WO2008016660A2 - Imidazoacridine compounds for treating leukemias - Google Patents

Abstract

A method of treating a patient suffering from certain types of leukemia, comprising administering to said patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. Definitions for the variables are provided therein.

Description

COMPOUNDS FOR TREATING LEUKEMIAS

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/835,028, filed August 2, 2006, U.S. Provisional Application No. 60/835,064, filed August 2, 2006, and U.S. Provisional Application No. 60/873,850, filed December 8, 2006. The entire teachings of these three prior applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION The treatment of cancers continues to suffer from dose-limiting toxicity of the active compounds, highlighting the need for the development of new anticancer drugs.

SUMMARY OF THE INVENTION

It has now been found that certain types of leukemia can be treated by administration of derivatives of imidazoacridines. Specifically, in leukemia cell viability assays (Examples 1 , 3 and 4) and in the xenograft experiments (Example 2), the compound of formula (III) (also known as Symadex®) showed efficacy equivalent to Daunorubicin in the MOLT4 cell lines, and in the MV -4-1 1 xenograft study. In addition, the compound of formula (III) has comparable efficacy to Daunorubicin against MOLT4 (T Cell Leukemia) cell lines and superior activity against the SR (large cell immunoblastic lymphoma) cell line. The compound of formula (III) was also effective in assays involving the K562 (Chronic Myelogenous Leukemia), RPMI (Multiple Myeloma), CCRF-CEM (Acute Lymphoblastic Leukemia), and HL-60 (Acute Promyelocytic Leukemia) cell lines (Example 1 ). The compound of formula (III) was as effective as Daunorubicin in MV -4-11 xenograft studies (Example 2). MV-4-11 xenograft studies also demonstrated a statistical therapeutic effect on tumor volume observed in Symadex®-treated cohort as compared to vehicle. At the higher dosing regimen (15 mg/kg) of Symadex® in the xenograft study, complete tumor response was observed in two animals. No effect on body weight was observed in either Symadex® cohorts, whereas a decrease in body weight was observed with continued treatment of Daunorubicin. In vitro viability studies of B-CLL cells (chronic lymphocyte leukemia) showed that the compound of formula (III) has lower toxicity and similar potency when compared to Clofarabine and Fludarabine (Example 3).

Accordingly, in one embodiment, the present invention is a method of treating a patient suffering from acute myeloid leukemia (AML), comprising administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In another embodiment, the present invention is a method of treating a patient suffering from an acute lymphocytic leukemia (ALL), comprising administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention is a method of treating a patient suffering from a chronic myeloid leukemia (CML), comprising administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention is a method of treating a patient suffering from chronic lymphocytic leukemia (CLL), comprising administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention is a method of treating a patient suffering from an acute myeloid leukemia (AML) characterized by a FLT3 mutation, comprising administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention is a method of treating a patient suffering from a hairy cell leukemia (HCL), comprising administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof. In another embodiment, the present invention is a method of treating a patient suffering from multiple myeloma, comprising administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof.

The imidazoacridines of the present invention are described by formula (I):

(J^

wherein

R is -OH or a C1-C6 alkoxy group;

R^a and R^b, is each independently hydrogen or an optionally substituted alkyl; R² is -H or an C1-C6 alkyl; and n is a whole number between 2 and 5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot showing cell viability plot (as % control) for cell lines treated with Symadex®.

FIG. 2 is a plot showing cell viability plot (as % control) for cell lines treated with daunorubicin.

FIG. 3 is a plot representing tumor volumes measured in xenograft experiments.

FIG. 4 is a bar plot comparing of the initial and final mean tumor volumes of measured in xenograft experiments. FIG. 5 is a plot representing relative percent body weights measured in xenograft experiments.

FIG. 6 is a plot showing viability of B-CLL cells from patient #5 treated ex- vivo with Symadex® using a trypan blue exclusion assay. - A -

FIG. 7 is a plot showing viability of B-CLL cells from patient #28 treated ex- vivo with Symadex® using a trypan blue exclusion assay.

FIG. 8 is a plot showing viability of B-CLL cells from patient #28 treated ex- vivo with Symadex® using a MTS assay. FIG. 9 is a plot showing viability of B-CLL cells from patient #30 treated ex- vivo with Symadex® using a trypan blue exclusion assay.

FIG. 10 is a plot showing viability of B-CLL cells from patient #30 treated ex-vivo with Symadex® using a MTS assay.

FIG. 11 is a plot showing viability of B-CLL cells from patient #31 treated ex-vivo with Symadex® using a MTS assay.

FIG. 12 is a plot showing viability of AML cells treated ex-vivo with Symadex® using a trypan blue exclusion assay.

FIG. 13 is a plot showing viability of AML cells treated ex-vivo with Symadex® using a MTS assay. FIG. 14 is a plot showing viability of AML cells treated ex-vivo with

Symadex® using a MTS assay.

FIG. 15 is a plot that shows the Symadex® activity as a FLT3 inhibitor.

FIG. 16 are plots that illustrate cell viability of FLT3-overexpressing cells RS4(11) in assays employing Symadex®. FIG. 17 are plots that illustrate cell viability of FLT3-overexpressing cells

MV 4(11) in assays employing Symadex®.

