WO2023175615A1 - Arts mimetic componds and combinations thereof for treating high-risk neuroblastoma - Google Patents

Arts mimetic componds and combinations thereof for treating high-risk neuroblastoma Download PDF

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WO2023175615A1
WO2023175615A1 PCT/IL2023/050269 IL2023050269W WO2023175615A1 WO 2023175615 A1 WO2023175615 A1 WO 2023175615A1 IL 2023050269 W IL2023050269 W IL 2023050269W WO 2023175615 A1 WO2023175615 A1 WO 2023175615A1
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arts
independently
pharmaceutically acceptable
alkyl
xiap
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French (fr)
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Sarit Larisch
Amos LOH HONG PHENG
Zhi Xiong CHEN
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Carmel-Haifa University Economic Corporation Ltd.
Singapore Health Services Pte Ltd
National University Of Singapore
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4174—Arylalkylimidazoles, e.g. oxymetazolin, naphazoline, miconazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C07D235/10—Radicals substituted by halogen atoms or nitro radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the invention relates to the field of cancer therapy. More particularly, the invention relates to novel ARTS mimetic compounds that specifically downregulate XIAP thereby inducing apoptosis, specifically, in neural malignant cells.
  • the invention further provides combined synergistic compositions comprising the ARTS mimetic compounds, methods and uses thereof in treating neuroblastoma.
  • Neuroblastoma a malignant embryonal tumor of the sympathetic nervous system, is the leading cause of death in children less than 5 years old and accounts for 15% of all childhood cancer mortalities (Bowen and Chung, 2009; Kholodenko et al., 2018). Due to its histologic and biologic heterogeneity, neuroblastoma exhibits a broad range of clinical disease phenotypes, from spontaneous regression to aggressive metastatic disease (Castleberry, 1997). The latter is categorized clinically as high risk disease, and is associated with limited response to existing treatment options and poor survival outcomes despite intensive multimodal therapy (Maris et al., 2007). Therefore, new treatment strategies are urgently needed for high-risk neuroblastoma.
  • apoptosis of neuronal precursors is crucial in determining the final number of terminally differentiated cells.
  • Aberrant developmental apoptosis is implicated in the development of embryonal nervous system tumors including neuroblastoma (Martin et al., 1988; Oppenheim, 1991).
  • the putative cellular source of neuroblastoma is thought to be primitive sympathetic neural precursors of sympathoadrenal lineage which have failed to undergo apoptosis in response to developmentally-timed trophic factor withdrawal signals, and thus, persist as tumor-initiating cells (Bailey and Kulesa, 2014; Beckwith and Perrin, 1963).
  • XIAP the best-characterized member of inhibitor of apoptosis (IAP) family, is an important regulator of neuronal culling during neural crest development and neuroectodermal differentiation (limura et al., 2016; Werner et al., 2015).
  • XIAP-binding proteins include second mitochondria-derived activator of caspase/direct inhibitor of apoptosis protein-binding protein with low PI (SMAC/DIABLO), high temperature requirement protein A2 (Omi/HtrA2), XAF1, and apoptosis-related protein in the TGF ⁇ signaling pathway (ARTS) (Hegde et al., 2002; Larisch et al., 2000; Liston et al., 2001; Saelens et al., 2004). ARTS was previously shown as promoting the degradation of XIAP (Gotttfried et al., 2004). Still further, Edison et al.
  • SMAC mimetics which are pan-IAP antagonists, have been widely studied and shown to be of significant therapeutic value either alone or in combination with other chemotherapeutics for treating a variety of cancers (Owens et al., 2013). These compounds inhibit both XIAP and c-IAPs, though with higher affinity and potency for c-IAPs than XIAP (Cossu et al., 2009).
  • ARTS mimetics are a class of newly-described molecules that specifically target XIAP (Mamriev et al., 2020). These ARTS mimetic compounds were identified in a screen for highest affinity binding molecules that bind to the unique binding site of ARTS in BIR3/XIAP, which is distinct from the SMAC binding site.
  • WO 2017/077535 by part of the present inventors, discloses ARTS mimetic compounds and uses thereof in induction of differentiation in breast cancer cells and restoring the normal-like phenotype of the cells.
  • XIAP alone in treating neuroblastoma. There is need in the art for specific and effective therapeutic strategies for treating high-risk neuroblastoma.
  • the present disclosure provides at least one apoptosis related protein in the TGF-beta signaling pathway (ARTS) mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition comprising at least one of said ARTS mimetic compound, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof, in some embodiments, the neoplastic disorder is neuroblastoma.
  • ARTS TGF-beta signaling pathway
  • the ARTS mimetic compound interacts and binds the Baculoviral IAP Repeat (BIR) domain 3 of X-linked inhibitor of apoptosis protein (XIAP), thereby leading to proteasomal degradation of XIAP.
  • BIR3 domain of XIAP comprises residues 265-330 of XIAP.
  • amino acid sequence of XIAP is in some embodiments as denoted by SEQ ID NO: 8, or any homologs or variants thereof.
  • a further aspect of the present disclosure relates to a combined composition
  • a combined composition comprising: (a) an effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle comprising the same.
  • the ARTS mimetic compound interacts and binds the BIR3 domain of XIAP, thereby leading to proteasomal degradation of XIAP.
  • the combined composition of the present disclosure further comprises (b), an effective amount of at least one anti-cancer agent.
  • the agent is at least one of a topoisomerase inhibitor and an alkaloid agent.
  • a kit comprising:
  • an effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof interacts and binds the BIR3 domain of XIAP, thereby leading to proteasomal degradation of XIAP.
  • the ARTS mimetic compound is provided in a first dosage form.
  • the disclosed kit further comprises as a further component (b), an effective amount of at least one anti-cancer agent.
  • the agent is at least one of a topoisomerase inhibitor and alkaloid agent.
  • the at least one ant-cancer agent is provided in the disclosed kit in a second dosage form.
  • a further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof. More specifically, the disclosed method comprises the step of administering to the subject a therapeutically effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition and/or kit comprising at least one of the ARTS mimetic compound/s.
  • the ARTS mimetic compound interacts and binds the BIR3 of XIAP, thereby leading to proteasomal degradation of XIAP.
  • the method of the present disclosure is applicable for a neoplastic disorder such as neuroblastoma.
  • a further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof.
  • the therapeutic method/s comprise the step of administering to the subject a therapeutically effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition and/or kit comprising at least one of said ARTS mimetic compound.
  • the ARTS mimetic compound interacts and binds the BIR3 of XIAP, thereby leading to inhibition of XIAP and/or proteasomal degradation of XIAP.
  • the subject is a subject treated with at least one anti-cancer agent.
  • the agent is at least one of a topoisomerase inhibitor and alkaloid agent.
  • the therapeutic methods disclosed herein are specifically applicable for a neoplastic disorder such as neuroblastoma.
  • FIG. 1A-1E XIAP is overexpressed in high-risk neuroblastoma and induces apoptosis when knocked down
  • Fig. 1A(i)-lA(ii). Immunoblot analysis of N-Myc and XIAP expressions in eight human neuroblastoma cell lines and non-cancerous normal tissue-derived cell lines (THLE3: liver-derived cells; HS5: bone marrow-derived cells) (Fig. 1A(i)). P-actin is used as internal control. (Fig. 1A(ii)). Scatterplot of corresponding densitometry ratio for N-Myc and XIAP expression relative to P-actin with linear trend fit (R 2 correlation 0.76).
  • Fig. 1C Immunoblot analysis of BE(2)-C and KELLY cell lines transduced with lentivirus encoding shRNAs targeting XIAP (shXIAP 78 and 79) or nontargeting control virus (sh.SC/?). P-actin is used as internal control. Control virus sh.SC/? served as negative control.
  • Apoptotic population was evaluated by flow cytometry analysis of cells subjected to AV/PI staining. Control virus sh.SC/? served as negative control.
  • Fig. 2A Summary of IAP antagonists’ efficacy in killing neuroblastoma cells.
  • the IAP antagonists were ranked based on the average potency of killing (IC 50 ) and specificity of killing towards only neuroblastoma cells: (1) A4, (2) B3, (3) BV6, (4) LCL161, (5) CUDC-427, (6) Debio 1143.
  • Graphical representation showing the fold change of caspase-3/7 activity relative to vehicle controls after treatment with 10 pM of either A4, B3 or BV6 at indicated timings in neuroblastoma cell lines, BE(2)-C (Fig. 2C(i)), and KELLY (Fig. 2C(ii)), and non-cancerous normal tissue cell line, HS5(Fig. 2C(iii)) (mean ⁇ SD; n 3; *, P ⁇ 0.05).
  • Fig. 2D Morphology of cells 48 hours post-treatment with 10 pM of either A4, B3 or BV6. Images were taken using a light microscope with 10X magnification enlarged (Scale: 1 cm: 200 pm).
  • FIG. 3A-3D Degradation of XIAP, not inhibition, is necessary for targeting high- risk neuroblastoma cells
  • Cells were transfected with XIAP NanoLuc® fusions and treated with fixed NanoBRETTM tracer and various concentrations of unlabeled A4 or BV6 as a competitive compound.
  • Raw BRET ratios were then converted to milliBRET units (mBU) and plotted to determine apparent intracellular affinity of A4 or BV6 to XIAP. Values were expressed mean ⁇ SD of three independent experiments.
  • Degradation profiles of endogenous XIAP in response to A4 or BV6 treatment of neuroblastoma cells BE(2)-C (Fig. 3B(i) and 3B(ii)) and KELLY (Fig. 3B(iii) and 3B(iv)).
  • Cells were treated with increasing dose of A4 or BV6 and luminescence was measured continuously in real-time for 2 hours.
  • Luminescence values were normalized to DMSO vehicle control and degradation profiles were generated using GraphPad Prism. Values were expressed as mean ⁇ SD of three independent experiments.
  • BRET response curves which indicate the binding of ubiquitin to XIAP upon treatment with XIAP-specific antagonist A4 in neuroblastoma cells.
  • Neuroblastoma cells in neuroblastoma cells, BE(2)-C (Fig. 3C(i)) and KELLY (Fig. 3C(ii)), with endogenous luciferase-tagged XIAP were transfected with Halo-tag ubiquitin and treated with fluorescent Halo-tag ligand followed by the addition of various concentrations of A4.
  • Raw BRET ratios were then converted to milliBRET units (mBU) and plotted to determine the binding of ubiquitin to XIAP. Values were expressed mean ⁇ SD of three independent experiments.
  • Fig. 4A Assignment of the 1H-15NHSQC spectrum of XIAP. The assigned cross peaks in the spectrum were labeled with residue name and sequence number.
  • Fig. 4B The ⁇ - ⁇ N-HSQC spectra of XIAP in the absence (black) and presence (gray) of A4. The spectra were collected as described in experimental procedures. Residues with chemical shift perturbations after A4 treatment were indicated with residue name and sequence number.
  • Figure 5A-5D Generation of luciferase-tagged XIAP using CRISPR knock-in gene editing
  • Fig. 5A Schematic representation of XIAP structure containing HiBiT and the combination with LgBiT resulted in active functional luciferase producing luminescence.
  • Fig. SB Graphical representation of luminescence measured in neuroblastoma cells, KELLY and BE(2)-C after CRISPR knock-in of HiBiT, a small subunit of highly sensitive luciferase. Three guide RNAs were used for optimization. Signal >3 folds represent positive signal.
  • Fig. SC Agarose gel analysis of neuroblastoma cells containing HiBiT using guide RNA 1. The detection of bands was performed via multiplex PCR using three primers as described in experimental procedures. Presence of HiBiT was indicated by the presence of two bands at size 21 Ibp and 139 bp.
  • HiBiT - LgBiT signal- a graphical representation of luminescence measured in neuroblastoma cells after stably transfection with large subunit LgBiT.
  • the combination of HiBiT and LgBiT forms the active luciferase which produces luminescence. The absence of either subunit would result in negative luminescent signal.
  • Figure 6A-6C Time course of XIAP degradation in response to XIAP-specific (A4) and pan-IAP (BV6) antagonists
  • Luminescence values were normalized to DMSO vehicle control and degradation profiles were generated using GraphPad Prism. Values were expressed as mean ⁇ SD of three independent experiments.
  • Fig. 7A-7B Pharmacokinetic curve demonstrating the concentrations of A4 in mice plasma (Fig. 7A) and tumor (Fig. 7B) over time (mean ⁇ SD).
  • Neuroblastoma PDXs were treated with 10 mg/kg A4 via intraperitoneal injection.
  • the concentrations of A4 in mice plasma were determined by LCMS/ MS in MRM positive mode.
  • FIG 8A-8C Treatment with XIAP-specific antagonist A4 prolongs and improves overall survival in high-risk neuroblastoma patient-derived xenografts (PDXs)
  • PDXs neuroblastoma patient-derived xenografts
  • FIG. 8C(i)-8C(ii) Bioluminescence imaging of tumor growth in A4-treated mice (Fig. 8C(i)) and vehicle-treated mice (Fig. 8C(ii)).
  • XIAP-specific antagonist A4 works synergistically with and promotes effective dose reduction of vincristine and topotecan in vitro
  • Combination index (CI) demonstrating synergism between A4 with Vincristine (Fig. 9A(i)) and A4 with Topotecan (Fig.9A(ii)).
  • Increasing doses of A4 with either Vincristine or Topotecan were added to neuroblastoma cell lines at a fixed ratio based on the IC 50 values of the individual drugs for 48h (A4: Vincristine; BE(2)-C 1:1, KELLY 1:0.5 and A4:Topotecan; BE(2)-C 1:6, KELLY 1:16), respectively.
  • CI was generated using CompuSyn software by Chou-Talalay.
  • Cl 60-90 represents the average combination index at 60-90% cell death.
  • CI ⁇ 1 denotes synergistic effect
  • CI >1 denotes antagonistic effect.
  • Dose reduction index (DRI) demonstrating the fold differences of A4 effectively reducing the dose of vincristine (Fig. 9B(i) and 9B(ii)) or topotecan (Fig. 9C(i) and 9B(ii)) when used in combination with these agents.
  • Increasing doses of A4 with either vincristine or topotecan were added to neuroblastoma cell lines at a fixed ratio based on the IC50 values of the individual drugs for 48h (A4:Vincristine; BE(2)-C 1:1, KELLY 1:0.5 and A4:Topotecan; BE(2)-C 1:6, KELLY 1:16), respectively.
  • DRI was generated using CompuSyn software by Chou-Talalay.
  • DRI 60-90 represents the average dose reduction index at 60-90% cell death.
  • DRI ⁇ 1 denotes unfavorable dose reduction and DRI >1 denotes favorable dose reduction.
  • FIG. 10 Mechanisms mediated by various IAP antagonists, pan-IAP antagonist (SMAC mimetic) BV6 and XIAP-specific antagonist (ARTS mimetic) A4 in high- risk neuroblastoma
  • the figure schematically illustrates and compares the effect of specific targeting of XIAP in high-risk neuroblastoma cell with targeting c-IAPs.
  • the use of ARTS-mimetic compounds that specifically lead to proteasomal degradation of XIAP, and apoptosis of the high-risk neuroblastoma cell whereas the use of c-IAPs results in negligible apoptosis of the cells.
  • Neuroblastoma is one of several neural crest-derived cancers that share a common developmental biology requiring XIAP. Reduced XIAP expression is required for developmental apoptosis to initiate. Hence, tumor-initiating cells of prenatal embryonal cancers including neuroblastoma which fail to undergo developmental apoptosis are often found to have high XIAP expression, suggesting a dependency on XIAP for survival (Potts et al., 2003; Wright et al., 2007). Despite the key role played by XIAP in neural crest development, the translational potential of exploiting XIAP antagonism as a treatment strategy for neuroblastoma has not been investigated.
  • the present disclosure shows that aggressive, high-risk MYCN-amplified neuroblastoma cells tend to express a higher level of XIAP.
  • XIAP expression has been associated with high-risk biological features and poor survival in acute myeloid leukemia as well as other neuroectodermal cancers like melanoma, gastrointestinal and pulmonary neuroendocrine tumors - all highly aggressive malignancies with poor prognoses (Dizdar et al., 2017; Emanuel et al., 2008; Sung et al., 2009; Tamm et al., 2004).
  • the inventors also demonstrated that the silencing of XI AP in high XI AP- expressing and MYCN-amplified neuroblastoma cells resulted in significant apoptosis, suggesting the addiction and dependency on XIAP for survival, thus highlighting the potential of targeting XIAP as a treatment strategy for high-risk neuroblastoma.
  • A4 was also best tolerated by normal cell lines representing liver and bone marrow, which are common sites of neuroblastoma metastasis. Furthermore, the study disclosed herein provides the first proof-of-concept on the efficacy of XIAP-specific antagonist against neuroblastoma tumors in vivo, having determined the murine pharmacokinetic profile of A4 and demonstrated its utility in PDX models. Encouragingly, A4 remained stable with good availability in plasma, and was shown to prolong and improve the overall survival of high-risk neuroblastoma PDXs at the tested dose of lOmg/kg.
  • A4 showed evidence for synergism with standard-of-care cytotoxic agents. Notably, A4 was able to reduce the doses of these cytotoxic agents when used in combination. This offers potential benefit in toxicity reduction if used in the treatment of high-risk neuroblastoma which currently involves intensive multimodal therapy.
  • pan-IAP antagonists tested in this study including LCL-161, CUDC-427, Debiol 143 and BV6 targeted mainly c-IAPs, induced high toxicity in normal cells and were ineffective towards XIAP-dependent, M-aYmCpN! ified neuroblastomas.
  • A4 demonstrates promising efficacy in prolonging the survival of high-risk, MYCN- amplified neuroblastoma patient-derived xenografts despite having weaker binding affinity for XIAP than Smac mimetic BV6. This indicates that the mechanism of action for A4 is distinct, and that degradation of XIAP is more important for therapeutic success than binding-mediated inhibition. Notably, both ARTS and A4 promote degradation of XIAP, whereas Smac and Smac-mimetics do not. This finding lends support to the development of more potent derivatives from first-generation A4.
  • the inventors showed that the ARTS mimetic A4 predominantly degrades XIAP while SMAC mimetic BV6 inhibits XIAP. Similar results were shown for other cancers (Mamriev et al., 2020). This difference in mechanism for suppressing XIAP resulted in different responses in neuroblastoma cells. Notably, similar observations have been described for receptor tyrosine kinase (RTK) targeting, where inhibitors had to be sustained at saturating concentration for extended periods to effect signalling suppression while equivalent effects could be achieved in a much shorter time via compounds that degrade RTK (Burslem et al., 2018).
  • RTK receptor tyrosine kinase
  • XIAP could have another functional role in protecting from apoptosis, independent from the one disrupted by SMAC-binding.
  • SMAC mimetic BV6 the IBM motif, located at the BIR3 domain of XIAP, also known as the SMAC binding pocket
  • other XIAP domains could also exhibit functionality.
  • the RING domain of XIAP has previously been shown to function as an E3 ubiquitin ligase involved in ubiquitination (Galban and Duckett, 2010). Upon release from the mitochondria, the main function of SMAC is to bind and inhibit XIAP.
  • SNIPERs non-genetic lAP-based protein erasers
  • XIAP could still be functionally protective in spite of binding to compounds that disrupt its binding and inhibition of caspases. However, this function remains to be investigated. Additionally, the minimal or lack of XIAP degradation induced by BV6 could be attributed to its preferential binding to c-IAPs (Cossu et al., 2009; Sun et al., 2008). This could have resulted in sequestration of BV6 from binding and targeting XIAP and thus, triggering less apoptotic killing. To overcome the effects of sequestration, a higher dose would be required but may not be feasible due to the toxicity of BV6 to non-cancerous cells.
  • the present disclosure provides at least one apoptosis related protein in the TGF-beta signaling pathway (ARTS) mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition comprising at least one of said ARTS mimetic compound, for use in in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof, in some embodiments, the neoplastic disorder is neuroblastoma.
  • ARTS TGF-beta signaling pathway
  • the ARTS mimetic compound interacts and binds the Baculo viral IAP Repeat (BIR) domain 3 of X- linked inhibitor of apoptosis protein (XIAP), thereby leading to proteasomal degradation of XIAP.
  • BIR3 domain 3 of XIAP comprises residues 265- 330 of XIAP, as denoted by SEQ ID NO: 10, or any homologs or variants thereof.
  • amino acid sequence of XIAP is in some embodiments, the amino acid sequence as denoted by SEQ ID NO: 8, or any homologs and variants thereof.
  • the present disclosure provides a compound having the general formula (I): or a pharmaceutically acceptable salt, solvate, hydrate, or physiologically functional derivative thereof including any stereoisomer thereof; wherein
  • R 1 may be independently selected from H, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl;
  • X being a heteroatom independently selected from N-containing group, O and S;
  • R 3 and R 3 ', independently of each other may be selected independently from H, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl;
  • Rs may be -L1 -R 7 -L2-R 8 ;
  • R 7 may be independently selected from C 1 -C 12 alkylene, C 2 -C 12 alkenylene, C 2 -C12 alkynylene, a ring system containing five to twelve atoms
  • R 8 may be independently selected from H, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, a ring system containing five to twelve atoms, each optionally substituted;
  • R 6 may be independently selected from H, halogen, CN, NO 2 , C 1 -C 12 alkoxy, C 1 -C 12 alkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl.
  • At least one of the ARTS mimetic compounds for use in accordance with the present disclosure has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof. More specifically, in some embodiments, Formula I being: (Formula I); wherein:
  • R 1 is independently selected from H, C 1 -C 3 alkyl
  • X is a heteroatom independently selected from O
  • R 3 and R 3 ' independently of each other may be selected independently from H, C 1 -C 3 alkyl
  • R 5 is -L1-R 7 -L2-R 8 ;
  • R 7 is a carbocyclic ring or a heterocyclic ring;
  • R 8 is a mono cyclic ring system having 5 to 12, optionally substituted with at least one of OH, CF 3 , halogen, -
  • the present disclosure provides compounds, specifically, the compounds a defined in Formula I, acting as ARTS mimetic compounds.
  • the invention provides compounds, specifically, the compounds a defined in Formula I, for use as XIAP antagonists.
  • a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents.
  • R 1 may be H.
  • the N-containing group is selected from N, NH, NH 2 , tertiary amine (tertiary alkyl amine), quaternary ammonium (quaternary alkyl ammonium).
  • the N-containing group may be connected (bonded) with one R 3 group, at times with two R 3 groups, at times with three R 3 groups. It should be noted that in accordance with these embodiments, R 3 may selected independently from each other. For example, the N-containing group may be connected to three R 3 groups forming for example quaternary alkyl ammonium.
  • X is O.
  • R 3 and R 3 ' independently of each other may be selected independently from may be H, straight or branched C 1 -C 12 alkyl. In some further embodiments, R 3 may be straight C 1 -C 12 alkyl. In yet some other embodiments, R 3 may be methyl, ethyl. In yet some further embodiments, R 3 may be methyl.
  • L2 is -(CH 2 ) n -. In some further embodiments, n is 1. In some other embodiments, L2 is -(CH 2 )-.
  • R 7 may be a ring system containing five to twelve atoms, optionally substituted.
  • the ring system of R 7 may be an aryl (aromatic ring) or aliphatic ring (non-aromatic ring).
  • R 7 may be C 5 -C 12 saturated cycloalkyl, C 5 -C 12 saturated cycloalkylene, C 5 -C 12 aryl or C 5 -C 12 arylene.
  • the ring system of R 7 may contain at least two carbon atoms and may include at least one heteroatom ring.
  • R 7 may be heteroaryl, heteroarylene, heterocycloalkylene or heterocycloalkyl. In some further embodiments, R 7 may be C 2 -C 12 heterocycloalkyl ring, C 2 -C 12 heteroaryl or C2- C 12 heteroarylene. In some embodiments, the heteroatom may be N, O, S. In yet some further embodiments, the heteroatom may be N, O. In some other embodiments, the heteroatom may be N. In some other embodiments, R 7 is piperidine. In some other embodiments, R 7 is piperazine.
  • R 8 may be H, straight or branched C 1 -C 12 alkyl or five to twelve atom ring system. In some further embodiments, R 8 may be straight or branched C 1 -C 12 alkyl or five to twelve atom ring system each optionally substituted. In yet some further embodiments, R 8 may be C 1 -C 5 alkyl. In some further embodiments, R 8 may be straight C 1 -C 5 alkyl substituted with OH. In some other embodiments, R 8 may be an aromatic ring, non-aromatic ring (aliphatic ring).
  • R 8 may be C 5 -C 12 saturated cycloalkyl, C 5 -C 12 saturated cycloalkylene, C 5 -C 12 aryl or C 5 -C 12 arylene.
  • the ring system of R 8 may contain at least two carbon atoms and may include at least one heteroatom ring.
  • R 8 may be heteroarylene, heteroaryl, heterocycloalkylene or heterocycloalkyl.
  • R 8 may be C 2 -C 12 heterocycloalkyl ring or C 2 -C 12 heterocyclic aromatic ring (aryl or arylene).
  • the heteroatom may be N, O, S
  • R 8 is an aromatic ring containing six atoms. In some other embodiments, R 8 may be an aromatic ring. In some other embodiments, R 8 is phenyl.
  • R 6 may be connected (bonded, attached) to any position of the ring.
  • the ring may be substituted with one R 6 , at time with two R 6 . at times with three R 6 , at times with four R 6 .
  • R 6 may be independently selected from the group consisting of H, halogen, CN, NO 2 .
  • R 6 may be independently selected from H, halogen.
  • R 6 may be H.
  • R 6 may be Cl.
  • R 6 may be an electron withdrawing group.
  • the ARTS mimetic compound of the general formula (I) is further characterized by at least one of:
  • a compound of the invention has the general formula (II) including any stereoisomer or salt thereof: salt thereof:
  • a compound of the invention has the general formula (III) including any stereoisomer or salt thereof:
  • a compound of the invention has the general formula (IV) including any stereoisomer or salt thereof:
  • a compound of the invention has the general formula (V) including any stereoisomer or salt thereof:
  • a compound of the invention has the general formula (VI) including
  • a compound of the invention has the general formula (VII) including any stereoisomer or salt thereof:
  • the present disclosure provides 1,5-di-carbonyl compounds.
  • the compounds contain one or more nitrogen atoms.
  • the compounds contain at least one ring structure.
  • the ring structure containing at least one nitrogen atom.
  • the 1 compounds contain a heterocyclic ring structure containing five to twelve atoms, the ring structure containing at least two carbon atoms and at least one heteroatom being N, O or S.
  • the present disclosure provides in accordance with the second aspect, a compound having the general formula (VIII): or a pharmaceutically acceptable salt or hydrate thereof including any stereoisomer thereof; wherein:
  • R 9 and R 10 may be the same or may be different and may be independently selected from each other from a ring system containing five to twelve atoms, each optionally substituted;
  • R 11 may be independently selected from H, straight or branched C 1 -C 12 alkyl, straight or branched C 2 -C 12 alkenyl, straight or branched C 2 -C 12 alkynyl;
  • a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents,
  • R 9 and R 10 may be a ring system containing five to twelve atoms. In some further embodiments, the ring system of R 9 and R 10 may contain at least two carbon atoms. In some other embodiments, the ring system of R 9 and R 10 may be an aromatic ring, non-aromatic ring, fused ring or the like. In some further embodiments, R 9 and R 10 may be C 5 -C 12 saturated cycloalkyl, C 5 -C 12 saturated cycloalkylene, C 5 -C 12 aryl or C 5 -C 12 arylene. In some other embodiments, the ring system of R 9 and R 10 may include at least one heteroatom ring.
  • R 9 and R 10 may be heteroaryl or heterocycloalkyl. In some further embodiments, R 9 and R 10 may be C 2 -C 12 hetero cycloalkyl or C 2 -C 12 hetero aromatic ring (aryl or arylene). It should be noted that according with some embodiments, R 9 and R 10 may be independently from each other contain different carbon atoms. In some embodiments, the heteroatom may be N, O, S. In some embodiments, R 9 and R 10 may be independently from each other selected from C 5 -C 12 aryl. In some embodiments, R 9 and R 10 may be C6 aryl, optionally substituted.
  • L3 and L4 may be -(CH 2 ) p -.
  • R11 is H or straight C 1 -C 12 alkyl. In some further embodiments, R 11 is straight C 1 -C 12 alkyl. In some other embodiments, R 11 is methyl.
  • L3 and L4 are a bond.
  • Rn is methyl
  • specific examples of compounds or pharmaceutically acceptable salts or hydrates including any stereoisomer thereof of the compounds of Formula VIII include, without limitation: limitation:
  • the present disclosure provides compounds containing one or more nitrogen atoms.
  • the compounds contain at least one ring structure.
  • the ring structure containing at least one nitrogen atom.
  • the compounds contain a heterocyclic ring structure containing five to twelve atoms, the ring structure containing at least two carbon atoms and at least one heteroatom being N, O or S. in some other embodiments, the compounds contain an amide group.
  • the present disclosure provides a compound having a general formula: or a pharmaceutically acceptable salt or hydrate thereof including any stereoisomer thereof; wherein:
  • R 14 and R 15 may be a ring system containing five to twelve atoms. In some further embodiments, the ring system of R 14 and R 15 may contain at least two carbon atoms. In some other embodiments, the ring system of R 14 and R 15 may be an aromatic ring or a non-aromatic ring. In some further embodiments, R 14 and R 15 may be C 5 -C 12 saturated cycloalkyl, C 5 -C 12 saturated cycloalkylene, C 5 -C 12 aryl or C 5 -C 12 arylene. In some other embodiments, the ring system of R 14 and R 15 may include at least one heteroatom ring.
  • R 14 and R 15 may be C 2 -C 12 hetero cycloalkyl or C 2 -C 12 hetero aromatic ring. It should be noted that according with some embodiments, R 14 and R 15 may be independently from each other contain different carbon atoms. In some embodiments, the heteroatom may be N, O, S. In some embodiments, R 14 and R 15 may be independently from each other selected from C 5 -C 12 aryl optionally substituted. In some embodiments, R 14 and R 15 may be independently from each other selected from isoquinoline or phyel each independently from the other optionally substituted. In some embodiments, L5, may be -(CH 2 ) q -.
  • each of q may be 0.
  • L5 may be a bond.
  • a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents.
  • the invention provides the compounds of any one of the compounds of Formulas I, II, III, IV, V, VI, VII, as well as the compounds of formulas VIII, IX as described herein and any analogs or derivative thereof including any stereoisomer or salt thereof or any vehicle, matrix, nano- or micro-particle, or composition comprising the same.
  • the at least one ARTS mimetic compound for use according to the present disclosure comprises a compound that has the structure of formula (g).
  • the compound is 3-[2-(4-Benzyl-piperazin-l-yl)- acetylamino]-5-chloro-1H-indole-2-carboxylic acid methyl ester, said compound has the structure of Formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
  • A4 ARTS mimetic A4 small molecule
  • the "A4" molecule may also be referred to herein by the chemical name: methyl 5-chloro-3-[[2-[4-(phenylmethyl) piperazin- 1 -yl ]amino]-1H-indolc-2-caiboxylat.
  • the compound may include any stereoisomer or salt thereof, for example the stereoisomer having the structure:
  • the invention provides an effective amount of the compounds of the invention, specifically, any one of the compounds of Formulas I, II, III, IV, V, VI, VII, as well as the compounds of formulas VIII, IX as described herein and any analogs or derivative thereof including any stereoisomer or salt thereof or any vehicle, matrix, nano- or micro-particle, or composition comprising the same as described herein above for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a neoplastic disorder affecting neural system and/or neural cell/s, specifically, cancer, in a subject in need thereof.
  • the inventors found that compounds having at least one amine group and at least one carbonyl group act as ARTS mimetics compounds. Specifically, the inventors found that 1,2 and 1,5 di-carbonyl compounds act as ARTS mimetics.
  • the ARTS mimetic compound for use in the preset disclosure is a compound having the general formula (X) or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, wherein formula (X) is: wherein
  • R 1 , R 2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 ’-R 3 ’- L 1 ”-R 3 ” and R 2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 ’-R 4 ’- L 2 ”-R4”; or
  • the ARTS mimetic compound for use is having the general formula (XI)
  • the ARTS mimetic compound for use in accordance with the present disclosure is a compound having the general formula (Xlb):
  • the R1 is L1’-R 3 '-L1”-R 3 ”.
  • the R1 is at least one of:
  • L1’, L1 ’’and R 3 ” are each absent and R 3 ' is an optionally substituted
  • L1’, L1”and R 3 ” are each absent and R 3 ' is:
  • the ARTS mimetic compound for use, according to the present disclosure is a compound having the general formula (Xlla) or (Xllb):
  • the ARTS mimetic compound for use of the present disclosure is a compound having the general formula (XIIc), (Xlld) or (Xlle):
  • L1 and R3 are each as defined above, wherein R is one or more of H, OH,
  • the ARTS mimetic compound for use in accordance with the present disclosure is a compound having the general formula (XIIc), or (Xlle):
  • the ARTS mimetic compound for use in accordance with the present disclosure is a compound having the formula (3.1), (3.2), (3.3); N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide ;
  • the present disclosure provides the use of 1 ,2-di-carbonyl compounds that serve herein as ARTS mimetics, in upregulating p53 levels.
  • the present disclosure provides a compound comprising at least one oxalamide moiety.
  • the present disclosure also encompasses pharmaceutically acceptable salt, solvate, hydrate or any stereoisomer of the compounds described herein.
  • R 1 , R 2 are each, independently from each other, absent or H, alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 ’-R 3 ’-L 1 ”-R 3 ” and R 2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 ’-R4’-L 2 ”-R4”; or
  • each one of L1’, L1”, L 2 ’ and L 2 ” is each independently from each other, may be optionally substituted by one or more of C 1 -C 5 alkoxy, C 1 -C 5 carboxylic acid, -(CH 2 ) m -OH, -(CH 2 ) m -SH, -(CH 2 ) m -NH 2 , -(CH 2 ) m -halogen and m is an integer selected from 0, 1, 2, 3, 4, 5.
  • R 1 , R 2 are each, independently from each other, a ring system containing five to twelve atoms.
  • a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents as further described herein.
  • R 1 , R 2 are each, independently from each other, a monocyclic or polycyclic ring system containing five to twelve atoms, optionally substituted with one or more substitutes.
  • each one of R 3 ' , R 3 ”, R 4 ’ and R 4 ” may be independently from each other a ring system containing five to twelve atoms, each optionally substituted.
  • each one of R 3 ' , R 3 ”, R 4 ’ , R 4 ” is independently from each other, a monocyclic or polycyclic ring system containing five to twelve atoms, optionally substituted with one or more substitutes.
