PHENOTHIAZINE-PYRIMIDINE-2,4(1 H, 3H)-DIONE COMPOUNDS FOR THE TREATMENT OF CANCER TECHNICAL FIELD

The invention relates to the design and synthesis of novel thioridazine derivatives that are thought to have anticancer effects greater than thioridazine.

PRIOR ART

It is known that cancer recurs after discontinuation of chemotherapy, as chemotherapeutic agents used in cancer treatment do not completely eradicate cancer cells. This happens because chemotherapeutic agents do not affect reagent-resistant cancer stem cells as much as they have effects on cancer cells. It has been recently discovered that thioridazine, which had been previously used as an antipsychotic agent, apoptosizes (programmed cell death) or differentiates (transformation into normal cells) cancer stem cells. Researches indicate that the anticancer effect of thioridazine stems from its abilitiy to antagonize D2-type dopaminergic receptors, which are overexpressed in cancer stem cells.

Tuynder et al. reported in 2004 that thioridazine apoptosized or differentiated human erythroleukemia and tamoxifen-resistant mammary cancer cells through down-regulation of translationally controlled tumor protein (TCTP). In this regards, Tuynder et al. proposed that the down-regulation of TCTP could relate to the antagonistic effects of thioridazine on dopamine receptors expressed by cancer cells. Recently, Amson et al. reported that the down- regulation of TCTP increased the ubiquitination and proteolytic degredation of the M/HDM-2 (murine/human double-minute-2) protein, and therefore, leading to an increase in free P53 protein, which ultimately activates the y-secretase pathway that apoptosizes cancer cells. Additionally, the down-regulation of TCTP activates GTP binding onto the Rheb protein, independent of the P53 pathway, which triggers the mTOR (mammalian target rapamycin) pathway leading to the differentiation of cancer stem cell into cancer cells. Sachlos et al. published in 2012, also patented their studies (patent # WO 2013/143000 A1) that thioridazine, out of 590 compounds that they studied, effectively differentiated hematopoietic cancer stem cells [obtained from acute myeloid leukemia (AML) patients] into mature cancer cells (EC50 <10 μΜ), while. normal hematopoietic stem cells were not affected by thioridazine. In the same publication Sachlos et al. reported that human pluripotent stem cells as well as human mammary stem cells express all types of dopamine receptors, while normal human pluripotent and human mammary cells lack these membrane- bound dopamine receptors, therefore, leading to the conclusion that thioridazine _. exerts its anticancer effect by binding to the dopamine receptors expressed by cancer stem cells. In addition, Sachlos et al. reported that 7OH-DPAT (an agonist of D2-type dopamine receptor family) increased the number of AML cells while SKF38393 (an agonist of D1-type dopamine receptor family) decreased the number of AML cells, and therefore, leading the research group to conclude that the anticancer effect of thioridazine is mainly driven by the antagonism of D2-type dopamine receptor family. Lastly, Sachlos et al. reported that Oct-4 expression significantly dropped in cancer stem cells treated with thioridazine, while Oct-4 expression level was not effected in normal cells treated with thioridazine.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to novel compounds derived from thioridazine that are designed to exert strong anticancer effect. The main objective of the study is to synthesize novel phenothiazine derivatives, that are predicted to have greater affinity and specificity towards the D2 dopaminergic receptor with less side effects than thioridazine, to determine their anticancer effects on cancer and cancer stem cell lines.

LIST OF FIGURES

Figure 1. Structures of thioridazine (Mellaril®, R+S enantiomeric mixture) Figure 2. Pharmacophore structure of 10 -H-phenothiazine

Figure 3. Structure of 1 -(2-methylthio-10H-phenothiazine-10-yl- propyl)pyrimidine2,4(1 -/,3/-/)-dione

Figure 4. Structure of 3-(2-methylthio-10 - -phenothiazine-10-yl- propyl)pyrimidine2,4(1H,3H)-dione

DETAILED DESCRIPTION OF THE INVENTION

The therapeutic index of thioridazine, a phenothiazine pharmacophore derivative, is quite low. Indeed, its use has been banned in many countries due to agranulocytosis, arythmia, and sudden death cases reported in the past. Thioridazine not only binds to dopamine receptors but also muscarinic, serotonin, and a-adrenergic receptors, unavoidably giving rise to side effects. Despite its side effects, thioridazine has been recently placed into reconsideration for preclinical studies in AML stem cell line.

The aim of our invention is to synthesize novel derivatives of thioridazine, namely 1-(2-methylthio-10H-phenothiazine-10-yl-propyl)- pyrimidine-2,4(1H,3/-/)-dione (Figure 3) and 3-(2-methylthio-10H-phenothiazine- 10-yl-propyl)pyrimidine-2,4(1/-/,3H)-dione (Figure 4), which were designed to more strongly bind D2 dopamine receptor and yet with less side effects as compared to thioridazine, and determine their anticancer effects on cancer and cancer stem cell lines. Currently there is no record of the aforementioned phenothiazine derivatives in literature.

It is thought that the proposed novel compounds may be used in the treatment of cancer with lesser cytotoxic side effects at therapeutic doses due possibly to a greater therapeutic index as they are expected to selectively bind the D2 dopamine receptor with higher affinity. The proposed compounds are thought to exert their effects on MCF-7 (ER+/PR+) human mammary ductal adenocarcinoma and MCF-7 (ER+/PR+;CD44+/CD24-) human mammary cancer stem celMines.

