WO2023104809A2

WO2023104809A2 - 2-oxothiazole compositions for treatment of t-cell acute lymphoblastic leukaemia

Info

Publication number: WO2023104809A2
Application number: PCT/EP2022/084623
Authority: WO
Inventors: Astrid Jullumstrø FEUERHERM; Felicity Jane ASHCROFT; Berit Johansen
Original assignee: Coegin Pharma AB
Current assignee: Coegin Pharma AB
Priority date: 2021-12-06
Filing date: 2022-12-06
Publication date: 2023-06-15
Anticipated expiration: 2024-06-06
Also published as: GB202117609D0; WO2023104809A3

Abstract

A compound of formula (I) wherein R₆ is H, C_1-6alkyl, -(CH₂)_pCOOH, -(CH₂)_pCOOC_1-6alkyl, - (CH₂)_pCONH₂, -(CH₂)_pCONHC_1-6alkyl, and -(CH₂)_pCON(C_1-6alkyl)₂; R¹¹ is H or C_1-6 alkyl; R₅ is -OC_1-10alkyl, -SC_1-10alkyl, -C_1-12alkyl, or OAr²; wherein Ar²is phenyl, optionally substituted with one or more halo; each p is 0 to 3; or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof; for use in the treatment of T-cell acute lymphocytic leukemia.

Description

2-OXOTHIAZOLE COMPOSITIONS FOR TREATMENT OF T-CELL ACUTE LYMPHOBLASTIC LEUKAEMIA

This invention relates to compositions for use in the treatment of T-cell induced acute lymphoblastic leukaemia (T-cell ALL). Acute lymphoblastic leukaemia is also known as acute lymphocytic leukaemia. In particular, the invention relates to the use of various 2-oxothiazole compounds in the treatment of T-cell ALL. The invention also relates to methods of treating, e.g. reducing symptoms associated with T-cell ALL in patients comprising administration of the compounds of the invention to the patient.

Background

Leukaemias account for approximately 8% of all human malignancies. Human leukaemia can be divided into four subgroups based on the cell lineage and clinical manifestation: acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL), chronic myeloid leukaemia (CML) and chronic lymphoid leukaemia (CLL). Acute leukaemias (AML and ALL) are most prevalent amongst young children and are associated with aggressive and often fatal diseases, unless treated immediately.

ALL represents 12% of all leukaemia cases, and approximately 80% of all leukaemia cases amongst children. The majority of ALL patients do not display an identifiable genetic or environmental cause. Genetic mutations, hereditary links and exposure to carcinogenic factors are however believed to be the most prominent risk factors. In ALL, early haemopoietic cells, called blast cells, accumulate in the bone marrow. This is also reflected in the clinical manifestation of ALL, characterised by anaemia and leukopenia as the bone marrow fails to exert normal function.

ALL is classified into two main subtypes: B-cell lymphoblastic leukaemia (B-cell ALL) and T-cell lymphoblastic leukaemia (T-cell ALL). T-cell ALL occurs more often in adults than in children and represents about 25% of cases in adults.

When T cells are affected by leukaemia, this is called T-lymphoblastic leukaemia or T cell leukaemia. B-cell lymphoblastic leukemia is a form of lymphoid leukemia in which too many B-cell lymphoblasts (immature white blood cells) are found in the blood and bone marrow. It is the most common type of acute lymphoblastic leukemia (ALL).

As ALL represents a heterogeneous group of diseases without an identifiable genetic or environmental cause, there currently isn’t a targeted treatment. Both types of ALL are treated in the same way. We have however surprisingly found that the compounds of the present case are more active in the context of T-cells than B- cells. This opens the door for targeting of subtypes of ALL.

Various 2-oxothiazoles have previously been reported as CPLA₂ inhibitors. EP-A-3784235 describes the use of 2-oxathiazoles for the treatment of fibrotic diseases.

EP-A-2951164 describes the use of 2-oxothiazoles for the treatment of chronic inflammatory disorders such as rheumatoid arthritis and psoriasis and for the treatment of hyperproliferative disorders such as cancer. The data in EP2951164 is for basal-like breast cancer.