  • the ring system of each one of R 3 ', R 3 ”, R 4 ’ , R 4 may be independently from each other an aryl, heteroaryl or aliphatic ring (non- aromatic ring).
  • each one of R 3 ' , R 3 ”, R 4 ’ , R 4 ” may be independently from each other C 5 -C 12 saturated cycloalkyl, C 5 -C 12 saturated cycloalkylene, C 5 -C 12 aryl, C 5 - C 12 heteroaryl or C 5 -C 12 arylene.
  • the ring system of each one of R 3 ', R 3 ”, R 4 ’ , R 4 ” may independently from each other contain at least two carbon atoms and may include at least one heteroatom ring.
  • each one of R 3 ' , R 3 ”, R 4 ’ , R 4 ” may be independently from each other heteroaryl, heteroarylene, heterocycloalkylene or heterocycloalkyl.
  • each one of R 3 ' , R 3 ”, R 4 ’ , R 4 ” may be independently from each other C 2 -C 12 heterocycloalkyl ring, C 2 -C 12 heteroaryl or C 2 -C 12 heteroarylene.
  • the heteroatom in a heteroaryl ring may be N, O, S.
  • the heteroatom in a heteroaryl ring may be N, O.
  • the heteroatom in a heteroaryl ring may be N.
  • each one of R 3 ', R 3 ”, R4’, R4” is absent, H, an optionally substituted aryl or an optionally substituted heteroaryl.
  • each one of R 3 ', R 3 ”, R 4 ’ , R4” is absent, an optionally substituted aryl or an optionally substituted heteroaryl.
  • each one of R 3 ', R 3 ”, R4’, R4” is absent or may be independently from each other H, an optionally substituted aromatic ring or an optionally substituted heteroaromatic ring.
  • each one of R 3 ', R 3 ”, R4’, R4” is absent or may be independently from each other an aromatic or heteroaromatic five to eleven membered ring.
  • each one of R 3 ', R 3 ”, R4’, R 4 ” is absent or may be independently from each other H, phenyl, 1 -naphthyl, 2-naphthyl, and 4-biphenyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl (furanyl), quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4- thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, in
  • each one of R 3 ', R 3 ”, R4’, R 4 ” is absent or may be independently from each other H, phenyl, l-methyl-1H-benzo[d]imidazole, benzoimidazole, pyridine, pyrrole, 1 -methyl- 1H-imidazole or 1H-imidazole.
  • each one L1’, L1”, L 2 ’ and L 2 ” may be substituted by one or more of C 1 -C 5 alkoxy, C 1 -C 5 carboxylic acid, -(CH 2 ) m -OH, -(CH 2 ) m -SH, -(CH 2 ) m -NH 2 , or -(CH 2 ) m -halogen;
  • each one of L2’, L2” is absent or may be -(CH 2 ) n --
  • each one of L2’, L2” may be optionally substituted with -(CH 2 ) m -OH.
  • n may be 0 to 3
  • at times n may be 1 to 3
  • at times n may be 2 to 3
  • m may be 1 to 3.
  • R 1 is L 1 ’-R 3 '-L 1 ”-R 3 ”, L 1 ’ and L 1 ”are each absent, R 3 ' is an optionally substituted aromatic or heteroaromatic five to eleven membered ring and R 3 ” is absent.
  • R 1 is L 1 ’-R 3 '-L 1 ”-R 3
  • L 1 ’ and L 1 are each absent
  • R 3 ' is an optionally substituted aryl or optionally substituted heteroaryl and R 3 ” is absent.
  • R 1 is L 1 ’-R 3 '-L 1 ”-R 3
  • L 1 ’ and L 1 are each absent
  • R 3 ' is an optionally substituted phenyl, benzoimidazole, l-methyl-1H-benzo[d]imidazole, pyridine, pyrrole, 1 -methyl- 1H-imidazole or 1H-imidazole and R 3 ” is absent.
  • R 1 is L 1 ’-R 3 '-L 1 ”-R 3
  • L 1 ’ and L 1 are each absent
  • R1 is L1’-R 3 ’- L1 ’-R 3
  • L1’ and L1 are each absent
  • R1 is L1’-R 3 ’-L1 ’-R 3
  • L 1 ’ and L 1 are each absent
  • R 3 ' is 1 -methyl- 1H-benzo[d] imidazole, optionally substituted with halogen, or CF 3
  • R 3 ” is absent.
  • R1 is L1’-R 3 '-L1”-R 3
  • L 1 ’ and L 1 are each absent
  • R 3 ' is 1 -methyl- 1H-benzo[d] imidazole, optionally substituted with, CF 3
  • R 3 ” is absent.
  • Rl is L1’-R 3 '-L1”-R 3
  • L1’ is absent
  • n is an integer selected from any one of 0, 1, 2, 3, 4, 5
  • m is an integer selected from 0, 1, 2, 3, 4, 5.
  • R1 is L1’-R 3 ’-L1 ’-R 3
  • L1’ is absent
  • R1 is L1’-R 3 '-L1”-R 3
  • L1’ is absent
  • n is an integer selected from any one of 0, 1, 2, 3, 4, 5
  • m is an integer selected from 0, 1, 2, 3, 4, 5.
  • R1 is L1’-R 3 '-L1”-R 3
  • L1’ is absent
  • R1 is L1’-R 3 '-L1”-R 3
  • L1’ is absent
  • the compound of the present disclosure having general formula (X) have the general formula (Xa), (Xb), (Xc), (Xd), (Xe), (Xf), (Xg), (Xh), (Xi), (Xj) (Xk) or (XI):
  • R3 is an optionally substituted aryl or an optionally substituted heteroaryl.
  • R3 is an optionally substituted 1 -methyl- 1H-imidazole.
  • R3 is 1 -methyl- 1H-imidazole.
  • R 2 is L 2 ’ -R 4 ’-L 2 ” -R4”.
  • L 2 is an optionally substituted -(CH 2 ) n
  • n is an integer selected from 1, 2, 3, 4, 5
  • R4’ is an optionally substituted aryl or an optionally substituted heteroaryl and L 2 ” and R 4 ” are each absent.
  • L 2 is an optionally substituted -(CH 2 ) n
  • n is an integer selected from 1, 2, 3, 4, 5
  • R4’ is an optionally substituted phenyl and L 2 ” and R 4 ” are each absent.
  • R 2 is L 2 ’ -R4’-L 2 ” -R4
  • L 2 ’ is an optionally substituted -(CH 2 )2
  • R4’ is an optionally substituted aryl or an optionally substituted heteroaryl and L 2 ” and R 4 ” are each absent.
  • R 2 is L 2 ’ -R4’-L 2 ” -R4
  • L 2 ’ is an optionally substituted -(CH 2 ) 2
  • R4’ is an optionally substituted phenyl and L 2 ” and R 4 ” are each absent.
  • R 2 is L 2 ’ -R4’-L 2 ” -R4
  • L 2 ’ is -(CH 2 ) 2 substituted with OH
  • R4’ is an optionally substituted aryl or an optionally substituted heteroaryl
  • L 2 ” and R 4 ” are each absent.
  • R 2 is L 2 ’ -R4’-L 2 ” -R4
  • L 2 ’ is -(CH 2 )2 substituted with OH
  • R4’ is an optionally substituted phenyl and L 2 ” and R 4 ” are each absent.
  • R4 is a phenyl optionally substituted with OH.
  • a compound of the invention has the general formula (XI), L 2 ’ is (CH 2 ) n -, optionally substituted by one or more of C 1 -C 5 alkoxy, C 1 -C 5 carboxylic acid, -(CH 2 ) m -OH, -(CH 2 ) m -SH, -(CH 2 ) m -NH 2 , or -(CH 2 ) m -halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5.
  • a compound of the invention has the general formula (XI), L 2 ’ is (CH 2 ) n -, substituted by OH, n is an integer selected from any one of 0, 1, 2, 3, 4, 5. In some embodiments, a compound of the invention has the general formula (XI), L 2 ’ is (CH 2 ) n -, substituted by OH, n is 2.
  • a compound of the invention has the general formula (XIa) or (Xlb):
  • R1 is L1’ -R 3 ”-L1”- R 3 ” as defined above.
  • R1 is L1’-R 3 ' -L1”-R 3 ”, L1’, L1”and R 3 ” are each absent and R 3 ' is an
  • R1 is L1’-R 3 ’-L1 ’-R 3 ”, L1’, L1”and R 3 ” are each absent and R 3 ' is an optionally substituted:
  • R1 is L1’-R 3 '-L1”-R 3 ”, L1’, LL ’and R 3 ” are each absent and R 3 ' is:
  • R1 is L1’-R 3 ’-L1 ’-R 3 ”, R 3 ' is an optionally substituted phenyl and R 3 ” is an optionally substituted:
  • the compound of the present disclosure having general formula (Xlla) or (Xllb):
  • specific examples of compounds or pharmaceutically acceptable salts or hydrates of the compounds of Formula X, XI include, without limitation: N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(1-methyl-2-(trifluoromethyl)-1H- benzo [d] imidazol-5 -yl)oxalamide
  • the present disclosure provides 1,5-di-carbonyl compounds.
  • the compounds contain one or more nitrogen atoms.
  • the compounds contain at least one ring structure.
  • the ring structure containing at least one nitrogen atom.
  • the compounds contain a heterocyclic ring structure containing five to twelve atoms, the ring structure containing at least two carbon atoms and at least one heteroatom being N, O or S.
  • the present disclosure provides in accordance with the second aspect, a compound having the general formula (XIII): or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof ; wherein R 9 and R 10 may be the same or may be different and may be independently selected from each other from a ring system containing five to twelve atoms, each optionally substituted;
  • R11 may be independently selected from H, straight or branched C 1 -C 12 alkyl, straight or branched C 2 -C 12 alkenyl, straight or branched C 2 -C 12 alkynyl;
  • R 9 and R 10 may be a ring system containing five to twelve atoms. In some further embodiments, the ring system of R 9 and R 10 may contain at least two carbon atoms. In some other embodiments, the ring system of R 9 and R 10 may be an aromatic ring, non-aromatic ring, fused ring or the like. In some further embodiments, R 9 and R 10 may be C 5 -C 12 saturated cycloalkyl, C 5 -C 12 saturated cycloalkylene, C 5 -C 12 aryl or C 5 -C 12 arylene. In some other embodiments, the ring system of R 9 and R 10 may include at least one heteroatom ring.
  • R 9 and R 10 may be heteroaryl or heterocycloalkyl. In some further embodiments, R 9 and R 10 may be C 2 -C 12 hetero cycloalkyl or C 2 -C 12 hetero aromatic ring (aryl or arylene). It should be noted that according with some embodiments, R 9 and R 10 may be independently from each other contain different carbon atoms. In some embodiments, the heteroatom may be N, O, S. In some embodiments, R 9 and R 10 may be independently from each other selected from C 5 -C 12 aryl. In some embodiments, R 9 and R 10 may be C6 aryl, optionally substituted.
  • L5 and L6 may be -(CH 2 ) p -.
  • specific examples of compounds or pharmaceutically acceptable salts or hydrates of the compounds of Formula XIII include, without limitation:
  • the present disclosure provides compounds containing one or more nitrogen atoms.
  • the compounds contain at least one ring structure.
  • the ring structure containing at least one nitrogen atom.
  • the compounds contain a heterocyclic ring structure containing five to twelve atoms, the ring structure containing at least two carbon atoms and at least one heteroatom being N, O or S. in some other embodiments, the compounds contain an amide group.
  • the present disclosure provides a compound having a general formula: or a pharmaceutically acceptable salt or hydrate thereof; wherein:
  • X, Y independently may be each independently selected from each other from NH, CH; R 14 and R 15 may be the same or may be different and may be independently from each other selected from a ring system containing five to twelve atoms, each optionally substituted;
  • R 14 and R 15 may be a ring system containing five to twelve atoms. In some further embodiments, the ring system of R 14 and R 15 may contain at least two carbon atoms. In some other embodiments, the ring system of R 14 and R 15 may be an aromatic ring or a non-aromatic ring. In some further embodiments, R 14 and R 15 may be C 5 -C 12 saturated cycloalkyl, C 5 -C 12 saturated cycloalkylene, C 5 -C 12 aryl or C 5 -C 12 arylene. In some other embodiments, the ring system of R 14 and R 15 may include at least one heteroatom ring.
  • R 14 and R 15 may be C 2 -C 12 hetero cycloalkyl or C 2 -C 12 hetero aromatic ring. It should be noted that according with some embodiments, R 14 and R 15 may be independently from each other contain different carbon atoms. In some embodiments, the heteroatom may be N, O, S. In some embodiments, R 14 and R 15 may be independently from each other selected from C 5 -C 12 aryl optionally substituted. In some embodiments, R 14 and R 15 may be independently from each other selected from isoquinoline or phyel each independently from the other optionally substituted.
  • L8 may be -(CH 2 ) q -.
  • each of q may be 0.
  • L8 may be a bond.
  • a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents.
  • specific examples of compounds or pharmaceutically acceptable salts or hydrates of the compounds of Formula XIV include, without limitation:
  • each variable can be a different moiety selected from the Markush group defining the variable.
  • the two R groups can represent different moieties selected from the Markush group defined for R.
  • alkyl by itself or as part of another substituent, means, unless otherwise stated, as used herein refers to a linear (straight), branched saturated hydrocarbon and can have a number of carbon atoms optionally designated (i.e., C 1 -Ce means one to six carbons).
  • C1-C12 alkyl or “C1-C12 alkylene” refers to a linear (straight), branched saturated hydrocarbon having from 1 to 12 carbon atoms, in some embodiments, contain from 2 to 8 carbons, in yet some embodiments from 2 to 5 carbons, in yet some further embodiments, from 1 to 3 carbon atoms.
  • alkyl refers to an alkyl end chain and alkylene refers to a middle chain alkyl.
  • Representative C 1 -C 12 alkyl groups include, but are not limited to, methyl, ethyl, propyl, n-propyl, isopropyl, cyclopropyl, n- butyl, butyl, sec-butyl, iso-butyl, tert-butyl, cyclobutyl, n-pentyl, pentyl, iso-pentyl, neopentyl, tert-pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, cycloheptyl, octyl, sec-octyl (1 -methylheptyl), and cyclooctyl as well as homologs and isomers of, for example, n- pentyl, n-hexy
  • haloalkyl as used herein can include alkyl structures that are substituted with one or more halo groups or with combinations thereof, for example, “C1-C12 haloalkyl” refers to a C 1 -C 12 alkyl as defined above, with one or more hydrogens substituted by halogen atoms.
  • alkenyl refers to a linear (straight), branched unsaturated hydrocarbon having from 2 to 20 carbon atoms and at least one carbon-carbon double bond.
  • C 2 -C 12 alkenyl or "C 2 -C 12 alkenylene'' as used herein refers to a linear, branched unsaturated hydrocarbon having from 2 to 12 carbon atoms and at least one carbon-carbon double bond, in some embodiments from 3 to 8 carbons, in yet some further embodiments, from 3 to 5 carbon atoms and at least one double bond.
  • alkenyl refers to an alkyl end chain and alkenylene refers to a middle chain alkyl.
  • alkenyl groups include, but are not limited to, groups such as ethenyl (i.e., vinyl), prop-l-enyl (i.e., allyl), but-l-enyl, pent-l-enyl, penta- 1,4-dienyl, and the like.
  • C 2 -C 12 haloalkenyl refers to a C2-Ci2alkenyl as defined above, with one or more hydrogens substituted by halogen atoms.
  • alkynyl refers to a linear (straight), branched unsaturated hydrocarbon having from 2 to 20 carbon atoms and at least one carbon-carbon triple bond.
  • C 2 -C 12 alkynyl or "C 2 -C 12 alkynylene'' as used herein refers to a linear, branched unsaturated hydrocarbon having from 2 to 12 carbon atoms in certain embodiments, from 3 to 8 carbons, and at least one triple bond (at least one carbon-carbon triple bond).
  • alkynyl refers to an alkyl end chain and alkynylene refers to a middle chain alkyl.
  • alkynyl groups include, but are not limited to, groups such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
  • C 2 -C 12 haloalkynyl refers to a C 2 -C 12 alkynyl as defined above, with one or more hydrogens substituted by halogen atoms.
  • alkoxy refers to an alkyl group bonded to an oxygen atom.
  • C 1 -C 12 alkoxyl refers to a C 1 -C 12 alkyl group linked to an oxygen.
  • the alkyl group may include one to twelve carbon atoms, at times between one to eight carbon atoms, at times one to five carbon atoms and at times one to three carbon atoms.
  • alkoxy groups include, but are not limited to, groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy, tert-butyloxy, pentyloxy, or hexyloxy, and the like.
  • halo or “halogen” (halide) independently or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • halide by itself or as part of another substituent, refers to a fluoride, chloride, bromide, or iodide atom.
  • a ring system containing five to twelve atoms refers to a mono- or multi- cyclic ring system having 5 to 12 atoms.
  • the ring system containing five to twelve atoms may be saturated, unsaturated or aromatic rings and the like including for example cycloalkyl, heterocycloalkyl, aryl, arylene, aromatic, heteroaromatic rings.
  • a ring system containing five to twelve atoms may contain two rings (bicyclic, etc.), for example aromatic rings and in such case the aromatic rings of the aryl group may be joined at a single point (e.g., biphenyl), or fused (e.g., naphthyl).
  • a ring system containing five to twelve atoms is a carbocyclic ring or heterocyclic ring.
  • the term “carbocyclic ring” refers to cyclic compounds containing only carbon atoms.
  • the carbocyclic ring may be optionally substituted by one or more substituents, and may be saturated, unsaturated or aromatic.
  • the term “heterocyclic ring” refers to cyclic compounds where one or more carbons are substituted by heteroatoms. Exemplary heteroatoms include, but not limited to, nitrogen, sulfur, and oxygen.
  • the heterocyclic ring may be optionally substituted, and may be saturated, unsaturated or aromatic.
  • saturated' as used herein means that the compound does not contain double or triple bonds.
  • the term “unsaturated' as used herein means that the compound contains at least one double or triple bond.
  • aromatic as used herein means that the compound contains alternating double and single bonds.
  • aryl refers to polyunsaturated, aromatic ring systems having between 5 to 12 atoms which can be a single ring or multiple rings (e.g., 1 to 2 rings) which are fused together or linked covalently.
  • aryl refers to cyclic, aromatic hydrocarbon groups that have 1 to 2 aromatic rings, including monocyclic or bicyclic groups having between 5 to 12 atoms. Non-limiting examples include phenyl, biphenyl or naphthyl.
  • the aryl group may be optionally substituted by one or more substituents, e.g., 1 to 5 substituents, at any point of attachment. The substituents can themselves be optionally substituted.
  • C 5 -C 12 aromatic refers to aromatic ring systems having 5 to 12 carbon atoms, such as phenyl, naphthalene and the like.
  • heteroaryl refers to aryls as defined above where one or more carbons are substituted by heteroatoms.
  • exemplary heteroatoms include, but not limited to, nitrogen, sulfur, and oxygen.
  • heteromatic refers to refers to a monocyclic or multi-cyclic (fused) aromatic ring system, where one or more of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
  • heteroaryl used interchangeably with the term “heteroaryl” denotes a heterocyclic aromatic ring systems containing 5 to 12 atoms, with at least one, preferably two carbon atoms and one or more heteroatoms selected from nitrogen, oxygen and sulfur.
  • Non-limiting examples include furan, thipohene, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole, thiazolem benzofurna, indole, benzothiophene, benzoimidazole, indazole, benzoxazole, benzoisoxazole, benzothiazole, isobenzfuran, isoidole, purine, pyridine, pyrazine, pyrimidine, pyrisazine, quinoline, quinozaline, quinazoline, isoquinoline, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4- triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl,
  • C 5 -C 12 saturated cycloalkyl refers to a saturated mono- or multi- cyclic ring system having 5 to 12 carbon atoms, preferably having 5 to 7 carbon atoms.
  • Example of "C 5 -C 12 cycloalkyl” groups include, but are not limited to cyclopentyl, cyclohexyl and cycloheptyl.
  • heterocycloalkyl or “heterocyclyl” or the term “heterocyclic” refers to a monocyclic or multi-cyclic non-aromatic ring system having 5 to 12 members, preferably having 5 to 7 carbon atoms, where one or more, in certain embodiments, 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur.
  • heteroalkyl examples include, but are not limited to, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, pyrrolidine, morpholine, tetrahydrothiopyran, tetrahydrothiophene, and the like.
  • heterocycloalkyl also encompasses non-aromatic ring being unsaturated or having one or more degrees of unsaturation containing one or more heteroatomic substitutions selected from S, SO, SO 2 , O, or N.
  • heterocyclic ring(s) or cycloalkyl ring(s).
  • heterocyclic examples include, but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3- dioxane, piperidine, pyrrolidine, morpholine, tetrahydrothiopyran, tetrahydrothiophene, and the like.
  • N-containing group is used herein a chemical group containing a nitrogen atom for example as amino group.
  • amino as used herein encompass primary, secondary, tertiary or quaternary amines where the point of attachment is through the nitrogen atom which is substituted.
  • the "N-containing group” include N, NH, NH 2 , tertiary amine (tertiary alkyl amine), quaternary ammonium (quaternary alkyl ammonium).
  • the nitrogen atom may be substituted with alkyl.
  • the substituent may be the same or may be different.
  • bonds denotes a covalent bond.
  • the bond may be between two similar atoms or between different atoms.
  • Non-limiting examples include C-C, C-S, C- O, C-N. S-O, S-N, N-0 and the like. It should be noted that a bond as defined above, for example, C-S encompasses both C-S and S-C and this holds for the bonds as defined herein.
  • substituted refers to substitution with the named substituent or substituents, multiple degrees of substitution being allowed unless otherwise stated.
  • carbon number refers to the carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like.
  • the invention also embraces solvates, pharmaceutically acceptable prodrugs, pharmaceutically active metabolites, and pharmaceutically acceptable salts of compounds of the formula (I) or formula (X) or any variations detailed herein.
  • the present disclosure also includes any or all of the stereochemical forms, including any enantiomeric or diastereomeric forms, and any tautomers or other forms of the compounds described.
  • solvate refers to an aggregate of a molecule with one or more solvent molecules, such as hydrate, alcoholate (aggregate or adduct with alcohol), and the like.
  • salts refers to salts derived from organic and inorganic acids of a compound described herein.
  • Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, hydrochloride, bromide, hydrobromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, camphorsulfonate, napthalenesulfon
  • pharmaceutically acceptable salt also includes hydrates of a salt of a compound described herein.
  • hydrate refers to a compound formed by the addition of water. The hydrates may be obtained by any known method in the art by dissolving the compounds in water and recrystallizing them to incorporate water into the crystalline structure.
  • the compounds of the present invention may have the ability to crystallize in more than one form, a characteristic, which is known as polymorphism, and it is understood that such polymorphic forms (“polymorphs”) are within the scope of formulae (I).
  • Polymorphism generally can occur as a response to changes in temperature or pressure or both and can also result from variations in the crystallization process. Polymorphs can be distinguished by various physical characteristics known in the art such as x-ray diffraction patterns, solubility, and melting point.
  • the compounds described herein may contain one or more chiral atoms, or may otherwise be capable of existing as two enantiomers or as two or more diastereomers. Accordingly, the compounds of this invention include mixtures of enantiomers as well as purified enantiomers or enantiomerically enriched mixtures. Furthermore, the compounds of this invention include mixtures of diastereomers, as well as purified stereoisomers or diastereomerically enriched mixtures. Also included within the scope of the invention are the individual isomers of the compounds of the invention, as defined above, as well as any wholly or partially mixtures thereof. The present invention also covers the individual isomers of the compounds represented by the formulas above as mixtures with isomers thereof in which one or more chiral centers are inverted.
  • stereoisomer as used herein is meant to encompass an isomer that possess identical constitution as a corresponding stereoisomer, but which differs in the arrangement of its atoms in space from the corresponding stereoisomer.
  • stereoisomers may be enantiomers, diastereomers and/or cis-trans (E/Z) isomers.
  • a composition comprising a fatty acid amide of the invention may comprise single enantiomers, single diastereomers as well as mixtures thereof at any ratio (for example racemic mixtures, non racemic mixtures, mixtures of at least two diastereomers and so forth).
  • the invention encompasses any stereoisomer of a fatty acid amide of the invention achieved through in vivo or in vitro metabolism, or by any type of synthetic rout.
  • Certain of the compounds described herein may contain one or more chiral atoms, or may otherwise be capable of existing as two enantiomers or as two or more diastereomers.
  • the compounds of this invention include mixtures of enantiomers as well as purified enantiomers or enantiomerically enriched mixtures.
  • the compounds of this invention include mixtures of diastereomers, as well as purified stereoisomers or diastereomerically enriched mixtures.
  • the individual isomers of the compounds of the invention as defined above, as well as any wholly or partially mixtures thereof.
  • the present invention also covers the individual isomers of the compounds represented by the formulas above as mixtures with isomers thereof in which one or more chiral centers are inverted.
  • the compounds of the present invention may form tautomers. It is understood that all tautomers and mixtures of tautomers of the compounds of the present invention, are included within the scope of the compounds of the present invention.
  • solvate refers to an aggregate of a molecule with one or more solvent molecules, such as hydrate, alcoholate (aggregate or adduct with alcohol), and the like.
  • physiologically functional derivative used herein relates to any physiologically acceptable derivative of a compound as described herein.
  • the physiologically functional derivatives also include prodrugs of the compounds of the invention.
  • prodrugs may be metabolized in vivo to a compound of the invention.
  • These prodrugs may or may not be active themselves and are also an object of the present invention.
  • a “pharmaceutically acceptable prodrug” is a compound that may be converted under physiological conditions to the specified compound or to a pharmaceutically acceptable salt of such compound.
  • a “pharmaceutically active metabolite” is a pharmacologically active product produced through metabolism in the body of a specified compound or salt thereof. Metabolites of a compound may be identified using routine techniques known in the art and their activities determined using tests such as those described herein. Prodrugs and active metabolites of a compound may be identified using routine techniques known in the art.
  • any of the ARTS mimetic compound as disclosed herein above specifically the compound having the general formula (I) or formula (X), or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, or any composition thereof or any vehicle, matrix, nano- or micro-particle comprising the same, for use in a method for inducing apoptosis in a cell.
  • Still further aspects of the present disclosure relate to any of the ARTS mimetic compound as disclosed herein above, specifically the compound having the general formula (I) or formula (X) or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, or any composition thereof or any vehicle, matrix, nano- or micro-particle comprising the same, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a pathological disorder in a subject in need thereof.
  • the present disclosure provides different ARTS mimetic compounds that specifically mimic the C domain of ARTS, specifically in binding thereof to its binding site within the BIR3 domain of XIAP.
  • ARTS'' apoptosis-related protein in the TGF- ⁇ signaling pathway
  • ARTS acts as a tumor suppressor protein that functions as an antagonist of XIAP and thereby promotes apoptosis.
  • ARTS protein refers to the human ARTS, as denoted by SEQ ID NO. 6, and any homologs or variants thereof. More specifically, the human ARTS protein comprises an amino acid sequence of 274 amino acid residues as denoted by GenBank Accession No. AF176379, encoded by a nucleic acid sequence of SEQ ID NO. 7, and any homologs or variants thereof.
  • the ARTS mimetic compound/s of the invention leads to inhibition of XIAP and/or ubiquitin proteasome system (UPS) mediated degradation of X-linked-Inhibitor of Apoptosis (XIAP) in a cell. More specifically, as shown by the Examples, the ARTS mimetic compounds of the invention act as XIAP antagonists, leading to UPS mediated degradation of XIAP.
  • UPS ubiquitin proteasome system
  • ARTS binds to XIAP through a domain comprising 27 residues covering the C-terminus of ARTS. This interaction induces auto degradation of XIAP.
  • the ARTS mimetic compound/s of the invention target BRI3 domain of XIAP mimicking the ability of ARTS to enhance XIAP degradation.
  • IAPs denotes a family of proteins that harbor between one to three copies of a baculovirus IAP repeat (BIR) domain that enable interaction with activated caspases. It was previously suggested that the BIR domains of certain IAPs, in particular XIAP, have the ability to directly inhibit caspase activity in vitro.
  • BIR baculovirus IAP repeat
  • X-linked inhibitor of apoptosis protein also known as inhibitor of apoptosis protein 3 (IAP3) and baculoviral IAP repeat-containing protein 4 (BIRC) denotes a protein known to stop an apoptotic process and thus inhibit cell death.
  • XIAP is produced by a gene named XIAP gene located on the X chromosome.
  • XIAP is also called human lAP-like Protein (hILP), because it is not as well conserved as the human IAPS: hIAP-1 and hIAP-2 -XIAP is the most potent human IAP protein currently identified.
  • XIAP belongs to a family of apoptotic suppressor proteins. Members of this family share a conserved motif termed, baculovirus IAP repeat (BIR domain), which is necessary for their anti-apoptotic function. XIAP acts as a direct caspase inhibitor by directly binding to the active site pocket of CASP3 and CASP7 and obstructs substrate entry. It further inactivates CASP9 by keeping it in a monomeric, inactive state.
  • BIR domain baculovirus IAP repeat
  • the invention relates to the human XIAP protein (GenBank Accession Nos. NP_001158, NP_001191330, as denoted by SEQ ID NO: 8, and any homologs and variants thereof) encoded by the XIAP gene (GenBank Accession Nos. NM_001167, NM_001204401, as denoted by SEQ ID NO: 9), and any homologs and variants thereof.
  • an antagonist is a compound that competes with a specific protein, a ligand for example, on binding to another protein, a receptor for example. Such binding usually, induces a specific biological response or action that is blocked by the competing antagonist.
  • Antagonists have affinity but no efficacy for their cognate binding protein and binding will disrupt the interaction and inhibit the function of such cognate protein.
  • the ARTS mimetic compounds of the present disclosure bind XIAP and lead to inhibition of XIAP and/or mediate ubiquitin proteasome system (UPS) degradation of XIAP anti-apoptotic protein, thereby reducing survival of the cells.
  • UPS ubiquitin proteasome system
  • ubiquitin proteasome system denotes a multi component system that identifies and degrades unneeded, damaged or misfolded proteins by breaking peptide bonds (proteolysis) of the protein in the cytoplasm of cells.
  • degradation of a protein via the UPS involves two discrete and successive steps. In the first step, proteins are tagged for degradation with a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules.
  • conjugation of ubiquitin a highly evolutionarily conserved 76 amino acid residue polypeptide, to the protein substrate proceeds via a three-step cascade mechanism involving El , E2 and E3 enzymes.
  • ubiquitin moieties By successively adding activated ubiquitin moieties to internal lysine residues on the previously conjugated ubiquitin molecule, a polyubiquitin chain is synthesized that is subsequently recognized by the downstream 26S proteasome complex.
  • protease complex a large, protease complex, referred to as the 26S proteasome that does not recognize nonmodified substrates.
  • the proteasomes are multicatalytic protease protein complexes found in all cells that degrades polyubiquitinated proteins to short peptides by breaking peptide bonds (proteolysis). Following degradation of the substrate, short peptides derived from the substrate are released, along with reusable ubiquitin.
  • ubiquitin-proteasome system plays a central and complex role in regulating apoptosis by directly targeting key cell death proteins, including caspases.
  • the term "apoptosis” refers to a regulated network of biochemical events which lead to a selective form of cell suicide and is characterized by readily observable morphological and biochemical phenomena.
  • Cells undergoing apoptosis show characteristic morphological and biochemical features. These features include chromatin aggregation or condensation, DNA fragmentation, nuclear and cytoplasmic condensation, partition of cytoplasm and nucleus into membrane bound vesicles (apoptotic bodies) which contain ribosomes, morphologically intact mitochondria and nuclear material. Cytochrome C release from mitochondria is seen as an indication of mitochondrial dysfunction accompanying apoptosis.
  • the ARTS mimetic compounds used by the present disclosure specifically, the "A4" compound and/or the "B3” compound bind a unique domain of XIAP and lead to inhibition of XIAP and/or degradation of XIAP. It should be further understood that the ARTS mimetic compounds of the present disclosure, specifically, any one of the A4 and/or the B3 compounds lead to apoptosis of neuronal cells as shown by the present disclosure. In some specific embodiments, the A4 compound leads to apoptosis of neuroblastoma cells that exhibit an advanced stage of tumorigenicity.
  • the "A4" compound may lead to reversion of the cancerous phenotype of neural cells. Still further, in some embodiments, the "A4" compound may revert neuroblastoma cells in a subject to cells having a normal phenotype. In more specific embodiments, the A4 compound may revert cells of an early-stage cells to display a normal phenotype in a subject. In some embodiments, the reversion of the cells is due to induction of differentiation in the early- stage cells by the A4 compound. As indicated above, apoptosis is a tightly controlled form of active cell death that is necessary for development and organismal homeostasis.
  • caspases for cysteine-aspartate protease.
  • the activity of caspases is tightly regulated and the cell maintains several “checkpoints” to control their activity.
  • the first level of regulation is intrinsic to caspases themselves. Caspases are initially transcribed as weakly active zymogens, which only upon proper stimulation are cleaved to form the active enzyme.
  • the second level of caspase regulation is achieved by inhibitors, namely the family of proteins called lAPs (Inhibitor of Apoptosis Protein) as described above.
  • the at least one ARTS mimetic compound for use according to the present disclosure is applicable for use in methods of treating neoplastic disorder affecting the neural system and/or neural cell.
  • such disorder is neuroblastoma.
  • the neuroblastoma is high-risk neuroblastoma.
  • the disclosed uses are applicable for neuroblastoma patients displaying overexpression of MYCN, or having amplified MYCN.
  • MYCN v-Myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog
  • MYCN is located on chromosome 2 and is a member of the MYC family of protooncogenes.
  • MYCN is known to be amplified in a subset of aggressive human cancers, including neuroblastoma, medulloblastoma, and small cell lung cancer. Amplification of MYCN is associated with poor prognosis and treatment resistance in these cancers.
  • MYCN is also involved in normal development, particularly in the development of the nervous system. It is important for the proliferation and differentiation of neural stem cells and plays a role in the formation of the neural crest, which gives rise to many different types of cells, including neurons, glial cells, and adrenal gland cells.
  • the ARTS mimetic compound for use, according to the present disclosure prolongs the overall survival (OS) of the treated subjects.
  • the invention provides the at least one ARTS mimetic compound for use in a method that further comprises the step of administering to the treated subject an effective amount of at least one anti-cancer agent.
  • the anti-cancer agent that may be used with the at least one ARTS mimetic compound may be at least one of at least one topoisomerase inhibitor and at least one alkaloid agent.
  • the topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
  • the alkaloid agent is anti-mitotic and/or antimicrotubule alkaloid agent, optionally, vincristine, or any derivative or formulation thereof.
