TECHNICAL FIELD

The present invention relates generally to compositions and methods for treating cancer. Specifically the presently disclosed subject matter generally relates to the epitrancriptomic regulation of ribonucleic acid (RNA) methylation through small-molecule inhibitors of the RNA m6A demethylase AlkBH5.

Provided herein are compounds are reported that specifically modulate RNA methylation by inhibition of the RNA demethylase AlkBH5. Furthermore, the subject compounds and compositions are useful for the treatment of cancer, such as glioblastoma, astrocytoma, acute myeloid leukemia, acute monocytic leukemia, chronic myelogenous leukemia, T acute lymphoblastic leukemia and the like.

BACKGROUND ART

Chemical modifications of RNA have recently been identified to have an impact on several critical cellular functions, such as proliferation, survival and differentiation, mostly through regulation of RNA stability (Helm et ah, 2017) [6] The most common modification in messenger RNA is N6-methyladenosine (m6A) (Roundtree et ah, 2017) [19]. It has been shown that m6A modifications of RNA affect its splicing, intracellular distribution, translation, and cytoplasmic degradation, playing thus a crucial role in regulating cell differentiation, neuronal signaling, carcinogenesis and immune tolerance (Maity et ah, 2016) [14] The m6A presence in RNA is regulated by specific enzymes, i.e. the RNA methyltransferases, RNA methyl ases and RNA reader proteins.

The N-methylation of the adenosine is a reversible process, catalysed by specific proteins. (Figure 1). Those include the RNA methyltransferase enzyme complex METTL3/METTL14/WTAP (Scholler et al, 2018) [21] consisting of the following three components: METTL3 (methyltransferase-like 3) (Bokar et.ak, 1998) [2], METTL14 (methyltransferase-like 14) (Liu et al, 2014) [9], and WTAP (Wilm's tumour- 1 -associated protein) (Horiuchi et al, 2013) [12]; RNA m6A methyltransferase Mettll6 (Pendleton et al, 2017) [18]; the RNA demethylases FTO (fat mass and obesity-associated protein) (Jia, et al, 2011) [13] and AlkBH5 (AlkB family member 5) (Zheng, et al., 2013) [29], called“erasers”. The fate of the RNA in post-transcriptomic processes is also directed by the“reader” enzymes that recognize specific m6A methylation in RNA. Several RNA reader enzymes have been identified, including YTHDF1 (YTH N6-Methyladenosine RNA Binding Protein 1), YTHDF2 (YTH N6-Methyladenosine RNA Binding Protein 2) YTHDF3 (YTH N6- Methyladenosine RNA Binding Protein 3), YTHDC1 (YTH domain-containing protein 1) and YTHDC2 (YTH domain-containing protein 2) (Park et al., 2017) [17]. These three types of enzymes collectively coordinate the m6A RNA methylome in the eukaryotic cell.

The methyl group from m6A can be removed by two RNA demethylases, Fat mass and obesity associated protein (FTO) and a-ketoglutarate dependent dioxygenase homo log 5 (ALKBH5), (Jia et ah, 2011; Thalhammer et al, 2011 [13]; Zheng et ah, 2013 [29]). Both the FTO and ALKBH5 RNA demethylases belong to the AlkB subfamily of the non-heme Fe(II)/2-oxoglutarate (20G) dependent dioxygenase superfamily. Members from the 20G dioxygenase superfamily act on diverse substrates involved in the regulation of protein biosynthesis (Xu et al, 2014) [30] Several crystal structures of the ALKBH5 catalytic domain have been reported, either in a form bound to 2-oxoglutarate-bound or in form bound to an inhibitor (Aik, et ah, 2014 [1]; Feng et ah, 2014; Xu et al, 2014 [30]). The corresponding enzymatic assay results have showed that the ALKBH5 catalytic domain can demethylate m6A containing ssRNA and ssDNA and that citrate is a weak ALKBH5 inhibitor (Xu et al, 2014b [30]). The available structural data facilitate the rational design of new specific ALKBH5 inhibitors and activators based on the established binding pocket of this protein.