EP3174873 also describes the use of 2-oxothiazole compounds for the treatment of chronic inflammatory disorders and hyperproliferative disorders such as cancer.

WO 2020/229688 describes a combination therapy for various proliferative disorders based on 2-oxothiazoles and a PI3K inhibitor. Again, the primary target is breast cancer. The combination therapy is tested on a CCRF-CEM cell line.

The present inventors sought new methods for treating T-cell ALL, in particular as a monotherapy. It was surprisingly found that certain 2-oxothiazoles can be used in the targeted treatment of T-cell ALL. The present inventors have shown experimentally that the compounds of the invention have utility in the treatment of T-cell ALL. For example, 2-oxothiazole compounds targeting the phospholipase activity of CPLA2 reduced the viability of the CCRF-CEM cell line (T-cell ALL model) in a dose-dependent manner. The compounds of the invention surprisingly target T-cell ALL over B-cell malignancies.

Summary of Invention

Thus viewed from one aspect the invention provides a compound of formula (I)

wherein R₆ is H, C_1-6alkyl, -(CH2)_PCOOH, -(CH2)_PCOOC_1-6alkyl, - (CH₂)_PCONH2, -(CH₂)_PCONHC_1-6alkyl, and -(CH₂)_PCON(C_1-6alkyl)₂;

R¹¹ is H or C_1-6 alkyl;

R₅ is -OC_1-10alkyl, -SC_1-10alkyl, -C_1-12alkyl, or OAr²; wherein Ar² is phenyl, optionally substituted with one or more halo; each p is 0 to 3; or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof; for use in the treatment of T-cell ALL.

Viewed from another aspect the invention provides a method of treating T- cell ALL comprising administering to an animal, preferably a mammal, e.g. human, in need thereof, an effective amount of a compound of formula (I) or a salt, ester, solvate, N-oxide, or prodrug thereof as hereinbefore described.

Viewed from another aspect the invention provides use of a compound of formula (I) or a salt, ester, solvate, N-oxide, or prodrug thereof as hereinbefore described for use in the manufacture of a medicament for treating T-cell ALL.

Viewed from another aspect the invention provides a method of treating T- cell ALL comprising screening ALL patients to determine if they have T-cell ALL, and administering a compound of formula (I) or a salt, ester, solvate, N-oxide, or prodrug thereof as hereinbefore described to patients who have T-cell ALL.

Detailed Description

This invention involves the use of compounds of formula (I) or a salt, ester, solvate, N-oxide, or prodrug thereof in the treatment of T-cell ALL.

T-cell ALL This invention targets T-cell ALL. ALL represents 12% of all leukaemia cases, and approximately 80% of all leukaemia cases amongst children. T-cell ALL is a subtype of ALL and represent about 25% of ALL cases in adults.

Leukaemias arise from haematopoietic stem cells (HSC). Leukaemic cells fail to go through terminal differentiation, and become arrested at earlier stages of maturation. In this state, the cells maintain their ability to proliferate, resulting in an accumulation of immature cells. The arrested differentiation can thus be considered causative of leukaemia.

A lymphoid stem cell becomes a lymphoblast cell and then one of three types of lymphocytes (white blood cells):

•B lymphocytes that make antibodies to help fight infection.

•T lymphocytes that help B lymphocytes make the antibodies that help fight infection.

•Natural killer cells that attack cancer cells and viruses.

T-cell acute lymphoblastic leukemia (T-ALL) is biologically distinct from its B lymphoblastic (B-ALL) counterpart and shows different kinetic patterns of disease response.

The present inventors have realised that the compounds of the invention offer a possible new route to the targeted treatment of T-cell ALL. In this way, the targeted compounds of the invention may allow B-cells to continue to produce antibodies and fight infection.

Current treatment of ALL mainly consists of chemotherapy, which in some cases is followed by stem cell transplantation. The initial response to treatment is indicative of the overall outcome for the patient. This type of treatment (and all known ALL treatments) are non-specific. The side effects of treatment are significant. It would be useful if a targeted therapy could allow B-cells to survive and continue to function whilst T-cells are targeted. The immune response of a patient with such a targeted therapy would be markedly improved.

