Patent Description:
Glucose is the main source of energy in eukaryotic organisms and plays a central role in metabolism and cellular homeostasis. Glucose transporters are a wide group of membrane proteins that facilitate the transport of glucose over a plasma membrane. Because tumors are fast growing, they need the proteins that carry nutrients into the cells to function at full capacity. Therefore, an important strategy for cancer treatment would be to block these proteins. Since the GLUT family is one of the major group of membrane transport proteins that transport glucose and other substances into cells, inhibiting these proteins should be important in stopping the spread of cancer. In addition, GLUT also plays a key role in T lymphocyte activation. Inhibition of glucose transport can modulate immune response and have implication in the treatment of a wide variety of immune related diseases from graft rejection to various autoimmune diseases.

<CIT> discloses novel types of hybrid cyclic libraries based on the immunophilin ligand family of natural products cyclosporine A, FK506 and rapamycin, and synthetic methods of making same. <CIT> discloses methods for treating cancer by administering nanoparticles that comprise rapamycin or a derivative thereof.

The present invention is based on the seminal discovery of rapafucin compounds that inhibit cell proliferation and T cell activation.

Described herein are any one of the following compounds:
<CHM>
<IMG>.

n is an integer selected from <NUM> to <NUM>;
R<NUM> is selected from the group consisting of
<CHM>
and
<CHM>
when R<NUM> is
<CHM>
R<NUM>', R<NUM>', R<NUM>', R<NUM>', and R<NUM>' are independently selected from the group consisting of OH, NH<NUM>, SH, CN, H, OAc, and OMe,
when R<NUM> is
<CHM>
A, B, X, Y, and Z are independently C or N,
when R<NUM> is
<CHM>
R<NUM>', R<NUM>', R<NUM>', R<NUM>', and R<NUM>' are independently selected from the group consisting of OH, NH<NUM>, SH, H, OAc, and OMe,
when R<NUM> is
<CHM>
A, X, Y, and Z=CHn' (n'=<NUM>-<NUM>), O, N, S, whenever appropriate, individually or in combination;
R<NUM>, R<NUM>, and R<NUM> are independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, phenyl, OH, NH<NUM>, SH, and CN;
R<NUM>, R<NUM>, R<NUM>, and R<NUM> are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, phenyl, OH, NH<NUM>, SH, and CN;
R<NUM>, R<NUM>, R<NUM>, and R<NUM> are independently selected from the group consisting of hydrogen and methyl;
R<NUM> and R<NUM> are independently selected from the group consisting of OH, NH<NUM>, SH, CN, and hydrogen;
"- - -" represents a double bond with E or Z configuration; and
wherein amino acids with residues <NUM>-<NUM> are selected from the group consisting of
<CHM>
<CHM>
<CHM>
<CHM>
and
<CHM>
or residue <NUM>, <NUM>, <NUM>, or <NUM> together with the adjacent nitrogen form
<CHM>
for use in the treatment of cancer in a subject. In one aspect, the cancer is an alimentary/gastrointestinal tract cancer, a liver cancer, a skin cancer, a breast cancer, a pancreatic cancer, an ovarian cancer, a prostate cancer, a lymphoma, a leukemia, a kidney cancer, a lung cancer, a muscle cancer, a bone cancer, bladder cancer, a brain cancer, eye or ocular cancer, rectal cancer, colon cancer, cervical cancer, bladder cancer, oral cancer, benign and malignant tumors, stomach cancer, corpus uteri, testicular cancer, renal cancer, throat cancer, acute lymphocytic leukemia, acute myelogenous leukemia, Ewing's Sarcoma, Kaposi's Sarcoma, basal cell carcinoma and squamous cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, hemangioendothelioma, Wilms Tumor, neuroblastoma, mouth/pharynx cancer, esophageal cancer, larynx cancer, neurofibromatosis, tuberous sclerosis, hemangiomas, or lymphangiogenesis. In one aspect, the cancer is metastatic cancer. In one aspect, the invention compound is administered intravenously. In one aspect, the invention provides that a chemotherapeutic compound or a compound of Table <NUM> is also administered either prior to, simultaneously with or following administration of an invention compound.

