The tumor size of mice receiving the test compound was compared with that of control mice (i.e., mice administered with a vehicle containing no test compound). After 15 days, the tumor size of mice receiving P3 was reduced by more than 50% compared to mice not receiving P3 (Fig. 3).

In some aspects, there is a compound (such as compound P1 to P10) of formula I and/or formula II that can be used to inhibit the proliferation and/or migration of cancer cells. In some aspects, cancer is bladder cancer, blood cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, gastrointestinal cancer, head cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, gastric cancer, testicular cancer, tongue cancer or uterine cancer. On the other hand, cancer is breast cancer. On the other hand, cancer is prostate cancer. In a specific embodiment, cancer is metastatic cancer, for example, there is the risk of transferring or migrating to bone or is in the cancer of the risk of transferring or migrating to bone.

In some embodiments, the present invention also provides a composition comprising one or more compounds of Formula I and/or Formula II (e.g., P1 to P2) in a pharmaceutically acceptable formulation. Thus, the use of one or more compounds described herein in the preparation of a medicament is also contemplated. Such a composition can be used to treat a variety of cancers. In some embodiments, the treatment is for metastatic cancer, such as lung cancer, breast cancer, or prostate cancer.

The compounds described herein can 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, polymer microcapsules or microvesicles, liposomes, and injectable or infusible solutions. The preferred form depends on the mode of administration and the specific disease being targeted. The composition also preferably contains a pharmaceutically acceptable carrier, vehicle, or adjuvant, as is well known in the art.

Acceptable formulation ingredients used in pharmaceutical preparations are nontoxic to recipients at the dosages and concentrations employed. In addition to the compounds described herein, the compositions can contain ingredients used to alter, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or penetration of the composition. Suitable materials for formulating pharmaceutical compositions include, but are not limited to, amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (e.g., acetate, borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, mannose, or dextrin); proteins (e.g., serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium), preservatives ( such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, 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 enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants (see Remington's Pharmaceutical Sciences). Sciences, 18th ed., (A. R. Gennaro, ed.), 1990, Mack Publishing Company), which is incorporated herein by reference.

The formulation components are present in concentrations acceptable to the site of administration. Buffering agents are advantageously used to maintain the composition at a physiological pH or slightly lower, generally in the pH range of about 4.0 to about 8.5 or about 5.0 to 8.0. The pharmaceutical composition may comprise a TRIS buffer having a pH of about 6.5 to 8.5, or an acetate buffer having a pH of about 4.0 to 5.5, and may further comprise sorbitol or a suitable substitute thereof.

Pharmaceutical compositions for in vivo administration are generally sterile. Sterilization can be accomplished by filtration through a sterile filter membrane. If the composition is lyophilized, sterilization can be performed before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. In some embodiments, the parenteral composition is placed in a container with a sterile access port, such as an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle, or a sterile prefilled syringe ready for injection.

The above compositions can be administered using conventional delivery methods, including but not limited to intravenous, intraperitoneal, oral, intralymphatic, subcutaneous administration, intraarterial, intramuscular, intrapleural, intrathecal, and by local catheter perfusion. The present invention also contemplates the discussed local administration to tumors or metastases. When the composition is administered by injection, it can be administered by continuous infusion or by single or multiple boluses. For parenteral administration, the agent can be administered in a pyrogen-free, parenterally acceptable aqueous solution comprising the desired compound in a pharmaceutically acceptable carrier. A particularly suitable carrier for parenteral injection is sterile distilled water, wherein one or more anticancer agents are formulated as sterile isotonic solutions that are suitably preserved.

After the pharmaceutical composition of the present invention has been formulated, it can be stored in a bottle as a solution, suspension, gel, emulsion, solid or as a dehydrated or lyophilized powder. Such a formulation can be stored in a ready-to-use form or in a form (e.g., lyophilized) to be reconstituted before administration.

If desired, stabilizers commonly used in pharmaceutical compositions, such as sucrose, trehalose, or glycine, may be used. Typically, such stabilizers are added in small amounts, such as about 0.1% to about 0.5% (weight/volume). Surfactant stabilizers, such as Tween®-20 or Tween®-80 (ICI Americas, Inc., Bridgewater, N.J., USA), may also be added in conventional amounts.

The ingredients used to prepare the pharmaceutical compositions are preferably of high purity and substantially free of potentially harmful contaminants (e.g., at least National Food (NF) grade, generally at least analytical grade, more generally at least pharmaceutical grade). In addition, compositions for in vivo use are generally sterile. To the extent that a given compound must be synthesized prior to use, the resulting product is generally substantially free of any potentially toxic agents. Compositions for parenteral administration are also sterile, substantially isotonic, and prepared under GMP conditions.