Specific and efficient ALKBH5 inhibitors or activators would enable to examine more closely the physiological and pathological processes related to the m6A demethylation of RNA (Deng et al, 2018) [5] It has been shown that the ALKBH5 overexpression significantly promotes cell proliferation in human cervical cancer cell line SiHa (Wang, X. et al. 2017) [25] Furthermore, the cell motility was also increased by ALKBH5. Thus, reducing m6A level could promote cervical cancer cell proliferation, indicating that increasing m6A level might have anti-cancer effects in cervical cancer. However, contrary to this, according to The cBioPortal for Cancer Genomics database (Gao et al, 2013) [11], ALKBH5 is expressed at a low level in acute myeloid leukemia (AML). Consequently, ALKBH5 may exert a tumor- suppressor function in AML. ALKBH5 is also inducible by hypoxia-inducible factor 1 (HIF- 1) in different cells (Thalhammer et al, 2011) [23] Intratumoural hypoxia is however commonly found in cancers and is an essential microenvironment for cancer progression. ALKBH5 has been therefore reported to promote tumorigenesis and proliferation in glioblastoma stem- like cells (GSCs) (Zhang, S. et al. 2017) [28] and breast cancer stem cells (BCSCs) (Zhang, C. et al. 2016) [ ]. The functional importance of m6A mRNA modification in GSC self-renewal and glioblastoma tumor progression has been demonstrated by functional studies through manipulating expression of METTL3 or METTL14, or pharmacologically inhibiting activity of FTO in GSCs (Cui et al, 2017) [10]. Therefore, the compounds inhibiting AlkBH5 activity can act as suppressors of cancer (Yanhong, S. et al, 2018) [26]

SUMMARY OF INVENTION

The present invention is related to a method of cancer cure by of modulating the RNA methylation at 6-position of adenine (m6A) using effective amount of a compound having binding and/or inhibition of RNA m6A demethylase AlkBH5.

The "summary of invention" heading is not intended to be restrictive or limiting. The invention also includes all aspects described in the detailed description or figures as originally filed. The original claims appended hereto also define aspects that are contemplated as the invention and are incorporated into this summary by reference.

In addition to the foregoing, the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations specifically mentioned above. For example, although aspects of the invention may have been described by reference to a genus or a range of values for brevity, it should be understood that each member of the genus and each value or sub-range within the range is intended as an aspect of the invention. Likewise, various aspects and features of the invention can be combined, creating additional aspects which are intended to be within the scope of the invention. Although the applicant(s) invented the full scope of the claims appended hereto, the claims appended hereto are not intended to encompass within their scope the prior art work of others. Therefore, in the event that statutory prior art within the scope of a claim is brought to the attention of the applicants by a Patent Office or other entity or individual, the applicant(s) reserve the right to exercise amendment rights under applicable patent laws to redefine the subject matter of such a claim to specifically exclude such statutory prior art or obvious variations of statutory prior art from the scope of such a claim. Variations of the invention defined by such amended claims also are intended as aspects of the invention.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is disclosed further with references to accompanying drawings where:

FIG. 1 illustrates a dynamic and reversible m6A methylation in RNA (SAM - S-adenosyl-L- methionine; SAH - S-adenosyl-L-homocystein) described in the prior art (see Niu, et al, 2013) [16];

FIG. 2 illustrates the binding site of the compound (III) according to invention; FIG. 3 illustrates the binding site of the compound (IV) according to invention;

FIG. 4 illustrates the inhibitory effect IE of the compound (III) on the demethylation of the probe RNA by AlkBH5;

FIG. 5 illustrates the inhibitory effect IE of the compound (IV) on the demethylation of the probe RNA by AlkBH5;

FIG. 6 illustrates the inhibitory effect INH% of the compound (III) at 100 mM concentration on the glioblastoma cell culture A- 172;

FIG. 7 illustrates the inhibitory effect INH% of the compound (IV) at 100 mM concentration on the glioblastoma cell culture A- 172;

FIG. 8 illustrates the inhibitory effect INH% of the compound (III) at 10 mM concentration on the Childhood T acute lymphoblastic leukemia cell culture CCRF-CEM;

FIG. 9 illustrates the inhibitory effect INH% of the compound (IV) at 100 mM concentration on the Childhood T acute lymphoblastic leukemia cell culture CCRF-CEM;

FIG. 10 illustrates the inhibitory effect INH% of the compound (III) at 100 mM concentration on the Childhood T acute lymphoblastic leukemia cell culture JURKAT;

FIG. 11 illustrates the inhibitory effect INH% of the compound (IV) at 100 mM concentration on the Childhood T acute lymphoblastic leukemia cell culture JURKAT.