FIG. 18 are plots that illustrate cell viability of FLT3 deficient cells HL60 in assays employing Symadex®.

DETAILED DESCRIPTION OF THE INVENTION

It has now been discovered that administration of certain derivatives of imidazoacridines can treat and/or alleviate the symptoms of certain leukemias.

Specifically, it has been discovered that certain leukemias can be treated by administering to a patient suffering from an inflammatory disease a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof: (J)

In formula (I), R represents a hydroxy or an alkoxy group. Preferably, R is a C1-C6 alkoxy group. Alternatively, R is an -OH or -OCH₃.

R^a and R^b, which may be identical or different, represent hydrogen or an optionally substituted alkyl. Preferably, R^a and R^b are Cl -C3 alkyls. More preferably, R^a and R^b are each independently ethyl. Alternatively, R^a and R^b are each independently methyl.

The substituents on R^a and R^b can be a hydroxyl, a C1-C4 hydroxyalkyl, an amino, a N-alkyl-amino or a N,N'-dialkylamino group. Such N-alkyl groups preferably containing 1-4 carbon atoms. Examples of such substituents are hydroxyethyl, aminoethyl, N-alkylaminoethyl and N.N'-dialkylaminoethyl.

Parameter n can be 2 to 5. Preferably, n is 2 or 3.

R² can represent hydrogen or a C1-C6 alkyl. Preferably, R² is a hydrogen or a C1-C4 alkyl. More preferably, R² is a -H. In some preferred embodiments, R is -OH or -OCH₃, R^a and R^b are identical and represent C1-C6 alkyl groups, preferably, methyl or ethyl; n is 2 or 3; R² represents hydrogen or a straight chain C1-C4 alkyl. Preferably, R² is an -H.

Examples of compounds of formula (I) include compounds (HA) through (UG):

In a most preferred embodiment, the compound of formula (I) is 5- [[(diethylamino)ethyl]amino]-8-hydroxyimidazo [4,5,1 -de]-acridine-6-one, whose structure is shown in formula (III):

The term "alkyl", as used herein, unless otherwise indicated, includes straight or branched saturated monovalent hydrocarbon radicals, typically Cl-ClO, preferably C1-C6. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, and t-butyl.

The terms "alkoxy", as used herein, means an "alkyl-O-" group, wherein alkyl, is defined above.

Compounds (HA) through (HG) and (III) can be synthesized according to a variety of synthetic schemes disclosed in U.S. Pat. Nos. 5,231,100 and 6,229,015, incorporated herein by reference in their entirety. One example of such a scheme is shown below:

(Scheme I) Compound (III) is known under the trade name of Symadex® and is also referred to as C-131 1.

Symadex® is the lead compound in clinical development from a new series of agents, the imidazoacridinones. It was previously reported to be a topoisomerase II (TOP2) cleavable complex inhibitor and DNA damaging agent. Contemporary analyses of Symadex® suggest alternative structural parallelisms with the molecular scaffolds now seen as common motifs in receptor tyrosine kinase (RTK) inhibitors.

In a kinase inhibitor screening program by Invitrogen and MDS Pharma, Symadex® clusters with RTK inhibitors; it was found to selectively inhibit wild- type (WT) FLT3 and its D835 Y mutant, CSFlR, FGFR2, PDGFR, FGFRl , and KIT.

As used herein, the term "leukemia" is a cancer of the blood or bone marrow characterized by an abnormal proliferation of blood cells, usually white blood cells (leukocytes). It is part of the broad group of diseases called hematological neoplasms.

Four major types of leukemias can be defined. First, leukemia is clinically and pathologically split into its acute and chronic forms.

Acute leukemia is characterized by the rapid growth of immature blood cells. This crowding makes the bone marrow unable to produce healthy blood cells. Acute forms of leukemia can occur in children and young adults. (In fact, it is a more common cause of death for children in the US than any other type of malignant disease.) Immediate treatment is required in acute leukemias due to the rapid progression and accumulation of the malignant cells, which then spill over into the bloodstream and spread to other organs of the body. If left untreated, the patient will die within months or even weeks.

Chronic leukemia is distinguished by the excessive build up of relatively mature, but still abnormal, blood cells. Typically taking months to years to progress, the cells are produced at a much higher rate than normal cells, resulting in many abnormal white blood cells in the blood. Chronic leukemia mostly occurs in older people, but can theoretically occur in any age group. Whereas acute leukemia must be treated immediately, chronic forms are sometimes monitored for some time before treatment to ensure maximum effectiveness of therapy. Furthermore, the diseases are classified according to the type of abnormal cell found most in the blood. When leukemia affects lymphoid cells (lymphocytes and plasma cells), it is called lymphocytic leukemia. When myeloid cells (eosinophils, neutrophils, and basophils) are affected, the disease is called myeloid or myelogenous leukemia.