  • the invention further encompasses the at least one ARTS mimetic compound for use, wherein the mimetic compound is comprised within at least one composition.
  • the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
  • a further aspect of the present disclosure relates to a combined composition comprising: (a) an effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle comprising the same.
  • the combined composition of the present disclosure further comprises (b), an effective amount of at least one anti-cancer agent.
  • the agent is at least one of a topoisomerase inhibitor and an alkaloid agent.
  • the at least one of said ARTS mimetic compound of the disclosed combined composition has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, or any vehicle, matrix, nano- or micro-particle comprising the same: (Formula I); wherein:
  • R 1 is independently selected from H, C 1 -C 3 alkyl
  • X is a heteroatom independently selected from O
  • R 3 and R 3 3 independently of each other may be selected independently from H, C 1 -C 3 alkyl
  • R5 is -L1-R 7 -L2-RS
  • the ARTS mimetic compound of the combined composition of the present disclosure or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, has the general formula of any one of:
  • the compound used herein (a) has the structure of formula (II) (Formula II) wherein R 1 , R 2 , R 6 , R 7 , R 8 , L1 and L2 are as defined herein above.
  • the compound used herein (b), has the structure of In yet some further alternative embodiments, the compound used herein (c), has the
  • the compound used herein (d) has the same
  • the at least one ARTS mimetic compound of the combined composition of the present disclosure is 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5- chloro-1H-indole-2-carboxylic acid methyl ester, also referred to herein as "A4".
  • the compound has the structure of Formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
  • the at least one ARTS mimetic compound of the combined composition is a compound having the general formula (X), or a pharmaceutically acceptable salt or hydrate thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, wherein
  • R 1 , R 2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 ’-R 3 ’-L 1 ”-R 3 ” and R 2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 ’-R4’-L 2 ”-R4”; or
  • the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
  • the ARTS mimetic compound of the combined composition is having the general formula (XIIc), or (Xlle):
  • the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
  • the ARTS mimetic compound is having the formula (3.1), (3.2), (3.3);
  • composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
  • At least one of the ARTS mimetic compounds of the disclosed combined composition is (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl- 1H-imidazole-2-carbonyl)phenyl)oxalamide.
  • this compound has the structure the formula (3.2), a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
  • he combined composition of the present disclosure comprises at least one topoisomerase inhibitor.
  • the topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
  • he combined composition of the present disclosure comprises at least one alkaloid agent.
  • the alkaloid agent is anti-mitotic and/or anti-microtubule alkaloid agent.
  • vincristine or any derivative or formulation thereof.
  • the combined composition of the present disclosure comprises an effective amount of 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro- 1H-indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof.
  • the combined composition of the present disclosure comprises an effective amount of 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro- 1H-indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
  • the combined composition of the present disclosure comprises an effective amount of (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)- N2-(4-(1-methyl-1H-imidazole-2-carbonyl)phenyl)oxalamide (formula (3.2)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof.
  • the combined composition disclosed herein comprises an effective amount of (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide (formula (3.2)), a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
  • compositions of the present disclosure further comprise at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
  • the anti-cancer agent used by the present disclosure and in the combined therapeutic compositions, kits and methods is at least one antimitotic and anti-microtubule alkaloid agent.
  • Antimicrotubule agents are plant-derived antimitotic chemicals that block cell proliferation by acting on the polymerization dynamics of spindles, which are essential for the proper spindle function of microtubules.
  • Microtubules an important part of the intracellular cytoskeleton structure and have unique polymerization dynamics that are critical for many cellular functions including cell division.
  • Vinca alkaloids and taxanes are two different classes of antimicrotubule agents that cause microtubule dysfunction.
  • Vinca alkaloids such as vincristine and vinblastine bind to tubulin dimers and prevent them from polymerization.
  • taxanes such as paclitaxel and docetaxel have opposite mechanisms of action. These stabilizing agents bind to microtubules and prevent them from depolymerization. The suppression of spindle microtubule dynamics results in cell-cycle arrest through slowing or blocking of mitosis at the metaphase-anaphase transition that leads to the induction of apoptotic cell death.
  • the anti-cancer agent used by the present disclosure and in the combined therapeutic compositions, kits and methods is at least one Vinca alkaloid.
  • the anti-cancer agent used by the present disclosure and in the combined therapeutic compositions, kits and methods is vincristine or any derivatives thereof.
  • Vincristine as used herein, also known as leurocristine and marketed under the brand name Oncovin among others, is a chemotherapeutic agent administered intravenously and used to treat acute lymphocytic leukemia, acute myeloid leukemia, Hodgkin's disease, neuroblastoma, and small cell lung cancer among others.
  • Vincristine has the formula C46H56N4O10,
  • the anti-cancer agent used by the present disclosure and in the combined therapeutic compositions, kits and methods is at least one topoisomerase inhibitor.
  • topoisomerase inhibitors are chemical compounds that block the action of topoisomerases, which are broken into two broad subtypes: type I topoisomerases (Topi) and type II topoisomerases (TopIl). Topoisomerase plays important roles in cellular reproduction and DNA organization, as they mediate the cleavage of single and double stranded DNA to relax supercoils, untangle catenanes, and condense chromosomes in eukaryotic cells. Topoisomerase inhibitors influence these essential cellular processes.
  • topoisomerase inhibitors prevent topoisomerases from performing DNA strand breaks while others, deemed topoisomerase poisons, associate with topoisomerase-DNA complexes and prevent the re-ligation step of the topoisomerase mechanism.
  • These topoisomerase-DNA-inhibitor complexes are cytotoxic agents, as the un-repaired single- and double stranded DNA breaks they cause can lead to apoptosis and cell death.
  • the anti-cancer agent used by the present disclosure and in the combined therapeutic compositions, kits and methods is Topotecan.
  • Topotecan sold under the brand name Hycamtin among others, is a chemotherapeutic agent medication that is a topoisomerase inhibitor. It is a synthetic, water-soluble analog of the natural chemical compound camptothecin having the Formula C23H23N3O5.
  • compositions provided by the invention optionally further comprise at least one pharmaceutically acceptable excipient or carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.
  • the compositions provided by the invention comprise an effective amount of any of the ARTS mimetic compounds of the invention, specifically, the A4 compound including any stereoisomer or salt thereof, as well as any vehicle, matrix, nano- or micro-particle comprising the same.
  • Nanoscale drug delivery systems using micellar formulations, liposomes and nanoparticles are emerging technologies for the rational drug delivery, which offers improved pharmacokinetic properties, controlled and sustained release of drugs and, more importantly, lower systemic toxicity.
  • a particularly desired solution allows for externally triggered release of encapsulated compounds. Externally controlled release can be accomplished if drug delivery vehicles, such as micelles, liposomes or polyelectrolyte multilayer capsules, incorporate nanoparticle (NP) actuators.
  • NP nanoparticle
  • DDS Controlled drug delivery systems
  • the present disclosure further encompasses the use of various nanostructures, including micellar formulations, liposomes, polymers, dendrimers, silicon or carbon materials, polymeric nanoparticles and magnetic nanoparticles, as carriers in drug delivery systems.
  • the term "nanostructure” or “nanoparticle” is used herein to denote any microscopic particle smaller than about 100 nm in diameter.
  • the carrier is an organized collection of lipids.
  • micellar formulations or liposomes it is to be understood to mean any biocompatible lipid that can assemble into an organized collection of lipids (organized structure).
  • the lipid may be natural, semi-synthetic or fully synthetic lipid, as well as electrically neutral, negatively or positively charged lipid.
  • the lipid may be a naturally occurring phospholipid.
  • the ARTS mimetic compounds and combinations of the present disclosure may be associated with any of the nanostructures described above, specifically, any of the micellar formulations, liposomes, polymers, dendrimers, silicon or carbon materials, polymeric nanoparticles and magnetic nanoparticles disclosed herein above.
  • association may be used interchangeably with the term “entrapped' , “attachment” , “linked', “embedded' , “absorbed' and the like, and contemplates any manner by which the at least one ARTS mimetic compounds and combinations of the disclosure is held.
  • compositions provided by the present disclosure optionally further comprise at least one pharmaceutically acceptable excipient or carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.
  • a drug is transported to the place of action, hence, its influence on vital tissues and undesirable side effects can be minimized. Accumulation of therapeutic compounds in the target site increases and, consequently, the required doses of drugs are lower. This modern form of therapy is especially important when there is a discrepancy between the dose or the concentration of a drug and its therapeutic results or toxic effects.
  • Cell-specific targeting can be accomplished by attaching drugs to specially designed carriers.
  • Various nanostructures, including liposomes, polymers, dendrimers, silicon or carbon materials, and magnetic nanoparticles, have been tested as carriers in drug delivery systems. Polymeric nanoparticles are one technology being developed to enable clinically feasible oral delivery.
  • compositions used in the methods and kits of the invention, described herein after may be adapted for administration by systemic, parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes.
  • Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).
  • systemic administration means the administration of a compound, drug or other material other than directly into the central blood system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • the compounds of the present invention which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • the pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred method of preparation are vacuum-drying and freeze drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions used to treat subjects in need thereof according to the invention generally comprise a buffering agent, an agent who adjusts the osmolarity thereof, and optionally, one or more pharmaceutically acceptable carriers, excipients and/or additives as known in the art.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • the carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Local administration to the area in need of treatment may be achieved by, for example, local infusion during surgery, topical application, direct injection into the specific organ, etc.
  • compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions used to treat subjects in need thereof according to the invention may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas.
  • the compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media.
  • Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran.
  • the suspension may also contain stabilizers.
  • the pharmaceutical compositions of the present invention also include, but are not limited to, emulsions and liposome-containing formulations.
  • formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
  • the unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient.
  • a further aspect of the present disclosure relates to a kit comprising: In one component of the disclosed kit (a), an effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof.
  • the ARTS mimetic compound interacts and binds the BIR3 domain of XIAP, thereby leading to inhibition of XIAP and/or proteasomal degradation of XIAP.
  • the ARTS mimetic compound is provided in a first dosage form.
  • the disclosed kit further comprises as a further component (b), an effective amount of at least one anti-cancer agent.
  • the agent is at least one of a topoisomerase inhibitor and alkaloid agent.
  • the at least one ant-cancer agent is provided in the disclosed kit in a second dosage form.
  • At least one of the ARTS mimetic compound is a compound that has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or wherein:
  • R 1 is independently selected from H, C 1 -C 3 alkyl
  • X is a heteroatom independently selected from O
  • R 3 and R 3 ' independently of each other may be selected independently from H, C 1 -C 3 alkyl
  • R5 is -L1-R 7 -L2-R 8 ;
  • At least one of the ARTS mimetic compounds comprised in the disclosed kit is 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole- 2-carboxylic acid methyl ester, has the structure of formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
  • At least one of the ARTS mimetic compounds of the disclosed kit has the general formula (X), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof: wherein
  • R 1 , R 2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 ’-R 3 ’-L 1 ”-R 3 ” and R 2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 ’-R4’-L 2 ”-R4”; or
  • the ARTS mimetic compound of the disclosed kits, as disclosed herein is having the general formula (XIIc), or (Xlle):
  • the ARTS mimetic compound is having the formula (3.1), (3.2), (3.3); N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide ;
  • the ARTS mimetic compound of the disclosed kit is (S)- N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide.
  • the topoisomerase inhibitor of the disclosed kits is Topotecan, or any derivative or formulation thereof.
  • the alkaloid agent of the disclosed kits is anti-mitotic and/or anti-microtubule alkaloid agent, optionally, vincristine, or any derivative or formulation thereof.
  • the present disclosure provides a kit comprising an effective amount of 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2- carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof.
  • the present disclosure provides a kit an effective amount of 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2- carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
  • the present disclosure provides a kit comprising an effective amount of (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H- imidazole-2-carbonyl)phenyl)oxalamide (formula (3.2)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof.
  • the present disclosure provides a kit comprising an effective amount of (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H- imidazole-2-carbonyl)phenyl)oxalamide (formula (3.2)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
  • a further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof. More specifically, the disclosed method comprises the step of administering to the subject a therapeutically effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition and/or kit comprising at least one of the ARTS mimetic compound/s.
  • the ARTS mimetic compound interacts and binds the BIR3 domain of XI AP, thereby leading to inhibition of XIAP and/or proteasomal degradation of XIAP.
  • the method of the present disclosure is applicable for a neoplastic disorder such as neuroblastoma.
  • At least one of the ARTS mimetic compound/s used by the therapeutic methods disclosed herein is a compound that has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof: (Formula I); wherein:
  • R 1 is independently selected from H, C 1 -C 3 alkyl
  • X is a heteroatom independently selected from O
  • R 3 and R 3 3 independently of each other may be selected independently from H, C 1 -C 3 alkyl
  • R5 is -L1-R 7 -L2-RS
  • At least one of the ARTS mimetic compound/s used by the methods of the present disclosure is a compound of the general formula (I). In some embodiments, such compound is further characterized by at least one of:
  • the at least one ARTS mimetic compound/s, or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, used by the therapeutic methods of the present disclosure is a compound that has the general formula of any one of:
  • the compound used in the therapeutic methods disclosed herein is the compound used in the therapeutic methods disclosed herein.
  • the compound used in the therapeutic methods disclosed herein (b) has the structure of formula (III) wherein R 1 , R 3 , R 6 , R 7 , R 8 , L1 and L2 are as defined by the present disclosure.
  • the compound used in the therapeutic methods disclosed herein (c), has the structure of formula (IV)
  • the compound used in the therapeutic methods disclosed herein (d), has the structure of formula (VI)
  • the ARTS mimetic compound used by the disclosed therapeutic methods is 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole- 2-carboxylic acid methyl ester. More specifically, this ARTS mimetic compound has the structure of formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
  • At least one of the ARTS mimetic compounds used by the therapeutic methods disclosed herein us a compound that has the general formula (X), a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof: wherein
  • R 1 , R 2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is L 1 ’-R 3 ’-L 1 ”-R 3 ” and R 2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
  • R 1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R 2 is L 2 ’-R4’-L 2 ”-R4”; or
  • the ARTS mimetic compound of the composition for use, as disclosed herein is having the general formula (XIIc), or (Xlle):
  • the ARTS mimetic compound is having the formula (3.1), (3.2), (3.3); N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide ;
  • the ARTS mimetic compound applicable in the therapeutic methods disclosed herein is a compound that has the structure of the formula (3.2), a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
  • the method of the invention may use any of the ARTS mimetic compound/s as defined by the invention.
  • the therapeutic method of the invention may use the ARTS mimetic compound referred to herein as A4 or any derivatives thereof.
  • proliferative disorder is a disorder displaying hyper proliferation. This term means cell division and growth that is not part of normal cellular turnover, metabolism, growth, or propagation of the whole organism. Unwanted proliferation of cells is seen in tumors and other pathological proliferation of cells, does not serve normal function, and for the most part will continue unbridled at a growth rate exceeding that of cells of a normal tissue in the absence of outside intervention.
  • hypo proliferative disease A pathological state that ensues because of the unwanted proliferation of cells is referred herein as a "hyper proliferative disease” or “hyper proliferative disorder.”
  • proliferative disorder cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ.
  • compositions and methods of the present invention may be used in the treatment of non-solid and solid tumors.
  • the neoplastic disorder affecting the neural system and/or neural cell treated by the disclosed therapeutic methods is a high-risk neuroblastoma.
  • Neuroblastoma is a cancer affecting the nerve tissue that is most frequently originated from one of the adrenal glands but may also develop in the neck, chest, abdomen, or spine. Symptoms may include bone pain, a lump in the abdomen, neck, or chest, or a painless bluish lump under the skin. Typically, neuroblastoma occurs due to genetic mutation/s during early development. Neuroblastoma is the most common cancer in babies and the third-most common cancer in children after leukemia and brain cancer. About 90% of cases occur in children less than 5 years old, and it is rare in adults. Neuroblastoma is classified and staged using two main classification systems.
  • the International Neuroblastoma Risk Group Staging System (INRGSS) was designed specifically for the International Neuroblastoma Risk Group (INRG) pre-treatment classification system and uses the results of imaging tests taken before surgery. Knowledge regarding the presence or absence of image-defined risk factors (IDRF) is required for this staging system.
  • the INRGSS defines the following stages:
  • Stage L1 The tumor is located only in the area where it started; no IDRFs are found on imaging scans, such as a CT or MRI scan.
  • Stage L2 The tumor has not spread beyond the area where it started and the nearby tissue; IDRFs are found on imaging scans, such as a CT or MRI scan.
  • Stage M The tumor has spread to other parts of the body (except stage MS).
  • Stage MS The tumor has spread to only the skin, liver, and/or bone marrow (less than 10% bone marrow involvement) in a patient younger than 18 months.
  • INSS International Neuroblastoma Staging System Committee
  • Stage 1 The tumor can be removed completely during surgery. Lymph nodes attached to the tumor removed during surgery may or may not contain cancer, but other lymph nodes near the tumor do not.
  • Stage 2A The tumor is located only in the area it started and cannot be completely removed during surgery. Nearby lymph nodes do not contain cancer.
  • Stage 2B The tumor is located only in the area where it started and may or may not be completely removed during surgery, but nearby lymph nodes do contain cancer.
  • Stage 3 The tumor cannot be removed with surgery. It has spread to regional lymph nodes (lymph nodes near the tumor) or other areas near the tumor, but not to other parts of the body.
  • Stage 4 The original tumor has spread to distant lymph nodes (lymph nodes in other parts of the body), bones, bone marrow, liver, skin, and/or other organs, except for those listed in stage 4S.
  • distant lymph nodes lymph nodes in other parts of the body
  • bones bones, bone marrow, liver, skin, and/or other organs, except for those listed in stage 4S.
  • Stage 4S The original tumor is located only where it started (as in stage 1, 2A, or 2B), and it has spread only to the skin, liver, and/or bone marrow, in infants younger than one.
  • the spread to the bone marrow is minimal (usually less than 10% of cells examined show cancer).
  • a combination of clinical, pathologic, and genetic markers is used to predict the clinical behavior of the tumor and to predict responsiveness to treatment. These markers are used to define risk.
  • each neuroblastoma is classified into 1 of 4 categories: very low-risk, low-risk, intermediaterisk, or high-risk, (i) The stage of the disease according to the INRG staging system; (ii) The child's age at the time of diagnosis; (iii) Histologic category, such as maturing ganglioneuroma versus ganglioneuroblastoma, intermixed versus ganglioneuroblastoma, or nodular versus neuroblastoma; (iv)Grade, or how cells of the tumor are differentiated; (v) MYCN gene status; (vi) Chromosome 1 Iq status; and (vii)Tumor cell ploidy, which is the DNA content of tumor cells.
  • Low-risk neuroblastoma as used herein, is defined herein as a disease characterized by one or more of the following features: Stage 1 disease; Stage 2A or 2B disease in which more than 50% of the tumor was surgically removed, except for a child with MYCN amplification; Stage 4S disease, no MYCN amplification, favorable histopathology, and hyperdiploidy, meaning having extra chromosomes.
  • Intermediate-risk neuroblastoma is defined herein as a disease characterized by one or more of the following features: Stage 2 A or 2B disease with no MYCN amplification in which less than 50% of the tumor was removed with surgery; Stage 3 disease in children younger than 18 months and no MYCN amplification; Stage 3 disease in children older than 18 months, no MYCN amplification, and favorable histopathology; Stage 4 disease in children younger than 12 months.
  • the present disclosure is particularly applicable for treating neuroblastoma patients displaying amplified and/or overexpressed MYCN.
  • the ARTS mimetic compound used by the therapeutic methods disclosed herein prolongs the overall survival (OS) of the treated subjects.
  • OS overall survival
  • OS refers to the length of time from the start of a particular treatment or intervention until a patient's death from any cause. OS is often used as a primary endpoint in clinical trials and is an important measure of the effectiveness of a treatment.
  • the ARTS mimetic compounds disclosed herein, and specifically, A4 and/or B3, extended the life-time of the patients.
  • the disclosed treatment extends the disease-free period.
  • the disclosed treatment using the ARTS mimetic compounds may reduce the relapse of the disease.
  • the therapeutic methods disclosed herein may further offer a combined treatment.
  • the method further comprises the step of administering to said subject an effective amount of at least one anti-cancer agent.
  • the anti-cancer agent used by the therapeutic methods of the present disclosure may be at least one of a topoisomerase inhibitor and alkaloid agent.
  • the topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
  • the alkaloid agent is anti-mitotic and/or anti-microtubule alkaloid agent, optionally, vincristine, or any derivative or formulation thereof.
  • a further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof.
  • the therapeutic method/s comprise the step of administering to the subject a therapeutically effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition and/or kit comprising at least one of said ARTS mimetic compound.
  • the ARTS mimetic compound interacts and binds the BIR3 domain of XIAP, thereby leading to proteasomal degradation of XIAP.
  • the subject is a subject treated with at least one anti-cancer agent.
  • the agent is at least one of a topoisomerase inhibitor and alkaloid agent.
  • the therapeutic methods disclosed herein are specifically applicable for a neoplastic disorder such as neuroblastoma.
  • the ARTS mimetic compound is 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2- carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and the alkaloid agent is vincristine, or any derivative or formulation thereof.
  • the ARTS mimetic compound is 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H- indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and the topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
  • the ARTS mimetic compound is (S)-N 1 -(1 -hydroxy-3-phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1H-imidazole- 2-carbonyl)phenyl)oxalamide (formula (3.2)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and the alkaloid agent is vincristine.
  • the ARTS mimetic compound is (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H- imidazole-2-carbonyl)phenyl)oxalamide (formula (3.2)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and the topoisomerase inhibitor is Topotecan.
  • the methods provided herein involve administration of the ARTS mimetic compound/s of the invention in a therapeutically effective amount.
  • effective amount is that determined by such considerations as are known to the man of skill in the art. The amount must be sufficient to prevent or ameliorate tissue damage caused by proliferative disorders. Dosing is dependent on the severity of the symptoms and on the responsiveness of the subject to the active drug, specifically, the antagonist of the invention. Medically trained professionals can easily determine the optimum dosage, dosing methodology and repetition rates. In any case, the attending physician, taking into consideration the age, sex, weight and state of the disease of the subject to be treated, will determine the dose. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient.
  • dosage is calculated according to body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the compositions and combined composition of the invention in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the ARTS mimetic compound used by the method of the invention is administered in maintenance doses, once or more daily.
  • terapéuticaally effective amount means an amount of the ARTS mimetic compound/s, a composition comprising the same which provides a medical benefit as noted by the clinician or other qualified observer. Regression of a tumor in a patient is typically measured with reference to the diameter of a tumor. Decrease in the diameter of a tumor indicates regression. Complete regression is also indicated by failure of tumors to reoccur after treatment has stopped.
  • treatment or prevention refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, proliferative disorder symptoms or undesired side effects of such proliferative disorder related disorders. More specifically, treatment or prevention includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms.
  • disease As used herein, “disease”, “disorder”, “condition” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms.
  • the present invention relates to the treatment of subjects, or patients, in need thereof.
  • patient or “subject in need” it is meant any organism who may be affected by the above- mentioned conditions, and to whom the treatment methods herein described are desired, including humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and murine subjects, rodents, domestic birds, aquaculture, fish and exotic aquarium fish. It should be appreciated that the treated subject may be also any reptile or zoo animal. More specifically, the methods and compositions of the invention are intended for mammals.
  • mammalian subject is meant any mammal for which the proposed therapy is desired, including human, equine, canine, and feline subjects, most specifically humans.
  • the method of the invention may be performed using administration via injection, drinking water, feed, spraying, oral gavage and directly into the digestive tract of subjects in need thereof. It should be further noted that particularly in case of human subject, administering of the compositions of the invention to the patient includes both self-administration and administration to the patient by another person.
  • the invention provides methods for treating proliferative disorders, and further relates to disorders associated or related to cancer.
  • associated and “related”, when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology.
  • phrase “combination therapy” or “adjunct therapy” or in defining use of a compound described herein, specifically, the ARTS mimetic compounds of the invention, and one or more other active pharmaceutical agents, specifically, the alkaloid agents such as vincristine, and/or topoisomerase inhibitors, such as topotecan, is intended to embrace administration of each agent in a sequential manner in a regimen that will provide beneficial effects of the drug combination, and is intended as well to embrace coadministration of these agents in a substantially simultaneous manner, such as in a single formulation having a fixed ratio of these active agents, or in multiple, separate formulations for each agent.
  • Another aspect of the invention further relates to a method for treating, inhibiting, preventing, ameliorating or delaying the onset of a proliferative disorder combining the therapeutic use of the ARTS mimetic compounds of the invention with alkaloid agents such as vincristine, and/or topoisomerase inhibitors, such as topotecan.
  • alkaloid agents such as vincristine, and/or topoisomerase inhibitors, such as topotecan.
  • the term "synergism” refers to interaction of discrete agents (as drugs), such that the total effect is greater than the sum of the individual effects.
  • compositions comprising an effective amount of the ARTS mimetic compounds of the invention or any combinations thereof with alkaloid agents such as vincristine, and/or topoisomerase inhibitors, such as topotecan.
  • compositions of the present invention can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already affected by a proliferative disorder (neural neoplasm, specifically, neuroblastoma), in an amount sufficient to cure or at least partially arrest the condition and its complications.
  • An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the condition and the general state of the patient's own immune system, but generally range from about 0.001 to about 1000 mg/Kg. In some specific embodiments, an effective amount of the disclosed ARTS mimetic compounds may range between about 0.
  • 01 to about 100 mg/Kg between about 0.1 to about 100 mg/Kg, between about 1 to about 100 mg/Kg, between about 2 to about 100 mg/Kg, between about 3 to about 100 mg/Kg, between about 4 to about 100 mg/Kg, between about 5 to about 100 mg/Kg, between about 6 to about 100 mg/Kg, between about 7 to about 100 mg/Kg, between about 8 to about 100 mg/Kg, between about 9 to about 100 mg/Kg, between about 10 to about 100 mg/Kg, between about 20 to about 100 mg/Kg, between about 30 to about 100 mg/Kg, between about 40 to about 100 mg/Kg, or between about 50 to about 100 mg/Kg.
  • an effective amount of the disclosed ARTS mimetic compounds may be 10 mg/Kg.
  • Single or multiple administrations on a daily, weekly or monthly schedule can be carried out with dose levels and pattern being selected by the treating physician.
  • the administration of the compositions of the invention may be periodic, for example, the periodic administration may be effected twice daily, three time daily, or at least one daily for at least about three days to three months.
  • the advantages of lower doses are evident to those of skill in the art. These include, inter alia, a lower risk of side effects, especially in long-term use, and a lower risk of the patients becoming desensitized to the treatment.
  • treatment using the compositions of the invention may be effected following at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 30, 60, 90 days of treatment, and proceeding on to treatment for life.
  • the compositions of the invention may include a prophylactic effective amount of the active ingredient.
  • prophylactically effective amount is intended to mean that amount of a pharmaceutical composition that will prevent or reduce the risk of occurrence or recurrence of the biological or medical event that is sought to be prevented in a tissue, a system, animal or human by a researcher, veterinarian, medical doctor or other clinician.
  • the compositions of the invention are administered to a patient who is at risk of developing the disease state to enhance the patient's resistance. Such an amount is defined to be a “prophylactically effective dose”.
  • the precise amounts again depend upon the patient's state of health and general level of immunity, but generally range from 0.001 to 1000 mg per dose, about 0. 01 to 900 mg per dose, about 0.1 to 800 mg per dose, about 1 to 1000 mg per dose, about 1 to 800 mg per dose, about 1 to 700 mg per dose.
  • compositions comprising, “comprising”, “includes”, “including”, “having” and their conjugates mean “including but not limited to”. This term encompasses the terms “consisting of” and “consisting essentially of”.
  • Consisting essentially of means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • Human neuroblastoma cell lines (SK-N-SH, SK-N-AS, NB1, CHP212, NLF) were cultured in RPMI-1640, KELLY in RPMI-1640 with 25mM HEPES, BE(2)-C in DMEM/F12 (1:1) with 15mM HEPES and IMR-32 in MEM/EBSS with 1% Non- Essential Amino Acids (NEAA) and 1 % sodium pyruvate.
  • Human non-cancerous normal cell lines, HS5 (bone marrow origin) and THLE3 (liver origin) were cultured in DMEM/High Glucose with 1% sodium pyruvate and 1% sodium bicarbonate, and Bronchial Epithelial Cell Growth Medium (BEGM), respectively. All media were supplemented with 10% fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • Patient-derived neuroblastoma cells NBL27-0218A, NBL16-0118, NBL01-1116, NBL07-0317, NBL01-1116
  • Neuroblastoma patient-derived xenografts were generated from MYCN- amplified tumor samples of patients recruited with written parental consent and child assent, under SingHealth Duke NUS ORB protocol 2014/2079 (Modeling, Analysis and Translational Therapeutics for Tumors of Childhood), and implanted orthotopically in the retroperitoneal space of NOD/SCID mice. All experiments are performed under the approval of the Institutional Animal Care and Use Committee (SingHealth Duke NUS IACUC #1066) in compliance with the law and guidelines stated.
  • IAP antagonists were provided in powder and reconstituted in dimethyl sulfoxide (DMSO) (MP Biomedicals).
  • A4 and B3 were obtained from Carmel - Haifa University Economic Corporation Ltd.
  • CUDC-427 was obtained from Curis, Inc.
  • LCL161 was obtained from Novartis (Singapore) Pte Ltd.
  • Debiol 143 was obtained from Debiopharm International SA
  • BV6 was obtained from Genentech, Inc.
  • A4, B3, and BV6 were stored in -80°C after reconstitution into stock concentration of 40 mM (A4 and B3) and 20 mM (BV6), respectively.
  • LCL161, CUDC-427 and Debiol 143 were stored in -20°C after reconstitution into stock concentration of 40 mM.
  • Tissue microarray Tissue microarray (TMA) and Immunohistochemistry
  • TMA analysis was performed on neuroblastoma patients’ samples obtained from KK Women’s and Children’s Hospital. Ethical permission was obtained from the SingHealth Cental Institutional Review Board (ORB 2012/450/F, 2019/2136).
  • TMAs of tumor specimens were constructed in triplicate from formalin-fixed paraffin-embedded tissue blocks using a 1 mm-wide diameter punch (Estigen) and a manual tissue-arraying instrument (Beecher Instruments). 4 pm- thick unstained sections of TMA blocks were treated with high pH H2 buffer (Leica Biosystem) for 20 minutes and stained with anti- XIAP antibody (sc-55550) (Santa Cruz Biotechnology) at dilution 1:500.
  • DAB substrate was used as the chromogen and nuclei were counterstained with hematoxylin.
  • the expression of XIAP on TMA was reviewed and scored by pathologist; with a scoring of 0 represents negative/null XIAP expression and a highest scoring of 3 represents high XIAP expression. Scoring results were tabulated and analyzed with the patients’ underlying clinical information.
  • Cell viability was measured in real time using RealTime-GloTM MT Cell Viability Assay as according to manufacturer’s instructions from Promega. Cells were seeded at IX 10 4 cells per well and treated with a range of doses of indicated antagonists, followed by continuous bioluminescence reading every 24 hours for 3 days using luminescent plate reader, Varioskan (SkanIT software). IC50 was determined using GraphPad Prism software.
  • Lentiviruses targeting XIAP were generated by transfecting 293FT cells with shXIAP- encoding plasmid (Sigma) and 3 rd generation lenti viral packaging plasmids (pLPl, pLP2, and pLP/VSVG) using Lipofectamine® 2000 (Thermo Fisher Scientific). Supernatants containing the lentivirus were collected and pelleted. sh.SC/? plasmid encoding nontargeting virus (SCR) was used as a negative control.
  • shXIAP and sh.SC/? plasmids were purchased from Sigma-Aldrich MISSION® shRNA libraries with the sequences stated in key resources table. Lentivirus produced was used to infect neuroblastoma cells followed by downstream experiments of western blotting, clonogenic and apoptotic assays.
  • Cells were seeded in 6-well plate at a confluency of at least 30% on the day of lentiviral transduction. Cells were transduced with lentivirus encoding either control or targeting XIAP, followed by 24 hours incubation and subsequent selection by puromycin for 2 to 3 weeks. Stably selected cells were harvested for staining once they have reached desired confluency. Cells were stained with crystal violet containing methanol followed by repeated washing with water and drying. Pictures of stained cells were taken using a Brother DCP-L2540DW scanner.
  • Caspase-3/7 levels as an indication for apoptosis activity were performed using a Caspase-Gio® 3/7 assay kit from Promega according to manufacturer’s instructions.
  • Cells were seeded at 1 X 10 4 cells per well and treated with 10 pM of IAP antagonists for various indicated timings, followed by bioluminescence measurement using luminescent plate reader, Tecan Infinite®200 Pro.
  • Graphical representation of the bioluminescence was expressed as a fold change relative to the vehicle control after general normalization to time 0 hour.
  • the activity of apoptosis was also measured by flow cytometry using the Dead Cell Apoptosis Kit with Annexin V FITC and PI (Thermo Fisher Scientific) according to manufacturer’s instructions.
  • Cells harvested from lentiviral infection were subjected to Annexin V (2.5 pL) and PI (40 ng/mL, 0.5 pL) staining, followed by flow cytometric analysis using BD LSRFortessaTM cell analyzer with excitation/emission spectra of 494 nm/518 nm for FITC and 535 nm/617 nm for PI.
  • NanoBiT luciferase Endogenous-tagging of XIAP with luciferase was performed using Promega’s NanoBiT luciferase technology.
  • the NanoBiT luciferase consists of two subunits - HiBiT and LgBiT.
  • HiBiT was first introduced at endogenous XIAP locus using CRISPR knock-in gene editing following Promega manufacturer’s instructions. Alt-R® S.p.
  • Cas9 Nuclease V3, Alt-R® transactivating CRISPR RNA (tracrRNA), Alt-R® CRISPR RNA (crRNA), Ultramer single-stranded oligo DNA nucleotides (ssODN), and nuclease-free duplex buffer were purchased from Integrated DNA Technologies (Martin et al.). The sequences used for CRISPR knock-in can be found in key resources table.
  • gRNA Guide RNA
  • RNP Ribonucleoprotein
  • the cells were assayed for HiBiT insertion using Nano-Gio HiBiT lytic detection system from Promega to determine the bioluminescence via Tecan Infinite® 200 Pro. The detection of more than 3 -fold luminescence measurement indicated the presence of HiBiT.
  • the presence of HiBiT at XIAP locus was subsequently confirmed via multiplex PCR using three primers as stated in key resources table. After the confirmation, the HiBiT-containing cells were subsequent subjected to transfection with LgBiT expression vector (Promega) using FuGENE® HD transfection reagent following manufacturer’s instructions.