Compounds of the Invention

In a first embodiment, the invention provides 2-oxothiazole compounds of formula (I)

wherein R₆ is H, C_1-6alkyl, -(CH2)_PCOOH, -(CH2)_PC00C_1-6alkyl, - (CH₂)_PCONH2, -(CH₂)_PCONHC_1-6alkyl, -(CH2)_PCON(C_1-6alkyl)₂,

R¹¹ is H or C_1-6 alkyl;

It is preferred if R₆ is -COOC_1-6alkyl, or -CONHC_1-6alkyl, e.g. -COOC_1- ₂alkyl, or -CONHCi-2alkyl.

It is preferred if R¹¹ is H or methyl, preferably H.

It is preferred if p is 0 or 1, especially 0.

It is preferred if the R5 group is in the para position on the ring.

It is preferred if R5 is -OCmoalkyl, -SC_4-10alkyl, -C_4-10alkyl, or OAr²; wherein Ar² is phenyl, optionally substituted with one halo. Halo means halogen and is preferably Cl or F, especially F.

It is preferred if R₅ is -OC_4-10alkyl, -SC_4-10alkyl, or -C_4-10alkyl.

It is preferred if R₅ is -OC_4-10alkyl.

In a more preferred embodiment, compounds of use in the invention are of formula (II):

wherein R₆ is -(CH2)_PCOOC_1-6alkyl, or -(CH2)_PCONHC_1-6alkyl;

R¹¹ is H or methyl;

R₅ is -OC_1-10alkyl, -SC_1-10alkyl, -C_1-12alkyl, or OAr²;

Ar² is phenyl, optionally substituted with one halo; each p is 0 to 1; or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof; for use in the treatment of T-cell ALL.

In a most preferred embodiment, compounds of use in the invention are of formula (III):

wherein R₆ is -COOC_1-6alkyl, or -CONHC_1-6alkyl;

R¹¹ is H or methyl;

R₅ is -OC_1-10alkyl, -SC_1-10alkyl, -C_1-12alkyl, or OAr²;

Ar² is phenyl, optionally substituted with one halo; or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof; for use in the treatment of T-cell ALL.

In a most preferred embodiment, compounds of use in the invention are of formula (IV):

wherein R₆ is -COOCi -ealkyl;

R¹¹ is H; R5 is -OC_1-10alkyl, -SC_1-10alkyl, or -C_1-12alkyl; or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof; for use in the treatment of T-cell ALL.

In a most preferred embodiment, compounds of use in the invention are of formula (V):

wherein R₆ is -COOC_1-2alkyl;

R¹¹ is H;

R₅ is -OC_4-10alkyl; or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof; for use in the treatment of T-cell ALL.

Highly preferred compounds for use in the invention are depicted below.

or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof; for use in the treatment of T-cell ALL.

Especially preferred compounds are:

Where possible, the compounds of the invention can be administered in salt, hydrate or solvate form, especially salt form.

Typically, a pharmaceutical acceptable salt may be readily prepared by using a desired acid. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. For example, an aqueous solution of an acid such as hydrochloric acid may be added to an aqueous suspension of a compound of formula (I) and the resulting mixture evaporated to dryness (lyophilised) to obtain the acid addition salt as a solid. Alternatively, a compound of formula (I) may be dissolved in a suitable solvent and the acid may be added in the same solvent or another suitable solvent. The resulting acid addition salt may then be precipitated directly, or by addition of a less polar solvent such as diisopropyl ether or hexane, and isolated by filtration.

Suitable addition salts are formed from inorganic or organic acids which form non-toxic salts and examples are hydrochloride, hydrobromide, hydroiodide, sulphate, bi sulphate, nitrate, phosphate, hydrogen phosphate, acetate, trifluoroacetate, maleate, malate, fumarate, lactate, tartrate, citrate, formate, gluconate, succinate, pyruvate, oxalate, oxaloacetate, trifluoroacetate, saccharate, benzoate, alkyl or aryl sulphonates (e.g. methanesulphonate, ethanesulphonate, benzenesulphonate or p-toluenesulphonate) and isethionate. R₆presentative examples include trifluoroacetate and formate salts, for example the bis or tris trifluoroacetate salts and the mono or diformate salts, in particular the tris or bis trifluoroacetate salt and the monoformate salt.