Combining these two classifications provides a total of four main categories. Acute lymphocytic leukemia (also known as Acute Lymphoblastic Leukemia, or ALL) is the most common type of leukemia in young children. This disease also affects adults, especially those age 65 and older. Acute myelogenous leukemia (also known as Acute Myeloid Leukemia, or

AML) occurs more commonly in adults than in children. This type of leukemia was previously called acute nonlymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) most often affects adults over the age of 55. It sometimes occurs in younger adults, but it almost never affects children. Chronic myelogenous leukemia (CML) occurs mainly in adults. A very small number of children also develop this disease.

The most common forms in adults are AML and CLL, whereas in children ALL is more prevalent.

Yet another type of leukemia is Hairy Cell Leukemia (HCL). Hairy cell leukemia is an incurable, indolent blood disorder in which mutated, partly matured B cells accumulate in the bone marrow. Its name is derived from the shape of the cells, which look like they are covered with short, fine, hair-shaped projections. Unlike any other leukemia, HCL is characterized by low white blood cell counts.

Patients with hairy cell leukemia who are symptom-free typically do not receive immediate treatment. They engage in "watchful waiting" with routine bloodwork and exams every three to six months to monitor disease progression and identify any new symptoms.

Treatment is generally considered necessary when the patient shows signs and symptoms such as low blood cell counts (e.g., infection-fighting neutrophil count below 1.0 K/ul), frequent infections, unexplained bruises, anemia, or fatigue that is significant enough to disrupt the patient's everyday life. Certain types of leukemia are characterized by mutations \nflt3 gene. FLT3 gene product (Fms-like tyrosine kinase; other names include CD 135, FLK2 (Fetal liver kinase 2), STKl (Stem cell kinase I)) is a class III receptor tyrosine kinase (RTK) structurally related to the receptors for platelet derived growth factor (PDGF), colony stimulating factor 1 (CSFl), and KIT ligand (KL). These RTKs contain five immunoglobulin-like domains in the extracellular region and an intracelular tyrosine kinase domain split in two by a specific hydrophilic insertion (kinase insert). FLT3, closely related to PDGF receptors and c-Kit is, however, not inhibited by the small molecule inhibitors of PDGF and c-Kit; (G Del Zotto et al., J. Biol Regulators Homeostatic Agents 15 : 103- 106, 2001 )

Activating mutations of the flt3 gene, located on 13ql2.2, have been identified in a proportion of acute myelogenous leukemia (AML) patients. FLT3 is the most commonly mutated gene in AML, and is constitutively activated by acquired mutation in approximately 30%— 35% of AML. In 20%-25% of cases of AML₅ there are internal tandem duplications (ITD) of a small number of amino acid residues in the juxtamembrane domain of FLT3, and in 10 % there are activating mutations in other FLT3 domains (mainly in exon 14). These mutations activate the FLT3 kinase activity constitutively, and result in increased cellular proliferation and viability. AML patients with FLT3 mutations have a poor prognosis. This progress had led to the development of small molecules that specifically inhibit the abnormally activated FLT3 kinase.

A FLT3 mutation that causes AML or related myeloid or lymphocytic hematological malignancies can be identified by methods well-known in the art. These methods include polymerase chain reaction (PCR)-based amplification techniques, gene sequencing or differential gene expression with mRNA based microarrays. To date, for example, several primer pairs for the detection of FLT3 mutations have been described. They include, for example, the following primer sequences: for the detection of ITD mutations in the juxtamembrane domain: forward primer: 5-CAATTTAGGTATGAAAGCCAGC-3 (SEQ ID NO. 1); reverse primer: 5-CTTTCAGCATTTTGACGGCAACC-3 (SEQ ID NO. 2); for the detection of D835 mutations in the kinase domain: forward primer: 5- CCGCCAGGAACGTGCTTG-3 (SEQ ID NO. 3); reverse primer: 5- GCAGCCTCACATTGCCCC-3 (SEQ ID NO. 4). Detailed descriptions on methodology, incorporated herein by reference, are readily available in textbooks of molecular medicine (see, for example, H Kiyoi and T Naoe, Methods in Molecular Medicine, H. Hand et al., Eds, Humana Press, Chapter 12, pp. 189-197, 2005, the relevant portions of which are incorporated herein by reference). Specific sequencing and microarray methods that identify either point mutations directly or indirectly via informatic reconstruction of expressed sequence tags and other gene fragment constructs also have become routinely available for application to patient blood and tissue samples. Guidance on how these methodologies have been used to determine the status of FLT3 mutations is given by J Jiang et al. (Blood 104. 1855- 1858, 2004); M. Beran et al, (Leukemia Res. 28:547-550, 2004); and F. Kuchenbauer et al. (Hematologica 90:1617-1625, 2005). The entire teachings of these publications is incorporated herein by reference.

Multiple myeloma is a cancer of plasma cells. Plasma cells are a type of white blood cell present in bone marrow, the soft, blood-producing tissue that fills in the center of most bones. Plasma cells usually make up less than 5 percent of the cells in bone marrow. But with multiple myeloma, a group of abnormal plasma cells (myeloma cells) multiplies, raising the percentage of plasma cells to more than 10 percent of the cells in the bone marrow. The result can be erosion of a patient's bones. The disease also interferes with the function of your bone marrow and immune system, which can lead to anemia and infection. Multiple myeloma may also cause kidney problems.