For the compounds described herein, alone or as part of a pharmaceutical composition, the dosage is from about 0.001 mg/kg body weight to 1 mg/kg body weight, preferably from about 1 μg/kg body weight to 100 μg/kg body weight, and most preferably from 1 μg/kg body weight to 10 μg/kg body weight. In some aspects, the compounds described herein can be infused to a patient at a rate of 20, 25, 30, 35, 40 μg/kg/minute to 30, 35, 40, 45, 50 μg/kg/minute (including all values and ranges therebetween) for up to 8 hours, including 1, 2, 3, 4, 5, 6, 7 or 8 hours, in a daily dose. The compounds described herein can be administered orally at about 1, 10, 20, 30, 40, 50, 60 micrograms per kilogram to 50, 60, 70, 80, 90, 100 micrograms per kilogram of body weight per day, or about 1, 10, 20, 30, 40, 50, 60 milligrams per kilogram to 50, 60, 70, 80, 90, 100 milligrams per kilogram of body weight per day. In some aspects, the compounds described herein can be administered at about 0.01 mg/kg to 10 mg/kg of body weight per day.

The therapeutically effective dosage will be readily determined by one skilled in the art and will depend on the course and severity of the disease, the patient's health status and response to treatment, the patient's age, weight, height, sex, previous medical history and the judgment of the treating physician.

In some methods of the present invention, the cancer cells are tumor cells. The cancer cells may be present in a patient. The patient may have a solid tumor. In such cases, embodiments may also involve performing surgery on the patient, for example, by resecting all or part of the tumor. The composition may be administered before, after, or concurrently with the surgery. In other embodiments, the composition may be administered to the patient directly, endoscopically, intratracheally, intratumorally, intravenously, intralesionally, intramuscularly, intraperitoneally, regionally, transdermally, topically, intraarterially, intravesically, or subcutaneously. The therapeutic composition can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more. They can 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 every 1, 2, 3, 4, 5, 6, 7 days, or every 1, 2, 3, 4, 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.

The method for treating cancer can also include administering chemotherapy or radiotherapy to the patient, which can be administered more than 1 time. Chemotherapy includes but is not limited to cisplatin (CDDP), carboplatin, procarbazine, nitrogen mustard, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan (bisulfan), nitrosoureas, dactinomycin D, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, taxotere, paclitaxel, trans-platinum, 5-fluorouracil, vincristine, vinblastine, methotrexate, gemcitabine, oxaliplatin, irinotecan, topotecan or any analog thereof or derivative variant. Radiotherapy includes but is not limited to X-ray irradiation, UV irradiation, γ-irradiation, electron beam radiation or microwave. In addition, as part of the inventive method, microtubule stabilizers can be administered to cells or patients, including but not limited to taxanes. It is particularly contemplated that any compound or derivative or analog can be used for these conjoint therapies.

Various chemical definitions for this compound are provided below.

As used herein, the term "fluoro" refers to -F; the term "cyano" refers to -CN; the term "methyl" refers to -CH ₃ ; the term "difluoromethyl" refers to -CF ₂ H; the term "trifluoromethyl" refers to -CF ₃ ; the term "cyclopropyl" refers to a three-membered saturated cycloalkyl ring; the term "cyclobutyl" refers to a four-membered saturated cycloalkyl ring; the term "β-tetrahydrofuran" refers to a five-membered saturated heterocyclic ring having O as the heteroatom and being substituted on the carbon beta to the heteroatom.

As used herein, the term "halogen" refers to -F, -Cl, -Br, or -I; the term "thiol" refers to -SH; the term "cyano" refers to -CN; the term "azido" refers to -N ₃ ; and the term "hydroxy" refers to -OH.

Unless otherwise noted, the term "alkyl" itself or as part of other substituents refers to a linear (i.e., non-branched) or branched carbon chain, which can be fully saturated, monounsaturated, or polyunsaturated. Unsaturated alkyl is an alkyl with one or more double or triple bonds. Saturated alkyl includes an alkyl with one or more carbon-carbon double bonds (alkenyl) and an alkyl with one or more carbon-carbon triple bonds (alkynyl). Groups such as -CH ₃ (methyl), -CH ₂ CH ₃ (ethyl), -CH ₂ CH ₂ CH ₃ (n-propyl), -CH (CH ₃ ) ₂ (isopropyl), -CH ₂ CH 2 CH ₂ CH ₃ ( _n -butyl), -CH (CH ₃ ) CH ₂ CH ₃ (sec-butyl), -CH ₂ CH (CH ₃ ) ₂ (isobutyl), -C (CH ₃ ) ₃ (tert-butyl), -CH ₂ C (CH ₃ ) ₃ (neopentyl) are non-limiting examples of alkyl.