Compounds of formula (I) may be manufactured using known chemical synthetic routes. The manufacture of the compounds of the invention typically involves known literature reactions. Variations of the substituents on the heterocyclic rings and manipulation of the side chain binding the carbonyl can be achieved using all manner of synthetic techniques which the skilled man will know. In particular, reference is made to WO2011/039365, WO2014/118195, and WO20 16/016472 which all describe synthetic pathways to compounds of this invention. Compounds of the invention can therefore be prepared following the teaching in these references.

By treating or treatment is meant at least one of:

(i) preventing or delaying the appearance of clinical symptoms of the disease developing in a mammal;

(ii) inhibiting the disease i.e. arresting, reducing or delaying the development of the disease or a relapse thereof or at least one clinical or subclinical symptom thereof, or (iii) relieving or attenuating one or more of the clinical or subclinical symptoms of the disease.

The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician. In general a skilled man can appreciate when “treatment” occurs. It is particularly preferred if the composition of the invention is used therapeutically, i.e. to treat a condition which has manifested rather than prophylactically. It may be that the composition of the invention is more effective when used therapeutically than prophylactically.

The compounds of the invention can be used on any animal subject, in particular a mammal and more particularly to a human or an animal serving as a model for a disease (e.g., mouse, monkey, etc.).

In order to treat a disease an effective amount of the active agent needs to be administered to a patient. A “therapeutically active amount” means the amount of a composition that, when administered to an animal for treating a state, disorder or condition, is sufficient to effect such treatment. The “therapeutically effective amount” will vary depending on the composition or combination product, the disease and its severity and the age, weight, physical condition and responsiveness of the subject to be treated and will be ultimately at the discretion of the attendant doctor.

It may be that to treat T-cell ALL according to the invention the composition of the invention has to be readministered at certain intervals. Suitable dosage regimes can be prescribe by a physician.

While it is possible that, for use in the methods of the invention, a compound of formula I may be administered as the bulk substance, it is preferable to present the active ingredient in a pharmaceutical formulation, for example, wherein the agent is in admixture with a pharmaceutically acceptable carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

The term “carrier” refers to a diluent, excipient, and/or vehicle with which an active compound is administered. The pharmaceutical compositions of the invention may contain combinations of more than one carrier. Such pharmaceutical carriers are well known in the art. The pharmaceutical compositions may also comprise any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), and/or solubilising agent(s) and so on. The compositions can also contain other active components, e.g. other drugs for the treatment of T-cell ALL.

It will be appreciated that pharmaceutical compositions for use in accordance with the present invention may be in the form of oral, parenteral, transdermal, sublingual, topical, implant, nasal, or enterally administered (or other mucosally administered) suspensions, capsules or tables, which may be formulated in conventional manner using one or more pharmaceutically acceptable carriers or excipients. The compositions of the invention could also be formulated as nanoparticle formulations.

However, for the treatment of T-cell ALL, the composition will preferably be administered orally or by parenteral or intravenous administration, such as injection. The composition or combination product may therefore be provided in the form of a tablet or solution for injection.

The pharmaceutical composition of the invention may contain from 0.01 to 99% weight - per volume of the active material.

A therapeutically effective amount of the compound of the present invention can be determined by methods known in the art. The therapeutically effective quantities will depend on the age and on the general physiological condition of the patient, the route of administration and the pharmaceutical formulation used.

The therapeutic doses will generally be between 10 and 20000 mg/day and preferably between about 30 and 15000 mg/day. Other ranges may be used, including, for example, 50-500 mg/day, 50-300 mg/day, 100-200 mg/day.

Administration may be once a day, twice a day, or more often, and may be decreased during a maintenance phase of the disease or disorder, e.g. once every second or third day instead of every day or twice a day. The dose and the administration frequency will depend on the clinical signs, which confirm maintenance of the remission phase, with the reduction or absence of at least one or more preferably more than one clinical signs of the acute phase known to the person skilled in the art.