DETAILED DESCRIPTION OF INVENTION

Disclosed herein are compounds and methods of modulating the RNA methylation through inhibition of the RNA m6A demethylase AlkBH5. In some variations of the invention, the compound is administered in a composition that also includes one or more pharmaceutically acceptable diluents, adjuvants, or carriers.

The compound can be a small molecule. In some embodiments, RNA m6A demethylase AlkBH5 inhibitor has a structure of Formula (I),

wherein: R1 is independently selected from the group consisting of H, alkyl, aryl, aralkyl, acyl, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, carbamoyl, alkylcarbamoyl, and dialkylcarbamoyl, aminoalkyl, aminoalaryl, R2 is independently selected from the group consisting of H, alkyl, aryl, aralkyl, aminoalkyl, aminoalaryl; or a pharmaceutically acceptable salt thereof.

In some embodiments, the RNA m6A demethylase AlkBH5 inhibitor compound has a structure of Formula (II)

wherein Rl, R2 and R3 are independently selected from the group consisting of H, alkyl, aryl, alkylenearyl, acyl, alkoxycarbonyl, aryloxycarbonyl, alkylenearyloxycarbonyl, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, and alkyleneamino; or a pharmaceutically acceptable salt thereof. In some embodiments, Rl and R2 are independently selected from the group consisting of alkyleneamino and hydrogen, where the amino group of the alkyleneamino moiety can be further substituted with one or two alkyl or alkylenearyl (e.g., a benzyl) groups.

In some embodiments, the METTL3/METT14/WTAP complex activator compound has a structure of Formula (III)

wherein R1 and R2 are independently selected from the group consisting of H, alkyl, aryl, alkylenearyl, acyl, alkoxycarbonyl, aryloxycarbonyl, alkylenearyloxycarbonyl, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, and alkyleneamino; or a pharmaceutically acceptable salt thereof In some embodiments, R1 and R2 are independently selected from the group consisting of alkyleneamino and hydrogen, where the amino group of the alkyleneamino moiety can be further substituted with one or two alkyl or alkylenearyl (e.g., a benzyl) groups. In a specific embodiment, R1 is methyl and R2 is hydrogen. In some embodiments, the METTL3/METT14/WTAP complex activator compound has a structure of Formula (IV)

wherein R1 and R2 are independently selected from the group consisting of H, alkyl, aryl, alkylenearyl, acyl, alkoxycarbonyl, aryloxycarbonyl, alkylenearyloxycarbonyl, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, and alkyleneamino; or a pharmaceutically acceptable salt thereof. In some embodiments, R1 and R2 are independently selected from the group consisting of alkyleneamino and hydrogen, where the amino group of the alkyleneamino moiety can be further substituted with one or two alkyl or alkylenearyl (e.g., a benzyl) groups. In a specific embodiment, R1 is methyl and R2 is hydrogen.

TERMS

As used herein, the term "alkyl" refers to straight chained and branched hydrocarbon groups containing carbon atoms, typically methyl, ethyl, and straight chain and branched propyl and butyl groups. Unless otherwise indicated, the hydrocarbon group can contain up to 20 carbon atoms.

The term "alkyl" includes "bridged alkyl," i.e., a C.sub.6-C.sub.l6 bicyclic or polycyclic hydrocarbon group, for example, norbomyl, adamantyl, bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, or decahydronaphthyl. Alkyl groups optionally can be substituted, for example, with hydroxy (OH), halo, amino, and sulfonyl.