The apoptotic and differentiation pathways triggered by the proposed compounds will be determined by monitoring p53 and Oct-4 mRNA gene expressions using Quantitatif Real Time PCR. Here, our primary objective is to prove the anticancer effect of the proposed compounds on cancer stem lines. Having a great sequence homology between human D2 and human D3 dopamine receptors (78%) makes it difficult to develop more specific novel drugs for these receptors. Ideally, we think that the compounds proposed (or at least one of those) may also be used as neuroleptic agents for illumination of physiological and behavioral moods if they proove to be highly D2 dopamine receptor specific.

To the best of knowledge, it is not well documented in the literature regarding the binding affinity values of either thioridazine or any phenothiazine derivatives against D2-type dopamine receptors, nor a structural insight into the mechanism of action was reported at a molecular level.

An X-ray structure of human D3 receptor in complex with eticlopride (a selective inverse agonist of D2 and D3 dopamine receptors which is not structurally related to thioridazine), known to exhibit the greatest homology to D2 dopamine receptor, is currently available at Protein Data Bank (PDB ID:3PBL, Swiss-Prot sequence code: P35462.2).

Although there is currently no X-ray nor NMR structure of D2 dopamine receptor deposited at the Protein Data Bank, Platania et al. were able to compare 3 ns explicit solvent molecular dynamics structures of human D2L and D3 dopamine receptors embedded in POPC (palmitoyl-oleoyl phosphatidyl choline) bilayer membrane and found that the binding site of D21. is greater than, that of D3 dopamine receptor. Platania et al. proposed that molecules bulkier than eticlopride, such as phenothiazine derivatives, may have a greater affinity towards D21. dopamine receptor, a finding that should be taken into consideration for drug development.

D3 and D2L dopamine receptors share 73.4% aminoacid sequence, homology. The aminoacid sequence of human D2L dopamine receptor is available at Protein Blast and Swiss-Prot Data Bank. In our prelimary study, we determined aminoacid sequential differences between D3 ve D2 dopamine receptors by UCSF Chimera v1.7 software (Pettersen et al.) and implemeted homology modeling to illuminate the 3D strucutre of the transmembrane region of human D2 dopamine receptor as compared to the D3 dopamine receptor by using Discovery Studio Client (DSC) v.3.0 software (Accelrys, San Diego, CA, USA) followed by short minimization.

In general, the binding site of the D3 dopamine receptor is hydrophobic. As compared to the D3 dopamine receptor, the D2L dopamine receptor possesses a VAL180 to GLU181 mutation with a carboxyl (COO ) tail that is ought to form H-bonding interaction with a suitable ligand. This is thought to be a unique property of the D2L receptor that is crucial in the developent of novel and specific antagonists/inverse agonists, in that, chemical addition of a H-bond donor, such as a N-H group, to the alkyl bridge of phenothiazine is thought to enable the novel phenothiazine derivative to form a H-bond with GLU 181 -COO ^* .

There are two disulfide brigdes available in the E3 and TM3-E3 regions of the D2L dopamine receptors. Particularly, the disulfide bond in the TM3-E2 regions makes a contribution to stability of the D2L dopamine receptor, while the other disulfide bond in the E3 region contributes to a sharp U-turn in the E3 region.

It was determined by Autodock_vina v1.3 (Trott et al.) that thioridazine binds to the D2L dopamine receptor with high affinity. Therefore, in spirit of thioridazine, development of a novel antagonist/inverse agonist that have greater affinity and speficity (than thioridazine) towards the D2L dopamine receptor as well as determination of anticancer activity of such novel compounds in human cancer cell lines is aimed in this project.

Computational dock and AMBER molecular Dynamics (Case et al.) results revealed that the C=O and N-H groups in the pyrimidine-2,4(1 H,3H)- dione substituent of designed compounds, 1-(2-methylthio-10H-phenothiazine- 10-yl-propyl)-pyrimidine-2,4(1 H,3H)-dione and 3-(2-methylthio-10H- phenothiazine-10-yl-propyl)pyrimidine-2,4(1 H,3H)-dione (AG°= -7.6 kcal/mol), make specific and high affinity H-bond interactions with the backbone amide hydrogen of CYS182 and the γ-COO- group of GLU181 in the binding site of D2L dopamine receptor.

It was also determined that the same compounds did not make specific

H-bond interactions in the binding site of D3 dopamine receptor, giving rise to slightly lower binding affinity (AG°= -7.4 kcal/mol). However, it is experimentally expected that these compounds might have much lower binding affinity towards the D3 dopamine receptor.

Therefore, the first objective of this project is to synthesize, purify and characterize the compounds 1-(2-methylthio-10H-phenothiazine-10-yl-propyl)- pyrimidine-2,4(1 H,3H)-dione and 3-(2-methylthio-10H-phenothiazine-10-yl- propyl)pyrimidine-2,4(1H,3H)-dione. The second objective of the project is to determine the anticancer activities of the synthesized compounds on MCF-7 (ER+/PR+) human mammary ductal adenocarcinoma and MCF-7 (ER+/PR+;CD44+/CD24-) human mammary cancer stem cell lines.