It is within the scope of the present invention to administer the compounds described herein to a subject that has been exposed to, is being exposed to, or will be exposed to one or more other anti-proliferative compounds. Ideally, however the compounds are administered as a monotherapy, i.e. not in combination with any other cancer targeting pharmaceutical.

Alternatively, the compounds of the invention might be used in combination with radiotherapy, cryotherapy, phototherapy, laser therapy or radiation therapy.

In a further embodiment the invention relates to the a compound of formula (X)

or a salt thereof; for use in the treatment of multiple myeloma or acute myeloid leukemia (AML).

Viewed from another aspect the invention provides a method of treating multiple myeloma or acute myeloid leukemia (AML) comprising administering to an animal, preferably a mammal, e.g. human, in need thereof, an effective amount of a compound of formula (X) or a salt, ester, solvate, N-oxide, or prodrug thereof as hereinbefore described.

Viewed from another aspect the invention provides use of a compound of formula (X) or a salt thereof for use in the manufacture of a medicament for treating multiple myeloma or acute myeloid leukemia (AML).

The inventors have further investigated cPLA2a enzyme as a potential target for an alternative treatment in hematological cancers specifically multiple myeloma by means of database screening, cell profiling and assessment of viability and apoptosis in MM cell lines in response to cPLA2a inhibitor (Methyl 2-(2-(4- heptyloxy)phenoxy)acetyl) thiazole-4-carboxylate).

In order to assess whether cancers of a specific origin or mutational background were more sensitive to the effects of inhibiting cPLA2a, we tested how cPLA2a inhibitor, AVX420, affected the growth of a panel of 66 cancer cell lines (Oncolines panel TM). Hematological cancer cell lines were the most sensitive to treatment with both the cPLA2a inhibitors tested and the only group for whom the average IC50 was significantly different from the average for the entire panel. The average IC50 values for solid cancers were 19.5 μM for AVX420. In contrast, the average IC50 values were significantly lower in blood cancer cell lines than solid cancer cell lines with IC50 values of 8.5 μM for AVX420 . A growing body of evidence has shown possible implications of cPLA2a in the development of solid as well as hematological cancers. Our finding, suggest that the cPLA2 enzyme may have a distinct and important role in blood cancers.

The invention is described further below with reference to the following non- limiting examples.

Examples

The following compound was used in the experiments:

Compound 1

Viability assay

The cell line CCRF-CEM served as a model system to investigate the role of CPLA2 as a possible therapeutic target in the context of T-cell ALL. The CCRF- CEM (ATCC CCL-119) cell line is derived from T lymphoblasts from peripheral blood of a child with acute leukaemia. The cells were grown according to the recommendations of the supplier.

Cell viability was measured using the non-toxic redox dye resazurin. To identify the half maximum inhibitory effect (IC50), the concentration in which 50% of the cells are dead, dose response relationships were investigated with increasing concentrations of inhibitors.

Cell cultures were seeded at 4:0 x 10⁵ cells/mL 72 hours prior to the experiments to ensure that the cells were actively proliferating. Cells were seeded in 96 well plates at 5 x 10⁴ cells/well in 80 μL in either CGM or in starvation medium (SM) supplemented with 0.5% FBS. The outermost wells were filled with medium instead of cells to avoid possible edge effects. The cells were incubated for 1 hour to allow them to settle prior to treatment with inhibitors. Working stocks of the inhibitors were made by diluting the inhibitors in dimethyl sulfoxide (DMSO). The final concentration of inhibitors were made by addition of SM. The concentration of DMSO in the treatments and control was standardised to 0.2%.

The cells were treated with compound 1 for 24, 48 and 72 hours in both medium supplemented with 10% and 0.5% FBS (fetal bovine serum). The viability was then measured using resazurin and dose-response curves were generated. IC50 values were then determined based on the dose-response curves (Table 1).