An "alkoxy" group is an alkyl group having an oxygen substituent, e.g., -O-alkyl.

The term "alkenyl" refers to straight chained and branched hydrocarbon groups containing carbon atoms having at least one carbon-carbon double bond. Unless otherwise indicated, the hydrocarbon group can contain up to 20 carbon atoms. Alkenyl groups can optionally be substituted, for example, with hydroxy (OH), halo, amino, and sulfonyl.

As used herein, the term "alkylene" refers to an alkyl group having a further defined substituent. For example, the term "alkylenearyl" refers to an alkyl group substituted with an aryl group, and "alkyleneamino" refers to an alkyl groups substituted with an amino group. The amino group of the alkyleneamino can be further substituted with, e.g., an alkyl group, an alkylenearyl group, an aryl group, or combinations thereof The term "alkenylene" refers to an alkenyl group having a further defined substituent.

As used herein, the term "aryl" refers to a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, e.g., phenyl or naphthyl. Unless otherwise indicated, an aryl group can be unsubstituted or substituted with one or more, and in particular one to four groups independently selected from, for example, halo, alkyl, alkenyl, OCF.sub.3, NO. sub.2, CN, NC, OH, alkoxy, amino, CO.sub.2H, CO.sub.2alkyl, aryl, and heteroaryl. Exemplary aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, chlorophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, 2,4-methoxychlorophenyl, and the like. An "aryloxy" group is an aryl group having an oxygen substituent, e.g., -O-aryl.

As used herein, the term "acyl" refers to a carbonyl group, e.g., C(O). The acyl group is further substituted with, for example, hydrogen, an alkyl, an alkenyl, an aryl, an alkenylaryl, an alkoxy, or an amino group. Specific examples of acyl groups include, but are not limited to, alkoxycarbonyl (e.g., C(O)— Oalkyl); aryloxycarbonyl (e.g., C(O)— Oaryl); alkylenearyloxycarbonyl (e.g., C(O)— Oalkylenearyl); carbamoyl (e.g., C(O)— NH.sub.2); alkylcarbamoyl (e.g., C(O)— NH(alkyl)) or dialkylcarbamoyl (e.g., C(O)— NH(alkyl).sub.2).

As used herein, the term "amino" refers to a nitrogen containing substituent, which can have zero, one, or two alkyl, alkenyl, aryl, alkylenearyl, or acyl substituents. An amino group having zero substituents is—NH.sub.2.

As used herein, the term "halo" or "halogen" refers to fluoride, bromide, iodide, or chloride.

As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid or inorganic acid. Examples of pharmaceutically acceptable nontoxic acid addition salts include, but are not limited to, salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid lactobionic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.

A method of treating glioblastoma, astrocytoma, acute myeloid leukemia, acute monocytic leukemia, chronic myelogenous leukemia, T acute lymphoblastic leukemia in a patient in need thereof, comprising administering to the patient a compound of Formula (I)

or a pharmaceutically acceptable salt thereof.

A method of treating glioblastoma, astrocytoma, acute myeloid leukemia, acute monocytic leukemia, chronic myelogenous leukemia, T acute lymphoblastic leukemia in a patient in need thereof, comprising administering to the patient a compound of Formula (II) A method of treating glioblastoma, astrocytoma, acute myeloid leukemia, acute monocytic leukemia, chronic myelogenous leukemia, T acute lymphoblastic leukemia in a patient in need thereof, comprising administering to the patient a compound of Formula (III)

A method of treating glioblastoma, astrocytoma, acute myeloid leukemia, acute monocytic leukemia, chronic myelogenous leukemia, T acute lymphoblastic leukemia in a patient in need thereof, comprising administering to the patient a compound of Formula (IV)

(IV), or a pharmaceutically acceptable salt thereof.

EXAMPLES

The following Examples have been included to provide illustrations of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill will appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications and alterations can be employed without departing from the spirit and scope of the presently disclosed subject matter. Example 1. Computational Modeling, Pharmacophore Generation, Virtual and Functional Screening.