Unless otherwise indicated, the term "heteroalkyl," by itself or in combination with other terms, refers to a linear or branched chain having at least one carbon atom and at least one heteroatom selected from O, N, S, P, and Si. In some embodiments, the heteroatom is selected from O and N. The heteroatom may be located at any interior position of the heteroalkyl group or at the position where the alkyl group is attached to the rest of the molecule. A maximum of two heteroatoms may be consecutive. The following groups are non-limiting examples of heteroalkyl groups: trifluoromethyl _{, -CH2F} , _-CH2Cl , _-CH2Br , _-CH2OH , _-CH2OCH3 , _-CH2OCH2CF3 , -CH2OC( _{O ) CH3 ,} -CH2NH2, _-CH2NHCH3 , -CH2N( _CH3 ) _{2 , -CH2CH2Cl} , _-CH2CH2OH , _{CH2CH2OC (} O _{) CH3 , -CH2CH2NHCO2C ( CH3} ) _{3 , and -CH2Si} ( _{CH3 ) 3 .}

The terms "cycloalkyl" and "heterocyclyl," by themselves or in combination with other terms, refer to cyclic "alkyl" and "heteroalkyl," respectively. Additionally, for heterocyclyl, a heteroatom can occupy the position at which the heterocycle is attached to the rest of the molecule.

The term "aryl" refers to a polyunsaturated, aromatic hydrocarbon substituent. Aryl can be monocyclic or polycyclic (e.g., 2 to 3 rings fused together or covalently linked). The term "heteroaryl" refers to an aryl group containing 1 to 4 heteroatoms selected from N, O, and S. Heteroaryl can be connected to the rest of the molecule through carbon or heteroatom. Examples of aryl and heteroaryl 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-pyrimidinyl, 4-pyrimidinyl, 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-mentioned aryl and heteroaryl ring systems are selected from the group consisting of the acceptable substituents described below.

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 the group consisting of halogen, nitro, cyano, hydroxy, amino, sulfhydryl, formyl, carboxyl, oxo, carbamoyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, alkoxy, _alkylthio , alkylamino, (alkyl) amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. In some aspects, the optional substituents are further substituted by one or more substituents independently selected from the group consisting of halogen, nitro, cyano, hydroxyl, amino, thiol, formyl, carboxyl, carbamoyl, unsubstituted alkyl, unsubstituted heteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl) ₂ amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, unsubstituted cycloalkyl, unsubstituted heterocyclyl, unsubstituted aryl, or unsubstituted heteroaryl. Exemplary optional substituents include, but are not limited to, -OH, oxygen (=O), -Cl, -F, Br, C _1-4 alkyl, phenyl, benzyl, -NH ₂ , -NH (C _1-4 alkyl), -N (C 1-4 alkyl) ₂ , -NO ₂ , -S (C _1-4 alkyl), -SO ₂ (C _1-4 alkyl), -CO ₂ (C _1-4 alkyl) and -O (C _1-4 alkyl) _.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention that is substantially non-toxic to organisms. Typical pharmaceutically acceptable salts include those prepared by reacting a compound of the present invention with an inorganic acid or organic acid or an organic base, depending on the substituents present on the compound of the present invention.

Non-limiting examples of inorganic acids that can be used to prepare pharmaceutically acceptable salts include hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like. Non-limiting examples of organic acids that can be used to prepare pharmaceutically acceptable salts include aliphatic monocarboxylic acids and aliphatic 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 include hydrochlorides, hydrobromides, nitrates, sulfates, pyrosulfates, hydrogensulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, hydroiodides, hydrofluorides, acetates, propionates, formates, oxalates, citrates, lactates, p-toluenesulfonates, methanesulfonates, maleates, and the like.

Suitable pharmaceutically acceptable salts can also be formed by reacting the reagents of the present invention with organic bases such as methylamine, ethylamine, ethanolamine, lysine, ornithine, etc. Pharmaceutically acceptable salts include salts formed between carboxyl or sulfonic acid groups present on some compounds of the present invention and inorganic or organic cations, such as sodium, potassium, ammonium or calcium, and organic cations such as isopropylammonium, trimethylammonium, tetramethylammonium and imidazolium.

It will be appreciated that the particular anion or cation forming part of any salt of the invention is not critical so long as the salt as a whole is pharmaceutically acceptable.

Other examples of pharmaceutically acceptable salts and methods for their preparation and use are provided in Handbook of Pharmaceutical Salts: Properties, Selection and Use (2002), which is incorporated herein by reference.

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. Additionally, the compositions of the invention can be used to implement the methods of the invention.

I. Examples

The following examples and accompanying drawings are incorporated to illustrate preferred embodiments of the present invention. It will be understood by those skilled in the art that the techniques disclosed in the examples or accompanying drawings represent techniques that the inventors have found to work well in the practice of the present invention and are therefore considered to constitute preferred embodiments of its practice. However, based on this disclosure, it will be understood by those skilled in the art that many changes may be made in the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtaining the same or similar results.