Table 1. IC50 values calculated for compound 1

Compound 1 displayed a dose-dependent reduction in viability under all the investigated conditions. For 0.5% FBS, the inhibition was also found to be time dependent, with increasing inhibition over time.

Cell proliferation assay

Compound 1 was also tested against a panel of 66 cancer cell lines in a cell proliferation assay. T-cell ALL was represented by the cell lines CCRF-CEM and Jurkat. IC50, LD50 and GI50 values were calculated for each cell line.

Cell preparation. All cell lines have been licensed from the American Type Culture Collection (ATCC) Manassas, Virginia (US). Master and working cell banks (MCB and WCB) were prepared by subculturing in ATCC-recommended media and freezing according to ATCC recommended protocols www.atcc.org). Cell line stocks for the assays were prepared from the WCB. The MCB, WCBs and assay stocks were prepared within respectively 3, 6 and 9 passages of the ATCC vial. Compound preparation. Solid powders of reference compounds were stored as indicated by supplier. Compounds were weighed on a calibrated balance and dissolved in 100 % DMSO. DMSO samples were stored at room temperature. At the day of the experiment, the compound stock was diluted in 3.16 fold steps in 100 % DMSO to obtain a 9-point dilution series. This was further diluted 31.6 times in 20mM sterile Hepes buffer pH 7.4. A volume of 5 μl was transferred to the cells to generate a test concentration range from 3.16x10^-5 M to 3.16x10^-9 M in duplicate. The final DMSO concentration during incubation was 0.4 % in all wells. If a compound showed very potent activity, the testing range was expanded to ensure a full dose-response curve could be measured in duplicate.

Cell proliferation assay. An assay stock was thawed and diluted in its ATCC recommended medium and dispensed in a 384-well plate, depending on the cell line used, at a concentration of 200 - 3200 cells per well in 45 μl medium. For each used cell line the optimal cell density is used. The margins of the plate were filled with phosphate-buffered saline. Plated cells were incubated in a humidified atmosphere of 5 % CO2 at 37 °C. After 24 hours, 5 μl of compound dilution was added and plates were further incubated for another 72 hours. After 72 hours, 25 μl of ATPlite IStep™ (PerkinElmer) solution was added to each well, and subsequently shaken for 2 minutes. After 10 minutes of incubation in the dark, the luminescence was recorded on an Envision multimode reader (PerkinElmer).

Controls

T= 0 signal. On a parallel plate, 45 μl cells were dispensed and incubated in a humidified atmosphere of 5 % CO2 at 37 °C. After 24 hours 5 μl DMSO-containing Hepes buffer and 25 μl ATPlite IStep™ solution were mixed, and luminescence measured after 10 minutes incubation (= luminescence_t=0). R₆ference compound. The IC50 of the reference compound doxorubicin is measured on a separate plate. The IC50 is trended. If the IC50 is out of specification (0.32 - 3.16 times deviating from historic average) the assay is invalidated. Cell growth control. The cellular doubling times of all cell lines are calculated from the t = 0 hours and t = 72 hours growth signals of the untreated cells. If the doubling time is out of specification (0.5 – 2.0 times deviating from historic average) the assay is invalidated. Maximum signals. For each cell line, the maximum luminescence was recorded after incubation for 72 hours without compound in the presence of 0.4% DMSO (= luminescence_{untreated,t=72h}). IC50s were calculated by non-linear regression using IDBS XLfit 5. The percentage growth after 72h (%-growth) was calculated as follows: 100% x (luminescence_{t = 72h} / luminescence_{untreated,t=72h}). This was fitted to the 10log compound concentration (conc) by a 4-parameter logistics curve: %-growth = bottom + (top – bottom) / (1+ 10(logIC50 –conc)*hill)), where hill is the Hill-coefficient, and bottom and top the asymptotic minimum and maximum cell growth that the compound allows in that assay. NCI60 parameters. The LD50, the concentration at which 50% of cells die, is the concentration where luminescence_{t = 72h} = ½ x luminescence_{t = 0h}. The GI₅₀, the concentration of 50% growth inhibition, is the concentration where cell growth is half maximum. This is concentration associated with the signal: ((luminescence_{untreated,t=72h} – luminescence_t=0) /2) + luminescence_t=0 Of the cancer cell lines tested, it was found that T-cell leukaemia was the most sensitive blood cancer to compound 1. Furthermore, it was found that amongst the leukaemia cell lines tested, T-cell ALL cancer cell lines were the most sensitive to compound 1. Table 2. Cell proliferation assay results for compound 1

Cell viability assay

The following cell lines (RPMI8226, JJN3, IH1 INA6) served as a models to investigate the role of cPLA₂ in B-cell malignancies.