In order to generate a productive pharmacophore, computational docking of the prospective RNA m6A demethylase AlkBH5 binding compounds was carried out using the complex crystal structures. The structure of the RNA m6A demethylase AlkBH5 was chosen as describing the potential target binding site for a small-molecule inhibitor. The crystal structure of this complex (PDB: 4061) had been measured by X-ray diffraction with resolution 1.9 A (Xu C et al, 2014) [27] The raw crystal structures were corrected and hydrogen atoms were automatically added to the protein using Schrodinger’s Protein Preparation Wizard of Maestro 10.7 (Sastry et al., 2013) [20]

AutoDock 4.2 (Morris et al, 2009) [15] was used for the docking studies to find out binding modes and binding energies of ligands to the receptor. The number of rotatable bonds of ligand was set by default by AutoDock Tools 1.5.6 (Morris et al., 2009) [15] However, if the number was greater than 6, then some of rotatable bonds were made as non-rotatable, otherwise calculations can be inaccurate. The active site was surrounded with a grid-box sized 65 x 65 x 65 points with spacing of 0.375 A. The AutoDock 4.2 force field was used in all molecular docking simulations.

The structure of ligand molecules was optimized using the density functional theory B3LYP method (Stephens et al., 1994) [...] with 6-31G basis set.

As reported by Wang P et al. [25], there are several distinct regions of probable interactions between the ligand and enzyme. As confirmed by our molecular docking calculations, the amino group of the adenosyl fragment of SAM is hydrogen bonded with Asp377 of the Mettl3 (cf. Figure 2). The binding is further supported by another bond between the adenine N1 atom and an adjacent peptide bond NH group. The adenine ring is sandwiched between Phe534 and Asn549, while many polar contacts help to hold the hydroxyl groups on the ribose as well as the amino and carboxyl groups of SAM. The terminal amino group of SAM is acting as hydrogen bond donor to the Asp395 of the catalytic center of enzyme.

Based on this structure we proceeded with the search of effectively bound small molecule fragments. In variance to the crystal structure of SAM itself, these compounds tend to be bound to the region of the AlkBH5 protein involving Met392, Lys513, Tyr518, Glu532, His538 and Asn539 (Figure 3). A virtual screening on FIMM compound library was carried out using nitrogen-containing heterocycles as base structures. The docking free energies AG and ligand efficiencies LE of the best binding compounds are given in Table 1.

Table 1. The compounds with the highest docking efficiencies to RNA m6A demethylase AlkBH5.

Example 2. Screening of computationally predicted RNA m6A demethylase AlkBH5 ligands in enzyme inhibition assay.

The enzymatic assay was modified from Huang et al. 2015 (Huang et ah, 2015) [31]. The experiments were conducted in reaction buffer (50 mM (millimolar) Tris-HCl, pH 7.5, 300 mM (micromolar) 20G, 280 mM (NH^Fe^O^ and 2 mM L-ascorbic acid). The reaction mixture contained 200 ng (nanogram) methylated N ⁶ -adenine RNA probe (5’- CUUGUCAm6ACAGCAGA-3’, Dharmacon) SEQ. 1 and lOnM (nanomolar) ALKBH5 protein. Reactions were incubated on 96-well plate for 2h at RT. After that, the amount of m6A that was measured using EpiQuik m6A RNA methylation Quantification Colorimetric Kit (Epigentek).

The inhibitory effect IE of compounds on RNA probe demethylation by AlkBH5 was calculated as the enhancement of the m6A amount as compared to the negative control (DMSO) relative to the difference between m6A amounts of the positive control (max inhibition) and the negative control (eq. (1)): IE = - (1)

cinh(. ^max ) ~ ^c DMSO

where C _M , C _M (max) and CDMSO are the amounts of m6A at a given concentration of the inhibitor, maximum inhibition and in the case of DMSO, respectively. The dependence of the IE on the inhibitor concentration for the compound (III) is shown on Fig. 4 and for the compound (IV) on Fig. 5. The inhibitory concentrations are IC50 = 0.840 mM (micromolar) for the compound (III) and IC50 = 1.79 mM (micromolar) for the compound (IV), respectively. Therefore, both compounds are efficient inhibitors of the RNA m6A demethylase AlkBH5.