Signs and symptoms of the disease can vary from person to person. One of the most common symptoms, however, is bone pain. A common sign is the presence of abnormal proteins, which can be produced by myeloma cells, in the blood or urine. These proteins, which are antibodies or parts of antibodies, are called monoclonal, or M, proteins.

The term "patient" means a warm blooded animal, such as for example rat, mice, dogs, cats, guinea pigs, and primates such as humans. The terms "treat" or "treating" include any treatment, including, but not limited to, alleviating symptoms, eliminating the causation of the symptoms either on a temporary or permanent basis, or preventing or slowing the appearance of symptoms and progression of the named disorder or condition. The term "therapeutically effective amount" means an amount of the compound, which is effective in treating the named disorder or condition. In certain embodiments, therapeutically effective amount means an amount sufficient to effect remyelination of nerve cells in a patient. In one embodiment, the compounds of the invention are administered chronically to the patient in need thereof. For example, the chronic administration of the compound is daily, weekly, biweekly, or monthly over a period of at least one year, at least two years, at least three or more years.

The dosage range at which the disclosed imidazoacridines, including compounds of formula (I), formulae (IIA) - (IIG) and formula (III), exhibit their ability to act therapeutically can vary depending upon the severity of the condition, the patient, the formulation, other underlying disease states that the patient is suffering from, and other medications that may be concurrently administered to the patient. Generally, the compounds described herein will exhibit their therapeutic activities at dosages of between about 0.1 mg/m2 free base equivalent per square meter of body surface area/single dose to about 1000 mg/m2 free base equivalent per square meter of body surface area/single dose. For example, the single dosage range can be between 10-800 mg/m2, 100-700 mg/m2, 400-600 mg/m2, 420-550 mg/m2 or 440-500 mg/m2 or the single dose can be 480 mg/m2. In one embodiment of the disclosed method, the imidazoacridinones are administered once a week for three consecutive weeks followed by one week without any administration. In another embodiment of the disclosed method, the imidazoacridinones are administered once a week. Commonly, the imidazoacridinones disclosed herein can be administered intravenously. Alternatively, the imidazoacridinones described herein are administered with a metronomic dosing regimen, i.e., repetitively and continuously with no extended interruptions. For example, total daily dosage for metronomic dosing would range between about 10 mg (5 mg bid) to 2000 mg (1000 mg bid). For example, the dosage range can be between 10-50 mg (5-25 mg bid), 50-150 mg (25- 75 mg bid), 150-250 mg (75-125 mg bid), 250-450 mg (125-225 mg bid), 450-750 mg (225-375 mg bid), 750-1000 mg (375 -500 mg bid), 1000-1250 mg (500-625 mg bid), 1250-1500 mg (625-750 mg bid), 1500 -1750 mg (750-875 mg bid) or 1750-2000 mg (875-1000 mg bid). The dosage can be 180 mg (90 mg bid) or 360 mg (180 mg bid) or 600 mg (300 mg bid) or 900 mg (450 mg bid). Commonly, the imidazoacridinones disclosed herein can be administered orally. Oral administration is preferred for metronomic dosing.. The imidazoacridinones disclosed herein can be administered intravenously, orally or subcutaneously in the total daily doses described above daily, twice daily, weekly, bi-weekly or monthly. In another embodiment the imidazoacridinones are administered daily for two consecutive days, for three consecutive days, for four consecutive days, for five consecutive days, for six consecutive days. Daily dosing of the compounds of the present invention can be continued, in one embodiment, for one week. Daily dosing of the imidazoacridinones of the present invention can be continued, in another embodiment, for 5 days once per month. In other embodiments, daily dosing can be continued for one month to six months; for six months to one year; for one year to five years; and for five years to ten years. In another embodiment the compounds are administered weekly. Alternatively, the imidazoacridinones disclosed herein can be administered daily or twice daily for a period of consecutive days, optionally followed by a period without drug treatment. In one embodiment the imidazoacridinones are administered once or twice daily for 3-28 consecutive days. In another embodiment the imidazoacridinones are administered once or twice daily for 3-7 consecutive days, for 7-12 consecutive days, for 12-18 consecutive days, for 18-28 consecutive days. In one embodiment the imidazoacridinones are administered once or twice daily for five consecutive days, for ten consecutive days, for sixteen consecutive days, for twenty-one consecutive days, for 28 consecutive days. Once or twice daily dose of administration of the imidazoacridinones can be repeated, in one embodiment, for three weeks; and the period without drug treatment can be optional or lasting for 1 to 2 weeks. In other embodiments once or twice daily dose of administration of the imidazoacridinones can be repeated, in one embodiment, for 4 weeks; and the period without drug treatment can be optional or lasting for 1 to 2 weeks. In other embodiments, once or twice daily dose can be repeated for one month to six months; for six months to one year; for one year to five years; and for five years to ten years, wherein the period without the imidazoacridinones treatment (e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) follows the period of imidazoacridinones treatment. In other embodiments, the length of the treatment by repeated administration is determined by a physician. It is to be understood that the total daily doses described in the previous paragraph apply to the dosing regimens described in this paragraph. In treating a patient afflicted with a condition described above, all of the disclosed imidazoacridines can be administered in any form or mode which makes the compound bioavailable in therapeutically effective amounts. For example, compounds of formula (I), formulae (HA) - (HG) and formula (III) can be administered in a form of a pharmaceutically acceptable salt. The term "pharmaceutically acceptable salts" means either an acid addition salt or a basic addition salt, whichever is possible to make with the compounds of the present invention. "Pharmaceutically acceptable acid addition salt" is any non-toxic organic or inorganic acid addition salt of the base compounds represented by formula (I), formulae (HA) - (HG) and formula (III). Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acid and acid metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids which form suitable salts include the mono-, di- and tri-carboxylic acids. Illustrative of such acids are, for example, acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic, hydroxybenzoic, phenylacetic, cinnamic, salicyclic, 2-phenoxybenzoic, p-toluenesulfonic acid and sulfonic acids such as methanesulfonic acid and 2-hydroxyethanesulfonic acid. Either the mono- or di-acid salts can be formed, and such salts can exist in either a hydrated or substantially anhydrous form. In general, the acid addition salts of these compounds are more soluble in water and various hydrophilic organic solvents and which in comparison to their free base forms, generally demonstrate higher melting points. "Pharmaceutically acceptable basic addition salts" means non-toxic organic or inorganic basic addition salts of the compounds of formula (I), formulae (HA) - (HG) and formula (III). Examples are alkali metal or alkaline-earth metal hydroxides such as sodium, potassium, calcium, magnesium or barium hydroxides; ammonia, and aliphatic, alicyclic, or aromatic organic amines such as methylamine, trimethylamine and picoline. The selection of the appropriate salt may be important so that the ester is not hydrolyzed. The selection criteria for the appropriate salt will be known to one skilled in the art.