The cell titer gio (CTG) assay was used to measure cell viability in response to cPLA₂ inhibition; this is a luminescence-based assay that uses quantification of the amount of ATP as an indication of the number of viable cells in a well. The cells were harvested by centrifugation at 1500 rpm for 8 minutes, supernatant was discarded, and the cells were resuspended in experimental media. 10,000 cells per well were seeded in 96-well plates in at least two technical replicates and treated with increasing doses of the compound. After 72 hours, the CTG assay reagent was added according to the manufacturer’s instructions and luminescence recorded. Dose- response curves were plotted for calculating the IC-50 of each compound on cell viability. The results are presented in Table 3.

Table 3. Cell viability assay results

It was surprisingly found that compound 1 had a greater effect on cell proliferation for T-cell ALL compared to other types of leukaemia.

Claims

1. A compound of formula (I)

wherein R₆ is H, C_1-6alkyl, -(CH2)_PCOOH, -(CH2)_PC00C_1-6alkyl, - (CH₂)_PCONH2, -(CH₂)_PCONHC_1-6alkyl, and -(CH2)_PCON(C_1-6alkyl)2;

R¹¹ is H or C_1-6 alkyl;

R₅ is -OC_1-10alkyl, -SC_1-10alkyl, -C_1-12alkyl, or OAr²; wherein Ar² is phenyl, optionally substituted with one or more halo; each p is 0 to 3; or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof; for use in the treatment of T-cell acute lymphocytic leukemia.

2 A compound for use as claimed in claim 1, wherein said compound is:

wherein R₆ is -(CH2)_PCOOC_1-6alkyl, or -(CH2)_PCONHC_1-6alkyl;

R¹¹ is H or methyl;

R₅ is -OC_1-10alkyl, -SC_1-10alkyl, -C_1-12alkyl, or OAr²; Ar² is phenyl, optionally substituted with one halo; each p is 0 to 1; or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof.

3. A compound for use as claimed in claim 1 which is of formula (III):

wherein R₆ is -COOC_1-6alkyl, or -CONHC_1-6alkyl;

R¹¹ is H or methyl;

R₅ is -OC_1-10alkyl, -SC_1-10alkyl, -C_1-12alkyl, or OAr²;

Ar² is phenyl, optionally substituted with one halo; or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof.

4 A compound for use as claimed in claim 1 which is of formula (IV):

wherein R₆ is -COOC_1-6alkyl;

R¹¹ is H;

R₅ is -OC_1-10alkyl, -SC_1-10alkyl, or -C_1-12alkyl; or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof. A compound for use as claimed in claim 1 which is of formula (V):

wherein R₆ is -COOC_1-2alkyl;

R¹¹ is H;

R₅ is -OC_4-10alkyl; or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof. A compound for use as claimed in claim 1 which is of formula

or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof.

7. A compound for use as claimed in claim 1 which is of formula

or a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof.

8. A compound for use as claimed in any preceding claim wherein the compound is formulated with a pharmaceutically acceptable excipient.

9. A compound for use as claimed in any preceding claim wherein the compound is administered without any other anti-cancer agents.

10. A method of treating T-cell acute lymphocytic leukemia comprising administering to an animal, preferably a mammal, e.g. a human, in need thereof, an effective amount of a compound of formula (I) of a salt, ester, solvate, N-oxide, or prodrug thereof, e.g. a salt thereof as defined in claim 1 to 8.

11. Use of a compound of formula (I) as defined in claim 1 to 8 in the manufacture of a medicament for treating T-cell acute lymphocytic leukemia.