  • HaloTag®-Ubiquitin expression vector was first introduced via transient transfection into above-mentioned stably-transfected luciferase-tagged XIAP neuroblastoma cells using FuGENE® HD transfection reagent. 24 hours post-transfection, the cells were replated into white 96-well plate for overnight in the presence or absence of fluorescent NanoBRETTM HaloTag® 618 ligand (which binds specifically for ubiquitin).
  • Nano-Gio® VivazineTM live cell substrate for 1 hour before treatment with XIAP-specific antagonist, A4.
  • XIAP-specific antagonist A4.
  • dual-filtered luminescence was measured every 5 minutes for 6 hours using Tecan Infinite®200 Pro (donor at 460nm and acceptor at 618nm) and BRET ratio (values at 618nm/values at 460nm) was determined.
  • BRET response curve was plotted using GraphPad Prism software after general normalization by subtracting no ligand BRET values.
  • NanoLuc-XIAP expression vector provided in the kit was transiently transfected into neuroblastoma cells using FuGENE® HD transfection reagent. 24 hours post-transfection, the cells were replated into white 96-well plate (Corning #3600) in the presence or absence of fluorescent tracer (which binds specifically to NanoLuc-XIAP). The cells were then treated with IAP antagonists for 30 minutes before the addition of substrate for measurement.
  • ⁇ - ⁇ N-HSQC experiment was performed using 50 mM of A4 compound dissolved in DMSO and 0.5 mM of 15 N-labeled XIAP.
  • the ⁇ - ⁇ N-HSQC spectra of XIAP in the absence and presence of different amounts of A4 compounds were acquired, processed and analyzed.
  • the collected data were processed with NMRPipe (Delaglio et al., 1995) and analyzed with NMRView (Johnson, 2004).
  • XIAP-specific antagonist A4 Drug interactions between XIAP-specific antagonist A4 and vincristine or topotecan were determined using the combination index (CI) by Chou and Talalay which was established based on the mass-action law principle to derive the median-effect equation (Chou, 2010).
  • CI combination index
  • Talalay which was established based on the mass-action law principle to derive the median-effect equation (Chou, 2010).
  • 1 X 10 4 cells were seeded in 96-well plate and treated with increasing doses of A4 and vincristine/ topotecan (individually or in combination) at a fixed ratio according to the IC50 values of the individual drugs for 48h (A4: Vincristine; BE(2)-C 1:1, KELLY 1:0.5 and A4:Topotecan; BE(2)-C 1:6, KELLY 1:16).
  • Cell viability was measured and CI and dose reduction index (DRI) values were quantified using the CompuSyn software by Chou-Talalay
  • the testing dose of A4 10 mg/kg was injected intraperitoneally into the orthotopic neuroblastoma PDXs.
  • the concentrations of A4 in mouse plasma and tumor samples were determined and validated by a highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method with multiple reaction monitoring (MRM) mode.
  • the LC-MS/MS system consisted of Agilent 1290 ultra high-performance liquid chromatography (UHPLC) connected in tandem to Sciex QTRAP 5500 mass spectrometer system.
  • Chromatographic separation was optimized using a liquid-liquid extraction and a reversed phase separation on a Kinetex F5 column (100 mm x 2.1 mm, 2.6 pm) with isocratic elution.
  • Ethyl indole-2-carboxylate was used as the internal standard (IS).
  • MRM transitions 441.2/189.0 (A4) and 190.1/114.1 (IS) were monitored with a dwell time of 300 msec and analyst 1.6.2 software (Sciex) was used to quantify the peaks with l/x2 weighted linear regression.
  • XIAP is overexpressed in high-risk neuroblastoma and induces apoptosis when knocked down
  • N-Myc and XIAP expression are necessary for nerve growth factor (NGF)-withdrawal-mediated apoptosis in sympathoadrenal progenitor cells. Since dysregulation of this mechanism could prolong the survival of these progenitor cells and promote tumorigenesis (Nakagawara et al., 2018; Potts et al., 2003), the inventors examined whether neuroblastomas, which are of sympathoadrenal origin, would have a high endogenous level of XIAP especially those with MYCN amplification. To investigate if there is a correlation between XIAP and N-Myc protein levels, their expression has been screened across a panel of neuroblastoma cell lines.
  • THLE3 liver tissue- derived cell line and HS5, a bone marrow-derived cell line
  • HS5 bone marrow-derived cell line
  • IAP antagonists include: (1) CUDC-427, a phase 1 clinically-tested monovalent pan-IAP antagonist (Tolcher et al., 2016); (2) LCL161, a phase 2 clinically-tested monovalent pan-IAP antagonist (Infante et al., 2014); (3) Debio 1143, a phase 3 clinically-tested monovalent pan-IAP antagonist (Cai et al., 2011); (4) BV6, a pre-clinical bivalent pan-IAP antagonist (Gao et al., 2007); (5) A4 and (6) B3, both pre-clinical XIAP-specific antagonists (Mamriev et al., 2020).
  • a panel of neuroblastoma cell lines including neuroblastoma patient-derived cell lines and non- cancerous normal tissue-derived cell lines were used.
  • the cells were treated with increasing doses of each antagonist (0-100 pM), followed by real-time measurement of cell viability every 24 hours for 72 hours.
  • XIAP-specific antagonists A4 and B3 showed highest potency against neuroblastoma cells across the panel of cell lines with the lowest IC50 achieved (2 - 15 pM) (Table 2 and Figure 2A).
  • pan-IAP antagonists with selective IAP affinity and activity showed poorer average efficacy, ranked by mean IC50: BV6 with IC50 of 3 - 20 pM, LCL161 and CUDC-427 with IC5020 - 60 pM, and Debio 1143 with IC50 50 - >100 pM (Table 2 and Figure 2A).
  • BV6 was the most potent in suppressing neuroblastoma cells.
  • the overall IC50 range of A4, B3 and BV6 on neuroblastoma cells were comparable despite the different modes of action, their relative effects on non-cancerous cells have not been previously studied.
  • non-cancerous normal tissue-derived cell lines THLE3 and HS5 described earlier were utilized to evaluate toxicity and tolerability.
  • XIAP-specific antagonists, A4 and B3 were observed to be the least toxic to non-cancerous cells and were the most discriminatory in killing neuroblastoma cells (Table 2 and Figure 2A).
  • the pan-IAP antagonists especially LCL161, CUDC-427 and Debio 1143 were more toxic towards non-cancerous cells than neuroblastoma cells and thus, deemed unsuitable for use in neuroblastoma treatment.
  • A4, B3 and BV6 were selected as the more efficacious antagonists to be studied further in subsequent experiments (Figure 2A).
  • BE(2)-C (Fig. 2b(i)) and KELLY (Fig. 2B(i)) cells treated with A4 or B3 showed loss of XIAP expression over time with intact c-IAPl expression ( Figure 2B(i)-(iii)).
  • apoptosis was induced, indicated by increasing cleavage of PARP and caspase-3 as well as increased caspase-3/7 activity ( Figure 2B and 2C).
  • BE(2)-C and KELLY cells treated with BV6 showed loss of c-IAPl expression with no change in XIAP expression ( Figure 2B).
  • BV6 induced minimal or no apoptosis in BE(2)- C and KELLY cells despite the loss of c-IAPl expression (Figure 2B and 2C). This suggested that targeting of XIAP, and not c-IAPl, is required for the killing of high-risk neuroblastoma. Consistent with the cell viability findings, evaluation of cell morphological changes confirmed that BE(2)-C and KELLY cells were sensitive to cell killing by A4 and B3 but resistant to BV6 treatment ( Figure 2D).
  • XIAP-overexpressing KELLY cells treated with B V6 did not undergo apoptosis, similar to control cells ( Figure 2E(i)), since XIAP is less sensitive to BV6 ( Figure 2B(i)-(iii)). This renders the high XIAP-expressing cells resistant to BV6, with or without XIAP overexpression.
  • NLF a low XIAP-expressing, MYC-Namplified neuroblastoma cell line, was responsive to all three antagonists which could be mitigated by XIAP overexpression (Figure 2E).
  • BV6 is able to reduce XIAP expression but with less potency than A4 and B3 and may explain why certain neuroblastoma cell lines remain sensitive to BV6 while lines with high XIAP and MYCN expression only respond to A4 and B3.
  • XIAP is a viable target for the suppression of high-risk, resistant neuroblastoma.
  • IC 50 (jiM) indicating the effect of individual IAP antagonists on viability of neuroblastoma and normal tissue-derived cells measured every 24 hours up to 72 hours
  • Binding and degradation of XIAP by A4 is necessary for targeting high-risk neuroblastoma cells
  • ubiquitin-proteasome system As rapid degradation of proteins is often catalyzed by the ubiquitin-proteasome system (UPS), the inventors sought to determine whether the UPS is involved in the rapid degradation of XIAP upon treatment with A4. Using a NanoBRET quantitative ubiquitination assay to examine the binding of ubiquitin to XIAP upon treatment with A4, neuroblastoma cells were observed to exhibit a clear dose-dependent increase in BRET ratio, indicating an increase binding of fluorescent-labelled ubiquitin to the endogenous luminescent-tagged XIAP (Figure 3C(i), (ii)). To determine if ubiquitination of XIAP leads to its proteasomal degradation, MG- 132 was used to inhibit the proteasome.
  • XIAP-specific antagonist A4 prolongs and improves overall survival in high-risk neuroblastoma patient-derived xenografts (PDXs)
  • the ARTS mimetic A4 compound was the most potent compound in suppressing high-risk neuroblastoma in vitro. Therefore, the effect of targeting XIAP by A4 in vivo was next examined using PDX models of MYCN -amplified high-risk neuroblastoma.
  • Human primary tumor cells derived from AfTCA-amplified high-risk neuroblastoma patients were implanted orthotopically in the retroperitoneal peri-adrenal space of mice, which modeled the typical anatomic site and microenvironment of human disease.
  • PDXs were then subjected to A4 treatment for pharmacokinetic and survival analysis.
  • PDXs generated after 4 weeks of tumor implantation were randomly distributed into treatment (10 mg/kg A4) and vehicle control (DMSO; ImL/kg) groups and injected intraperitoneally twice a week for 3 weeks.
  • the mice were monitored over a period of 7 weeks in total from implantation to the end of treatment, and deaths within the monitoring period were recorded.
  • Kaplan-Meier curves of MYCN-amplified neuroblastoma PDXs showed a significantly longer overall survival in the treatment group, with more than 50% of mice alive at the end of the treatment period (Figure 8B(i)). In contrast, the majority of the mice in the vehicle control group did not survive (Figure 8B(ii)).
  • XIAP-specific antagonist A4 works synergistically with and promotes effective dose reduction of vincristine and topotecan in vitro
  • DRI dose reduction index
  • BV6 upon BV6 treatment, it preferentially binds c-IAPs over XIAP, resulting in possible sequestration of BV6 by c-IAPs and less caspase being free from XIAP (1).
  • BV6 rapidly triggers the autoubiquitination and degradation of c-IAPs which however showed minimal effect on neuroblastoma.
  • the binding and inhibition of XIAP by BV6 was also seen to be less effective in triggering apoptosis.
  • the BV6-bound XIAP could still be functional independent on the inhibition binding site (2), thus allowing XIAP to bind to other proteins that could mediate anti-apoptotic effect (2a).
  • BV6 mediates overall less or no apoptosis in these high-risk neuroblastoma.
  • XIAP-specific antagonist outperforms pan-IAP antagonist by specifically degrading and not inhibiting XIAP, thus presenting as a potential treatment strategy to overcome high-risk neuroblastoma.

Abstract

The present disclosure provides apoptosis related protein in the TGF-beta signaling pathway (ARTS) mimetic compounds, compositions and uses thereof for treating neoplastic disorders affecting the neural system and/or neural cell in a subject in need thereof, specifically neuroblastoma. The present disclosure further provides combined compositions of these ARTS mimetic compounds.

Description

ARTS MIMETIC COMPOUNDS AND COMBINATIONS THEREOF FOR TREATING HIGH-RISK NEUROBLASTOMA
FIELD OF THE INVENTION
The invention relates to the field of cancer therapy. More particularly, the invention relates to novel ARTS mimetic compounds that specifically downregulate XIAP thereby inducing apoptosis, specifically, in neural malignant cells. The invention further provides combined synergistic compositions comprising the ARTS mimetic compounds, methods and uses thereof in treating neuroblastoma.
BACKGROUND ART
References considered to be relevant as background to the presently disclosed subject matter are listed below:
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Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.
BACKGROUND OF THE INVENTION
Neuroblastoma, a malignant embryonal tumor of the sympathetic nervous system, is the leading cause of death in children less than 5 years old and accounts for 15% of all childhood cancer mortalities (Bowen and Chung, 2009; Kholodenko et al., 2018). Due to its histologic and biologic heterogeneity, neuroblastoma exhibits a broad range of clinical disease phenotypes, from spontaneous regression to aggressive metastatic disease (Castleberry, 1997). The latter is categorized clinically as high risk disease, and is associated with limited response to existing treatment options and poor survival outcomes despite intensive multimodal therapy (Maris et al., 2007). Therefore, new treatment strategies are urgently needed for high-risk neuroblastoma.
Developmental apoptosis of neuronal precursors is crucial in determining the final number of terminally differentiated cells. Aberrant developmental apoptosis is implicated in the development of embryonal nervous system tumors including neuroblastoma (Martin et al., 1988; Oppenheim, 1991). The putative cellular source of neuroblastoma is thought to be primitive sympathetic neural precursors of sympathoadrenal lineage which have failed to undergo apoptosis in response to developmentally-timed trophic factor withdrawal signals, and thus, persist as tumor-initiating cells (Bailey and Kulesa, 2014; Beckwith and Perrin, 1963).
XIAP, the best-characterized member of inhibitor of apoptosis (IAP) family, is an important regulator of neuronal culling during neural crest development and neuroectodermal differentiation (limura et al., 2016; Werner et al., 2015). Reduced levels of XIAP, rather than other IAPS, is a pre-condition for developmental apoptosis to proceed, and its upregulation has been found to be associated with chemotherapy resistance and unfavorable outcome in relapsed and advanced stage neuroblastoma (Holcik and Korneluk, 2001; Potts et al., 2003). Greater overexpression of XIAP over other IAPs has also been noted in other neuroectodermal cancers such as melanoma, and neuroendocrine tumors (Dizdar et al., 2017; Emanuel et al., 2008). Furthermore, the inventors previously showed that loss of XIAP intrinsic antagonist, XAF1, results in failure of caspase-mediated cell death in neuroblastoma and is associated with poorer survival and disease outcomes (Choo et al., 2016).
Several XIAP-binding proteins have been identified and these include second mitochondria-derived activator of caspase/direct inhibitor of apoptosis protein-binding protein with low PI (SMAC/DIABLO), high temperature requirement protein A2 (Omi/HtrA2), XAF1, and apoptosis-related protein in the TGFβ signaling pathway (ARTS) (Hegde et al., 2002; Larisch et al., 2000; Liston et al., 2001; Saelens et al., 2004). ARTS was previously shown as promoting the degradation of XIAP (Gotttfried et al., 2004). Still further, Edison et al. (2017) describe a mechanism by which the ARTS protein promotes proteasome-mediated degradation of XIAP and Bcl-2 and thereby stimulates cell death [WO 2013/121428]. Based on the known binding structure of these proteins to XIAP, several small molecule mimetics have been developed for use as potential treatment agents. For instance, SMAC mimetics, which are pan-IAP antagonists, have been widely studied and shown to be of significant therapeutic value either alone or in combination with other chemotherapeutics for treating a variety of cancers (Owens et al., 2013). These compounds inhibit both XIAP and c-IAPs, though with higher affinity and potency for c-IAPs than XIAP (Cossu et al., 2009). In contrast, ARTS mimetics are a class of newly-described molecules that specifically target XIAP (Mamriev et al., 2020). These ARTS mimetic compounds were identified in a screen for highest affinity binding molecules that bind to the unique binding site of ARTS in BIR3/XIAP, which is distinct from the SMAC binding site. WO 2017/077535 by part of the present inventors, discloses ARTS mimetic compounds and uses thereof in induction of differentiation in breast cancer cells and restoring the normal-like phenotype of the cells. However, there have been limited studies on the effectiveness of targeting XIAP alone in treating neuroblastoma. There is need in the art for specific and effective therapeutic strategies for treating high-risk neuroblastoma.
SUMMARY OF THE INVENTION
A first aspect, the present disclosure provides at least one apoptosis related protein in the TGF-beta signaling pathway (ARTS) mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition comprising at least one of said ARTS mimetic compound, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof, in some embodiments, the neoplastic disorder is neuroblastoma. More specifically, the ARTS mimetic compound interacts and binds the Baculoviral IAP Repeat (BIR) domain 3 of X-linked inhibitor of apoptosis protein (XIAP), thereby leading to proteasomal degradation of XIAP. In some embodiments the BIR3 domain of XIAP, comprises residues 265-330 of XIAP. It should be noted that the amino acid sequence of XIAP is in some embodiments as denoted by SEQ ID NO: 8, or any homologs or variants thereof.
A further aspect of the present disclosure relates to a combined composition comprising: (a) an effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle comprising the same. It should be noted that the ARTS mimetic compound interacts and binds the BIR3 domain of XIAP, thereby leading to proteasomal degradation of XIAP. The combined composition of the present disclosure further comprises (b), an effective amount of at least one anti-cancer agent. In more specific embodiments, the agent is at least one of a topoisomerase inhibitor and an alkaloid agent. A further aspect of the present disclosure relates to a kit comprising:
In one component of the disclosed kit (a), an effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof. The ARTS mimetic compound interacts and binds the BIR3 domain of XIAP, thereby leading to proteasomal degradation of XIAP. In some optional embodiments, the ARTS mimetic compound is provided in a first dosage form. The disclosed kit further comprises as a further component (b), an effective amount of at least one anti-cancer agent. In some embodiments, the agent is at least one of a topoisomerase inhibitor and alkaloid agent. Optionally, the at least one ant-cancer agent is provided in the disclosed kit in a second dosage form.
A further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof. More specifically, the disclosed method comprises the step of administering to the subject a therapeutically effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition and/or kit comprising at least one of the ARTS mimetic compound/s. It should be understood that the ARTS mimetic compound interacts and binds the BIR3 of XIAP, thereby leading to proteasomal degradation of XIAP. In some embodiments, the method of the present disclosure is applicable for a neoplastic disorder such as neuroblastoma.
A further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof. The therapeutic method/s comprise the step of administering to the subject a therapeutically effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition and/or kit comprising at least one of said ARTS mimetic compound. The ARTS mimetic compound interacts and binds the BIR3 of XIAP, thereby leading to inhibition of XIAP and/or proteasomal degradation of XIAP. It should be further noted that the subject is a subject treated with at least one anti-cancer agent. In more specific embodiments, the agent is at least one of a topoisomerase inhibitor and alkaloid agent. Still further, the therapeutic methods disclosed herein are specifically applicable for a neoplastic disorder such as neuroblastoma.
These and other aspects of the invention will become apparent by the hand of the following description. BRIEF DESCRIPTION OF THE DRAWINGS
In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
Figure 1A-1E. XIAP is overexpressed in high-risk neuroblastoma and induces apoptosis when knocked down
Fig. 1A(i)-lA(ii). Immunoblot analysis of N-Myc and XIAP expressions in eight human neuroblastoma cell lines and non-cancerous normal tissue-derived cell lines (THLE3: liver-derived cells; HS5: bone marrow-derived cells) (Fig. 1A(i)). P-actin is used as internal control. (Fig. 1A(ii)). Scatterplot of corresponding densitometry ratio for N-Myc and XIAP expression relative to P-actin with linear trend fit (R2 correlation = 0.76).
Fig. 1B(i)- 1B(iii). Representative images of XIAP immunohistochemistry staining in a tissue microarray (TMA) (n = 64) (20X), indicating staining intensity of XIAP with 0=null, l+= low, 2+=moderate, 3+=high (Fig. 1B(i)).
Fig. 1B(ii)-1B(iii). Graphical distribution of XIAP staining in MYCN-amplified neuroblastoma cases (n=14), (Fig. 1B(ii)) and non- MYCN-amplified neuroblastoma cases (n=50), (Fig. 1B(iii)).
Fig. 1C. Immunoblot analysis of BE(2)-C and KELLY cell lines transduced with lentivirus encoding shRNAs targeting XIAP (shXIAP 78 and 79) or nontargeting control virus (sh.SC/?). P-actin is used as internal control. Control virus sh.SC/? served as negative control.
Fig. 1D(i)-lD(ii). Graphical representation showing fold change of apoptotic-positive BE(2)-C ( Fig. 1D(i)) and KELLY (Fig. 1D(ii)) cells transduced with shRNAs targeting XIAP (shX/AP 78 and 79) relative to cells transduced with non-targeting control (sh.SC/?) (mean ± SD; n=3; *, P<0.05). Apoptotic population was evaluated by flow cytometry analysis of cells subjected to AV/PI staining. Control virus sh.SC/? served as negative control.
Fig. 1E(i)-lE(ii). Crystal violet staining to determine cell viability and colony formation after lentiviral-induced silencing of XIAP and selected with 1 pg/ml (BE(2)-C) (Fig. lE(i)) or 500 ng/ml puromycin (KELLY) (Fig. 1E(ii)) for several weeks. Control virus sh.SC/? served as negative control. Stained cells (shown in the photo in dark green) represent viable cells. Figure 2A-2E. Specific loss of XIAP expression, not c-IAPl, is required for mediating apoptosis in high-risk neuroblastoma cells
Fig. 2A. Summary of IAP antagonists’ efficacy in killing neuroblastoma cells. The IAP antagonists were ranked based on the average potency of killing (IC50) and specificity of killing towards only neuroblastoma cells: (1) A4, (2) B3, (3) BV6, (4) LCL161, (5) CUDC-427, (6) Debio 1143.
Fig. 2B(i)-2B(iii). Immunoblot analysis of c-IAPl, XIAP, PARP and cleaved caspase-3 expression in neuroblastoma cell lines, BE(2)-C (Fig. 2B(i)), and KELLY (Fig. 2B(ii)), and non-cancerous normal tissue cell line, HS5(Fig. 2B(iii)), after treatment with 10 pM of either A4, B3 or BV6 at indicated timings.
Fig. 2C(i)-2C(iii). Graphical representation showing the fold change of caspase-3/7 activity relative to vehicle controls after treatment with 10 pM of either A4, B3 or BV6 at indicated timings in neuroblastoma cell lines, BE(2)-C (Fig. 2C(i)), and KELLY (Fig. 2C(ii)), and non-cancerous normal tissue cell line, HS5(Fig. 2C(iii)) (mean ± SD; n=3; *, P<0.05).
Fig. 2D. Morphology of cells 48 hours post-treatment with 10 pM of either A4, B3 or BV6. Images were taken using a light microscope with 10X magnification enlarged (Scale: 1 cm: 200 pm).
Fig. 2E(i)-2E(ii). Immunoblot analysis of c-IAPl, XIAP, PARP and cleaved caspase-3 expression in neuroblastoma cell lines, KELLY (Fig. 2E(i)) and NLF (Fig. 2E(ii)) transfected with plasmid encoding either XIAP or empty vector, followed by treatment with 10 pM of A4, B3 or BV6 at indicated timings. P-actin is used as internal control.
Figure 3A-3D. Degradation of XIAP, not inhibition, is necessary for targeting high- risk neuroblastoma cells
Fig. 3A(i)-3A(ii). BRET response curves which indicate direct binding/ target engagement of either XIAP-specific antagonist A4 (Fig. 3A(i)) or pan-IAP antagonist BV6 (Fig. 3A(ii)) to XIAP in neuroblastoma cells. Cells were transfected with XIAP NanoLuc® fusions and treated with fixed NanoBRET™ tracer and various concentrations of unlabeled A4 or BV6 as a competitive compound. BRET signal was measured at 2 hours after treatment using a Tecan Infinite 200 Pro equipped with NanoBRET™ dual-filters (donor 460nm and acceptor 618nm) and determined via the formula (BRET ratio = values at 618nm/values at 460nm). Raw BRET ratios were then converted to milliBRET units (mBU) and plotted to determine apparent intracellular affinity of A4 or BV6 to XIAP. Values were expressed mean ± SD of three independent experiments.
Fig. 3B(i)-3B(iv). Degradation profiles of endogenous XIAP in response to A4 or BV6 treatment of neuroblastoma cells, BE(2)-C (Fig. 3B(i) and 3B(ii)) and KELLY (Fig. 3B(iii) and 3B(iv)). Cells were treated with increasing dose of A4 or BV6 and luminescence was measured continuously in real-time for 2 hours. Luminescence values were normalized to DMSO vehicle control and degradation profiles were generated using GraphPad Prism. Values were expressed as mean ± SD of three independent experiments. Fig. 3C(i)-3C(ii). BRET response curves which indicate the binding of ubiquitin to XIAP upon treatment with XIAP-specific antagonist A4 in neuroblastoma cells. Neuroblastoma cells in neuroblastoma cells, BE(2)-C (Fig. 3C(i)) and KELLY (Fig. 3C(ii)), with endogenous luciferase-tagged XIAP were transfected with Halo-tag ubiquitin and treated with fluorescent Halo-tag ligand followed by the addition of various concentrations of A4. BRET signal was measured every 5 minutes for 6 hours after treatment using a Tecan equipped with NanoBRET™ dual-filters (donor 460nm and acceptor 618nm) and determined via the formula (BRET ratio = values at 618nm/valucs at 460nm). Raw BRET ratios were then converted to milliBRET units (mBU) and plotted to determine the binding of ubiquitin to XIAP. Values were expressed mean ± SD of three independent experiments.
Fig. 3D(i)-3D(ii). Immunoblot analysis of XIAP expression upon A4 treatment with (+) or without (-) 10 pM proteasomal inhibitor MG-132 in neuroblastoma cells, BE(2)-C (Fig. 3D(i)) and KELLY (Fig. 3D(ii)). Cells were pre-treated with or without MG-132 for 2 hours prior the addition of A4 for 4 hours. β-actin is used as internal control.
Figure 4A-4B. NMR analysis on 1H-15N-HSQC spectra of XIAP in the absence and presence of A4
Fig. 4A. Assignment of the 1H-15NHSQC spectrum of XIAP. The assigned cross peaks in the spectrum were labeled with residue name and sequence number.
Fig. 4B. The ^-^N-HSQC spectra of XIAP in the absence (black) and presence (gray) of A4. The spectra were collected as described in experimental procedures. Residues with chemical shift perturbations after A4 treatment were indicated with residue name and sequence number. Figure 5A-5D. Generation of luciferase-tagged XIAP using CRISPR knock-in gene editing
Fig. 5A. Schematic representation of XIAP structure containing HiBiT and the combination with LgBiT resulted in active functional luciferase producing luminescence. Fig. SB. Graphical representation of luminescence measured in neuroblastoma cells, KELLY and BE(2)-C after CRISPR knock-in of HiBiT, a small subunit of highly sensitive luciferase. Three guide RNAs were used for optimization. Signal >3 folds represent positive signal.
Fig. SC. Agarose gel analysis of neuroblastoma cells containing HiBiT using guide RNA 1. The detection of bands was performed via multiplex PCR using three primers as described in experimental procedures. Presence of HiBiT was indicated by the presence of two bands at size 21 Ibp and 139 bp.
Fig. 5D. HiBiT - LgBiT signal- a graphical representation of luminescence measured in neuroblastoma cells after stably transfection with large subunit LgBiT. The combination of HiBiT and LgBiT forms the active luciferase which produces luminescence. The absence of either subunit would result in negative luminescent signal.
Figure 6A-6C. Time course of XIAP degradation in response to XIAP-specific (A4) and pan-IAP (BV6) antagonists
Fig. 6A(i)-6A(ii). Degradation profiles of endogenous XIAP in response to A4 treatment of neuroblastoma cells, BE(2)-C (Fig. 6A(i)) and KELLY (Fig. 6A(ii)).
Fig. 6B(i)-6B(ii). Degradation profiles of endogenous XIAP in response to BV6 treatment of neuroblastoma cells, BE(2)-C ((Fig. 6B(i)) and KELLY (Fig. 6B(ii)).
Fig. 6C(i)-6C(ii). Degradation curve of endogenous XIAP in response to BV6 treatment of neuroblastoma cells, BE(2)-C ((Fig. 6C(i)) and KELLY (Fig. 6C(ii)). at 8 hours. Luminescence values were normalized to DMSO vehicle control and degradation profiles were generated using GraphPad Prism. Values were expressed as mean ± SD of three independent experiments.
Figure 7A-7B. Pharmacokinetic profile of XIAP-specific antagonist A4
Fig. 7A-7B. Pharmacokinetic curve demonstrating the concentrations of A4 in mice plasma (Fig. 7A) and tumor (Fig. 7B) over time (mean ± SD). Neuroblastoma PDXs were treated with 10 mg/kg A4 via intraperitoneal injection. 150-200 pL of blood was taken from each mouse via cardiac puncture and tumors were harvested over the following time courses of 0, 0.5-, 1-, 2-, 4-, 24- and 48-hours post-dosing (n=3/time point). The concentrations of A4 in mice plasma were determined by LCMS/ MS in MRM positive mode.
Figure 8A-8C. Treatment with XIAP-specific antagonist A4 prolongs and improves overall survival in high-risk neuroblastoma patient-derived xenografts (PDXs)
Fig. 8A(i)-8A(ii). Immunoblot analysis of XIAP expressions in tumors harvested from treated neuroblastoma PDXs. (Fig. 8A(i))Neuroblastoma PDXs were treated with 10 mg/kg A4 via intraperitoneal injection. Tumors were harvested over the following time courses of 0, 0.5, 1, 2, 4, 24 and 48 hours post-dosing (n=3/time point). P-actin is used as internal control.
Fig. 8A(ii). Graphical representation of densitometry ratio for XIAP expression normalized to P-actin in tumors harvested from neuroblastoma PDXs as described in Fig. 8A(i). Values were expressed as mean ± SD with n=3/time point.
Fig. 8B. Kaplan-Meier survival plot of MYCN-amplified neuroblastoma PDXs (n=9/treatment group). Dark gray line indicates percentage survival of vehicle-treated mice (control) and light gray line is the percentage survival of A4-treated mice (10 mg/kg). The mice were monitored over a period of 7 weeks, including 4 weeks of stabilizing implantation and subsequent 3 weeks of treatment period. Log-rank (Mantelcox test) derived from Kaplan-Meier survival plot was used for statistical comparisons between groups with *P<0.05.
Fig. 8C(i)-8C(ii). Bioluminescence imaging of tumor growth in A4-treated mice (Fig. 8C(i)) and vehicle-treated mice (Fig. 8C(ii)).
Figure 9A-9C. XIAP-specific antagonist A4 works synergistically with and promotes effective dose reduction of vincristine and topotecan in vitro
Fig. 9A(i)-9A(ii). Combination index (CI) demonstrating synergism between A4 with Vincristine (Fig. 9A(i)) and A4 with Topotecan (Fig.9A(ii)). Increasing doses of A4 with either Vincristine or Topotecan were added to neuroblastoma cell lines at a fixed ratio based on the IC50 values of the individual drugs for 48h (A4: Vincristine; BE(2)-C 1:1, KELLY 1:0.5 and A4:Topotecan; BE(2)-C 1:6, KELLY 1:16), respectively. CI was generated using CompuSyn software by Chou-Talalay. Cl60-90 represents the average combination index at 60-90% cell death. CI <1 denotes synergistic effect; CI =1 denotes addictive effect and CI >1 denotes antagonistic effect.
Fig. 9B(i)-9B(ii)-9C(i)-9C(ii). Dose reduction index (DRI) demonstrating the fold differences of A4 effectively reducing the dose of vincristine (Fig. 9B(i) and 9B(ii)) or topotecan (Fig. 9C(i) and 9B(ii)) when used in combination with these agents. Increasing doses of A4 with either vincristine or topotecan were added to neuroblastoma cell lines at a fixed ratio based on the IC50 values of the individual drugs for 48h (A4:Vincristine; BE(2)-C 1:1, KELLY 1:0.5 and A4:Topotecan; BE(2)-C 1:6, KELLY 1:16), respectively. DRI was generated using CompuSyn software by Chou-Talalay. DRI60-90 represents the average dose reduction index at 60-90% cell death. DRI <1 denotes unfavorable dose reduction and DRI >1 denotes favorable dose reduction. Dose response curves of vincristine (Fig. 9B(ii)) and topotecan (Fig. 9C(ii)) treated alone or in combination with A4 in BE(2)-C and KELLY neuroblastoma cells.
Figure 10. Mechanisms mediated by various IAP antagonists, pan-IAP antagonist (SMAC mimetic) BV6 and XIAP-specific antagonist (ARTS mimetic) A4 in high- risk neuroblastoma
The figure schematically illustrates and compares the effect of specific targeting of XIAP in high-risk neuroblastoma cell with targeting c-IAPs. As shown by the figure, the use of ARTS-mimetic compounds that specifically lead to proteasomal degradation of XIAP, and apoptosis of the high-risk neuroblastoma cell, whereas the use of c-IAPs results in negligible apoptosis of the cells.
DETAILED DESCRIPTION OF EMBODIMENTS
Neuroblastoma is one of several neural crest-derived cancers that share a common developmental biology requiring XIAP. Reduced XIAP expression is required for developmental apoptosis to initiate. Hence, tumor-initiating cells of prenatal embryonal cancers including neuroblastoma which fail to undergo developmental apoptosis are often found to have high XIAP expression, suggesting a dependency on XIAP for survival (Potts et al., 2003; Wright et al., 2007). Despite the key role played by XIAP in neural crest development, the translational potential of exploiting XIAP antagonism as a treatment strategy for neuroblastoma has not been investigated.
The present disclosure shows that aggressive, high-risk MYCN-amplified neuroblastoma cells tend to express a higher level of XIAP. Interestingly, such an association of XIAP expression with high-risk features of disease had also been observed in previous studies. For instance, the upregulation of XIAP expression, but not other apoptotic regulators, has been associated with high-risk biological features and poor survival in acute myeloid leukemia as well as other neuroectodermal cancers like melanoma, gastrointestinal and pulmonary neuroendocrine tumors - all highly aggressive malignancies with poor prognoses (Dizdar et al., 2017; Emanuel et al., 2008; Sung et al., 2009; Tamm et al., 2004). The inventors also demonstrated that the silencing of XI AP in high XI AP- expressing and MYCN-amplified neuroblastoma cells resulted in significant apoptosis, suggesting the addiction and dependency on XIAP for survival, thus highlighting the potential of targeting XIAP as a treatment strategy for high-risk neuroblastoma.