Compounds of the present invention can be administered by a number of routes including orally, sublingually, buccally, subcutaneously, intramuscularly, intravenously, transdermally, intranasally, rectally, topically, and the like. One skilled in the art of preparing formulations can determine the proper form and mode of administration depending upon the particular characteristics of the compound selected for the condition or disease to be treated, the stage of the disease, the condition of the patient and other relevant circumstances. For example, see Remington's Pharmaceutical Sciences, 18^th Edition, Mack Publishing Co. (1990), incorporated herein by reference.

The compound of formula (I) of this invention may also be administered topically, and when done so the carrier may suitably comprise a solution, ointment or gel base. The base, for example, may comprise one or more of petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.

The solutions or suspensions may also include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylene diaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials. The invention is illustrated by the following examples, which are not intended to be limiting in any way.

EXEMPLIFICATION

Example 1 Svmadex® reduces viability of leukemia cell lines in in vitro experiments The activity of Symadex® against Leukemia cells in vitro was tested in a variety of cell lines. Daunorubicin was used as a control. Cell viability was monitored by the Trypan Blue Exclusion assay at 72 hours. Trypan blue exclusion (TBE) assay for cell viability

This assay was performed at 72 hours. 10 μl of cell solution was added to a 0.5 ml tube and 2.5 μl 0.4% Trypan blue stock in PBS (filtered) was added. 10 μl of this solution was added onto a hematocounter and counted immediately without a green filter at magnification 10.

The results are presented in FIG. 1 and FIG. 2.

As can be seen from FIG 1 and FIG. 2, Symadex® demonstrated higher potency then daunorubicin in the SR (large cell immunoblastic lymphoma) cell line. Symadex® had comparable efficacy to daunorubicin against the MOLT4 (T Cell Leukemia) cell line. Symadex® demonstrated lower potency in the K562 (Chronic Myelogenous Leukemia) RPMI (Multiple Myeloma), CCRF-CEM (Acute Lymphoblastic Leukemia), and HL-60 (Acute Promyelocytic Leukemia) cell lines.

Example 2 Xenograft studies demonstrate a statistical therapeutic effect on tumor volume observed in Svmadex®-treated cohorts

Tumors were created in seventy athymic nude mice by subcutaneous injection in the hind flank with My-4-11 cells. The day prior to the first administration of the test articles, all animals in the study were ranked by their measured tumor volumes and were then redistributed equally using an S-shaped distribution scheme into Ibur experimental groups which consisted of a daunorubicin positive control (2.5 mg/kg (group 1, n=10)), the Test compound at two doses (low dose of 5 mg/kg (group 2, n=10); and the high dose of 15 mg/kg (group 3, n=10)), and a vehicle control (group 4, n=10). Vehicle control article, positive control article and the Test compound were delivered by intraperitoneal injection every three days beginning on the first day Ibllowina animal redistribution (day 26). Tumors were measured with a vernier caliper two times a week until the completion of the study. At the completion of the study, terminal plasma samples were collected one hour post dosing by cardiac puncture, tumors were excised, weighed, and then flash frozen. The results are presented in FIG. 3, FIG. 4 and FIG. 5.

FIG. 3 shows tumor volumes represented as a percent of the initial measured volume. FIG. 4 shows comparison of the initial (Day 0) and final (Day 15) mean tumor volumes of the animals in each cohort that were alive on Day 15 (n=6, errors based on s.e.m.)- FIG. 5 shows relative percent body weights during the study for each cohort.