Example 3. Inhibitory effect of RNA m6A demethylase AlkBH5 inhibitor compounds on glioblastoma cell line A-172.

The A-172 cell line was obtained from the Glioma Tumor Cell Panel (ATCC® TCP-1018™). The HEK-293T cells (ATCC®ACS-4500™). Both HEK-293T and A-172 cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS and Pen/Strep. The cells were grown at 37 °C in the presence of 5% C02.

8 ^X 10 ³ HEK-293T and A-172 cells were seeded in 200 pL (micro liter) on a 16-well E-plate. Cells were incubated for 48 h with added compounds at given concentrations and 0.5% DMSO was used as a vehicle control. Cells viability were measured using real-time xCELLigence machine (RTCA xCELLigence).

The inhibitory effect of the compounds on the proliferation of the malignant A-172 cells was estimated relative to the proliferation of the normal HEK-293T cells. First, the cell counts n _c in the HEK-293T and A-172 cell cultures at a given concentration c of the inhibitor were normalized by the cell counts n DMSO in the case of negative control (DMSO). The normalized cell counts N _c

n _c (HEK-293T)

N _c ( HEK-293T) =

n _D MSO (HEK-293T) (3) were then used for the calculation of the inhibitory effect of a compound at given concentration as follows:

W _C (A- 172)

1NH% 100

N _C (HEK-293T) (4) The time dependence of the inhibitory effect INH% of compound (III) on the malignant A- 172 cells at 100 mM concentration is given in Fig. 6 and the time dependence of the inhibitory effect INH% of compound (IV) at 100 mM concentration in Fig. 7. In both cases, the proliferation of the glioblastoma cells A- 172 is suppressed as compared to the normal HEK- 293T cells.

Example 4. Inhibitory effect of RNA m6A demethylase AlkBH5 inhibitor compounds on Childhood T acute lymphoblastic leukemia cell line CCRF-CEM.

The CCRF-CEM cell line was obtained from the ATCC® TCP1010™ Leukemia Cell Line Panel. The JURKAT cells ATCC®CRL-2899™. Both the JURKAT and CCRF-CEM cells were grown in Roswell Park Memorial Institute medium 1640 (RPMI 1640) supplemented with 10% heat-inactivated FBS and Pen/Strep. The cells were grown at 37 °C in the presence of 5% C02.

lx 10 ⁵ JURKAT and CCRF-CEM cells were seeded in 1 mL on a 24-well plate. Cells were incubated for 48 h with added compounds at given concentrations and 0.5% DMSO was used as a vehicle control. Cells viability were measured using Countess Automated Cell Counter by Thermo Fisher Scientific Invitrogen.

Similarly to the glioblastoma cells, the inhibitory effect of the compounds on the proliferation of the Childhood T acute lymphoblastic leukemia cell line CCRF-CEM and cell line JURKAT cells was estimated relative to the proliferation of the normal HEK-293T cells. Again, the cell counts n _c in the JURKAT and CCRF-CEM cell cultures at a given concentration c of the inhibitor were normalized by the cell counts U _BMSO in the case of negative control (DMSO). The normalized cell counts N _c

n _c (CCRF-CEM)

N _c (CCRF-CEM)

nDMSO (CCRF-CEM) (5) n _c (JURKAT)

N _c (JURKAT) =

nDMSO (JURKAT) (6) were then used for the calculation of the inhibitory effects of a compound at given concentration as follows:

The time dependence of the inhibitory effect INH% of compound (III) on the malignant CCRF-CEM cells at 10 mM (micromolar) concentration is given in Fig. 8 and the time dependence of the inhibitory effect INH% of compound (IV) at 100 mM concentration in Fig. 9. The time dependence of the inhibitory effect INH% of compound (III) on the malignant JURKAT cells at 10 mM concentration is given in Fig. 10 and the time dependence of the inhibitory effect INH% of compound (IV) at 100 mM concentration in Fig. 11. In all cases, the proliferation of the leukemia cells CCRF-CEM is suppressed as compared to the normal HEK-293T cells.

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