Several small molecule compounds targeting pan-IAPs or XIAP alone were tested. SMAC mimetics with pan-IAP targeting activity have been widely studied and tested in many cancers while XIAP-specific targeting is a new treatment strategy not previously explored in neuroblastoma. The inventors found that the ARTS mimetic A4, an XIAP- specific antagonist, demonstrated the best efficacy across a panel of commercial and patient-derived neuroblastoma cell lines. It was especially effective against high XIAP- expressing cell lines which also happened to be MYCN-amplified, a classical hallmark of aggressive and resistant neuroblastoma. Moreover, its efficacy appeared to be stratified according to XIAP and N-Myc expression status. A4 was also best tolerated by normal cell lines representing liver and bone marrow, which are common sites of neuroblastoma metastasis. Furthermore, the study disclosed herein provides the first proof-of-concept on the efficacy of XIAP-specific antagonist against neuroblastoma tumors in vivo, having determined the murine pharmacokinetic profile of A4 and demonstrated its utility in PDX models. Encouragingly, A4 remained stable with good availability in plasma, and was shown to prolong and improve the overall survival of high-risk neuroblastoma PDXs at the tested dose of lOmg/kg. Though tumors did not show gross regression in size when A4 was used as a single agent, A4 showed evidence for synergism with standard-of-care cytotoxic agents. Notably, A4 was able to reduce the doses of these cytotoxic agents when used in combination. This offers potential benefit in toxicity reduction if used in the treatment of high-risk neuroblastoma which currently involves intensive multimodal therapy. In contrast, it was found that pan-IAP antagonists tested in this study including LCL-161, CUDC-427, Debiol 143 and BV6 targeted mainly c-IAPs, induced high toxicity in normal cells and were ineffective towards XIAP-dependent, M-aYmCpN! ified neuroblastomas.
A4 demonstrates promising efficacy in prolonging the survival of high-risk, MYCN- amplified neuroblastoma patient-derived xenografts despite having weaker binding affinity for XIAP than Smac mimetic BV6. This indicates that the mechanism of action for A4 is distinct, and that degradation of XIAP is more important for therapeutic success than binding-mediated inhibition. Notably, both ARTS and A4 promote degradation of XIAP, whereas Smac and Smac-mimetics do not. This finding lends support to the development of more potent derivatives from first-generation A4.
Intriguingly, the inventors showed that the ARTS mimetic A4 predominantly degrades XIAP while SMAC mimetic BV6 inhibits XIAP. Similar results were shown for other cancers (Mamriev et al., 2020). This difference in mechanism for suppressing XIAP resulted in different responses in neuroblastoma cells. Notably, similar observations have been described for receptor tyrosine kinase (RTK) targeting, where inhibitors had to be sustained at saturating concentration for extended periods to effect signalling suppression while equivalent effects could be achieved in a much shorter time via compounds that degrade RTK (Burslem et al., 2018). It was thus postulated that XIAP could have another functional role in protecting from apoptosis, independent from the one disrupted by SMAC-binding. Hence, besides the main binding pocket bound by SMAC mimetic BV6 (the IBM motif, located at the BIR3 domain of XIAP, also known as the SMAC binding pocket), other XIAP domains could also exhibit functionality. In this regard, the RING domain of XIAP has previously been shown to function as an E3 ubiquitin ligase involved in ubiquitination (Galban and Duckett, 2010). Upon release from the mitochondria, the main function of SMAC is to bind and inhibit XIAP. However, instead of inhibiting XIAP, XIAP-bound SMAC has been shown to be targeted by XIAP for ubiquitination and subsequent, degradation, thereby freeing XIAP (MacFarlane et al., 2002). Therefore, the ability of XIAP to be bound and yet still catalyze ubiquitination suggests that XIAP is still functional even when bound by its endogenous inhibitor. This possibility is supported by a recent study on the development of specific non-genetic lAP-based protein erasers (SNIPERs) (Ma et al., 2021). SNIPERs consist of an lAP-binding site to the BIR3 domain and a ligand for the target protein. Using its lAP-binding site, SNIPERs are able to bind to IAPS and bring the target protein into close proximity for degradation by utilizing the E3 ligase function of IAPs’ RING domain. Indeed, XIAP could still be functionally protective in spite of binding to compounds that disrupt its binding and inhibition of caspases. However, this function remains to be investigated. Additionally, the minimal or lack of XIAP degradation induced by BV6 could be attributed to its preferential binding to c-IAPs (Cossu et al., 2009; Sun et al., 2008). This could have resulted in sequestration of BV6 from binding and targeting XIAP and thus, triggering less apoptotic killing. To overcome the effects of sequestration, a higher dose would be required but may not be feasible due to the toxicity of BV6 to non-cancerous cells.
Taken together, binding and degradation of XIAP by ARTS mimetics is a novel and effective therapeutic strategy against high-risk neuroblastoma either alone or in combination with current standard-of-care agents (Figure 10).
Thus, as a first aspect, the present disclosure provides at least one apoptosis related protein in the TGF-beta signaling pathway (ARTS) mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition comprising at least one of said ARTS mimetic compound, for use in in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof, in some embodiments, the neoplastic disorder is neuroblastoma. More specifically, the ARTS mimetic compound interacts and binds the Baculo viral IAP Repeat (BIR) domain 3 of X- linked inhibitor of apoptosis protein (XIAP), thereby leading to proteasomal degradation of XIAP. In some embodiments the BIR3 domain 3 of XIAP, comprises residues 265- 330 of XIAP, as denoted by SEQ ID NO: 10, or any homologs or variants thereof. It should be noted that the amino acid sequence of XIAP is in some embodiments, the amino acid sequence as denoted by SEQ ID NO: 8, or any homologs and variants thereof.
In accordance with this aspect, the present disclosure provides a compound having the general formula (I):
Figure imgf000019_0001
or a pharmaceutically acceptable salt, solvate, hydrate, or physiologically functional derivative thereof including any stereoisomer thereof; wherein
R1 may be independently selected from H, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl; R2 may be independently selected from -C(=O)-X-R3, -S(=O)-X-R3';
X being a heteroatom independently selected from N-containing group, O and S; R3 and R3', independently of each other may be selected independently from H, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl;
Rs may be -L1 -R7-L2-R8; L1 and L2, independently of each other, may be selected independently from -(CH2)n-; - NH-C(=O)-(CH2)n-, -C(=O)-NH-(CH2)n-; -S-S-(CH2)n-; -O-(CH2)n-; -NH-(CH2)n-; C(=O)-(CH2)n; -S-(CH2)n-; -NH-S(=O)n-(CH2)n-; each n, being independently, may be 0 to 5; R7 may be independently selected from C1-C12 alkylene, C2-C12 alkenylene, C2-C12 alkynylene, a ring system containing five to twelve atoms, each optionally substituted;
R8 may be independently selected from H, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, a ring system containing five to twelve atoms, each optionally substituted;
R6 may be independently selected from H, halogen, CN, NO2, C1-C12 alkoxy, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl.
In some embodiments, at least one of the ARTS mimetic compounds for use in accordance with the present disclosure, has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof. More specifically, in some embodiments, Formula I being:
Figure imgf000020_0001
(Formula I); wherein:
R1 is independently selected from H, C1-C3 alkyl; R2 is independently selected from - C(=O)-X-R3, -S(=O)-X- R3'; X is a heteroatom independently selected from O; R3 and R3' independently of each other may be selected independently from H, C1-C3 alkyl;
R5 is -L1-R7-L2-R8; L1 and L2, independently of each other, are selected independently from -(CH2)n ; -NH-C(=O)-(CH2)n-, -C(=O)-NH-(CH2)n-; -S-S-(CH2)n-; -O-(CH2)n-; - NH-(CH2)n-; C(=O)-(CH2)n-; -S-(CH2)n-; -NH-S(=O)n-(CH2)n-; n is independently 1 to 3; R7 is a carbocyclic ring or a heterocyclic ring; R8 is a mono cyclic ring system having 5 to 12, optionally substituted with at least one of OH, CF3, halogen, -COOH, -NH2, CN, C1-C3alkyl; and R6 is independently selected from H, halogen, CN, NO2, C1-C3 alkoxy, straight or branched C1-C3 alkyl.
In some specific embodiments, the present disclosure provides compounds, specifically, the compounds a defined in Formula I, acting as ARTS mimetic compounds.
Still further, the invention provides compounds, specifically, the compounds a defined in Formula I, for use as XIAP antagonists.
Still further, in some embodiments that further define the compounds of the invention, a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents.
In some other embodiments, the substituents may be selected from OH, CF3, halogen, C(=O), -COOH, -NH2, CN, alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, C1- C12 haloalkyl, C2-C12 haloalkenyl, C2-C12 haloalkynyl, C1-C12 alkoxy, C1-C5 carboxyl, halogen, five to twelve ring system (aromatic or heteroaromatic ring).
In some embodiments R1 may be H. In some embodiments, R2 may be -C(=O)-X-R3.
In some embodiments, the N-containing group is selected from N, NH, NH2, tertiary amine (tertiary alkyl amine), quaternary ammonium (quaternary alkyl ammonium). In some other embodiments, the N-containing group may be connected (bonded) with one R3 group, at times with two R3 groups, at times with three R3 groups. It should be noted that in accordance with these embodiments, R3 may selected independently from each other. For example, the N-containing group may be connected to three R3 groups forming for example quaternary alkyl ammonium.
In some other embodiments X is O. In some further embodiments, R3 and R3', independently of each other may be selected independently from may be H, straight or branched C1-C12 alkyl. In some further embodiments, R3 may be straight C1-C12 alkyl. In yet some other embodiments, R3 may be methyl, ethyl. In yet some further embodiments, R3 may be methyl.
In some embodiments, L1 and L2 may be independently selected independently from each other from -NH-C(=O)-(CH2)n-, -(CH2)n-- According to these embodiments, the group -(CH2)n- wherein n=l to 5, may encompasses an alkyl, alkylene, alkenyl, alkenylene and alkynyl as defined herein having at most five carbon atoms. In some embodiments, each of n may be independently 1 to 3. In some other embodiments when n=0, L1 and L2 may be a bond. In some embodiments, L1 is -NH-C(=O)-(CH2)n-. In some further embodiments, n is 1. In some other embodiments, L1 is -NH-C(=O)- (CH2)-. In some embodiments, L2 is -(CH2)n-. In some further embodiments, n is 1. In some other embodiments, L2 is -(CH2)-.
In some embodiments, R7 may be a ring system containing five to twelve atoms, optionally substituted. In some other embodiments, the ring system of R7 may be an aryl (aromatic ring) or aliphatic ring (non-aromatic ring). In some further embodiments, R7 may be C5-C12 saturated cycloalkyl, C5-C12 saturated cycloalkylene, C5-C12 aryl or C5-C12 arylene. In some further embodiments, the ring system of R7 may contain at least two carbon atoms and may include at least one heteroatom ring. In some further embodiments, R7 may be heteroaryl, heteroarylene, heterocycloalkylene or heterocycloalkyl. In some further embodiments, R7 may be C2-C12 heterocycloalkyl ring, C2-C12 heteroaryl or C2- C12 heteroarylene. In some embodiments, the heteroatom may be N, O, S. In yet some further embodiments, the heteroatom may be N, O. In some other embodiments, the heteroatom may be N. In some other embodiments, R7 is piperidine. In some other embodiments, R7 is piperazine.
In some other embodiments, R8 may be H, straight or branched C1-C12 alkyl or five to twelve atom ring system. In some further embodiments, R8 may be straight or branched C1-C12 alkyl or five to twelve atom ring system each optionally substituted. In yet some further embodiments, R8 may be C1-C5 alkyl. In some further embodiments, R8 may be straight C1-C5 alkyl substituted with OH. In some other embodiments, R8 may be an aromatic ring, non-aromatic ring (aliphatic ring). In some further embodiments, R8 may be C5-C12 saturated cycloalkyl, C5-C12 saturated cycloalkylene, C5-C12 aryl or C5-C12 arylene. In some further embodiments, the ring system of R8 may contain at least two carbon atoms and may include at least one heteroatom ring. In some further embodiments, R8 may be heteroarylene, heteroaryl, heterocycloalkylene or heterocycloalkyl. In some further embodiments, R8 may be C2-C12 heterocycloalkyl ring or C2-C12 heterocyclic aromatic ring (aryl or arylene). In some embodiments, the heteroatom may be N, O, S In yet some further embodiments, R8 is an aromatic ring containing six atoms. In some other embodiments, R8 may be an aromatic ring. In some other embodiments, R8 is phenyl.
In some embodiments, R6 may be connected (bonded, attached) to any position of the ring. In some further embodiments, the ring may be substituted with one R6, at time with two R6. at times with three R6, at times with four R6. In some embodiments, R6 may be independently selected from the group consisting of H, halogen, CN, NO2. In some other embodiments, R6 may be independently selected from H, halogen. In some other embodiments, R6 may be H. In some further embodiments, R6 may be Cl. In some other embodiments, R6 may be an electron withdrawing group.
In yet some further embodiments, the ARTS mimetic compound of the general formula (I) is further characterized by at least one of:
In some embodiments (a), the R1 is H; in yet some further additional or alternative embodiments (b), the R2 is -C(=O)-X-R3; in some further additional or alternative embodiments (c), the X is O; in some further additional or alternative embodiments (d), the L1 and L2 independently selected independently from each other from -NH-C(=O)- (CH2)n-, -(CH2)n-; and in some further additional or alternative embodiments (e), the R6 is independently selected from the group consisting of H, halogen, CN, NO2.
In some embodiments with this aspect, a compound of the invention has the general formula (II) including any stereoisomer or salt thereof: salt thereof:
Figure imgf000023_0001
In some embodiments a compound of the invention has the general formula (III) including any stereoisomer or salt thereof:
Figure imgf000023_0002
In some embodiments a compound of the invention has the general formula (IV) including any stereoisomer or salt thereof:
Figure imgf000024_0001
In some embodiments a compound of the invention has the general formula (V) including any stereoisomer or salt thereof:
Figure imgf000024_0002
In some embodiments a compound of the invention has the general formula (VI) including
Figure imgf000024_0003
In some embodiments a compound of the invention has the general formula (VII) including any stereoisomer or salt thereof:
Figure imgf000025_0002
1n some further embodiments, a compound of the invention having Formula I, Formula II or Formula III is characterized by R1 is H, R2 is -C(=O)-X-R3, L1 is NH-C(=O)-(CH2)- , L2 is -(CH2)-, R7 is piperazine, R8 is phenyl, R6 is Cl, X is O and R3 is methyl. In some further embodiments, a compound of the invention having Formula IV, is characterized by R1 is H, R2 is -C(=O)-X- R3, L2 is -(CH2)-, R6 is Cl, R7 is piperazine, R8 is phenyl, X is O and R3 is methyl. In some further embodiments, a compound of the invention having Formula V, is characterized by R1 is H, R2 is -C(=O)-X-R3, L1 is NH-C(=O)-(CH2)-, L2 is -(CH2)-, R8 is phenyl, R6 is Cl, X is O and R3 is methyl. In some further embodiments, a compound of the invention having Formula VI, is characterized by R1 is H, R2 is - C(=O)-X-R3, L1 is NH-C(=O)-(CH2)-, L2 is -(CH2)-, R6 is Cl, X is O and R3 is methyl. In some further embodiments, a compound of the invention having Formula VII, is characterized by R1 is H, R2 is -C(=O)-X-R3, L1 is NH-C(=O)-(CH2)-, L2 is -(CH2)-, Re is Cl, X is O and R3 is methyl.
In some embodiments, specific examples of compounds or pharmaceutically acceptable salts or hydrates or any stereoisomer thereof of the compounds of Formula I- VII include, without limitation:
Figure imgf000025_0001
3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2-carboxylic acid methyl ester)
Figure imgf000026_0001
5-Bromo-3- { 2-[4-(2-hydroxy-ethyl)-piperazin- 1-yl] -acetylamino } - 1H-indole-2- carboxylic acid methyl ester
Figure imgf000026_0002
3-[2-(4-Benzyl-piperazin- 1 -yl)-acetylamino]-4-chloro- 1 H-indole-2-carboxylic acid methyl ester
Figure imgf000026_0003
(3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2-carboxylic acid methyl ester)
In accordance with the second aspect, the present disclosure provides 1,5-di-carbonyl compounds. In accordance with this aspect, the compounds contain one or more nitrogen atoms. In some other embodiments, the compounds contain at least one ring structure. In some other embodiments, the ring structure containing at least one nitrogen atom. In some further embodiments, the 1 compounds contain a heterocyclic ring structure containing five to twelve atoms, the ring structure containing at least two carbon atoms and at least one heteroatom being N, O or S. The present disclosure provides in accordance with the second aspect, a compound having the general formula (VIII):
Figure imgf000027_0001
or a pharmaceutically acceptable salt or hydrate thereof including any stereoisomer thereof; wherein:
R9 and R10 may be the same or may be different and may be independently selected from each other from a ring system containing five to twelve atoms, each optionally substituted;
R11 may be independently selected from H, straight or branched C1-C12 alkyl, straight or branched C2-C12 alkenyl, straight or branched C2-C12 alkynyl;
L3 and L4, independently of each other, may be selected independently from -(CH2)p-; - NH-C(=O)-(CH2)p-, -C(=O)-NH-(CH2)p-; -S-S-(CH2)p-; -O-(CH2)p-; -NH-(CH2)p-; C(=O)-(CH2)p; -S-(CH2)p-; -NH-S(=O)P-(CH2)p-; each p, being independently, may be 0 to 5.
In some embodiments, a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents, In some further embodiments, the substituents may be selected from OH, CF3, halogen, C(=O), -COOH, -NH2, CN, C(=O)- alkyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, C1-C12 alkoxy, C1-C5 carboxyl, halogen, aromatic or heteroaromatic ring.
In some embodiments, R9 and R10 may be a ring system containing five to twelve atoms. In some further embodiments, the ring system of R9 and R10 may contain at least two carbon atoms. In some other embodiments, the ring system of R9 and R10 may be an aromatic ring, non-aromatic ring, fused ring or the like. In some further embodiments, R9 and R10 may be C5-C12 saturated cycloalkyl, C5-C12 saturated cycloalkylene, C5-C12 aryl or C5-C12 arylene. In some other embodiments, the ring system of R9 and R10 may include at least one heteroatom ring. In some further embodiments, R9 and R10 may be heteroaryl or heterocycloalkyl. In some further embodiments, R9 and R10 may be C2-C12 hetero cycloalkyl or C2-C12 hetero aromatic ring (aryl or arylene). It should be noted that according with some embodiments, R9 and R10 may be independently from each other contain different carbon atoms. In some embodiments, the heteroatom may be N, O, S. In some embodiments, R9 and R10 may be independently from each other selected from C5-C12 aryl. In some embodiments, R9 and R10 may be C6 aryl, optionally substituted. In some embodiments, R9 and R10 may be C6 aryl optionally substituted with -C(=O)-alkyl. In some further embodiment, R9 and R10 may be C6 aryl optionally substituted with - C(=O)-CH3.
In some embodiments, L3 and L4, may be -(CH2)p-. According to these embodiments, the group -(CH2)p- wherein p=l to 5, may encompasses an alkyl, alkylene, alkenyl, alkenylene and alkynyl as defined herein having at most five carbon atoms. In some embodiments, each of p may be 0. At times, when p=0, L3 and L4 are a bond.
In some embodiments, R11 is H or straight C1-C12 alkyl. In some further embodiments, R11 is straight C1-C12 alkyl. In some other embodiments, R11 is methyl.
In some embodiments, R9 and R10 may be C6 aryl optionally substituted with -C(=O)- CH3.
In some embodiments, L3 and L4 are a bond. In some embodiments, Rn is methyl, R9 and R10 are C6 aryl substituted with -C(=O)-CH3 and L3 and L4 are a bond.
In some embodiments, specific examples of compounds or pharmaceutically acceptable salts or hydrates including any stereoisomer thereof of the compounds of Formula VIII include, without limitation: limitation:
Figure imgf000028_0001
I -Methyl- 1 H-pyrazolc-3,5-dicarboxylic acid bis-[(3-acetyl-phenyl)-amide]
Figure imgf000029_0001
2-Methyl-2H-pyrazole-3,4-dicarboxylic acid bis-[(3-acetyl-phenyl)-amide]
In accordance with the third aspect, the present disclosure provides compounds containing one or more nitrogen atoms. In some other embodiments, the compounds contain at least one ring structure. In some other embodiments, the ring structure containing at least one nitrogen atom. In some further embodiments, the compounds contain a heterocyclic ring structure containing five to twelve atoms, the ring structure containing at least two carbon atoms and at least one heteroatom being N, O or S. in some other embodiments, the compounds contain an amide group.
In accordance with the third aspect, the present disclosure provides a compound having a general formula:
Figure imgf000029_0002
or a pharmaceutically acceptable salt or hydrate thereof including any stereoisomer thereof; wherein:
X, Y independently may be each independently selected from each other from NH, CH; R14 and R15 may be the same or may be different and may be independently from each other selected from a ring system containing five to twelve atoms, each optionally substituted; L5 may be selected independently from -(CH2)q-; -NH-C(=O)-(CH2)q-, -C(=O)-NH- (CH2)q-; -S-S-(CH2)q-; -O-(CH2)q-; -NH-(CH2)q-; C(=O)-(CH2)q-; -S-(CH2)q-; -NH- S(=O)q-(CH2)q-; each q, being independently, may be 0 to 5;
In some embodiments, R14 and R15 may be a ring system containing five to twelve atoms. In some further embodiments, the ring system of R14 and R15 may contain at least two carbon atoms. In some other embodiments, the ring system of R14 and R15 may be an aromatic ring or a non-aromatic ring. In some further embodiments, R14 and R15 may be C5-C12 saturated cycloalkyl, C5-C12 saturated cycloalkylene, C5-C12 aryl or C5-C12 arylene. In some other embodiments, the ring system of R14 and R15 may include at least one heteroatom ring. In some further embodiments, R14 and R15 may be C2-C12 hetero cycloalkyl or C2-C12 hetero aromatic ring. It should be noted that according with some embodiments, R14 and R15 may be independently from each other contain different carbon atoms. In some embodiments, the heteroatom may be N, O, S. In some embodiments, R14 and R15 may be independently from each other selected from C5-C12 aryl optionally substituted. In some embodiments, R14 and R15 may be independently from each other selected from isoquinoline or phyel each independently from the other optionally substituted. In some embodiments, L5, may be -(CH2)q-. According to these embodiments, the group -(CH2)q- wherein q=0 to 5, may encompasses an alkyl, alkenyl and alkynyl as defined herein having at most five carbon atoms. In some embodiments, each of q may be 0. At times, when q=0, L5 may be a bond.
In some embodiments, a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents. In some further embodiments, the substituents may be selected from OH, CF3, halogen, NO2, C(=O), -COOH, -NH2, CN, C(=O)-C1-C12 alkyl, straight or branched C1- C12 alkyl, straight or branched C2-C12 alkenyl, straight or branched C2-C12 alkynyl, straight or branched C1-C12 alkylene, straight or branched C2-C12 alkenylene, straight or branched C2-C12 alkynylene, C1-C12 alkoxy, C1-C5 carboxyl, aromatic or heteroaromatic ring.
In some embodiments, specific examples of compounds or pharmaceutically acceptable salts or hydrates including any stereoisomer thereof of the compounds of Formula IX include, without limitation:
Figure imgf000031_0001
1-[(3,5-Dichloro-phenylcarbamoyl)-methyl]-piperidine-4-carboxylic acid (2-hydroxy- phenyl)-amide
Figure imgf000031_0002
2-[4-(8-Nitro-isoquinolin-5-ylamino)-piperazin-l-yl]-iV-phenyl-acetamide
It should be appreciated that in some aspects, the invention provides the compounds of any one of the compounds of Formulas I, II, III, IV, V, VI, VII, as well as the compounds of formulas VIII, IX as described herein and any analogs or derivative thereof including any stereoisomer or salt thereof or any vehicle, matrix, nano- or micro-particle, or composition comprising the same.
In more specific embodiments, the at least one ARTS mimetic compound for use according to the present disclosure, comprises a compound that has the structure of formula (g). In some embodiments, the compound is 3-[2-(4-Benzyl-piperazin-l-yl)- acetylamino]-5-chloro-1H-indole-2-carboxylic acid methyl ester, said compound has the structure of Formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000032_0002
It should be appreciated that such compound may be referred to herein by the present application as "A4" or "ARTS mimetic A4 small molecule" or the like. In some embodiments, the "A4" molecule may also be referred to herein by the chemical name: methyl 5-chloro-3-[[2-[4-(phenylmethyl) piperazin- 1 -yl ]amino]-1H-indolc-2-caiboxylat. It should be also noted that when referring to "A4", the compound may include any stereoisomer or salt thereof, for example the stereoisomer having the structure:
Figure imgf000032_0001
In some particular aspects, the invention provides an effective amount of the compounds of the invention, specifically, any one of the compounds of Formulas I, II, III, IV, V, VI, VII, as well as the compounds of formulas VIII, IX as described herein and any analogs or derivative thereof including any stereoisomer or salt thereof or any vehicle, matrix, nano- or micro-particle, or composition comprising the same as described herein above for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a neoplastic disorder affecting neural system and/or neural cell/s, specifically, cancer, in a subject in need thereof.
Still further, the inventors found that compounds having at least one amine group and at least one carbonyl group act as ARTS mimetics compounds. Specifically, the inventors found that 1,2 and 1,5 di-carbonyl compounds act as ARTS mimetics. In yet some further embodiments, the ARTS mimetic compound for use in the preset disclosure, is a compound having the general formula (X) or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, wherein formula (X) is:
Figure imgf000033_0001
wherein
R1, R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is L1’-R3’- L1”-R3” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R2 is L2’-R4’- L2”-R4”; or
R1 is L1’-R3’- L1”-R3” and R2 is L2’-R4’- L2”-R4”; wherein R3’, R3”, R4’, R4” is each independently from each other, absent or H, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, heteroaryl, heterocycloalkyl, a ring system containing five to twelve atoms, each optionally substituted; L1’, L1”, L2’ and L2” is each independently from each other, absent or -(CH2)n, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2- -S-, -S-S-, -S-(CH2)n-; n is an integer selected from any one of 0, 1, 2, 3, 4, 5, each one of L1’, L1”, L2’ and L2” independently from each other, may be optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m- halogen and m is an integer selected from 0, 1, 2, 3, 4, 5. In some embodiments of the ARTS mimetic compound for use in the preset disclosure, the L1’, L1”, L2’ and L2” are independently from each other selected from -(CH2)n-, C(=O), optionally -(CH2)n- substituted with -(CH2) m-OH and wherein n is an integer selected from 1, 2, or 3 and m is an integer selected from 1, 2 or 3.
In yet some further embodiments, the ARTS mimetic compound for use in accordance with the present disclosure, each one of R3', R3”, R4’, R4” is absent or may be independently from each other an aromatic or a heteroaromatic ring, each is independently from each other optionally substituted with OH, alkyl, halogen, CF3, NO2, C(=O).
In some further embodiments of the present disclosure, the ARTS mimetic compound for use, is having the general formula (XI)
Figure imgf000034_0001
Wherein R1 and L2’ is as defined above and wherein R is one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other and t is an integer selected from 1 to 5.
In some embodiments of the ARTS mimetic compound for use in the preset disclosure, the L2’ is (CH2)n-, or C(=O), optionally substituted by one or more of C1-C5alkoxy, C1- C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, or -(CH2)m-halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5.
In some further embodiments, the ARTS mimetic compound for use in accordance with the present disclosure, is a compound having the general formula (Xlb):
Figure imgf000035_0001
In some further embodiments of disclosed ARTS mimetic compound for use, the R1 is L1’-R3'-L1”-R3”.
In yet some further embodiments of disclosed ARTS mimetic compound for use, the R1 is at least one of:
(i) L1’, L1 ’’and R3 ” are each absent and R3' is an optionally substituted
Figure imgf000035_0002
(ii) L1’, L1”and R3” are each absent and R3' is:
Figure imgf000035_0003
(iii)L1’is absent R3' is an optionally substituted phenyl, R3” is an optionally substituted:
Figure imgf000035_0004
In some embodiments, the ARTS mimetic compound for use, according to the present disclosure, is a compound having the general formula (Xlla) or (Xllb):
Figure imgf000036_0001
wherein R is one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other and t is an integer selected from 1 to 5.
In some embodiments, the ARTS mimetic compound for use of the present disclosure, is a compound having the general formula (XIIc), (Xlld) or (Xlle):
Figure imgf000036_0002
Figure imgf000037_0001
wherein L1” and R3” are each as defined above, wherein R is one or more of H, OH,
CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other and t is an integer selected from 1 to 5.
In some embodiments, the ARTS mimetic compound for use in accordance with the present disclosure is a compound having the general formula (XIIc), or (Xlle):
Figure imgf000037_0002
Figure imgf000038_0001
wherein R3’ and R3 ” is each independently from the other, an optionally substituted aryl or an optionally substituted heteroaryl, and L1” is C(=O), wherein R is one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other and t is an integer selected from 1 to 5.
In yet some further embodiments, the ARTS mimetic compound for use in accordance with the present disclosure, is a compound having the formula (3.1), (3.2), (3.3);
Figure imgf000038_0002
N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide ;
Figure imgf000039_0001
(S)-N1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide (denoted herein as “B3”);
Figure imgf000039_0002
(R)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide.
More specifically, the present disclosure provides the use of 1 ,2-di-carbonyl compounds that serve herein as ARTS mimetics, in upregulating p53 levels. In accordance with this aspect, the present disclosure provides a compound comprising at least one oxalamide moiety. The present disclosure also encompasses pharmaceutically acceptable salt, solvate, hydrate or any stereoisomer of the compounds described herein.
In accordance with this aspect, the present disclosure provides the use of a compound having the general formula (X):
Figure imgf000040_0001
or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof; wherein
R1, R2 are each, independently from each other, absent or H, alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is L1’-R3’-L1”-R3” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R2 is L2’-R4’-L2”-R4”; or
R1 is L1’-R3’-L1”-R3” and R2 is L2’-R4’-L2”-R4”; wherein R3’, R3”, R4’, R4” is each independently from each other, absent or H, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, heteroaryl, heterocycloalkyl, a ring system containing five to twelve atoms, each optionally substituted; L1’, L1”, L2’ and L2” is each independently from each other, absent or -(CH2)n, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2-, -S-, -S-S-, or -S-(CH2)n-, each optionally substituted; n is an integer selected from any one of 0, 1, 2, 3, 4, 5 each may be independently from each other, optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m- halogen and m is an integer selected from 0, 1, 2, 3, 4, 5.
In some embodiments, each one of L1’, L1”, L2’ and L2” is each independently from each other, may be optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m-halogen and m is an integer selected from 0, 1, 2, 3, 4, 5.
In some examples, R1, R2 are each, independently from each other, a ring system containing five to twelve atoms. In some embodiments, a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents as further described herein.
In some examples, R1, R2 are each, independently from each other, a monocyclic or polycyclic ring system containing five to twelve atoms, optionally substituted with one or more substitutes.
In some embodiments, each one of R3' , R3”, R4’ and R4” may be independently from each other a ring system containing five to twelve atoms, each optionally substituted.
In some examples, each one of R3' , R3”, R4’ , R4” is independently from each other, a monocyclic or polycyclic ring system containing five to twelve atoms, optionally substituted with one or more substitutes.
In some other embodiments, the ring system of each one of R3', R3”, R4’ , R4”, may be independently from each other an aryl, heteroaryl or aliphatic ring (non- aromatic ring).
In some further embodiments , each one of R3' , R3”, R4’ , R4” may be independently from each other C5-C12 saturated cycloalkyl, C5-C12 saturated cycloalkylene, C5-C12 aryl, C5- C12 heteroaryl or C5-C12 arylene.
In some further embodiments, the ring system of each one of R3', R3”, R4’ , R4” may independently from each other contain at least two carbon atoms and may include at least one heteroatom ring.
In some further embodiments , each one of R3' , R3”, R4’ , R4” may be independently from each other heteroaryl, heteroarylene, heterocycloalkylene or heterocycloalkyl.
In some further embodiments , each one of R3' , R3”, R4’ , R4” may be independently from each other C2-C12 heterocycloalkyl ring, C2-C12 heteroaryl or C2-C12 heteroarylene. In some embodiments, the heteroatom in a heteroaryl ring may be N, O, S.
In yet some further embodiments, the heteroatom in a heteroaryl ring may be N, O.
In some other embodiments, the heteroatom in a heteroaryl ring may be N.
In some embodiments, each one of R3', R3”, R4’, R4”is absent, H, an optionally substituted aryl or an optionally substituted heteroaryl.
In some embodiments, each one of R3', R3”, R4’ , R4”is absent, an optionally substituted aryl or an optionally substituted heteroaryl.
In some further embodiments, each one of R3', R3”, R4’, R4”is absent or may be independently from each other H, an optionally substituted aromatic ring or an optionally substituted heteroaromatic ring.
In some further embodiments, each one of R3', R3”, R4’, R4”is absent or may be independently from each other an aromatic or heteroaromatic five to eleven membered ring.
In some other embodiments, each one of R3', R3”, R4’, R4” is absent or may be independently from each other H, phenyl, 1 -naphthyl, 2-naphthyl, and 4-biphenyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, furyl (furanyl), quinolyl, isoquinolyl, thienyl, imidazolyl, thiazolyl, indolyl, pyrryl, oxazolyl, benzofuryl, benzothienyl, benzthiazolyl, isoxazolyl, pyrazolyl, triazolyl, tetrazolyl, indazolyl, 1,2,4- thiadiazolyl, isothiazolyl, benzothienyl, purinyl, carbazolyl, benzimidazolyl, indolinyl, 1,2, 3, -oxadiazoyl, 1,2, 4, -oxadiazoyl, 1,2, 5, -oxadiazoyl, 1,3, 4, -oxadiazoyl, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2 oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1 oxo thiomorpholinyl, and 1,1-dioxo thiomorpholinyl, l-methyl-1H-benzo[d]imidazole, benzoimidazole, pyridine, pyrrole, 1 -methyl- 1H-imidazole or 1H-imidazole, each optionally substituted.
In some other embodiments, each one of R3', R3”, R4’, R4” is absent or may be independently from each other H, phenyl, l-methyl-1H-benzo[d]imidazole, benzoimidazole, pyridine, pyrrole, 1 -methyl- 1H-imidazole or 1H-imidazole.
In some embodiments, each one of R3', R3”, R4’ , R4” is absent or may be independently from each other an aromatic or a heteroaromatic ring, each is independently from each other optionally substituted with one or more of OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen.