Even though there is no significant difference between the drug-treated cohorts, there is an observed decrease in body weight in the daunorubicin treatment. Tumors were implanted 26 days prior to treatment. The dosing regimen was q3dx6 with intraperitoneal administration; the dosages for daunorubicin, "C-1311 Low", and "C-1311 High" were 5.9, 5, and 15 mg/kg, respectively.

The tumor volumes were measured at the time of sacrifice. The results are presented in Table 1 below. The values represent the percent of starting tumor volume prior to dosing. Two animals in the Symadex® (C-1311) 15 mg/kg cohort demonstrated complete tumor response.

Table 1

Statistical comparison of the tumor volumes (Day 10-15) of each cohort to the vehicle or daunorubicin cohorts using the Kolgomorov-Smirnov Test (a = 0.025) was performed. These values were based on the 5 dose maximum with 2 random sacrifices after 3 and 4 doses from each cohort. Results are presented in Table 2 below. Based on this comparison, there is significance between the vehicle and drug-treated cohorts; however there is no significant difference between Symadex® and daunorubicin-treated cohorts. Table 2

Example 3 Svmadex® reduces viability of B-CLL cells

The activity of C- 131 1 against CLL cells was tested in ex vivo cultures of B lymphocytes from 3 CLL patients. In the three experiments C-131 1 activity was compared to clofarabine, clofarbine and fludarabine or clofarbine, fludarabine and daunorubicin. Preliminary evidence suggests C-1311 has activity against cells from CLL patients and to be similar in potency to Clofarabine and Fludarabine against these isolated cells.

After preparation of B-CLL cell suspension by the methods known in the art, the following measurements were performed.

Preparation of Ex vivo cell culture

B cell solution was spun in 2 x 2.0 ml Eppendorf® tubes at 6000 rpm (4000 x g) at 8⁰C for 1 min and the supernatant was discarded. The cell pellet was resuspended in warm RPMI medium with 15% autologous serum at 1.5 — 2 x 10⁶ cells/ml. 1.0 ml B cells was placed into each well of 48-well for cell counting and TBE assay and if MTS was to be performed 200 μl B cells into wells of a 96 well plate for MTS assay at 96 hours.

Trypan blue exclusion (TBE) assay for cell viability

This assay was performed at 96 hours. 10 μl of cell solution was added to a 0.5 ml tube and 2.5 μl 0.4% Trypan blue stock in PBS (filtered) was added. 10 μl of this solution was added onto a hematocounter and counted immediately without a green filter at magnification 10. MT5 cell proliferation assay

MTS assay is an assay in which the bioreduction of the MTS reagent (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium)) by cells is being measured to assess metabolic activity of cells. CellTiter 96 Aqueous non-radioactive cell proliferation assay was performed at 96 hours.

Activity ofC1311 against B-CLL cells from patient # 5

B cells were treated with vehicle alone or C-1311 (lμM and 5μM) or Clofarabine (0.1 μM or 1 μM). The % viability of cells as compared to the control cells was monitored by the TBE assay at 24, 48, 72 and 96 hours. The results are shown in FIG. 6.

As can be seen, Symadex®, when compared to clofarabine in ex vivo B-CLL cells, is also effective and shows similar potency at higher doses.

Activity of Cl 311 against B-CLL cells from patient # 28

B cells were treated with vehicle alone, C-1311, clofarabine or fludarabine. Table 3 below shows the concentrations tested.

Table 3

The % viability of cells as compared to the control cells was monitored by the TBE assay or the MTS assay at 96 hours.

The results are shown in FIG. 7 and 8.

As can be seen, Symadex®, when compared to clofarabine and fludarabine against ex vivo B-CLL cells, is also effective and shows similar potency at higher doses.

Activity of C J 311 against B-CLLL cells from patient # 30

B cells were treated with vehicle alone, C-131 1, clofarabine, fludarabine or daunorubicin. Table 4 below shows the concentrations tested. Table 4

The % viability of cells as compared to the control cells was monitored by the TBE assay at 92 hours and the MTS assay at 96 hours.

The results are shown in FIG. 9 and 10.

Against this patient's ex vivo B-cells, Symadex®, when compared to clofarabine, fludarabine and daunorubicin was less effective.

Therefore, Symadex® demonstrated similar activity to fludarabine and/or clofarabine in B-CLL cells from two of three patients tested.

Activity ofCBll against B-CLLL cells from patient # 31

B cells were treated with vehicle alone, C-1311, clofarabine, fludarabine or daunorubicin. Table 5 below shows the concentrations tested. Table 5

The % viability of cells as compared to the control cells was monitored by the MTS assay at 96 hours.

The results are shown in FIG. 1 1

As can be seen, Symadex®, when compared to clofarabine, fludarabine and daunorubicin against ex vivo B-CLL cells, is also effective and shows similar potency at higher doses.

Therefore, Symadex® demonstrated similar activity to fludarabine and/or clofarabine in B-CLL cells from three of four patients tested.