In another embodiment, the above compounds are provided for use in the treatment of possible organ rejection in subjects receiving an organ transplant.

In another embodiment, the above compounds are provided for use in the treatment of autoimmune diseases.

An isolated compound from the above compounds is included in one embodiment of the invention. Further, a method of synthesizing a compound Formula A18 or E11 shown in <FIG> comprising synthetic scheme I or II is included in one embodiment. Further, a pharmaceutical composition comprising an invention compound is included in the invention.

Synthesis of A18. Reagents and Conditions: (a) Fmoc-AA-OH, HATU, DIPEA, DMF, RT, <NUM>; (b) <NUM>% Piperidine, DMF, RT, <NUM>; (c) HATU, DIPEA, DMF, RT, <NUM>; (d) Hoveyda-Grubbs catalyst 2nd generation (<NUM> mol%), <NUM>,<NUM>-dichloroethane, <NUM> microwave, <NUM>.

Synthesis of E11. Reagents and Conditions: (a) Fmoc-AA-OH, HATU, DIPEA, DMF, RT, <NUM>; (b) <NUM>% Piperidine, DMF, RT, <NUM>; (c) HATU, DIPEA, DMF, RT, <NUM>; (d) Hoveyda-Grubbs catalyst 2nd generation (<NUM> mol%), <NUM>,<NUM>-dichloroethane, <NUM> microwave, <NUM>.

Additional compounds that can be used to treat cancer, autoimmune disease and possible organ rejection are represented by the following generic structure:
<CHM>
wherein.

Also disclosed, but not claimed, is a compound that can be used to treat cancer, autoimmune disease and possible organ rejection represented by the following generic structure:
<CHM>
wherein.

The present invention is based on the identification of novel inhibitors of cellular proliferation.

As used herein, a "therapeutically effective amount" of a compound, is the quantity of a compound which, when administered to an individual or animal, results in a sufficiently high level of that compound in the individual or animal to cause a discernible inhibition of cellular proliferation. The exact dosage and frequency of administration depends on the particular compound of the invention used, the particular condition being treated, the severity of the condition being treated, the age, weight and general physical condition of the particular patient as well as the other medication, the patient may be taking, as is well known to those skilled in the art. Furthermore, said "therapeutically effective amount" may be lowered or increased depending on the response of the treated patient and/or depending on the evaluation of the physician prescribing the compounds of the instant invention. The effective daily amount ranges mentioned hereinabove are therefore only guidelines. The term "pharmaceutically acceptable salts" refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention, e.g., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.

As used herein, the term "cancer" or "cancerous growth" means the uncontrolled, abnormal growth of cells and includes within its scope all the well-known diseases that are caused by the uncontrolled and abnormal growth of cells. Non-limiting examples of common cancers include bladder cancer, breast cancer, ovarian cancer, pancreatic cancer, and gastric cancer, cervical cancer, colon cancer, endometrial cancer, head and neck cancer, lung cancer, melanoma, multiple myeloma, leukemia (e.g., myeloid, lymphocytic, myelocytic and lymphoblastic leukemias), non-hodgkin's lymphoma, prostate cancer, rectal cancer, and malignant melanomas.

In addition to invention compounds, one of skill in the art would recognize that chemotherapeutic agents can be used prior to, simultaneously with or following treatment with invention compounds. Illustrative agents include but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, <NUM>-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic antibodies or other proteins are also envisioned in combination therapies of the invention.

The following examples are intended to illustrate but not limit the invention.

A <NUM>,<NUM> compound and <NUM> pool rapafucin library were screened using the alamar blue cell viability assay with the human non-small cell lung cancer (NSCLC) cell line A549. At a final concentration of <NUM> per compound or <NUM> per pool of <NUM> rapafucins, we obtained over <NUM> hits that showed significant inhibition of A549 (<FIG>). Ten of the most potent pools of hits were selected and each of the individual compounds from each pool was synthesized, followed by retesting of each rapafucin from those pools. Several potent rapafucin hits were discovered to inhibit cell proliferation of A549. In order to identify the most potent rapafucin hits, the initial set of active compounds was subjected for a follow-up dose-dependent analysis. Structures of the two most potent rapafucins A18 and E11 are shown in <FIG>. Each was resynthesized on preparative scale, purified by silica gel chromatography, followed by HPLC purification, and subjected to a series of detailed structure characterization (schemes I and II; <FIG>).

Dose-dependent inhibition of cell proliferation by A18 and E11 was next evaluated in several other human cancer cell lines, including breast cancer HCC1954, pancreatic cancer PANC10. <NUM>, leukemia Jurkat T and colon cancer RKO (<FIG>). Two rapafucins were found to significantly inhibit the viability of those cancer cell lines with IC50 values ranging from <NUM> to <NUM> (Table <NUM>). In addition, A18 shows more potent anti-proliferation activity than E11 in most cancer cell lines except pancreatic cancer PANC10. These results suggested that two rapafucins A18 and E11 have broad-spectrum of anticancer activity. (<FIG> and <FIG>; Table <NUM>)
<IMG>.

To identify the molecular target of two rapafucins A18 and E11, a series of cell-based and biochemical studies were performed. Interestingly, it was found that the anti-proliferation effect of A18 and E11 can be slightly decreased when cells were cultured under high concentration of glucose (<FIG>). Compared to low concentration of glucose (<NUM>/L), IC50 values of A18 and E11 increased <NUM>-<NUM> fold when cultured HEK293T or HeLa cells in high concentration of glucose (<NUM>/L). Since constant uptake of glucose is mediated by a family of transporters known as the facilitative glucose transporters (GLUTs) in mammalian cells, it was speculated that A18 and E11 might work through blocking the transport of glucose through GLUTs. Indeed, glucose uptake assay showed that A18 and E11 significantly inhibit glucose transport in A549 cells (<FIG>). In addition, the inhibition of glucose transport induced by A18 and E11 occurred within <NUM> after the assay started (<FIG>), suggesting that the inhibitory activity is likely to be via a direct and fast mechanism. Furthermore, this assay revealed that only <NUM>% glucose uptake inhibition achieved for E11 when A549 cells were treated with drugs for <NUM>. This suggested that the binding of E11 to its target is slower than A18 and two compounds might have a different working mechanism.

One or to two rounds of structure-activity (SAR) studies were then performed by synthesizing new analogs using different amino acid building blocks. Initial SAR analysis (<FIG> and <FIG>) revealed that replacement of any amino acids at the tetrapeptide moiety in A18 cannot be tolerated. However, replacement of the fourth amino acid N-methyl-L---Alanine with N-methyl-L-norvaline or N-methyl-L-norleucine in E11 could slightly increase in activity. E11-<NUM>-<NUM>-<NUM> was named as E11 in the following context.

Direct action on glucose transporters was measured by monitoring uptake of <NUM>-labeled <NUM>-O-methylglucose, which is transported by glucose transporters but not metabolized further, allowing the assessment of initial rate of glucose uptake. Under such conditions, A18 and E11 significantly inhibited uptake of this labeled glucose analog with IC50 values of <NUM> and <NUM>, respectively (<FIG>). Initial uptake can also be assessed by measuring the uptake of <NUM>-labeled <NUM>-deoxy-D-glucose, which gets into the cell through glucose transporters and is phosphorylated by hexokinase but cannot be metabolized further due to the lack of oxygen at the second position. A18 and E11 blocked the uptake of this labeled glucose analog with similar potency (<FIG>). Compared to previously reported glucose transporter inhibitors, A18 and E11 are the first two compounds that have IC50 values below <NUM> (Table <NUM>).

A18 and E11were previously shown to have a broad spectrum of anticancer activity. If anticancer activity works through glucose transporter inhibition, it was speculated that the target of A18 and E11 is Glut1, as glut1 is responsible for basal glucose transport in almost all cell types and glut1 was upregulated in many cancer cells tested. To test this hypothesis, red blood cells (RBCs) were applied as a cell model because RBCs express Glut1 as their sole glucose transporter and have been frequently used in studying glucose transport. Indeed, the <NUM>H-labeled <NUM>-O-methylglucose uptake assays showed that A18 and E11 inhibited the glucose transport in RBCs with IC<NUM> values of <NUM> and <NUM>, respectively. To eliminate other possibilities, the glucose uptake assays were repeated in RBC-derived ghosts, in which all the intracellular proteins and enzymes were removed and only membrane-bound and membrane-associated proteins remained. Interestingly, the glucose uptake assays revealed that only A18 inhibited the glucose transport in RBCs---derived ghosts with an IC<NUM> of <NUM>. However, E11 totally lost its inhibitory activity, suggesting that E11 might work through binding to other intracellular protein first and then blocking glucose transport. (see <FIG>, <FIG> and <FIG>).

Up to now, at least <NUM> different isoforms of GLUTs have been identified in human cells. It was then asked whether A18 and E11 are specific inhibitors of GLUT1. To answer this question, colon cancer DLD-<NUM> wild type and GLUT1 gene knock out cell lines were chosen as a cell model (<FIG>). Interestingly, <NUM>H-labeled <NUM>-deoxy-D-glucose uptake and alamar blue cell viability assays showed that A18 still strongly inhibited the glucose transport and cell proliferation in both cell lines. However, E11 didn't show any inhibition in DLD-<NUM> GLUT1 gene knock out cells but kept inhibitory activity in wild type cells (<FIG> and <FIG>). This suggested that E11, but not A18, is a specific inhibitor of GLUT1. GLUTs that are most relevant to cancer are Glut1 and Glut3. To obtain additional evidence, Glut1 and Glut3 in HEK 293T cells were overexpressed and an alamar blue cell viability assay was performed again. As expected, E11 indeed didn't show any inhibition in GLUT3 overexpression cells but kept partial inhibitory activity in GLUT1 overexpression cells (<FIG> and Table <NUM>), strongly supporting the hypothesis that E11 is a specific inhibitor of GLUT1. (see <FIG>, <FIG>, <FIG>, <FIG>; Table <NUM>).

Given the underlying principle of the design of the rapafucin libraries, it was next explored whether the inhibition of GLUT1 by A18 or E11 is dependent on FKBP. A hallmark of FKBP dependence is that the cellular effects would be antagonized by another unrelated FKBP binding ligands with no or orthogonal biological activity as has been shown for FK506 and rapamycin. For unknown reasons, both FK506 and rapamycin were unable to antagonize inhibitory effects of A18 or E11 on <NUM>-labeled <NUM>-deoxy-D-glucose uptake (<FIG>). However, synthetic ligand of FKBP (SLF)(<FIG>) significantly impaired the inhibitory activity of E11 (<FIG>), suggesting that the activity of E11 requires FKBP.

After showing that GLUT1 was very likely to be the target of A18 and E11, the direct interaction of A18 and E11 to GLUT1 was then examined. A series of biotin or diazrine-alkyne rapafucin conjugates through different positions were synthesized. Glucose uptake assays showed that only a few of conjugates kept inhibitory activity in A549 cells (<FIG>). Using the most potent biotin-rapafucin conjugates (<FIG>), pulldown assays followed by Western blot using anti-GLUT1 antibodies were performed. It was found that the biotin-rapafucin conjugates are able to pull down GLUT1 from RBC-derived ghosts cell lysate (<FIG>). Importantly, the binding of the biotin-rapafucin probe to GLUT1 is competed by rapafucin. Moreover, the binding of the A18 probe to GLUT1 cannot be competed by E11 and vice versa, suggesting that two rapfucins might have a different binding position. Finally, as expected, the binding of the rapafucin probe to GLUT1 cannot be competed by FK506 and Rapamycin. Taken together, pulldown assays showed that rapafucin A18 and E11 can bind directly to GLUT1. (<FIG>, <FIG>, <FIG> and <FIG>).

Whether A18 and E11 killed cancer cells through cell death or a different pathway was investigated. There was no increase in phosphor-p53 level and active caspase <NUM>, <NUM> and <NUM> in HEK 293T cells, suggesting that A18 and E11 do not induce DNA damage or apoptosis (<FIG>). However, flow cytometric analysis revealed that A18 and E11 treatment led to cell cycle arrest. A18 and E11 treatment resulted in approximately <NUM>% more cells in S phase. This finding for the first time demonstrated that glucose transporter inhibitor treatment led to S phase cell cycle arrest.

Whether A18 and E11 treatment affect key cell growth signaling proteins was examined next. Western blot analysis revealed that A18 and E11 are capable of inducing phosphorylation of AMPK and causing mTOR inhibition. But it has no effects on the phosphorylation of ERK, AKT or JNK (<FIG>). As previously reported, AMPK is likely to act as the key link between the ATP reduction and the subsequent cancer cell inhibition. (<FIG> and <FIG>). Based on the data reported here, the working model for A18 and E11 was proposed as outlined in <FIG>. After A18 or E11 treatment, glucose supply in cancer cells dramatically decreased, followed by some key glycolytic enzymes and metabolites (ATP) decreased. These led to upregulation of phosphorylation of AMPK and downregulation of phosphorylation of S6K. All these changes induced cell cycle arrest, necrosis or senescence, and finally induced cancer cell inhibition.

Claim 1:
A compound with the following formula:
<CHM>
wherein
n is an integer selected from <NUM> to <NUM>;
R<NUM> is selected from the group consisting of
<CHM>
and
<CHM>
when R<NUM> is
<CHM>
R<NUM>', R<NUM>', R<NUM>', R<NUM>', and R<NUM>' are independently selected from the group consisting of OH, NH<NUM>, SH, CN, H, OAc, and OMe,
when R<NUM> is
<CHM>
A, B, X, Y, and Z are independently C or N,
when R<NUM> is
<CHM>
R<NUM>', R<NUM>', R<NUM>', R<NUM>', and R<NUM>' are independently selected from the group consisting of OH, NH<NUM>, SH, H, OAc, and OMe,
when R<NUM> is
<CHM>
A, X, Y, or Z=CHn' (n'=<NUM>-<NUM>), O, N, S, whenever appropriate, individually or in combination;
R<NUM>, R<NUM>, and R<NUM> are independently selected from the group consisting of H, methyl, ethyl, propyl, isopropyl, phenyl, OH, NH<NUM>, SH, and CN;
R<NUM>, R<NUM>, R<NUM>, and R<NUM> are independently selected from the group consisting of methyl, ethyl, propyl, isopropyl, phenyl, OH, NH<NUM>, SH, and CN;
R<NUM>, R<NUM>, R<NUM>, and R<NUM> are independently selected from the group consisting of hydrogen and methyl; R<NUM> and R<NUM> are independently selected from the group consisting of OH, NH<NUM>, SH, CN, and hydrogen;
"- - -" represents a double bond with E or Z configuration; and
the amino acids with residues <NUM> to <NUM> are selected from the group consisting of
<CHM>
<CHM>
<CHM>
<CHM>
<CHM>
, and
<CHM>
or residue <NUM>, <NUM>, <NUM>, or <NUM> together with the adjacent nitrogen form
<CHM>