In some embodiments, each one of R3', R3”, R4’ , R4” is absent or may be independently from each other an aromatic or a heteroaromatic ring, each is independently from each other optionally substituted with OH, alkyl, halogen, CF3, NO2, C(=O).
In some embodiments, each one L1’, L1”, L2’ and L2” may be substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, or -(CH2)m-halogen;
In some embodiments, each one of L1’, L1”, L2’ and L2” may be absent or independently from each other selected from -(CH2)n-, C(=O).
In some further embodiments, each one of L2’, L2” is absent or may be -(CH2)n--
In some further embodiments, each one of L2’, L2” may be optionally substituted with -(CH2)m-OH.
In some embodiments, n may be 0 to 3, at times n may be 1 to 3, at times n may be 2 to 3 and m may be 1 to 3.
In some other embodiments, each one of L1 ’or L1” is absent or may be C(=O). In some examples in which R1 is L1’-R3'-L1”-R3”, L1’ and L1 ”are each absent, R3' is an optionally substituted aromatic or heteroaromatic five to eleven membered ring and R3” is absent.
In some examples in which R1 is L1’-R3'-L1”-R3”, L1’ and L1 ”are each absent, R3' is an optionally substituted aryl or optionally substituted heteroaryl and R3” is absent.
In some examples, in which R1 is L1’-R3'-L1”-R3”, L1’ and L1 ” are each absent, R3' is an optionally substituted phenyl, benzoimidazole, l-methyl-1H-benzo[d]imidazole, pyridine, pyrrole, 1 -methyl- 1H-imidazole or 1H-imidazole and R3” is absent.
In some embodiments, in which R1 is L1’-R3'-L1”-R3”, L1’ and L1 ” are each absent, R3' is phenyl, benzoimidazole, l-methyl-1H-benzo[d]imidazole, pyridine, pyrrole, 1-methyl- 1H-imidazole or 1H-imidazole optionally substituted with one or more of OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen and R3” is absent.
In some embodiments, in which R1 is L1’-R3'-L1”-R3”, L1’ and L1 ” are each absent, R3' is phenyl, benzoimidazole, 1-methyl- 1H-benzo[d]imidazole, pyridine, pyrrole, 1-methyl- 1H-imidazole or 1H-imidazole optionally substituted with OH, alkyl, halogen, CF3, NO2, C(=O) and R3” is absent.
In some embodiments, in which R1 is L1’-R3’- L1 ’-R3”, L1’ and L1” are each absent, R3' is phenyl, benzoimidazole, l-methyl-1H-benzo[d]imidazole, optionally substituted with OH, alkyl, halogen, CF3, NO2, or C(=O) and R3” is absent.
In some embodiments, in which RI is L1’-R3’- L1 ’-R3”, L1’ and L1 ” are each absent, R3' is l-methyl-1H-benzo[d]imidazole, optionally substituted with OH, alkyl, halogen, CF3, NO2, or C(=O) and R3” is absent. In some embodiments, in which R1 is L1’-R3’-L1 ’-R3”, L1’ and L1 ” are each absent, R3' is 1 -methyl- 1H-benzo[d] imidazole, optionally substituted with halogen, or CF3, and R3” is absent.
In some embodiments, in which R1 is L1’-R3'-L1”-R3”, L1’ and L1 ” are each absent, R3' is 1 -methyl- 1H-benzo[d] imidazole, optionally substituted with, CF3, and R3” is absent.
In some examples, in which R1 is L1’-R3'-L1”-R3”, LL is absent, R3' and R3”are each independently from the other, an optionally substituted aromatic or heteroaromatic five to eleven membered ring and L1” is -(CH2)n, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2- -S-, -S-S-, or -S-(CH2)n-, each optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m- halogen, n is an integer selected from any one of 0, 1 , 2, 3, 4, 5 and m is an integer selected from O, 1, 2, 3, 4, 5.
In some examples in which Rl is L1’-R3'-L1”-R3”, L1’ is absent, R3' and R3”are each independently from the other, an optionally substituted aryl or an optionally substituted heteroaryl and L1” is -(CH2)n-, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2-, -S-, - S-S-, or -S-(CH2)n-, each optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m-halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5.
In some examples in which Rl is L1’-R3'-L1”-R3”, L1’ is absent, R3' and R3”are each independently from the other, an optionally substituted aryl or an optionally substituted heteroaryl and L1” is -(CH2)n-, or C(=O), each optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, or - (CH2)m-halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5. In some examples in which R1 is L1’-R3’-L1 ’-R3”, L1’ is absent, R3' and R3”are each independently from the other, an optionally substituted aryl or an optionally substituted heteroaryl and L1” is C(=O).
In some examples in which R1 is L1’-R3'-L1”-R3”, L1’ is absent, R3' and R3”are each independently from the other, an optionally substituted phenyl, benzoimidazole, 1- methyl-1H-benzo[d]imidazole, pyridine, pyrrole, 1 -methyl- 1H-imidazole or 1H- imidazole and L1” is -(CH2)n-, or C(=O), each optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, or - (CH2)m-halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5.
In some examples in which R1 is L1’-R3'-L1”-R3”, L1’ is absent, R3' and R3”are each independently from the other, an optionally substituted phenyl, benzoimidazole, 1- methyl-1H-benzo[d]imidazole, pyridine, pyrrole, 1 -methyl- 1H-imidazole or 1H- imidazole and L1 ” is C(=O).
In some examples in which R1 is L1’-R3'-L1”-R3”, L1’ is absent, R3' and R3”are each independently from the other, an optionally substituted phenyl or 1 -methyl- 1H-imidazole or 1H-imidazole and L1”is -(CH2)n-,C(=O), each optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, or - (CH2)m-halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5.
In some examples in which R1 is L1’-R3'-L1”-R3”, L1’ is absent, R3' is phenyl, R3”is 1 -methyl- 1H-imidazole and L1”is C(=O).
In some embodiments, the compound of the present disclosure having general formula (X) have the general formula (Xa), (Xb), (Xc), (Xd), (Xe), (Xf), (Xg), (Xh), (Xi), (Xj) (Xk) or (XI):
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
wherein L1”, R3” are as defined above and wherein R is one or more substituents, being one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other; t represents the number of substituents and may vary within the same compound being one or more identical substituents (for example one or more H) or different substituents.
In some examples, in compounds of Formula (X), (Xd), (Xe), (Xf), (Xg), (Xh), (Xi), (Xj) (Xk) or (XI), L1” is -(CH2)n-, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2-, -S-, -S- S-, or -S-(CH2)n-, each optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m-halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5. In some examples, in compounds of Formula (X), (Xd), (Xe), (Xf), (Xg), (Xh), (Xi), (Xj) (Xk) or (XI), L1” is -(CH2)n-, or C(=O), each optionally substituted by one or more ofC1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, or -(CH2) m- halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5.
In some examples, in compounds of Formula (X), (Xd), (Xe), (Xf), (Xg), (Xh), (Xi), (Xj) (Xk) or (Xl), L1” is C(=O).
In some examples, in compounds of Formula (X), (Xd), (Xe), (Xf), (Xg), (Xh), (Xi), (Xj) (Xk) or (XI), R3” is an optionally substituted aryl or an optionally substituted heteroaryl.
In some examples, in compounds of Formula (X), (Xd), (Xe), (Xf), (Xg), (Xh), (Xi), (Xj) (Xk) or (XI), R3” is an optionally substituted 1 -methyl- 1H-imidazole.
In some examples, in compounds of Formula (X), (Xd), (Xe), (Xf), (Xg), (Xh), (Xi), (Xj) (Xk) or (XI), R3” is 1 -methyl- 1H-imidazole.
In some examples, in compounds of Formula (X), (Xd), (Xe), (Xf), (Xg), (Xh), (Xi), (Xj) (Xk) or (XI), L1” is C(=O) and R3 ” is 1 -methyl- 1H-imidazole.
In some examples, in compounds of Formula (X), (Xd), (Xe), (Xf), (Xg), (Xh), (Xi), (Xj) (Xk) or (XI), R2 is L2’ -R4’-L2” -R4”.
In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ is (CH2)n-, or C(=O), each optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m- OH, -(CH2)m-SH, -(CH2)m-NH2, or -(CH2)m-halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5, R4’ is an optionally substituted aromatic or heteroaromatic five to eleven membered ring and L2” and R4” are each absent.
In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ is an optionally substituted -(CH2)n- or C(=O), n is an integer selected from 1, 2, 3, 4, 5, R4’ is an optionally substituted aromatic or heteroaromatic five to eleven membered ring and L2” and R4” are each absent.
In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ is an optionally substituted -(CH2)n- or C(=O), n is an integer selected from 1, 2, 3, 4, 5, R4’ is an optionally substituted aryl or an optionally substituted heteroaryl and L2” and R4” are each absent.
In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ is an optionally substituted -(CH2)n, n is an integer selected from 1, 2, 3, 4, 5, R4’ is an optionally substituted aryl or an optionally substituted heteroaryl and L2” and R4” are each absent.
In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ is an optionally substituted -(CH2)n, n is an integer selected from 1, 2, 3, 4, 5, R4’ is an optionally substituted phenyl and L2” and R4” are each absent.
In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ is an optionally substituted -(CH2)2, R4’ is an optionally substituted aryl or an optionally substituted heteroaryl and L2” and R4” are each absent.
In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ is an optionally substituted -(CH2)2, R4’ is an optionally substituted phenyl and L2” and R4” are each absent.
In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ is -(CH2)2 substituted with OH, R4’ is an optionally substituted aryl or an optionally substituted heteroaryl and L2” and R4” are each absent.
In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ is -(CH2)2 substituted with OH, R4’ is an optionally substituted phenyl and L2” and R4” are each absent.
In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ , L2” and R4” are each absent, R4’ is an aryl optionally substituted with OH, alkyl, halogen, CF3, NO2, C(=O). In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ , L2” and R4” are each absent, R4’ is a phenyl optionally substituted with OH, alkyl, halogen, CF3, NO2, C(=O).
In some examples in which R2 is L2’ -R4’-L2” -R4”, L2’ , L2” and R4” are each absent, R4’ is a phenyl optionally substituted with OH.
In some embodiments with this aspect, a compound of the invention has the general formula (XI)
Figure imgf000052_0001
wherein R1 and L2’ is as defined above and wherein R is one or more substituents as defined above, being one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other and t is as defined above.
In some embodiments, a compound of the invention has the general formula (XI), L2’ is (CH2)n-, or C(=O), each optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, or -(CH2)m-halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5.
In some embodiments, a compound of the invention has the general formula (XI), L2’ is (CH2)n-, optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, or -(CH2)m-halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5.
In some embodiments, a compound of the invention has the general formula (XI), L2’ is (CH2)n-, substituted by OH, n is an integer selected from any one of 0, 1, 2, 3, 4, 5. In some embodiments, a compound of the invention has the general formula (XI), L2’ is (CH2)n-, substituted by OH, n is 2.
In some embodiments with this aspect, a compound of the invention has the general formula (XIa) or (Xlb):
Figure imgf000053_0001
In some examples in the compounds of Formula (XI) (XIa), or (Xlb), R1 is L1’ -R3”-L1”- R3” as defined above.
In some examples, in the compounds of the disclosure having formula (X), (XI), (XIa), or (Xlb), R1 is L1’-R3' -L1”-R3”, L1’, L1”and R3” are each absent and R3' is an
Figure imgf000053_0002
In some examples, in the compounds of the disclosure having formula (X), (XI), (XIa), or (Xlb), R1 is L1’-R3’-L1 ’-R3”, L1’, L1”and R3” are each absent and R3' is an optionally substituted:
ring linked to formula (X), (XI), (XIa), or (Xlb).
In some examples, in the compounds of the disclosure having formula (X), (XI), (XIa), or (Xlb), R1 is L1’-R3’-L1 ’-R3”, L1’, LL ’and R3” are each absent and R3' is: alkyl, halogen, CF3, NO2, C(=O), the wavy line indicate bond to formula (X), (XI), (XIa), or (Xlb).
In some examples, in the compounds of the disclosure having formula (X), (XI), (XIa), or (Xlb), R1 is L1’-R3'-L1”-R3”, L1’, LL ’and R3” are each absent and R3' is:
, the wavy line indicate bond to formula (X), (XI), (XIa), or (Xlb).
In some examples, in the compounds of the disclosure having formula (X), (XI), (XIa), or (Xlb), R1 is L1’-R3'-L1”-R3”, L1’is absent R3' and R3 ” is each independently from the other optionally substituted aryl or an optionally substituted heteroaryl, and L1” is -(CH2)n-,C(= O), each optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, or -(CH2)m-halogen, n is an integer selected from any one of 0, 1, 2, 3, 4, 5, at times 1, 2, 3, 4, 5 and m is an integer selected from 0, 1, 2, 3, 4, 5. In some examples, in the compounds of the disclosure having formula (X), (XI), (XIa), or (Xlb), R1 is L1’-R3’-L1 ’-R3”, R3' is an optionally substituted phenyl and R3” is an optionally substituted:
In some examples, in the compounds of the disclosure having formula (X), (XI), (XIa), or (Xlb), R1 is L1’-R3'-L1”-R3”, L1’is absent R3' is an optionally substituted phenyl, R3” is an optionally substituted: and L1” is C(=O).
In some examples, the compound of the present disclosure having general formula (Xlla) or (Xllb):
OH (Xlla) or
Figure imgf000056_0001
wherein R is one or more substituents as defined above, being one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other;t represents the number of substituents and may vary within the same compound being one or more identical substituents (for example one or more H) or different substituents.
In some examples, the compound of the present disclosure having the general formula (XIIc):
Figure imgf000056_0002
wherein L1” and R3 ’ are each as defined above, R is one or more substituents as defined above, being one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other; t represents the number of substituents and may vary within the same compound being one or more identical substituents (for example one or more H) or different substituents.
In some examples, the compound of the present disclosure having the general formula
(Xlld):
Figure imgf000057_0001
(Xlld) wherein L1” and R3 ” are each as defined above, R is one or more substituents as defined above, being one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other; t represents the number of substituents and may vary within the same compound being one or more identical substituents (for example one or more H) or different substituents.
In some examples, the compound of the present disclosure having the general formula
(Xlle)
(Xlle);
Figure imgf000057_0002
wherein L1” and R3 ” are each as defined above, wherein R is one or more substituents as defined above, being one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other; t represents the number of substituents and may vary within the same compound being one or more identical substituents (for example one or more H) or different substituents.
In some embodiments, specific examples of compounds or pharmaceutically acceptable salts or hydrates of the compounds of Formula X, XI, include, without limitation:
Figure imgf000058_0001
N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(1-methyl-2-(trifluoromethyl)-1H- benzo [d] imidazol-5 -yl)oxalamide
Figure imgf000058_0002
(R)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(1-methyl-2-(trifluoromethyl)-1H- benzo [d] imidazol-5 -yl)oxalamide
Figure imgf000059_0001
(S)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(1-methyl-2-(trifluoromethyl)-1H- benzo [d] imidazol-5 -yl)oxalamide
Figure imgf000059_0002
N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(3-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide
Figure imgf000059_0003
(S)-N 1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(3-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide
Figure imgf000060_0001
(R)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(3-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide
Figure imgf000060_0002
N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide.
Figure imgf000061_0001
(S)-N 1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide (denoted herein as “B3”).
Figure imgf000061_0002
(R)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide
Figure imgf000061_0003
N 1 ,N2-bis(2-hydroxyphenyl)oxal amide In accordance with the second aspect, the present disclosure provides 1,5-di-carbonyl compounds. In accordance with this aspect, the compounds contain one or more nitrogen atoms. In some other embodiments, the compounds contain at least one ring structure. In some other embodiments, the ring structure containing at least one nitrogen atom. In some further embodiments, the compounds contain a heterocyclic ring structure containing five to twelve atoms, the ring structure containing at least two carbon atoms and at least one heteroatom being N, O or S.
The present disclosure provides in accordance with the second aspect, a compound having the general formula (XIII):
Figure imgf000062_0001
or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof ; wherein R9 and R10 may be the same or may be different and may be independently selected from each other from a ring system containing five to twelve atoms, each optionally substituted;
R11 may be independently selected from H, straight or branched C1-C12 alkyl, straight or branched C2-C12 alkenyl, straight or branched C2-C12 alkynyl;
L5 and L6, are each independently of each other, are absent or may be selected independently from -(CH2)p-; -NH-C(=O)-(CH2)p-, -C(=O)-NH-(CH2)p-; -S-S-(CH2)p-; - O-(CH2)p-; -NH-(CH2)p-; C(=O)-(CH2)p-; -S-(CH2)p-; -NH-S(=O)P-(CH2)p-; each p, being independently, may be 0 to 5; In some embodiments, each one of R9 and R10 being a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents, In some further embodiments, the substituents may be selected from OH, CF3, halogen, C(=O), -COOH, -NH2, CN, C(=O)- alkyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkynylene, C1-C12 alkoxy, C1-C5 carboxyl, halogen, aromatic or heteroaromatic ring.
In some embodiments, R9 and R10 may be a ring system containing five to twelve atoms. In some further embodiments, the ring system of R9 and R10 may contain at least two carbon atoms. In some other embodiments, the ring system of R9 and R10 may be an aromatic ring, non-aromatic ring, fused ring or the like. In some further embodiments, R9 and R10 may be C5-C12 saturated cycloalkyl, C5-C12 saturated cycloalkylene, C5-C12 aryl or C5-C12 arylene. In some other embodiments, the ring system of R9 and R10 may include at least one heteroatom ring. In some further embodiments, R9 and R10 may be heteroaryl or heterocycloalkyl. In some further embodiments, R9 and R10 may be C2-C12 hetero cycloalkyl or C2-C12 hetero aromatic ring (aryl or arylene). It should be noted that according with some embodiments, R9 and R10 may be independently from each other contain different carbon atoms. In some embodiments, the heteroatom may be N, O, S. In some embodiments, R9 and R10 may be independently from each other selected from C5-C12 aryl. In some embodiments, R9 and R10 may be C6 aryl, optionally substituted.
In some embodiments, L5 and L6, may be -(CH2)p-. According to these embodiments, the group -(CH2)p- wherein p=l to 5, may encompasses an alkyl, alkylene, alkenyl, alkenylene and alkynyl as defined herein having at most five carbon atoms. In some embodiments, each of p may be 0. At times, when p=0, L5 and L6 are each a bond.
In some embodiments, specific examples of compounds or pharmaceutically acceptable salts or hydrates of the compounds of Formula XIII include, without limitation:
Figure imgf000064_0001
N3 ,N5 -bis(3 -acetylphenyl) - 1 -methyl- 1 H-pyrazole-3 ,5 -dicarboxamide (5.1)
Figure imgf000064_0002
N4,N5-bis(3-acetylphenyl)-l-methyl-1H-pyrazole-4,5-dicarboxamide
In accordance with the third aspect, the present disclosure provides compounds containing one or more nitrogen atoms. In some other embodiments, the compounds contain at least one ring structure. In some other embodiments, the ring structure containing at least one nitrogen atom. In some further embodiments, the compounds contain a heterocyclic ring structure containing five to twelve atoms, the ring structure containing at least two carbon atoms and at least one heteroatom being N, O or S. in some other embodiments, the compounds contain an amide group. In accordance with the third aspect, the present disclosure provides a compound having a general formula:
Figure imgf000065_0001
or a pharmaceutically acceptable salt or hydrate thereof; wherein:
X, Y independently may be each independently selected from each other from NH, CH; R14 and R15 may be the same or may be different and may be independently from each other selected from a ring system containing five to twelve atoms, each optionally substituted;
L8 may be selected independently from -(CH2)q-; -NH-C(=O)-(CH2)q-, -C(=O)-NH- (CH2)q-; -S-S-(CH2)q-; -O-(CH2)q-; -NH-(CH2)q-; C(=O)-(CH2)q-; -S-(CH2)q-; -NH- S(=O)q-(CH2)q-; each q, being independently, may be 0 to 5;
In some embodiments, R14 and R15 may be a ring system containing five to twelve atoms. In some further embodiments, the ring system of R14 and R15 may contain at least two carbon atoms. In some other embodiments, the ring system of R14 and R15 may be an aromatic ring or a non-aromatic ring. In some further embodiments, R14 and R15 may be C5-C12 saturated cycloalkyl, C5-C12 saturated cycloalkylene, C5-C12 aryl or C5-C12 arylene. In some other embodiments, the ring system of R14 and R15 may include at least one heteroatom ring. In some further embodiments, R14 and R15 may be C2-C12 hetero cycloalkyl or C2-C12 hetero aromatic ring. It should be noted that according with some embodiments, R14 and R15 may be independently from each other contain different carbon atoms. In some embodiments, the heteroatom may be N, O, S. In some embodiments, R14 and R15 may be independently from each other selected from C5-C12 aryl optionally substituted. In some embodiments, R14 and R15 may be independently from each other selected from isoquinoline or phyel each independently from the other optionally substituted.
In some embodiments, L8, may be -(CH2)q-. According to these embodiments, the group -(CH2)q- wherein q=0 to 5, may encompasses an alkyl, alkenyl and alkynyl as defined herein having at most five carbon atoms. In some embodiments, each of q may be 0. At times, when q=0, L8 may be a bond.
In some embodiments, a ring system containing five to twelve atoms may be substituted with one or more substituents, in certain embodiments one, two, three or four substituents. In some further embodiments, the substituents may be selected from OH, CF3, halogen, NO2, C(=O), -COOH, -NH2, CN, C(=O)-C1-Ci2 alkyl, straight or branched C1- C12 alkyl, straight or branched C2-C12 alkenyl, straight or branched C2-C12 alkynyl, straight or branched C1-C12 alkylene, straight or branched C2-C12 alkenylene, straight or branched C2-CI2 alkynylene, C1-C12 alkoxy, C1-C5 carboxyl, aromatic or heteroaromatic ring.
In some embodiments, specific examples of compounds or pharmaceutically acceptable salts or hydrates of the compounds of Formula XIV include, without limitation:
Figure imgf000066_0001
1 - [(3 ,5 -Dichloro-phenylcarbamoyl)-methyl] -piperidine-4-carboxylic acid (2-hydroxy- phenyl)-amide
Figure imgf000067_0001
2-[4-(8-Nitro-isoquinolin-5-ylamino)-piperazin-l-yl]-A-phenyl-acetamide
For compounds of the disclosure in which a variable appears more than once, each variable can be a different moiety selected from the Markush group defining the variable. For example, where a structure is described having two R groups that are simultaneously present on the same compound; the two R groups can represent different moieties selected from the Markush group defined for R.
It is further appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment/example. Conversely, various features of the disclosure which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.
In the following text, when referring to a compound (such as ARTS mimetic compound) it is to be understood as also referring to the composition, uses (compound for use, composition for use), methods, combined therapies and kits disclosed herein. Thus, whenever providing a feature with reference to the compound, it is to be understood as defining the same feature with respect to the composition, uses (compound for use, composition for use), methods, combined therapies and kits, mutatis mutandis.
The term “alkyl” by itself or as part of another substituent, means, unless otherwise stated, as used herein refers to a linear (straight), branched saturated hydrocarbon and can have a number of carbon atoms optionally designated (i.e., C1-Ce means one to six carbons). The term “C1-C12 alkyl” or "C1-C12 alkylene" refers to a linear (straight), branched saturated hydrocarbon having from 1 to 12 carbon atoms, in some embodiments, contain from 2 to 8 carbons, in yet some embodiments from 2 to 5 carbons, in yet some further embodiments, from 1 to 3 carbon atoms. It should be noted that alkyl refers to an alkyl end chain and alkylene refers to a middle chain alkyl. Representative C1-C12 alkyl groups include, but are not limited to, methyl, ethyl, propyl, n-propyl, isopropyl, cyclopropyl, n- butyl, butyl, sec-butyl, iso-butyl, tert-butyl, cyclobutyl, n-pentyl, pentyl, iso-pentyl, neopentyl, tert-pentyl, cyclopentyl, hexyl, cyclohexyl, heptyl, cycloheptyl, octyl, sec-octyl (1 -methylheptyl), and cyclooctyl as well as homologs and isomers of, for example, n- pentyl, n-hexyl, and the like.
The term “haloalkyl” as used herein can include alkyl structures that are substituted with one or more halo groups or with combinations thereof, for example, “C1-C12 haloalkyl” refers to a C1-C12 alkyl as defined above, with one or more hydrogens substituted by halogen atoms.
The term “alkenyl” as used herein refers to a linear (straight), branched unsaturated hydrocarbon having from 2 to 20 carbon atoms and at least one carbon-carbon double bond. The term “C2-C12 alkenyl” or "C2-C12 alkenylene'' as used herein refers to a linear, branched unsaturated hydrocarbon having from 2 to 12 carbon atoms and at least one carbon-carbon double bond, in some embodiments from 3 to 8 carbons, in yet some further embodiments, from 3 to 5 carbon atoms and at least one double bond. It should be noted that alkenyl refers to an alkyl end chain and alkenylene refers to a middle chain alkyl. Examples of alkenyl groups, include, but are not limited to, groups such as ethenyl (i.e., vinyl), prop-l-enyl (i.e., allyl), but-l-enyl, pent-l-enyl, penta- 1,4-dienyl, and the like.
The term “C2-C12 haloalkenyl” as used herein refers to a C2-Ci2alkenyl as defined above, with one or more hydrogens substituted by halogen atoms.
The term “alkynyl” as used herein refers to a linear (straight), branched unsaturated hydrocarbon having from 2 to 20 carbon atoms and at least one carbon-carbon triple bond. The term “C2-C12 alkynyl” or "C2-C12 alkynylene'' as used herein refers to a linear, branched unsaturated hydrocarbon having from 2 to 12 carbon atoms in certain embodiments, from 3 to 8 carbons, and at least one triple bond (at least one carbon-carbon triple bond). It should be noted that alkynyl refers to an alkyl end chain and alkynylene refers to a middle chain alkyl. Examples of alkynyl groups, include, but are not limited to, groups such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
The term “C2-C12 haloalkynyl” as used herein refers to a C2-C12 alkynyl as defined above, with one or more hydrogens substituted by halogen atoms.
As used herein “alkoxy” refers to an alkyl group bonded to an oxygen atom. Similarly, the term “C1-C12 alkoxyl” as used herein refers to a C1-C12 alkyl group linked to an oxygen. At times, the alkyl group may include one to twelve carbon atoms, at times between one to eight carbon atoms, at times one to five carbon atoms and at times one to three carbon atoms. Examples of alkoxy groups, include, but are not limited to, groups such as methoxy, ethoxy, propyloxy, isopropyloxy, butyloxy, isobutyloxy, tert-butyloxy, pentyloxy, or hexyloxy, and the like.
The term “halo” or “halogen” (halide) independently or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. The term “halide” by itself or as part of another substituent, refers to a fluoride, chloride, bromide, or iodide atom.
As used herein, a ring system containing five to twelve atoms refers to a mono- or multi- cyclic ring system having 5 to 12 atoms. The ring system containing five to twelve atoms may be saturated, unsaturated or aromatic rings and the like including for example cycloalkyl, heterocycloalkyl, aryl, arylene, aromatic, heteroaromatic rings. A ring system containing five to twelve atoms may contain two rings (bicyclic, etc.), for example aromatic rings and in such case the aromatic rings of the aryl group may be joined at a single point (e.g., biphenyl), or fused (e.g., naphthyl). In some embodiments, a ring system containing five to twelve atoms is a carbocyclic ring or heterocyclic ring. The term “carbocyclic ring” refers to cyclic compounds containing only carbon atoms. The carbocyclic ring may be optionally substituted by one or more substituents, and may be saturated, unsaturated or aromatic. The term “heterocyclic ring” refers to cyclic compounds where one or more carbons are substituted by heteroatoms. Exemplary heteroatoms include, but not limited to, nitrogen, sulfur, and oxygen. The heterocyclic ring may be optionally substituted, and may be saturated, unsaturated or aromatic. The term “saturated' as used herein means that the compound does not contain double or triple bonds. The term “unsaturated' as used herein means that the compound contains at least one double or triple bond. The term “aromatic” as used herein means that the compound contains alternating double and single bonds.
As used herein, "aryl" refers to polyunsaturated, aromatic ring systems having between 5 to 12 atoms which can be a single ring or multiple rings (e.g., 1 to 2 rings) which are fused together or linked covalently. Unless otherwise specifically defined, the term “aryl” refers to cyclic, aromatic hydrocarbon groups that have 1 to 2 aromatic rings, including monocyclic or bicyclic groups having between 5 to 12 atoms. Non-limiting examples include phenyl, biphenyl or naphthyl. The aryl group may be optionally substituted by one or more substituents, e.g., 1 to 5 substituents, at any point of attachment. The substituents can themselves be optionally substituted. As used herein, "C5-C12 aromatic" refers to aromatic ring systems having 5 to 12 carbon atoms, such as phenyl, naphthalene and the like.
As used herein, the term “heteroaryl" refers to aryls as defined above where one or more carbons are substituted by heteroatoms. Exemplary heteroatoms include, but not limited to, nitrogen, sulfur, and oxygen. As used herein, "heteroaromatic" refers to refers to a monocyclic or multi-cyclic (fused) aromatic ring system, where one or more of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur. The term "heteroaromatic” used interchangeably with the term "heteroaryl" denotes a heterocyclic aromatic ring systems containing 5 to 12 atoms, with at least one, preferably two carbon atoms and one or more heteroatoms selected from nitrogen, oxygen and sulfur. Non-limiting examples include furan, thipohene, pyrrole, oxazole, thiazole, imidazole, pyrazole, isoxazole, thiazolem benzofurna, indole, benzothiophene, benzoimidazole, indazole, benzoxazole, benzoisoxazole, benzothiazole, isobenzfuran, isoidole, purine, pyridine, pyrazine, pyrimidine, pyrisazine, quinoline, quinozaline, quinazoline, isoquinoline, furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, isoxazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4- triazolyl, pyranyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,2,3-triazinyl, 1,2,4- triazinyl, 1,3,5-triazinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4- oxadiazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4- thiadiazolyl, tetrazolyl, thiadiazinyl, indolyl, isoindolyl, benzofuryl, benzothienyl, indazolyl, benzimidazolyl, benzothiazolyl, benzoisothiazolyl, benzoxazolyl, benzisoxazolyl, purinyl, quinazolinyl, quinolizinyl, quinolinyl, isoquinolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, azepinyl, diazepinyl, acridinyl, 1- methyl-1H-benzo[d]imidazole, 1H-benzo[d] imidazole and the like.
As used herein, "C5-C12 saturated cycloalkyl" refers to a saturated mono- or multi- cyclic ring system having 5 to 12 carbon atoms, preferably having 5 to 7 carbon atoms. Example of "C5-C12 cycloalkyl" groups include, but are not limited to cyclopentyl, cyclohexyl and cycloheptyl.
As used herein, "heterocycloalkyl" or "heterocyclyl" or the term "heterocyclic" refers to a monocyclic or multi-cyclic non-aromatic ring system having 5 to 12 members, preferably having 5 to 7 carbon atoms, where one or more, in certain embodiments, 1 to 3, of the atoms in the ring system is a heteroatom, that is, an element other than carbon, including but not limited to, nitrogen, oxygen or sulfur. Examples of "heteroalkyl" include, but are not limited to, pyran, 1,4-dioxane, 1,3-dioxane, piperidine, pyrrolidine, morpholine, tetrahydrothiopyran, tetrahydrothiophene, and the like. The term heterocycloalkyl" also encompasses non-aromatic ring being unsaturated or having one or more degrees of unsaturation containing one or more heteroatomic substitutions selected from S, SO, SO2, O, or N. "heterocyclic” ring(s) or cycloalkyl ring(s). Examples of "heterocyclic" include, but are not limited to, tetrahydrofuran, pyran, 1,4-dioxane, 1,3- dioxane, piperidine, pyrrolidine, morpholine, tetrahydrothiopyran, tetrahydrothiophene, and the like.
The term "N-containing group" is used herein a chemical group containing a nitrogen atom for example as amino group. The term "amino" as used herein encompass primary, secondary, tertiary or quaternary amines where the point of attachment is through the nitrogen atom which is substituted. For example, the "N-containing group” include N, NH, NH2, tertiary amine (tertiary alkyl amine), quaternary ammonium (quaternary alkyl ammonium). The nitrogen atom may be substituted with alkyl. In case of a tertiary amine or quaternary amines, the substituent may be the same or may be different.
The term "bond" as used herein denotes a covalent bond. The bond may be between two similar atoms or between different atoms. Non-limiting examples include C-C, C-S, C- O, C-N. S-O, S-N, N-0 and the like. It should be noted that a bond as defined above, for example, C-S encompasses both C-S and S-C and this holds for the bonds as defined herein.
The term “optionally substituted” refers to substitution with the named substituent or substituents, multiple degrees of substitution being allowed unless otherwise stated. The term substituted as used herein means that the compounds may contain one or more substituents, including, but not limited to, optionally substituted OH, CF3, halogen, C(=O), -COOH, -NH2, CN, alkyl, alkenyl, alkynyl, alkylene, straight alkenylene, alkynylene, haloalkyl, haloalkenyl, haloalkynyl, alkoxy, carboxyl, halogen, ring system including five to twelve atoms, aromatic or heteroaromatic ring, C(=O)- alkyl, alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -ORa, -
SRa, -OC(O)-Ra, -N(Ra)2, -C(O)Ra, -C(O)ORa, -OC(O)N(Ra)2, -C(O)N(Ra)2, -N(Ra)C(O)ORa, -N(Ra)C(O)Ra, - N(Ra)C(O)N(Ra)2,
N(Ra)C(NRa)N(Ra)2, -N(Ra)S(O)wRa (where w is 1 or 2), -S(O)„ORa (where w is 1 or 2), -S(O)wN(Ra)2 (where w is 1 or 2), or PO3(Ra)2, wherein each Ra is independently hydrogen or alkyl.
It should be noted that the carbon number, as used herein, refers to the carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like.
The invention also embraces solvates, pharmaceutically acceptable prodrugs, pharmaceutically active metabolites, and pharmaceutically acceptable salts of compounds of the formula (I) or formula (X) or any variations detailed herein. The present disclosure also includes any or all of the stereochemical forms, including any enantiomeric or diastereomeric forms, and any tautomers or other forms of the compounds described.
The term “solvate” refers to an aggregate of a molecule with one or more solvent molecules, such as hydrate, alcoholate (aggregate or adduct with alcohol), and the like.