Example 4 Svmadex® reduces viability of AML cells

The activity of C- 1311 against AML cells was tested in ex vivo cultures of total mononuclear cells from two AML patients. C-1311 activity was compared to clofarabine and daunorubicin. Preliminary evidence suggests C-131 1 has activity against cells from AML patients and to be similar in potency to clofarabine and daunorubicin against these isolated cells. After preparation of total mononuclear cell suspension by the methods known in the art, the following measurements were performed.

Preparation of Ex vivo cell culture

Total mononuclear cell solution was spun in 2 x 2.0 ml EppendorfD tubes at 6000 rpm (4000 x g) at 8⁰C for 1 min and the supernatant was discarded. The cell pellet was resuspended in warm RPMI medium with 15% autologous serum at 1.5 ~ 2 x 10⁶ cells/ml. 0.5 ml AML cells was placed into each well of 48-well for cell counting and TBE assay and if MTS was to be performed 180 μl AML cells into wells of a 96 well plate for MTS assay at 96 hours.

Activity of ClSIl against AML cells from patient # 7

AML cells were treated with vehicle alone, C- 1311 , clofarabine or daunorubicin. Table 6 below shows the concentrations tested.

Table 6

The % viability of cells as compared to the control cells was monitored by the TBE assay and the MTS assay at 96 hours. The results are shown in FIG. 12 and 13. Activity ofC1311 against AML cells from patient #JJ

AML cells were treated with vehicle alone, C-1311, daunorubicin or PKC412. Table 7 below shows the concentrations tested.

Table 7

The % viability of cells as compared to the control cells was monitored by the MTS assay at 96 hours. The results are shown in FIG. 14.

As can be seen, Symadex®, when compared to clofarabine and daunorubicin against ex-vivo AML cells, is also effective and shows very similar potency and when compared to PKC412 shows higher potency.

Example 5: Determination ofFLT3 tyrosine kinase activity in vitro

Testing of the compounds described in this invention was carried out using the SelectScreen™ platform from Invitrogen, Inc. (Carlsbad, CA, USA) and the details of its performance are readily viewed via the web by linking to: http://www.invitrogen.com/downloads/SelectScrn_Brochure.pdf. Briefly, the approach is based on treating each specific kinase with a unique substrate and optical reporter system in the presence of ATP at 100 micromolar or at the apparent optimal ATP concentration for each kinase. In controls, the substrate is phosphorylated and a baseline optimal response is recorded. Graded amounts of putative inhibitor are then added in separate increments to generate a dose response curve. The latter is obtained by fitting to a sigmoid saturation equation, such as the Hill equation, and the concentration of test article producing 50 per cent inhibition is then noted as the IC₅₀. An effective level of inhibition in the low nanomolar range is considered to qualify the test compound as potential drug or targeting agent against the specific kinase that it has inhibited. The IC₅₀ value is, therefore, a measure of potency. Another important feature is specificity. It is considered a desirable property when claiming efficacy to determine how many kinases are inhibited by the same molecule. The fewer number inhibited points toward specificity; the greater to inhibitory promiscuity.

For Symadex®, the in vitro kinase screens revealed not only high activity against FLT3 and its constitutively activate mutant FLT3 D835Y but also a high specificity, with at least a 2-log unit difference between the EC50 value for FLT3 and the corresponding inhibitory index for other and closely related protein tyrosine kinases, e.g. KDR, cKIT, PDGF, FGF, NTRK and others, in the TK and RTK domains.

The screening was carried out in two phases. First the compounds of this invention were tested against a subset, the primary kinase screen, to determine initial activity at 1 and 0.1 micromolar concentrations. If greater than 50% inhibition was observed (at 100 mM ATP) against all but INSR, the compounds proceeded to be tested against the broader Secondary Kinase Screen. Thereafter compounds with at least 50% inhibitory activity on any given kinase were re-examined with a 9 point dilution series to obtain the EC₅₀ against that kinase. Note should be made that activity against INSR, the insulin receptor, in the primary screen or against INSRR (insulin receptor-related receptor) or IGFl, the insulin like growth factor 1 , in the secondary screen would have disqualified the compounds from further development, because inhibition of an essential homeostatic set of receptors is a hallmark of undesirable side effects. None of the compounds of this invention inhibited these so called "housekeeping" receptors even at 10 micromolar concentration, a high enough concentration generally indicative of drug safety absent. The experiments described in this section employed Symadex® FIG. 15 shows its activity as a FLT3 inhibitor.

Example 6 Determination ofFLT3 protein tyrosine kinase activity targeting in cell culture

It is now understood that an important subset of human myeloid leukemia cells overexpress FLT3 as constitutively activated mutations. The most prevalent of these is the internal tandem duplication. Two cell lines commonly used to test the potency of FLT3 inhibitors on cell growth and viability are the RS4(11) and MV4(11 ). The RS4(1 1 ) cell line, also known as RS4; 11 , was established from a bone marrow patient with acute lymphoblastic leukemia. This female patient was 32 years of age. The cells lack surface and cytoplasmic immunoglobulin, and are negative for CDlO. The cells have a characteristic chromosome translocation (4;11)(q21;q23), and an isochromosome for the long arm of chromosome 7. These cells express the wild-type FLT3 receptor and are further characterized by expression of the MHC Class II antigens (HLA DR+); CD9+; CD24+. The MV4(1 1) cell line, also known as MV-4-11, was established from the blast cells of a 10 year old male with biphenotypic B myelomonocytic leukemia (ATCC). Other sources indicate this cell line is derived from acute monocytic leukemia (AML FAB M5). The cytogenetic analysis reveals that there are 48 chromosomes (+8, +19) and a (4;11)(q21 ;q23) translocation. These cells express a mutant form of FLT3 containing an internal tandem duplication (ITD), and are further characterized by expression of CD4 (40-96%); CDlO (4-11%); CDl 5 (96-99%). In addition, the activity of Symadex® was compared to mitoxantrone, daunorubicin and PKC412 in three cell lines with differential FLT3 status. HL60 cells (promyelocytic leukemia) were used as FLT3 negative control cells, as they are FLT3 and p53 deficient.