A. Materials and Methods

Cell lines and cell culture: MDA-MB-231 cells were grown in McCoy's 5A modified medium (Gibco) supplemented with 10% FBS (Hyclone). Py8119 cells were grown in F12K nutrient medium (Gibco) supplemented with 5% Fetal Clone II (Fisher Scientific). All cell lines were incubated at 37°C in a 5% _CO2 incubator.

Cell migration assay: The migration assay was performed in a transwell membrane filter insert in a 24-well tissue culture plate (BD Biosciences San Jose, CA, USA). The transwell membrane filter insert contained a polycarbonate membrane with a diameter of 6.5 mm, a pore size of 8 μm, and a thickness of 10 nm. 500 μl of breast cancer cell suspension was added to the upper side of the insert at a density of 10 × ¹⁰ cells/insert, and 750 μl of CM with or without other compounds was added to the well below. The cells were incubated at 37°C for 18 to 20 hours. Cells that did not migrate through the filter were removed using a cotton swab, and cells that migrated through the insert were fixed and stained with Hema 3 Stat Pack (Fisher Scientific). The number of migrated cells in 5 fields of view for each insert was counted under a light microscope at 10 × magnification.

Soft agar colony formation assay: For anchorage-independent cell growth, MDA-MB-231 cells were plated in complete medium supplemented with 50 μM compound (P1 to P10) containing 0.4% agarose on top of a 0.8% agarose base supplemented with complete medium. The cells were maintained for approximately 2 weeks and then stained with p-iodonitrotetrazolium violet (Sigma-Aldrich, St. Louis, MO). Images were captured using a scanner and the number of colonies was counted.

Animals: Four-week-old female athymic nude mice (Harlan Sprague–Dawley, Indianapolis, IN, USA) were used for mammary fat pad injections. Four- to five-week-old female C57bl/6 mice were used for intratibial injections. Animals were maintained under the care and supervision of the Laboratory Animal Research Facility at the University of Texas Health Science Center at San Antonio, TX. Animal protocols were approved and monitored by the Institutional Animal Care and Use Committee.

In vivo xenograft experiment: MDA-MB-231 cells were injected subcutaneously into the mammary fat pads of 4-week-old female nu/nu athymic nude mice. Each mouse received bilateral subcutaneous inoculation of 100 μl of a cell suspension containing approximately 1×10 ⁷ cells/ml in serum-free medium in the left and right inguinal mammary fat pad areas. Animals were randomly divided into 3 different groups and solid tumors were allowed to form to a volume of approximately 5 mm ³ before treatment began. 400 μmol of compound P3/500 μl of saline, or saline as a control, was administered intraperitoneally (IP) 3 times per week for 3 weeks. The growth of xenograft tumors was monitored twice a week, and the tumor size was measured in two dimensions using a caliper. Tumor volume was calculated using the equation V = (L×W ² )×0.5 (mm ³ ), where L is the tumor length and W is the tumor width.

Statistical analysis: Unless otherwise indicated in the figure legends, data are presented as mean ± S.E.M. of at least three measurements. Asterisks indicate the degree of significant difference compared to the control group (*, P < 0.05; **, P < 0.01; ***, P < 0.001). One-way analysis of variance (ANOVA) and Student's Newman-Keuls test were used to compare groups using GraphPad Prism 5.04 software (GraphPad).

Claims (8)

1. A compound of formula I, which is a chemical analog of the non-hydrolyzable ATP analog 5'-[γ-thio]adenosine triphosphate (ATPγS): R1 and R2 are independently selected from hydrogen, cyano, C1 to C3 alkyl, halogen or trifluoromethyl. 2. The compound according to claim 1, wherein R1 is selected from hydrogen, cyano, C1 to C3 alkyl, halogen or trifluoromethyl, and R2 is hydrogen or halogen. 3. The compound according to claim 2, wherein R1 is hydrogen, fluorine, methyl, cyano, or trifluoromethyl. 4. The compound according to claim 1, wherein R1 is cyano and R2 is hydrogen; R1 is hydrogen and R2 is hydrogen; R1 is trifluoromethyl and R2 is hydrogen; R1 is methyl and R2 is hydrogen; R1 is fluorine and R2 is fluorine. 5. Use of one or more compounds according to claim 1 in the preparation of a medicament for treating cancer patients. 6. The use according to claim 5, wherein the cancer is breast cancer. 7. The use according to claim 5, wherein one or more compounds according to claim 1 are administered intravenously. 8. The use according to claim 5, wherein one or more compounds according to claim 1 are administered orally.