As used herein the term "pharmaceutically acceptable salt" refers to salts derived from organic and inorganic acids of a compound described herein. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, hydrochloride, bromide, hydrobromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, camphorsulfonate, napthalenesulfonate, propionate, succinate, fumarate, maleate, malonate, mandelate, malate, phthalate, and pamoate. The term “pharmaceutically acceptable salt” as used herein also refers to a salt of a compound described herein having an acidic functional group, such as a carboxylic acid functional group, and a base. Exemplary bases include, but are not limited to, hydroxide of alkali metals including sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, organic amines such as unsubstituted or hydroxyl-substituted mono-, di- or trialkylamines, dicyclohexylamine; tributyl amine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis-, or tris-(2-OH-(C1-C6)-alkylamine), such as N,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D- glucamine; morpholine; thiomorpholine; piperidine; pyrrolidine; and amino acids such as arginine, lysine, and the like. The term “pharmaceutically acceptable salt” also includes hydrates of a salt of a compound described herein. The term “hydrate” refers to a compound formed by the addition of water. The hydrates may be obtained by any known method in the art by dissolving the compounds in water and recrystallizing them to incorporate water into the crystalline structure.
The compounds of the present invention, as defined above, may have the ability to crystallize in more than one form, a characteristic, which is known as polymorphism, and it is understood that such polymorphic forms ("polymorphs") are within the scope of formulae (I). Polymorphism generally can occur as a response to changes in temperature or pressure or both and can also result from variations in the crystallization process. Polymorphs can be distinguished by various physical characteristics known in the art such as x-ray diffraction patterns, solubility, and melting point.
Certain of the compounds described herein may contain one or more chiral atoms, or may otherwise be capable of existing as two enantiomers or as two or more diastereomers. Accordingly, the compounds of this invention include mixtures of enantiomers as well as purified enantiomers or enantiomerically enriched mixtures. Furthermore, the compounds of this invention include mixtures of diastereomers, as well as purified stereoisomers or diastereomerically enriched mixtures. Also included within the scope of the invention are the individual isomers of the compounds of the invention, as defined above, as well as any wholly or partially mixtures thereof. The present invention also covers the individual isomers of the compounds represented by the formulas above as mixtures with isomers thereof in which one or more chiral centers are inverted.
The term "stereoisomer" as used herein is meant to encompass an isomer that possess identical constitution as a corresponding stereoisomer, but which differs in the arrangement of its atoms in space from the corresponding stereoisomer. For example, stereoisomers may be enantiomers, diastereomers and/or cis-trans (E/Z) isomers. It should be understood that a composition comprising a fatty acid amide of the invention may comprise single enantiomers, single diastereomers as well as mixtures thereof at any ratio (for example racemic mixtures, non racemic mixtures, mixtures of at least two diastereomers and so forth). Furthermore, the invention encompasses any stereoisomer of a fatty acid amide of the invention achieved through in vivo or in vitro metabolism, or by any type of synthetic rout. Certain of the compounds described herein may contain one or more chiral atoms, or may otherwise be capable of existing as two enantiomers or as two or more diastereomers. Accordingly, the compounds of this invention include mixtures of enantiomers as well as purified enantiomers or enantiomerically enriched mixtures. Furthermore, the compounds of this invention include mixtures of diastereomers, as well as purified stereoisomers or diastereomerically enriched mixtures. Also included within the scope of the invention are the individual isomers of the compounds of the invention, as defined above, as well as any wholly or partially mixtures thereof. The present invention also covers the individual isomers of the compounds represented by the formulas above as mixtures with isomers thereof in which one or more chiral centers are inverted.
It is also noted that the compounds of the present invention may form tautomers. It is understood that all tautomers and mixtures of tautomers of the compounds of the present invention, are included within the scope of the compounds of the present invention.
The term “solvate” refers to an aggregate of a molecule with one or more solvent molecules, such as hydrate, alcoholate (aggregate or adduct with alcohol), and the like.
The term "physiologically functional derivative” used herein relates to any physiologically acceptable derivative of a compound as described herein.
The physiologically functional derivatives also include prodrugs of the compounds of the invention. Such prodrugs may be metabolized in vivo to a compound of the invention. These prodrugs may or may not be active themselves and are also an object of the present invention.
A “pharmaceutically acceptable prodrug” is a compound that may be converted under physiological conditions to the specified compound or to a pharmaceutically acceptable salt of such compound.
A “pharmaceutically active metabolite” is a pharmacologically active product produced through metabolism in the body of a specified compound or salt thereof. Metabolites of a compound may be identified using routine techniques known in the art and their activities determined using tests such as those described herein. Prodrugs and active metabolites of a compound may be identified using routine techniques known in the art. It should be appreciated that the present disclosure provides in some aspects thereof, any of the ARTS mimetic compound as disclosed herein above, specifically the compound having the general formula (I) or formula (X), or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, or any composition thereof or any vehicle, matrix, nano- or micro-particle comprising the same, for use in a method for inducing apoptosis in a cell.
Still further aspects of the present disclosure relate to any of the ARTS mimetic compound as disclosed herein above, specifically the compound having the general formula (I) or formula (X) or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, or any composition thereof or any vehicle, matrix, nano- or micro-particle comprising the same, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of a pathological disorder in a subject in need thereof.
The present disclosure provides different ARTS mimetic compounds that specifically mimic the C domain of ARTS, specifically in binding thereof to its binding site within the BIR3 domain of XIAP.
As used herein "ARTS'' (apoptosis-related protein in the TGF-β signaling pathway) is a septin-like mitochondrial protein derived from alternative splicing of the H5/PNUTL2/hCDCrel2a/2b gene. ARTS acts as a tumor suppressor protein that functions as an antagonist of XIAP and thereby promotes apoptosis.
It should be appreciated that in certain embodiments, as used herein in the specification and in the claim section below, ARTS protein refers to the human ARTS, as denoted by SEQ ID NO. 6, and any homologs or variants thereof. More specifically, the human ARTS protein comprises an amino acid sequence of 274 amino acid residues as denoted by GenBank Accession No. AF176379, encoded by a nucleic acid sequence of SEQ ID NO. 7, and any homologs or variants thereof.
In further embodiments, the ARTS mimetic compound/s of the invention and specifically the " A4" and "B3 " compounds as defined herein above, leads to inhibition of XIAP and/or ubiquitin proteasome system (UPS) mediated degradation of X-linked-Inhibitor of Apoptosis (XIAP) in a cell. More specifically, as shown by the Examples, the ARTS mimetic compounds of the invention act as XIAP antagonists, leading to UPS mediated degradation of XIAP.
As recently shown by the inventors, ARTS binds to XIAP through a domain comprising 27 residues covering the C-terminus of ARTS. This interaction induces auto degradation of XIAP. The ARTS mimetic compound/s of the invention target BRI3 domain of XIAP mimicking the ability of ARTS to enhance XIAP degradation.
As used herein the term “IAPs” denotes a family of proteins that harbor between one to three copies of a baculovirus IAP repeat (BIR) domain that enable interaction with activated caspases. It was previously suggested that the BIR domains of certain IAPs, in particular XIAP, have the ability to directly inhibit caspase activity in vitro.
X-linked inhibitor of apoptosis protein (XIAP), also known as inhibitor of apoptosis protein 3 (IAP3) and baculoviral IAP repeat-containing protein 4 (BIRC) denotes a protein known to stop an apoptotic process and thus inhibit cell death. In human, XIAP is produced by a gene named XIAP gene located on the X chromosome. XIAP is also called human lAP-like Protein (hILP), because it is not as well conserved as the human IAPS: hIAP-1 and hIAP-2 -XIAP is the most potent human IAP protein currently identified.
XIAP belongs to a family of apoptotic suppressor proteins. Members of this family share a conserved motif termed, baculovirus IAP repeat (BIR domain), which is necessary for their anti-apoptotic function. XIAP acts as a direct caspase inhibitor by directly binding to the active site pocket of CASP3 and CASP7 and obstructs substrate entry. It further inactivates CASP9 by keeping it in a monomeric, inactive state.
It should be noted that in certain embodiments, the invention relates to the human XIAP protein (GenBank Accession Nos. NP_001158, NP_001191330, as denoted by SEQ ID NO: 8, and any homologs and variants thereof) encoded by the XIAP gene (GenBank Accession Nos. NM_001167, NM_001204401, as denoted by SEQ ID NO: 9), and any homologs and variants thereof.
As indicated above, the present disclosure relates to the ARTS mimetic compounds of the invention that act as antagonist/s of XIAP. An antagonist is a compound that competes with a specific protein, a ligand for example, on binding to another protein, a receptor for example. Such binding usually, induces a specific biological response or action that is blocked by the competing antagonist. Antagonists have affinity but no efficacy for their cognate binding protein and binding will disrupt the interaction and inhibit the function of such cognate protein. Antagonists mediate their effects by binding to the active (orthosteric = right place) site or to allosteric (= other place) sites on any cognate protein, in this case, XIAP (or receptor, in case applicable), or they may interact at unique binding sites not normally involved in the biological regulation of the cognate protein.
As shown in the Examples, down regulation of XIAP is mediated by the ubiquitin - proteasome machinery (UPS).
Thus, in certain embodiments, the ARTS mimetic compounds of the present disclosure, bind XIAP and lead to inhibition of XIAP and/or mediate ubiquitin proteasome system (UPS) degradation of XIAP anti-apoptotic protein, thereby reducing survival of the cells. As used herein the term “ubiquitin proteasome system” denotes a multi component system that identifies and degrades unneeded, damaged or misfolded proteins by breaking peptide bonds (proteolysis) of the protein in the cytoplasm of cells. As appreciated in the art, degradation of a protein via the UPS involves two discrete and successive steps. In the first step, proteins are tagged for degradation with a small protein called ubiquitin. The tagging reaction is catalyzed by enzymes called ubiquitin ligases. Once a protein is tagged with a single ubiquitin molecule, this is a signal to other ligases to attach additional ubiquitin molecules.
More specifically, conjugation of ubiquitin, a highly evolutionarily conserved 76 amino acid residue polypeptide, to the protein substrate proceeds via a three-step cascade mechanism involving El , E2 and E3 enzymes. By successively adding activated ubiquitin moieties to internal lysine residues on the previously conjugated ubiquitin molecule, a polyubiquitin chain is synthesized that is subsequently recognized by the downstream 26S proteasome complex.
In the second step, degradation of polyubiquitinated substrates is carried out by a large, protease complex, referred to as the 26S proteasome that does not recognize nonmodified substrates. The proteasomes are multicatalytic protease protein complexes found in all cells that degrades polyubiquitinated proteins to short peptides by breaking peptide bonds (proteolysis). Following degradation of the substrate, short peptides derived from the substrate are released, along with reusable ubiquitin.
It should be noted that the ubiquitin-proteasome system (UPS) plays a central and complex role in regulating apoptosis by directly targeting key cell death proteins, including caspases. The term "apoptosis" refers to a regulated network of biochemical events which lead to a selective form of cell suicide and is characterized by readily observable morphological and biochemical phenomena. Cells undergoing apoptosis show characteristic morphological and biochemical features. These features include chromatin aggregation or condensation, DNA fragmentation, nuclear and cytoplasmic condensation, partition of cytoplasm and nucleus into membrane bound vesicles (apoptotic bodies) which contain ribosomes, morphologically intact mitochondria and nuclear material. Cytochrome C release from mitochondria is seen as an indication of mitochondrial dysfunction accompanying apoptosis.
It should be understood that in some embodiments, the ARTS mimetic compounds used by the present disclosure, specifically, the "A4" compound and/or the "B3" compound bind a unique domain of XIAP and lead to inhibition of XIAP and/or degradation of XIAP. It should be further understood that the ARTS mimetic compounds of the present disclosure, specifically, any one of the A4 and/or the B3 compounds lead to apoptosis of neuronal cells as shown by the present disclosure. In some specific embodiments, the A4 compound leads to apoptosis of neuroblastoma cells that exhibit an advanced stage of tumorigenicity. In yet some other alternative or additional embodiments, the "A4" compound may lead to reversion of the cancerous phenotype of neural cells. Still further, in some embodiments, the "A4" compound may revert neuroblastoma cells in a subject to cells having a normal phenotype. In more specific embodiments, the A4 compound may revert cells of an early-stage cells to display a normal phenotype in a subject. In some embodiments, the reversion of the cells is due to induction of differentiation in the early- stage cells by the A4 compound. As indicated above, apoptosis is a tightly controlled form of active cell death that is necessary for development and organismal homeostasis. Death by the apoptotic pathway is achieved among others, by the activation of a family of highly potent and specific proteases, termed caspases (for cysteine-aspartate protease). The activity of caspases is tightly regulated and the cell maintains several “checkpoints” to control their activity. The first level of regulation is intrinsic to caspases themselves. Caspases are initially transcribed as weakly active zymogens, which only upon proper stimulation are cleaved to form the active enzyme.
The second level of caspase regulation is achieved by inhibitors, namely the family of proteins called lAPs (Inhibitor of Apoptosis Protein) as described above. In some embodiments, the at least one ARTS mimetic compound for use according to the present disclosure, is applicable for use in methods of treating neoplastic disorder affecting the neural system and/or neural cell. In more specific embodiments, such disorder is neuroblastoma. In yet some further embodiments, the neuroblastoma is high-risk neuroblastoma. In yet some further embodiments, the disclosed uses are applicable for neuroblastoma patients displaying overexpression of MYCN, or having amplified MYCN. More specifically, as used herein, MYCN (v-Myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog) is a gene that codes for a transcription factor protein that plays a critical role in the development of the nervous system. MYCN is located on chromosome 2 and is a member of the MYC family of protooncogenes. MYCN is known to be amplified in a subset of aggressive human cancers, including neuroblastoma, medulloblastoma, and small cell lung cancer. Amplification of MYCN is associated with poor prognosis and treatment resistance in these cancers. Still further, MYCN is also involved in normal development, particularly in the development of the nervous system. It is important for the proliferation and differentiation of neural stem cells and plays a role in the formation of the neural crest, which gives rise to many different types of cells, including neurons, glial cells, and adrenal gland cells.
Still further, in some embodiments, the ARTS mimetic compound for use, according to the present disclosure, prolongs the overall survival (OS) of the treated subjects.
In some embodiments, the invention provides the at least one ARTS mimetic compound for use in a method that further comprises the step of administering to the treated subject an effective amount of at least one anti-cancer agent.
In some embodiments, the anti-cancer agent that may be used with the at least one ARTS mimetic compound may be at least one of at least one topoisomerase inhibitor and at least one alkaloid agent.
In some specific embodiments, the topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
In yet some further embodiments, the alkaloid agent is anti-mitotic and/or antimicrotubule alkaloid agent, optionally, vincristine, or any derivative or formulation thereof.
It should be understood that the invention further encompasses the at least one ARTS mimetic compound for use, wherein the mimetic compound is comprised within at least one composition. In some embodiments, the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s. A further aspect of the present disclosure relates to a combined composition comprising: (a) an effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle comprising the same. It should be noted that the ARTS mimetic compound interacts and binds the BIR3 domain of XIAP, thereby leading to inhibition of XIAP and/or proteasomal degradation of XIAP. The combined composition of the present disclosure further comprises (b), an effective amount of at least one anti-cancer agent. In more specific embodiments, the agent is at least one of a topoisomerase inhibitor and an alkaloid agent.
In some embodiments, the at least one of said ARTS mimetic compound of the disclosed combined composition has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, or any vehicle, matrix, nano- or micro-particle comprising the same:
Figure imgf000081_0001
(Formula I); wherein:
R1 is independently selected from H, C1-C3 alkyl; R2 is independently selected from - C(=O)-X-R3, -S(=O)-X-R3'; X is a heteroatom independently selected from O; R3 and R33 independently of each other may be selected independently from H, C1-C3 alkyl; R5 is -L1-R7-L2-RS; L1 and L2, independently of each other, are selected independently from -(CH2)n ; -NH-C(=O)-(CH2)n-, -C(=O)-NH-(CH2)n-; -S-S-(CH2)n-; -O-(CH2)n-; - NH-(CH2)n-; C(=O)-(CH2)n-; -S-(CH2)n-; -NH-S(=O)n-(CH2)n-; n is independently 1 to 3; R7 is a carbocyclic ring or a heterocyclic ring; R8 is a mono cyclic ring system having 5 to 12, optionally substituted with at least one of OH, CF3, halogen, -COOH, -NH2, CN, Ci-Csalkyl; and R6 is independently selected from H, halogen, CN, NO2, C1-C3 alkoxy, straight or branched C1-C3 alkyl.
In some further embodiments, the ARTS mimetic compound of the disclosed combined composition is a compound of the general formula (I) that is further characterized by at least one of: in some embodiments (a), the R1 is H; in yet some further additional or alternative embodiments (b), the R2 is -C(=O)-X-R3; in some further additional or alternative embodiments (c), the X is O; in some further additional or alternative embodiments (d), the L1 and L2 independently selected independently from each other from -NH-C(=O)-(CH2)n-, -(CH2)n-; and in some further additional or alternative embodiments (e), the R6 is independently selected from the group consisting of H, halogen, CN, NO2.
In some embodiments, the ARTS mimetic compound of the combined composition of the present disclosure, or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, has the general formula of any one of:
In some embodiments, the compound used herein (a), has the structure of formula (II)
Figure imgf000082_0001
(Formula II) wherein R1, R2, R6, R7, R8, L1 and L2 are as defined herein above.
In yet some alternative embodiments, the compound used herein (b), has the structure of
Figure imgf000082_0002
In yet some further alternative embodiments, the compound used herein (c), has the
Figure imgf000083_0001
Still further, in some alternative embodiments, the compound used herein (d), has the
Figure imgf000083_0002
In some embodiments, the at least one ARTS mimetic compound of the combined composition of the present disclosure is 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5- chloro-1H-indole-2-carboxylic acid methyl ester, also referred to herein as "A4". It should be noted that in some embodiments, the compound has the structure of Formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000084_0001
In yet some alternative or additional embodiments, the at least one ARTS mimetic compound of the combined composition is a compound having the general formula (X), or a pharmaceutically acceptable salt or hydrate thereof, or any vehicle, matrix, nano- or micro-particle comprising the same,
Figure imgf000084_0002
wherein
R1, R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is L1’-R3’-L1”-R3” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R2 is L2’-R4’-L2”-R4”; or
R1 is L1’-R3’-L1”-R3” and R2 is L2’-R4’-L2”-R4”; wherein R3’, R3”, R4’, R4” is each independently from each other, absent or H, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, heteroaryl, heterocycloalkyl, a ring system containing five to twelve atoms, each optionally substituted; L1’, L1”, L2’ and L2” is each independently from each other, absent or -(CH2)n, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2-,-S-, -S-S-, -S-(CH2)n-; n is an integer selected from any one of 0, 1, 2, 3, 4, 5, each one of L1’, L1”, L2’ and L2” independently from each other, may be optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m- halogen and m is an integer selected from 0, 1, 2, 3, 4, 5. It should be understood that the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
In some further embodiments, the ARTS mimetic compound of the of the combined composition in accordance with the preset disclosure, is a compound having the general formula (XI)
Figure imgf000085_0001
wherein R1 and L2’ is as defined above and wherein R is one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other and t is an integer selected from 1 to 5. In some embodiments, the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
In yet some further embodiment, the ARTS mimetic compound of the combined composition, as disclosed herein, is having the general formula (XIIc), or (Xlle):
Figure imgf000086_0001
wherein R3’ and R3 ” is each independently from the other, an optionally substituted aryl or an optionally substituted heteroaryl, and L1” is C(=O), wherein R is one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other and t is an integer selected from 1 to 5. In some embodiments, the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
In further embodiments of the composition for use as disclosed herein, the ARTS mimetic compound is having the formula (3.1), (3.2), (3.3);
Figure imgf000087_0001
N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide ;
Figure imgf000087_0002
(S)-N 1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide (denoted herein as “B3”);
Figure imgf000087_0003
(R)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide. In some embodiments, the composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
In some embodiments, at least one of the ARTS mimetic compounds of the disclosed combined composition is (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl- 1H-imidazole-2-carbonyl)phenyl)oxalamide. In some embodiments, this compound has the structure the formula (3.2), a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000088_0001
(S)-N 1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide, , also referred to herein as "B3".
In yet some further embodiments, he combined composition of the present disclosure comprises at least one topoisomerase inhibitor. In some embodiments, the topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
In yet some further embodiments, he combined composition of the present disclosure comprises at least one alkaloid agent. In yet some further embodiments, the alkaloid agent is anti-mitotic and/or anti-microtubule alkaloid agent. In some optional embodiments, vincristine, or any derivative or formulation thereof.
Still further, in some embodiments the combined composition of the present disclosure comprises an effective amount of 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro- 1H-indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof. In yet some further embodiments, the combined composition of the present disclosure comprises an effective amount of 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro- 1H-indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
Still further, in some alternative embodiments, the combined composition of the present disclosure comprises an effective amount of (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)- N2-(4-(1-methyl-1H-imidazole-2-carbonyl)phenyl)oxalamide (formula (3.2)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof.
In some embodiments, the combined composition disclosed herein comprises an effective amount of (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide (formula (3.2)), a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
In some embodiments, the compositions of the present disclosure further comprise at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
In some embodiments, the anti-cancer agent used by the present disclosure and in the combined therapeutic compositions, kits and methods, is at least one antimitotic and anti-microtubule alkaloid agent. Antimicrotubule agents are plant-derived antimitotic chemicals that block cell proliferation by acting on the polymerization dynamics of spindles, which are essential for the proper spindle function of microtubules. Microtubules, an important part of the intracellular cytoskeleton structure and have unique polymerization dynamics that are critical for many cellular functions including cell division. Vinca alkaloids and taxanes are two different classes of antimicrotubule agents that cause microtubule dysfunction. Vinca alkaloids such as vincristine and vinblastine bind to tubulin dimers and prevent them from polymerization. On the other hand, taxanes such as paclitaxel and docetaxel have opposite mechanisms of action. These stabilizing agents bind to microtubules and prevent them from depolymerization. The suppression of spindle microtubule dynamics results in cell-cycle arrest through slowing or blocking of mitosis at the metaphase-anaphase transition that leads to the induction of apoptotic cell death. In some embodiments, the anti-cancer agent used by the present disclosure and in the combined therapeutic compositions, kits and methods is at least one Vinca alkaloid. In yet some further specific embodiments, the anti-cancer agent used by the present disclosure and in the combined therapeutic compositions, kits and methods is vincristine or any derivatives thereof.
Vincristine, as used herein, also known as leurocristine and marketed under the brand name Oncovin among others, is a chemotherapeutic agent administered intravenously and used to treat acute lymphocytic leukemia, acute myeloid leukemia, Hodgkin's disease, neuroblastoma, and small cell lung cancer among others. Vincristine has the formula C46H56N4O10,
In some embodiments, the anti-cancer agent used by the present disclosure and in the combined therapeutic compositions, kits and methods, is at least one topoisomerase inhibitor. As used herein, topoisomerase inhibitors are chemical compounds that block the action of topoisomerases, which are broken into two broad subtypes: type I topoisomerases (Topi) and type II topoisomerases (TopIl). Topoisomerase plays important roles in cellular reproduction and DNA organization, as they mediate the cleavage of single and double stranded DNA to relax supercoils, untangle catenanes, and condense chromosomes in eukaryotic cells. Topoisomerase inhibitors influence these essential cellular processes. Some topoisomerase inhibitors prevent topoisomerases from performing DNA strand breaks while others, deemed topoisomerase poisons, associate with topoisomerase-DNA complexes and prevent the re-ligation step of the topoisomerase mechanism. These topoisomerase-DNA-inhibitor complexes are cytotoxic agents, as the un-repaired single- and double stranded DNA breaks they cause can lead to apoptosis and cell death.
In some embodiments, the anti-cancer agent used by the present disclosure and in the combined therapeutic compositions, kits and methods, is Topotecan.
Topotecan, sold under the brand name Hycamtin among others, is a chemotherapeutic agent medication that is a topoisomerase inhibitor. It is a synthetic, water-soluble analog of the natural chemical compound camptothecin having the Formula C23H23N3O5.
As mentioned herein before, the compositions provided by the invention optionally further comprise at least one pharmaceutically acceptable excipient or carrier. As used herein “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. As mentioned above, the compositions provided by the invention comprise an effective amount of any of the ARTS mimetic compounds of the invention, specifically, the A4 compound including any stereoisomer or salt thereof, as well as any vehicle, matrix, nano- or micro-particle comprising the same.
Of particular relevance are formulations of the ARTS mimetic compounds and combinations thereof may be adapted for use as a nano- or micro-particles. Nanoscale drug delivery systems using micellar formulations, liposomes and nanoparticles are emerging technologies for the rational drug delivery, which offers improved pharmacokinetic properties, controlled and sustained release of drugs and, more importantly, lower systemic toxicity. A particularly desired solution allows for externally triggered release of encapsulated compounds. Externally controlled release can be accomplished if drug delivery vehicles, such as micelles, liposomes or polyelectrolyte multilayer capsules, incorporate nanoparticle (NP) actuators. More specifically, Controlled drug delivery systems (DDS) have several advantages compared to the traditional forms of drugs. It should be therefore understood that the present disclosure further encompasses the use of various nanostructures, including micellar formulations, liposomes, polymers, dendrimers, silicon or carbon materials, polymeric nanoparticles and magnetic nanoparticles, as carriers in drug delivery systems. The term "nanostructure" or "nanoparticle" is used herein to denote any microscopic particle smaller than about 100 nm in diameter. In some other embodiments, the carrier is an organized collection of lipids. When referring to the structure forming lipids, specifically, micellar formulations or liposomes, it is to be understood to mean any biocompatible lipid that can assemble into an organized collection of lipids (organized structure). In some embodiments, the lipid may be natural, semi-synthetic or fully synthetic lipid, as well as electrically neutral, negatively or positively charged lipid. In some embodiments, the lipid may be a naturally occurring phospholipid.
It should be appreciated that the ARTS mimetic compounds and combinations of the present disclosure may be associated with any of the nanostructures described above, specifically, any of the micellar formulations, liposomes, polymers, dendrimers, silicon or carbon materials, polymeric nanoparticles and magnetic nanoparticles disclosed herein above. The term “association” may be used interchangeably with the term “entrapped' , “attachment” , “linked', “embedded' , “absorbed' and the like, and contemplates any manner by which the at least one ARTS mimetic compounds and combinations of the disclosure is held.
As mentioned herein before, the compositions provided by the present disclosure optionally further comprise at least one pharmaceutically acceptable excipient or carrier. As used herein “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated.
Still further, a drug is transported to the place of action, hence, its influence on vital tissues and undesirable side effects can be minimized. Accumulation of therapeutic compounds in the target site increases and, consequently, the required doses of drugs are lower. This modern form of therapy is especially important when there is a discrepancy between the dose or the concentration of a drug and its therapeutic results or toxic effects. Cell-specific targeting can be accomplished by attaching drugs to specially designed carriers. Various nanostructures, including liposomes, polymers, dendrimers, silicon or carbon materials, and magnetic nanoparticles, have been tested as carriers in drug delivery systems. Polymeric nanoparticles are one technology being developed to enable clinically feasible oral delivery.
The pharmaceutical composition of the invention can be administered and dosed by the methods of the invention, in accordance with good medical practice. More specifically, the compositions used in the methods and kits of the invention, described herein after, may be adapted for administration by systemic, parenteral, intraperitoneal, transdermal, oral (including buccal or sublingual), rectal, topical (including buccal or sublingual), vaginal, intranasal and any other appropriate routes. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s).
The phrases "systemic administration," "administered systemically," "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound, drug or other material other than directly into the central blood system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes. The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
Regardless of the route of administration selected, the compounds of the present invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
The pharmaceutical forms suitable for injection use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
In the case of sterile powders for the preparation of the sterile injectable solutions, the preferred method of preparation are vacuum-drying and freeze drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Pharmaceutical compositions used to treat subjects in need thereof according to the invention generally comprise a buffering agent, an agent who adjusts the osmolarity thereof, and optionally, one or more pharmaceutically acceptable carriers, excipients and/or additives as known in the art. Supplementary active ingredients can also be incorporated into the compositions. The carrier can be solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
Local administration to the area in need of treatment may be achieved by, for example, local infusion during surgery, topical application, direct injection into the specific organ, etc.
Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, lozenges (including liquid-filled), chews, multi- and nano-particulates, gels, solid solution, liposome, films, ovules, sprays or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
Pharmaceutical compositions used to treat subjects in need thereof according to the invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers. The pharmaceutical compositions of the present invention also include, but are not limited to, emulsions and liposome-containing formulations.
It should be understood that in addition to the ingredients particularly mentioned above, the formulations may also include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.
In particular embodiments, the unit dosage formulations are those containing a daily dose or sub-dose, as herein above recited, or an appropriate fraction thereof, of an active ingredient. A further aspect of the present disclosure relates to a kit comprising: In one component of the disclosed kit (a), an effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof. The ARTS mimetic compound interacts and binds the BIR3 domain of XIAP, thereby leading to inhibition of XIAP and/or proteasomal degradation of XIAP. In some optional embodiments, the ARTS mimetic compound is provided in a first dosage form. The disclosed kit further comprises as a further component (b), an effective amount of at least one anti-cancer agent. In some embodiments, the agent is at least one of a topoisomerase inhibitor and alkaloid agent. Optionally, the at least one ant-cancer agent is provided in the disclosed kit in a second dosage form.
In some embodiments, at least one of the ARTS mimetic compound is a compound that has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or
Figure imgf000095_0001
wherein:
R1 is independently selected from H, C1-C3 alkyl; R2 is independently selected from - C(=O)-X-R3, -S(=O)-X-R3'; X is a heteroatom independently selected from O; R3 and R3', independently of each other may be selected independently from H, C1-C3 alkyl; R5 is -L1-R7-L2-R8; L1 and L2, independently of each other, are selected independently from -(CH2)n ; -NH-C(=O)-(CH2)n-, -C(=O)-NH-(CH2)n-; -S-S-(CH2)n-; -O-(CH2)n-; - NH-(CH2)n-; C(=O)-(CH2)n-; -S-(CH2)n-; -NH-S(=O)n-(CH2)n-; n is independently 1 to 3; R7 is a carbocyclic ring or a heterocyclic ring; R8 is a mono cyclic ring system having 5 to 12, optionally substituted with at least one of OH, CF3, halogen, -COOH, -NH2, CN, C1-C5alkyl; and R6 is independently selected from H, halogen, CN, NO2, C1-C3 alkoxy, straight or branched C1-C3 alkyl.
In some specific embodiments, at least one of the ARTS mimetic compounds comprised in the disclosed kit is 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole- 2-carboxylic acid methyl ester, has the structure of formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000096_0001
In yet some alternative or additional embodiments, at least one of the ARTS mimetic compounds of the disclosed kit has the general formula (X), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000096_0002
wherein
R1, R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is L1’-R3’-L1”-R3” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R2 is L2’-R4’-L2”-R4”; or
R1 is L1’-R3’-L1”-R3” and R2 is L2’-R4’-L2”-R4”; wherein R3', R3”, R4’ , R4” is each independently from each other, absent or H, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, heteroaryl, heterocycloalkyl, a ring system containing five to twelve atoms, each optionally substituted; L1’, L1”, L2’ and L2” is each independently from each other, absent or -(CH2)n, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2-,-S-, -S-S-, -S-(CH2)n-; n is an integer selected from any one of 0, 1, 2, 3, 4, 5, each one of L1’, L1”, L2’ and L2” independently from each other, may be optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m- halogen and m is an integer selected from 0, 1, 2, 3, 4, 5.
In a further embodiment, the ARTS mimetic compound of the disclosed kits in accordance with the preset disclosure, is a compound having the general formula (XI)
Figure imgf000097_0001
wherein R1 and L2’ is as defined above and wherein R is one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other and t is an integer selected from 1 to 5.
In a further embodiment, the ARTS mimetic compound of the disclosed kits, as disclosed herein, is having the general formula (XIIc), or (Xlle):
Figure imgf000098_0001
wherein R3’ and R3 ” is each independently from the other, an optionally substituted aryl or an optionally substituted heteroaryl, and L1” is C(=O), wherein R is one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other and t is an integer selected from 1 to 5.
In further embodiments of the kits as disclosed herein, the ARTS mimetic compound is having the formula (3.1), (3.2), (3.3);
Figure imgf000098_0002
N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide ;
Figure imgf000099_0001
(S)-N 1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide (denoted herein as “B3”);
Figure imgf000099_0002
(R)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide.
In some specific embodiments, the ARTS mimetic compound of the disclosed kit is (S)- N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide.
In yet some further embodiments, the topoisomerase inhibitor of the disclosed kits is Topotecan, or any derivative or formulation thereof. In yet some further embodiments, the alkaloid agent of the disclosed kits is anti-mitotic and/or anti-microtubule alkaloid agent, optionally, vincristine, or any derivative or formulation thereof.
In some specific embodiments, the present disclosure provides a kit comprising an effective amount of 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2- carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof.
In some further specific embodiments, the present disclosure provides a kit an effective amount of 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2- carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
In some specific embodiments, the present disclosure provides a kit comprising an effective amount of (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H- imidazole-2-carbonyl)phenyl)oxalamide (formula (3.2)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof.
In some further specific embodiments, the present disclosure provides a kit comprising an effective amount of (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H- imidazole-2-carbonyl)phenyl)oxalamide (formula (3.2)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
A further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof. More specifically, the disclosed method comprises the step of administering to the subject a therapeutically effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition and/or kit comprising at least one of the ARTS mimetic compound/s. It should be understood that the ARTS mimetic compound interacts and binds the BIR3 domain of XI AP, thereby leading to inhibition of XIAP and/or proteasomal degradation of XIAP. In some embodiments, the method of the present disclosure is applicable for a neoplastic disorder such as neuroblastoma.
In some embodiments, at least one of the ARTS mimetic compound/s used by the therapeutic methods disclosed herein is a compound that has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000101_0001
(Formula I); wherein:
R1 is independently selected from H, C1-C3 alkyl; R2 is independently selected from - C(=O)-X-R3, -S(=O)-X-R3'; X is a heteroatom independently selected from O; R3 and R33 independently of each other may be selected independently from H, C1-C3 alkyl; R5 is -L1-R7-L2-RS; L1 and L2, independently of each other, are selected independently from -(CH2)n ; -NH-C(=O)-(CH2)n-, -C(=O)-NH-(CH2)n-; -S-S-(CH2)n-; -O-(CH2)n-; - NH-(CH2)n-; C(=O)-(CH2)n-; -S-(CH2)n-; -NH-S(=O)n-(CH2)n-; n is independently 1 to 3; R7 is a carbocyclic ring or a heterocyclic ring; R8 is a mono cyclic ring system having 5 to 12, optionally substituted with at least one of OH, CF3, halogen, -COOH, -NH2, CN, Ci-Cialkyl; and R6 is independently selected from H, halogen, CN, NO2, C1-C3 alkoxy, straight or branched C1-C3 alkyl.