When standard protocols for adherent cell culture are followed, according to the instructions supplied by the American Type Culture Collection for the two. line, the cell will proliferate with an approximate 30 hour doubling time. However, when exposed to graded amounts of Symadex®, the growth and viability of these cell lines is arrested. As shown in FIG. 16, the effective concentration to decrement cell viability by 50% (EC₅₀) for Symadex® in RS4(11) at 72 hrs of continuous exposure is 34 nM, which compares favorably to the positive control, PKC-412, a known FLT3 inhibitor. In the case of the more clinically significant MV4(11) line, which bears the internal tandem duplication and requires an active FLT3 signaling cascade for survival, Symadex® and PKC-412 were closely matched (see FIG. 17). When the drugs were used to arrest growth in a the HL-60 leukemia, which is known to neither express FLT3 nor require the FLT3 signaling cascade for growth or viability, the EC₅₀ of Symadex® rose to values greater than 10 micromolar, as did PKC-412, confirming that their cytotoxicity had been derived in large measure by attack on FLT3 (see FIG. 18). While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

What is claimed is:

1. A method of treating a patient suffering from acute myeloid leukemia (AML), comprising: administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof:

φ_}

wherein

R is -OH or a C1-C6 alkoxy group;

R^a and R^b, is each independently hydrogen or an optionally substituted alkyl;

R² is -H or an C1-C6 alkyl; and n is a whole number between 2 and 5.

The method of Claim 1, wherein the compound is the compound of formula (III) or a pharmaceutically acceptable salt thereof:

A method of treating a patient suffering from a acute lymphocytic leukemia (ALL), comprising: administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof:

φ_}

wherein

R is -OH or a C1-C6 alkoxy group;

R^a and R^b, is each independently hydrogen or an optionally substituted alkyl;

R² is -H or an C1-C6 alkyl; and n is a whole number between 2 and 5.

The method of Claim 3, wherein the compound is the compound of formula (III) or a pharmaceutically acceptable salt thereof:

A method of treating a patient suffering from a chronic myeloid leukemia (CML), comprising: administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof:

wherein

R is -OH or a C1-C6 alkoxy group;

R^a and R^b, is each independently hydrogen or an optionally substituted alkyl;

R² is -H or an C1-C6 alkyl; and n is a whole number between 2 and 5.

The method of Claim 5, wherein the compound is the compound of formula (III) or a pharmaceutically acceptable salt thereof:

A method of treating a patient suffering from chronic lymphocytic leukemia (CLL), comprising: administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof:

(J)₅

wherein

R is -OH or a C1-C6 alkoxy group;

R^a and R^b, is each independently hydrogen or an optionally substituted alkyl;

R² is -H or an C1-C6 alkyl; and n is a whole number between 2 and 5.

The method of Claim 7, wherein the compound is the compound of formula (III) or a pharmaceutically acceptable salt thereof:

A method of treating a patient suffering from an acute myeloid leukemia (AML) characterized by a FLT3 mutation, comprising: administering to the patient a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof:

wherein

R is -OH or a C1-C6 alkoxy group;

R^a and R^b, is each independently hydrogen or an optionally substituted alkyl;

R² is -H or an Cl -C6 alkyl; and n is a whole number between 2 and 5.

10. A method of Claim 9, wherein the compound is the compound of formula (III) or a pharmaceutically acceptable salt thereof:

11. A method of treating a patient suffering from a hairy cell leukemia (HCL), comprising: administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof:

₂)_nNR^BR^b _(I)} wherein

R is -OH or a C1-C6 alkoxy group;

R^a and R^b, is each independently hydrogen or an optionally substituted alkyl;

R² is -H or an C1-C6 alkyl; and n is a whole number between 2 and 5.

12. The method of Claim 11, wherein the compound is the compound of formula (III) or a pharmaceutically acceptable salt thereof:

13. A method of treating a patient suffering from multiple myeloma), comprising: administering to said patient a therapeutically effective amount of the compound of formula (I) or a pharmaceutically acceptable salt thereof:

(J)₅

wherein

R is -OH or a C1-C6 alkoxy group;

R^a and R^b, is each independently hydrogen or an optionally substituted alkyl;

R² is -H or an C1-C6 alkyl; and n is a whole number between 2 and 5.

14. The method of Claim 11, wherein the compound is the compound of formula (III) or a pharmaceutically acceptable salt thereof: