Example 2
Example 4
Compound 4a can be prepared according to the procedure described in Example 223 of U.S. Pat. No. 9,382,248.
Biological Studies
peIF4E Signaling Cellular Assay
Phosphorylated eIF4E is assayed using the CisBio peIF4E HTRF® assay kit (CisBio, catalogue No. 64EF4PEG). Cells are plated in 96-well tissue-culture treated plate in appropriate growth medium (90 μL). Compounds (10×) are diluted using 3-fold serial dilutions in cell culture medium and added to cells. Plates are incubated for 2 hrs at 37° C. The cell supernatant is carefully removed either by aspirating supernatant or by flicking the plate. Immediately 50 μL of supplemented lysis buffer (1×) is added and incubated for at least 30 minutes at room temperature under shaking. After homogenization by pipeting up and down, 16 μL of cell lysate is transferred from the 96-well cell-culture plate to a 384-well small volume white plate. 4 μL of premixed antibody solutions (vol/vol) is prepared in the detection buffer and added. The plate is covered with a plate sealer and incubated overnight at room temperature. The fluorescence emissions at two different wavelengths are read (665 nm and 620 nm) on a Wallac Victor2. Emission ratios are converted into percent inhibitions and imported into GraphPad Prism software. The concentration of compound necessary to achieve inhibition of enzyme activity by 50% (IC50) is calculated using concentrations ranging from 20 μM to 0.1 nM (12-point curve). IC50 values are determined using a nonlinear regression model available in GraphPad Prism 5.
The results of these assays are set forth in Table 1 below. To this end, IC50 values of less than 0.001 μM are labelled as “+++”, from 0.001 to 0.01 μM are labelled as “++”, and greater than 0.01 μM are labelled as “+” (NA means “not available”).
MNK Inhibition Decreases Expression of Immune Checkpoint Receptors and Ligands
Upon activation of T cell receptor (TCR) signaling, T cells proliferate, produce cytokines (e.g., IL-2) and induce the expression of immune checkpoint receptors. Programmed death 1 (PD-1) is an inhibitory checkpoint receptor expressed on the surface of activated T cells, as well as on myeloid cells. The ligand for PD-1, programmed death ligand-1 (PD-L1, B7-H1/CD274) is not expressed by T cells or normal epithelial cells, but is expressed by antigen presenting cells and overexpressed in several cancers. Interaction of PD-1 with PD-L1 results in an anti-proliferative effect on T cells and ultimately T cell exhaustion and apoptosis. To study the role of MNK in activated T cells and tumor cells, the effect of a MNK inhibitor on molecules of immune checkpoint control was examined.
PD-1 (CD279) Expression
To examine the effect of MNK inhibitors on PD-1 expression, Jurkat cells (Clone E6.1, ATCC, transformed T cells) were used, which express PD-1 when activated through T cell receptor (TCR) signaling. Briefly, Jurkat cells were grown in 1×RPMI with 1×Pen/Strep, and 10% FBS, then about 3×106 Jurkat cells were activated in presence of 1 μg/mL PHA (Sigma) and 50 ng/mL PMA (Sigma). Test Cells were treated simultaneously with various concentrations of an MNK inhibitor (0, 0.01, 0.1, 1, 3 and 10 μM). After 48 hours, culture supernatants were harvested and examined via sandwich ELISA for the presence of IL-2 using human IL-2 ELISA DuoSet (R&D Systems, Minneapolis, Minn.). The level of PD-1 on Jurkat cells was examined by incubating with human FcR block, then contacted with allophycocyanin (APC) conjugated anti-PD-1 antibody (5 μl per 100 μl volume of test, Biolegend, San Diego, CA) for 25 minutes at 4° C., without washing the cells, fixable dead cell stain (1:10,000; BD Biosciences, San Jose, CA) was added and incubated further for 10 minutes at 4° C. Cells were washed two times with flow buffer, and finally cells were fixed with fixation buffer for 15 minutes at 4° C. After fixation, cells were washed twice with flow buffer and re-suspended in flow buffer and assessed for fluorescence using BD Accuri C6 flow cytometer. Data were analyzed using the C6 cytometer software (BD Biosciences, San Jose, CA) or Attune Nxt Cytometer (Invitrogen, Carlsbad, CA).
Activation of Jurkat T cells with PHA and PMA induced the expression of PD-1 on the cell surface of about 25-30% of the stimulated Jurkat cells as compared to uninduced cells (Unstim) and induced a 1,000-fold increase in IL-2 cytokine production, respectively. Treatment of PHA/PMA activated Jurkat T cells with the MNK inhibitor resulted in a concentration dependent decrease in the expression of the immune inhibitory receptor PD-1, up to a 50% reduction at the highest concentration as compared to control. In addition, this reduction of PD-1 was not due to a block in Jurkat T cell activation per se since MNK inhibition by an MNK inhibitor did not alter cytokine production as measured by IL-2 levels. Lastly, MNK inhibition by various different MNK inhibitors had no effect on cell viability. In fact, various different MNK inhibitors in the Jurkat T cell assay showed the ability to downregulate immune checkpoint inhibitors without affecting cell viability. The results of these assays are set forth in Table 1 below. To this end, percentage of inhibition of PD-1 positive cells (10 μM) of more than 50% are labelled as “+++”, from 10% to 50% are labelled as “++”, and less than 10% are labelled as “+” (NA means “not available”).
Aqueous Solubility
Solubility, the phenomenon of dissolution of solute in solvent to give a homogenous system, is one of the important parameters to achieve a desired concentration of a compound in systemic circulation for desired (anticipated) pharmacological response. Compounds having good aqueous solubility are desirable because they result in good in vivo bioavailability owing to their high dissolution rate following administration to a subject. Compounds having good aqueous solubility also contribute to the ease of formulation development, formulation manufacture and stability of the formulation.
High Throughput Thermodynamic Solubility Procedure
In a 96-well plate, 10 mM DMSO stocks (50-100 uL) of each compound were dried under heated nitrogen flow using a SPE-96 plate dryer (upper flow rate=50 L/min, temperature=60° C., lower flow rate=20 L/min, temperature=80° C.). After DMSO had been completely removed, remaining materials were dissolved in test solvents including deionized water, Fasted-State Simulated Intestinal Fluid (FaSSIF, pH 6.5) and Fasted-State Simulated Gastric Fluid (FaSSGF, pH 1.6). Compounds prepared as the free base were assessed in conditions with and without one equivalent of TFA added. The theoretical maximum concentrations of the aqueous solutions were 10 mM. Each well was capped and incubated for a period of 18 hours at room temperature or 37° C. Mixing of the solutions during the incubation period was performed by either shaking the plate at 750 rpm or adding StirStix (stainless steel capillaries) and agitating the mixture using a rotary magnetic tumble stirrer. After the incubation period, aliquots from each well were filtered. Solubility of all samples were quantified following comparison to standards of known concentration by HPLC-UV.
The results of the aqueous solubility are set forth in Table 1 below.
The inventors have unexpectedly found that the dissolution rate of an Mnk inhibitor compound can be remarkably improved over comparative compound 4a by: (i) substituting the imidazopyridine in the compound with 3- or 4-piperidine; and (ii) by substituting the pyrimidine in the compound with lower alkyl, halogen or cyano. Such substitutions or structural features induce chirality in the molecules.
The improvement in the aqueous solubility of the compounds of the invention is especially significant in low pH environments, such as in gastric or stomach fluid. For example, as shown in Table 1, compounds 3D, 3K, 3L, 3X, 3Y, rac-1F, 1Fa, 1Fb, rac-2F, 2Fa and 2Fb exhibited excellent solubility in fasted state simulated gastric fluid (FaSSGF), which has a pH value of 1.6.
Importantly, in addition to improved aqueous solubility, the 3- and 4-piperidine-substituted compounds have also retained their anti-cancer potency in the form of their ability to inhibit pEIF4E signaling. In some cases, the anti-cancer potency is further manifested in the ability of these 3- and 4-piperidine-substituted compounds to inhibit PD-1, as can be seen in Table 1.
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