In some embodiments, at least one of the ARTS mimetic compound/s used by the methods of the present disclosure is a compound of the general formula (I). In some embodiments, such compound is further characterized by at least one of:
In some embodiments (a), the R1 is H; in yet some further additional or alternative embodiments (b), the R2 is -C(=O)-X-R3; in some further additional or alternative embodiments (c), the X is O; in some further additional or alternative embodiments (d), the L1 and L2 independently selected independently from each other from -NH-C(=O)- (CH2)n-, -(CH2)n-; and in some further additional or alternative embodiments (e), the Re is independently selected from the group consisting of H, halogen, CN, NO2. Still further, in some embodiments, the at least one ARTS mimetic compound/s, or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, used by the therapeutic methods of the present disclosure is a compound that has the general formula of any one of:
In some embodiments, the compound used in the therapeutic methods disclosed herein
(a), has the structure of formula (II)
Figure imgf000102_0001
(Formula II) wherein R1, R2, R6, R7, R8, L1 and L2 are as defined herein above.
In yet some alternative embodiments, the compound used in the therapeutic methods disclosed herein (b), has the structure of formula (III)
Figure imgf000102_0002
wherein R1, R3, R6, R7, R8, L1 and L2 are as defined by the present disclosure.
In yet some further alternative embodiments, the compound used in the therapeutic methods disclosed herein (c), has the structure of formula (IV)
Figure imgf000103_0001
Still further, in some alternative embodiments, the compound used in the therapeutic methods disclosed herein (d), has the structure of formula (VI)
Figure imgf000103_0002
In some specific embodiments, the ARTS mimetic compound used by the disclosed therapeutic methods is 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole- 2-carboxylic acid methyl ester. More specifically, this ARTS mimetic compound has the structure of formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000104_0001
In it some alternative or additional embodiments, at least one of the ARTS mimetic compounds used by the therapeutic methods disclosed herein us a compound that has the general formula (X), a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000104_0002
wherein
R1, R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is L1’-R3’-L1”-R3” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R2 is L2’-R4’-L2”-R4”; or
R1 is L1’-R3’-L1”-R3” and R2 is L2’-R4’-L2”-R4”; wherein R3’, R3”, R4’, R4” is each independently from each other, absent or H, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, heteroaryl, heterocycloalkyl, a ring system containing five to twelve atoms, each optionally substituted; L1’, L1”, L2’ and L2” is each independently from each other, absent or -(CH2)n, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2-,-S-, -S-S-, -S-(CH2)n-; n is an integer selected from any one of 0, 1, 2, 3, 4, 5, each one of L1’, L1”, L2’ and L2” independently from each other, may be optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m- halogen and m is an integer selected from 0, 1, 2, 3, 4, 5.
In a further embodiment, the ARTS mimetic compound of the of the composition for use in accordance with the preset disclosure, is a compound having the general formula (XI)
Figure imgf000105_0001
wherein R1 and L2’ is as defined above and wherein R is one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other and t is an integer selected from 1 to 5.
In yet some further embodiment, the ARTS mimetic compound of the composition for use, as disclosed herein, is having the general formula (XIIc), or (Xlle):
Figure imgf000106_0001
wherein R3’ and R3 ” is each independently from the other, an optionally substituted aryl or an optionally substituted heteroaryl, and L1” is C(=O), wherein R is one or more of H, OH, CF3, halogen, C(=O), -COOH, -NH2, alkyl, alkylene, C1-C12 alkoxy, a ring system containing five to twelve atoms, C1-C5 carboxyl, halogen, independently selected from each other and t is an integer selected from 1 to 5.
In further embodiments of the composition for use as disclosed herein, the ARTS mimetic compound is having the formula (3.1), (3.2), (3.3);
Figure imgf000106_0002
N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide ;
Figure imgf000107_0001
(S)-N 1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide ;
Figure imgf000107_0002
(R)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide..
Still further, in more specific embodiments, the ARTS mimetic compound applicable in the therapeutic methods disclosed herein is a compound that has the structure of the formula (3.2), a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000108_0001
(S)-N 1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide. In more specific embodiments, the method of the invention may use any of the ARTS mimetic compound/s as defined by the invention. In some particular embodiments, the therapeutic method of the invention may use the ARTS mimetic compound referred to herein as A4 or any derivatives thereof.
As used herein, "proliferative disorder" is a disorder displaying hyper proliferation. This term means cell division and growth that is not part of normal cellular turnover, metabolism, growth, or propagation of the whole organism. Unwanted proliferation of cells is seen in tumors and other pathological proliferation of cells, does not serve normal function, and for the most part will continue unbridled at a growth rate exceeding that of cells of a normal tissue in the absence of outside intervention. A pathological state that ensues because of the unwanted proliferation of cells is referred herein as a "hyper proliferative disease" or "hyper proliferative disorder." It should be noted that the term “proliferative disorder”, “cancer”, “tumor” and “malignancy” all relate equivalently to a hyperplasia of a tissue or organ. In general, the compositions and methods of the present invention may be used in the treatment of non-solid and solid tumors.
In some embodiments, the neoplastic disorder affecting the neural system and/or neural cell treated by the disclosed therapeutic methods is a high-risk neuroblastoma.
Neuroblastoma (NB) is a cancer affecting the nerve tissue that is most frequently originated from one of the adrenal glands but may also develop in the neck, chest, abdomen, or spine. Symptoms may include bone pain, a lump in the abdomen, neck, or chest, or a painless bluish lump under the skin. Typically, neuroblastoma occurs due to genetic mutation/s during early development. Neuroblastoma is the most common cancer in babies and the third-most common cancer in children after leukemia and brain cancer. About 90% of cases occur in children less than 5 years old, and it is rare in adults. Neuroblastoma is classified and staged using two main classification systems.
The International Neuroblastoma Risk Group Staging System (INRGSS)was designed specifically for the International Neuroblastoma Risk Group (INRG) pre-treatment classification system and uses the results of imaging tests taken before surgery. Knowledge regarding the presence or absence of image-defined risk factors (IDRF) is required for this staging system. The INRGSS defines the following stages:
Stage L1: The tumor is located only in the area where it started; no IDRFs are found on imaging scans, such as a CT or MRI scan. Stage L2: The tumor has not spread beyond the area where it started and the nearby tissue; IDRFs are found on imaging scans, such as a CT or MRI scan. Stage M: The tumor has spread to other parts of the body (except stage MS). Stage MS: The tumor has spread to only the skin, liver, and/or bone marrow (less than 10% bone marrow involvement) in a patient younger than 18 months.
International Neuroblastoma Staging System Committee (INSS) system, is another staging system previously used for neuroblastoma. The following is a brief summary of each INSS stage:
Stage 1: The tumor can be removed completely during surgery. Lymph nodes attached to the tumor removed during surgery may or may not contain cancer, but other lymph nodes near the tumor do not. Stage 2A: The tumor is located only in the area it started and cannot be completely removed during surgery. Nearby lymph nodes do not contain cancer. Stage 2B: The tumor is located only in the area where it started and may or may not be completely removed during surgery, but nearby lymph nodes do contain cancer. Stage 3: The tumor cannot be removed with surgery. It has spread to regional lymph nodes (lymph nodes near the tumor) or other areas near the tumor, but not to other parts of the body.
Stage 4: The original tumor has spread to distant lymph nodes (lymph nodes in other parts of the body), bones, bone marrow, liver, skin, and/or other organs, except for those listed in stage 4S.
Stage 4S: The original tumor is located only where it started (as in stage 1, 2A, or 2B), and it has spread only to the skin, liver, and/or bone marrow, in infants younger than one. The spread to the bone marrow is minimal (usually less than 10% of cells examined show cancer). In the INRG classification system, a combination of clinical, pathologic, and genetic markers is used to predict the clinical behavior of the tumor and to predict responsiveness to treatment. These markers are used to define risk. Using the following factors, each neuroblastoma is classified into 1 of 4 categories: very low-risk, low-risk, intermediaterisk, or high-risk, (i) The stage of the disease according to the INRG staging system; (ii) The child's age at the time of diagnosis; (iii) Histologic category, such as maturing ganglioneuroma versus ganglioneuroblastoma, intermixed versus ganglioneuroblastoma, or nodular versus neuroblastoma; (iv)Grade, or how cells of the tumor are differentiated; (v) MYCN gene status; (vi) Chromosome 1 Iq status; and (vii)Tumor cell ploidy, which is the DNA content of tumor cells.
The following factors are used to determine risk:
(i) The stage of the disease according to the INSS system; (ii) The child's age at the time of diagnosis; (m)MYCN gene status; (iv) Tumor ploidy (this is only important for children younger than 18 months); and (v) Tumor histopathology according to the International Neuroblastoma Pathologic Classification (INPC) system.
Low-risk neuroblastoma as used herein, is defined herein as a disease characterized by one or more of the following features: Stage 1 disease; Stage 2A or 2B disease in which more than 50% of the tumor was surgically removed, except for a child with MYCN amplification; Stage 4S disease, no MYCN amplification, favorable histopathology, and hyperdiploidy, meaning having extra chromosomes.
Intermediate-risk neuroblastoma, as used herein, is defined herein as a disease characterized by one or more of the following features: Stage 2 A or 2B disease with no MYCN amplification in which less than 50% of the tumor was removed with surgery; Stage 3 disease in children younger than 18 months and no MYCN amplification; Stage 3 disease in children older than 18 months, no MYCN amplification, and favorable histopathology; Stage 4 disease in children younger than 12 months. In some embodiments, the present disclosure is particularly applicable for treating neuroblastoma patients displaying amplified and/or overexpressed MYCN.
In some embodiments, the ARTS mimetic compound used by the therapeutic methods disclosed herein prolongs the overall survival (OS) of the treated subjects. Overall survival (OS), as used herein, refers to the length of time from the start of a particular treatment or intervention until a patient's death from any cause. OS is often used as a primary endpoint in clinical trials and is an important measure of the effectiveness of a treatment. In the present disclosure, the ARTS mimetic compounds disclosed herein, and specifically, A4 and/or B3, extended the life-time of the patients. In yet some further embodiments, the disclosed treatment extends the disease-free period. In yet some further embodiments, the disclosed treatment using the ARTS mimetic compounds may reduce the relapse of the disease.
In some embodiments, the therapeutic methods disclosed herein may further offer a combined treatment. Thus, in some embodiments, the method further comprises the step of administering to said subject an effective amount of at least one anti-cancer agent.
In some embodiments, the anti-cancer agent used by the therapeutic methods of the present disclosure may be at least one of a topoisomerase inhibitor and alkaloid agent.
In some specific embodiments of the disclosed therapeutic methods, the topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
In some specific embodiments of the disclosed therapeutic methods, the alkaloid agent is anti-mitotic and/or anti-microtubule alkaloid agent, optionally, vincristine, or any derivative or formulation thereof.
A further aspect of the present disclosure relates to a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof. The therapeutic method/s comprise the step of administering to the subject a therapeutically effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition and/or kit comprising at least one of said ARTS mimetic compound. The ARTS mimetic compound interacts and binds the BIR3 domain of XIAP, thereby leading to proteasomal degradation of XIAP. It should be further noted that the subject is a subject treated with at least one anti-cancer agent. In more specific embodiments, the agent is at least one of a topoisomerase inhibitor and alkaloid agent. Still further, the therapeutic methods disclosed herein are specifically applicable for a neoplastic disorder such as neuroblastoma.
In some embodiments of the disclosed combined therapeutic methods, the ARTS mimetic compound is 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2- carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and the alkaloid agent is vincristine, or any derivative or formulation thereof. In yet some further embodiments of the disclosed combined therapeutic methods, the ARTS mimetic compound is 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H- indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and the topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
In some embodiments of the disclosed combined therapeutic methods, the ARTS mimetic compound is (S)-N 1 -(1 -hydroxy-3-phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1H-imidazole- 2-carbonyl)phenyl)oxalamide (formula (3.2)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and the alkaloid agent is vincristine. In some further embodiments of the disclosed combined therapeutic methods, the ARTS mimetic compound is (S)-N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H- imidazole-2-carbonyl)phenyl)oxalamide (formula (3.2)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and the topoisomerase inhibitor is Topotecan.
The methods provided herein involve administration of the ARTS mimetic compound/s of the invention in a therapeutically effective amount. The term "effective amount" as used herein is that determined by such considerations as are known to the man of skill in the art. The amount must be sufficient to prevent or ameliorate tissue damage caused by proliferative disorders. Dosing is dependent on the severity of the symptoms and on the responsiveness of the subject to the active drug, specifically, the antagonist of the invention. Medically trained professionals can easily determine the optimum dosage, dosing methodology and repetition rates. In any case, the attending physician, taking into consideration the age, sex, weight and state of the disease of the subject to be treated, will determine the dose. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. In general, dosage is calculated according to body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the compositions and combined composition of the invention in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the ARTS mimetic compound used by the method of the invention is administered in maintenance doses, once or more daily. As use herein "therapeutically effective amount" means an amount of the ARTS mimetic compound/s, a composition comprising the same which provides a medical benefit as noted by the clinician or other qualified observer. Regression of a tumor in a patient is typically measured with reference to the diameter of a tumor. Decrease in the diameter of a tumor indicates regression. Complete regression is also indicated by failure of tumors to reoccur after treatment has stopped.
The present invention provides methods for treating proliferative disorder. The term “treatment or prevention” refers to the complete range of therapeutically positive effects of administrating to a subject including inhibition, reduction of, alleviation of, and relief from, proliferative disorder symptoms or undesired side effects of such proliferative disorder related disorders. More specifically, treatment or prevention includes the prevention or postponement of development of the disease, prevention or postponement of development of symptoms and/or a reduction in the severity of such symptoms that will or are expected to develop. These further include ameliorating existing symptoms, preventing- additional symptoms and ameliorating or preventing the underlying metabolic causes of symptoms.
As used herein, “disease”, “disorder”, “condition” and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms.
The present invention relates to the treatment of subjects, or patients, in need thereof. By “patient” or “subject in need” it is meant any organism who may be affected by the above- mentioned conditions, and to whom the treatment methods herein described are desired, including humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and murine subjects, rodents, domestic birds, aquaculture, fish and exotic aquarium fish. It should be appreciated that the treated subject may be also any reptile or zoo animal. More specifically, the methods and compositions of the invention are intended for mammals. By “mammalian subject” is meant any mammal for which the proposed therapy is desired, including human, equine, canine, and feline subjects, most specifically humans. It should be noted that specifically in cases of non-human subjects, the method of the invention may be performed using administration via injection, drinking water, feed, spraying, oral gavage and directly into the digestive tract of subjects in need thereof. It should be further noted that particularly in case of human subject, administering of the compositions of the invention to the patient includes both self-administration and administration to the patient by another person.
The invention provides methods for treating proliferative disorders, and further relates to disorders associated or related to cancer. It is understood that the interchangeably used terms "associated" and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology.
The phrase “combination therapy” or “adjunct therapy” or in defining use of a compound described herein, specifically, the ARTS mimetic compounds of the invention, and one or more other active pharmaceutical agents, specifically, the alkaloid agents such as vincristine, and/or topoisomerase inhibitors, such as topotecan, is intended to embrace administration of each agent in a sequential manner in a regimen that will provide beneficial effects of the drug combination, and is intended as well to embrace coadministration of these agents in a substantially simultaneous manner, such as in a single formulation having a fixed ratio of these active agents, or in multiple, separate formulations for each agent.
Another aspect of the invention further relates to a method for treating, inhibiting, preventing, ameliorating or delaying the onset of a proliferative disorder combining the therapeutic use of the ARTS mimetic compounds of the invention with alkaloid agents such as vincristine, and/or topoisomerase inhibitors, such as topotecan.
Therefore, a combined therapy is further established by the invention.
The term "synergism" refers to interaction of discrete agents (as drugs), such that the total effect is greater than the sum of the individual effects.
The invention provides pharmaceutical compositions comprising an effective amount of the ARTS mimetic compounds of the invention or any combinations thereof with alkaloid agents such as vincristine, and/or topoisomerase inhibitors, such as topotecan.
In certain embodiments, the compositions of the present invention can be administered for prophylactic and/or therapeutic treatments. In therapeutic application, compositions are administered to a patient already affected by a proliferative disorder (neural neoplasm, specifically, neuroblastoma), in an amount sufficient to cure or at least partially arrest the condition and its complications. An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the condition and the general state of the patient's own immune system, but generally range from about 0.001 to about 1000 mg/Kg. In some specific embodiments, an effective amount of the disclosed ARTS mimetic compounds may range between about 0. 01 to about 100 mg/Kg, between about 0.1 to about 100 mg/Kg, between about 1 to about 100 mg/Kg, between about 2 to about 100 mg/Kg, between about 3 to about 100 mg/Kg, between about 4 to about 100 mg/Kg, between about 5 to about 100 mg/Kg, between about 6 to about 100 mg/Kg, between about 7 to about 100 mg/Kg, between about 8 to about 100 mg/Kg, between about 9 to about 100 mg/Kg, between about 10 to about 100 mg/Kg, between about 20 to about 100 mg/Kg, between about 30 to about 100 mg/Kg, between about 40 to about 100 mg/Kg, or between about 50 to about 100 mg/Kg. In yet some further embodiments, an effective amount of the disclosed ARTS mimetic compounds may be 10 mg/Kg. Single or multiple administrations on a daily, weekly or monthly schedule can be carried out with dose levels and pattern being selected by the treating physician. Additionally, the administration of the compositions of the invention, may be periodic, for example, the periodic administration may be effected twice daily, three time daily, or at least one daily for at least about three days to three months. The advantages of lower doses are evident to those of skill in the art. These include, inter alia, a lower risk of side effects, especially in long-term use, and a lower risk of the patients becoming desensitized to the treatment. In another embodiment, treatment using the compositions of the invention, may be effected following at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 30, 60, 90 days of treatment, and proceeding on to treatment for life.
It should be noted that the treatment of different proliferative conditions may indicate the use of different doses or different time periods, these will be evident to the skilled medical practitioner.
For prophylactic applications, the compositions of the invention may include a prophylactic effective amount of the active ingredient. The term “prophylactically effective amount” is intended to mean that amount of a pharmaceutical composition that will prevent or reduce the risk of occurrence or recurrence of the biological or medical event that is sought to be prevented in a tissue, a system, animal or human by a researcher, veterinarian, medical doctor or other clinician. In prophylactic applications, the compositions of the invention are administered to a patient who is at risk of developing the disease state to enhance the patient's resistance. Such an amount is defined to be a “prophylactically effective dose”. In this use, the precise amounts again depend upon the patient's state of health and general level of immunity, but generally range from 0.001 to 1000 mg per dose, about 0. 01 to 900 mg per dose, about 0.1 to 800 mg per dose, about 1 to 1000 mg per dose, about 1 to 800 mg per dose, about 1 to 700 mg per dose.
All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.
The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. As used herein the term "about" refers to ± 10 %.
The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". This term encompasses the terms "consisting of" and "consisting essentially of". The phrase "consisting essentially of" means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method. Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It should be noted that various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.
As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.
Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.
Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.
It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.
The following examples are representative of techniques employed by the inventors in carrying out aspects of the present invention. It should be appreciated that while these techniques are exemplary of preferred embodiments for the practice of the invention, those of skill in the art, in light of the present disclosure, will recognize that numerous modifications can be made without departing from the spirit and intended scope of the invention.
EXAMPLES
Experimental procedures
Table 1
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Cell Lines and Cell Culture
Human neuroblastoma cell lines (SK-N-SH, SK-N-AS, NB1, CHP212, NLF) were cultured in RPMI-1640, KELLY in RPMI-1640 with 25mM HEPES, BE(2)-C in DMEM/F12 (1:1) with 15mM HEPES and IMR-32 in MEM/EBSS with 1% Non- Essential Amino Acids (NEAA) and 1 % sodium pyruvate. Human non-cancerous normal cell lines, HS5 (bone marrow origin) and THLE3 (liver origin) were cultured in DMEM/High Glucose with 1% sodium pyruvate and 1% sodium bicarbonate, and Bronchial Epithelial Cell Growth Medium (BEGM), respectively. All media were supplemented with 10% fetal bovine serum (FBS). Patient-derived neuroblastoma cells (NBL27-0218A, NBL16-0118, NBL01-1116, NBL07-0317, NBL01-1116) were generated and kindly provided by Dr. Amos Loh Hong Pheng’s laboratory from the VIVA-KKH Paediatric Brain and Solid Tumour Programme, Singapore. They were cultured in DMEM/F12 (1:1) supplemented with 15% FBS and other supplements as previously described (Hee et al., 2020). All cell lines were obtained from American Type Culture Collection (ATCC) unless stated otherwise and were cultured and maintained at 37°C in 5% CO2 humidified incubator.
Animal Models
Neuroblastoma patient-derived xenografts (PDXs) were generated from MYCN- amplified tumor samples of patients recruited with written parental consent and child assent, under SingHealth Duke NUS ORB protocol 2014/2079 (Modeling, Analysis and Translational Therapeutics for Tumors of Childhood), and implanted orthotopically in the retroperitoneal space of NOD/SCID mice. All experiments are performed under the approval of the Institutional Animal Care and Use Committee (SingHealth Duke NUS IACUC #1066) in compliance with the law and guidelines stated.
Test compounds preparation
All IAP antagonists were provided in powder and reconstituted in dimethyl sulfoxide (DMSO) (MP Biomedicals). A4 and B3 were obtained from Carmel - Haifa University Economic Corporation Ltd., CUDC-427 was obtained from Curis, Inc., LCL161 was obtained from Novartis (Singapore) Pte Ltd., Debiol 143 was obtained from Debiopharm International SA, and BV6 was obtained from Genentech, Inc., A4, B3, and BV6 were stored in -80°C after reconstitution into stock concentration of 40 mM (A4 and B3) and 20 mM (BV6), respectively. LCL161, CUDC-427 and Debiol 143 were stored in -20°C after reconstitution into stock concentration of 40 mM.
Tissue microarray (TMA) and Immunohistochemistry
TMA analysis was performed on neuroblastoma patients’ samples obtained from KK Women’s and Children’s Hospital. Ethical permission was obtained from the SingHealth Cental Institutional Review Board (ORB 2012/450/F, 2019/2136). TMAs of tumor specimens were constructed in triplicate from formalin-fixed paraffin-embedded tissue blocks using a 1 mm-wide diameter punch (Estigen) and a manual tissue-arraying instrument (Beecher Instruments). 4 pm- thick unstained sections of TMA blocks were treated with high pH H2 buffer (Leica Biosystem) for 20 minutes and stained with anti- XIAP antibody (sc-55550) (Santa Cruz Biotechnology) at dilution 1:500. DAB substrate was used as the chromogen and nuclei were counterstained with hematoxylin. The expression of XIAP on TMA was reviewed and scored by pathologist; with a scoring of 0 represents negative/null XIAP expression and a highest scoring of 3 represents high XIAP expression. Scoring results were tabulated and analyzed with the patients’ underlying clinical information.
Western blotting
Whole cell lysates harvested were lysed in EBC buffer (pH 8.0 of 50 mM Tris, 0.5% NP- 40, 120 mM sodium chloride) containing protease inhibitors and protein concentration were quantified using the Bradford assay (Thermo Fisher Scientific). 60 -100 pg of proteins were separated by 12% gel SDS-PAGE followed by a wet-transfer onto PVDF membranes (BioRad). Membranes were probed overnight at 4°C with specific primary antibodies as stated in key resources table. Detection was performed using X-ray film with enhanced chemiluminescence method (Merck) Densitometric analysis of the blots for quantification of protein expression ratios was performed using ImageJ software.
Cell viability assays
Cell viability was measured in real time using RealTime-Glo™ MT Cell Viability Assay as according to manufacturer’s instructions from Promega. Cells were seeded at IX 104 cells per well and treated with a range of doses of indicated antagonists, followed by continuous bioluminescence reading every 24 hours for 3 days using luminescent plate reader, Varioskan (SkanIT software). IC50 was determined using GraphPad Prism software.
Lentivirus production and infection
Lentiviruses targeting XIAP were generated by transfecting 293FT cells with shXIAP- encoding plasmid (Sigma) and 3rd generation lenti viral packaging plasmids (pLPl, pLP2, and pLP/VSVG) using Lipofectamine® 2000 (Thermo Fisher Scientific). Supernatants containing the lentivirus were collected and pelleted. sh.SC/? plasmid encoding nontargeting virus (SCR) was used as a negative control. shXIAP and sh.SC/? plasmids were purchased from Sigma-Aldrich MISSION® shRNA libraries with the sequences stated in key resources table. Lentivirus produced was used to infect neuroblastoma cells followed by downstream experiments of western blotting, clonogenic and apoptotic assays.
Clonogenic assays
Cells were seeded in 6-well plate at a confluency of at least 30% on the day of lentiviral transduction. Cells were transduced with lentivirus encoding either control or targeting XIAP, followed by 24 hours incubation and subsequent selection by puromycin for 2 to 3 weeks. Stably selected cells were harvested for staining once they have reached desired confluency. Cells were stained with crystal violet containing methanol followed by repeated washing with water and drying. Pictures of stained cells were taken using a Brother DCP-L2540DW scanner.
Apoptotic assays
The determination of Caspase-3/7 levels as an indication for apoptosis activity were performed using a Caspase-Gio® 3/7 assay kit from Promega according to manufacturer’s instructions. Cells were seeded at 1 X 104 cells per well and treated with 10 pM of IAP antagonists for various indicated timings, followed by bioluminescence measurement using luminescent plate reader, Tecan Infinite®200 Pro. Graphical representation of the bioluminescence was expressed as a fold change relative to the vehicle control after general normalization to time 0 hour.
The activity of apoptosis was also measured by flow cytometry using the Dead Cell Apoptosis Kit with Annexin V FITC and PI (Thermo Fisher Scientific) according to manufacturer’s instructions. Cells harvested from lentiviral infection were subjected to Annexin V (2.5 pL) and PI (40 ng/mL, 0.5 pL) staining, followed by flow cytometric analysis using BD LSRFortessaTM cell analyzer with excitation/emission spectra of 494 nm/518 nm for FITC and 535 nm/617 nm for PI.
Generation of luciferase-tagged neuroblastoma cells using CRISPR/Cas-9 gene editing (Knock-in)
Endogenous-tagging of XIAP with luciferase was performed using Promega’s NanoBiT luciferase technology. The NanoBiT luciferase consists of two subunits - HiBiT and LgBiT. HiBiT was first introduced at endogenous XIAP locus using CRISPR knock-in gene editing following Promega manufacturer’s instructions. Alt-R® S.p. Cas9 Nuclease V3, Alt-R® transactivating CRISPR RNA (tracrRNA), Alt-R® CRISPR RNA (crRNA), Ultramer single-stranded oligo DNA nucleotides (ssODN), and nuclease-free duplex buffer were purchased from Integrated DNA Technologies (Martin et al.). The sequences used for CRISPR knock-in can be found in key resources table.
Guide RNA (gRNA) was prepared by 5 minutes 95 °C heating of reaction consisting of 1200 pmol of each crRNA and tracrRNA, and nuclease-free duplex buffer in a final volume of 50 pL, followed by cooling to room temperature. Ribonucleoprotein (RNP) complexes were prepared by incubating 120 pmol gRNA and 100 pmol Cas9 in a final volume of 10 pL for 10 minutes at room temperature. For nucleofection of neuroblastoma cells, 2 X 105 cells were first resuspended in 20 pL 4D-Nucleofector reagent (Nucleofector SF Solution + Supplement) (Lonza) with the subsequent addition of 2.5 pL RNP complex and 100 pmol donor ssODN. The reaction was then subjected to electroporation with the Lonza 4D Nucleofector using program CA-137 for BE(2)-C and CM- 130 for KELLY. Each sample was warmed and incubated with growth medium for 20 minutes before transferring the cell suspension to a 24-well plate. After recovering for a few days, the cells were assayed for HiBiT insertion using Nano-Gio HiBiT lytic detection system from Promega to determine the bioluminescence via Tecan Infinite® 200 Pro. The detection of more than 3 -fold luminescence measurement indicated the presence of HiBiT. The presence of HiBiT at XIAP locus was subsequently confirmed via multiplex PCR using three primers as stated in key resources table. After the confirmation, the HiBiT-containing cells were subsequent subjected to transfection with LgBiT expression vector (Promega) using FuGENE® HD transfection reagent following manufacturer’s instructions. Cells were then incubated for 24 hours, followed by hygromycin B (Thermo Fisher Scientific) selection for 1 to 2 weeks. Stably-transfected cells were harvested and assayed for HiBiT-LgBiT (NanoBiT luciferase) insertion using Nano-Gio live cell substrate from Promega to measure the bioluminescence via Tecan Infinite®200 Pro. Positive signals indicate successful tagging of luciferase to XIAP in neuroblastoma cells. Luciferase-tagged-XIAP neuroblastoma cells were used in subsequent downstream experiments including the monitoring of luminescence changes in real time after treatment with IAP antagonists. Kinetic measuring of bioluminescence was performed using Nano-Gio® Vivazine™ live cell substrate from Promega.
NanoBRET™ Ubiquitination Assay
The quantification of XIAP ubiquitination was performed via Promega’ s NanoBRET ubiquitination assay. Following manufacturer’s instructions, HaloTag®-Ubiquitin expression vector was first introduced via transient transfection into above-mentioned stably-transfected luciferase-tagged XIAP neuroblastoma cells using FuGENE® HD transfection reagent. 24 hours post-transfection, the cells were replated into white 96-well plate for overnight in the presence or absence of fluorescent NanoBRET™ HaloTag® 618 ligand (which binds specifically for ubiquitin). The cells were then incubated with Nano-Gio® Vivazine™ live cell substrate for 1 hour before treatment with XIAP-specific antagonist, A4. Using luciferase-tagged-XIAP as donor protein and fluorescent Halo- tagged ligand as acceptor, dual-filtered luminescence was measured every 5 minutes for 6 hours using Tecan Infinite®200 Pro (donor at 460nm and acceptor at 618nm) and BRET ratio (values at 618nm/values at 460nm) was determined. BRET response curve was plotted using GraphPad Prism software after general normalization by subtracting no ligand BRET values.
NanoBRET™ Target Engagement Assay
The quantification and determination of XIAP target engagement with test compounds was performed via Promega’ s NanoBRET XIAP target engagement assay. Following manufacturer’s instructions, NanoLuc-XIAP expression vector provided in the kit was transiently transfected into neuroblastoma cells using FuGENE® HD transfection reagent. 24 hours post-transfection, the cells were replated into white 96-well plate (Corning #3600) in the presence or absence of fluorescent tracer (which binds specifically to NanoLuc-XIAP). The cells were then treated with IAP antagonists for 30 minutes before the addition of substrate for measurement. Using NanoLuc-XIAP as donor protein and fluorescent tracer as acceptor, dual-filtered luminescence was measured via Tecan Infinite®200 Pro (donor at 460nm and acceptor at 618nm) and BRET ratio (values at 618nm/values at 460nm) was determined. BRET response curve was plotted using GraphPad Prism software after general normalization by subtracting no tracer BRET values.
Nuclear magnetic resonance (NMR) spectroscopy on XIAP
Uniformly 13C,15N-labeled XIAP of 0.8-1.0 mM concentration was used for NMR studies. NMR experiments were carried out at 298 K (25 °C) on a Bruker Avance spectrometer with a proton frequency of 600 MHz or 700 MHz and equipped with a cryoprobe. Sequence-specific assignment was obtained based on the following experiments including ^-^N-HSQC, HNCA, HNCACB, HN(CO)CA, HN(CO)CACB and HNCO. Spectra were acquired using standard pulses from Topspin (version 2.1). To determine XIAP interaction with small molecule A4, ^-^N-HSQC experiment was performed using 50 mM of A4 compound dissolved in DMSO and 0.5 mM of 15N-labeled XIAP. The ^-^N-HSQC spectra of XIAP in the absence and presence of different amounts of A4 compounds were acquired, processed and analyzed. The collected data were processed with NMRPipe (Delaglio et al., 1995) and analyzed with NMRView (Johnson, 2004).
Drug Interactions Assay
Drug interactions between XIAP-specific antagonist A4 and vincristine or topotecan were determined using the combination index (CI) by Chou and Talalay which was established based on the mass-action law principle to derive the median-effect equation (Chou, 2010). 1 X 104 cells were seeded in 96-well plate and treated with increasing doses of A4 and vincristine/ topotecan (individually or in combination) at a fixed ratio according to the IC50 values of the individual drugs for 48h (A4: Vincristine; BE(2)-C 1:1, KELLY 1:0.5 and A4:Topotecan; BE(2)-C 1:6, KELLY 1:16). Cell viability was measured and CI and dose reduction index (DRI) values were quantified using the CompuSyn software by Chou-Talalay. Pharmacokinetics analysis of XIAP-specific antagonist A4
The testing dose of A4 10 mg/kg was injected intraperitoneally into the orthotopic neuroblastoma PDXs. 150-200 pL of blood was taken from mice via cardiac puncture and tumors were harvested over the following time courses of 0, 0.5, 1, 2, 4, 24 and 48 hours post-dosing (n=3/time point). The concentrations of A4 in mouse plasma and tumor samples were determined and validated by a highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method with multiple reaction monitoring (MRM) mode. The LC-MS/MS system consisted of Agilent 1290 ultra high-performance liquid chromatography (UHPLC) connected in tandem to Sciex QTRAP 5500 mass spectrometer system. Chromatographic separation was optimized using a liquid-liquid extraction and a reversed phase separation on a Kinetex F5 column (100 mm x 2.1 mm, 2.6 pm) with isocratic elution. Ethyl indole-2-carboxylate was used as the internal standard (IS). MRM transitions 441.2/189.0 (A4) and 190.1/114.1 (IS) were monitored with a dwell time of 300 msec and analyst 1.6.2 software (Sciex) was used to quantify the peaks with l/x2 weighted linear regression.
In Vivo Experiments
Tumor samples collected from neuroblastoma PDXs were infected with luciferase lentivirus. Tumor analysis was performed by intraperitoneal injection of D-luciferin solution (150 mg/kg) (PerkinElmer) into the mice and imaged with an IVIS spectrum. Bioluminescent signals were then quantified using Living Imaging 4.4 (Caliper Life Sciences). After 4 weeks of tumor implantation into retroperitoneal space of mice, tumorbearing mice were randomly assigned to receive either vehicle (DMSO; ImL/kg) or XIAP-specific antagonist A4 (10 mg/kg) (n=9 per group). All agents were administered by intraperitoneal injection twice weekly for consecutive three weeks. Survival data was recorded and Kaplan-Meier curves were plotted using GraphPad Prism software.
Quantification and statistical analysis
Statistical analysis was performed using GraphPad Prism software version 5.0 (La Jolla, CA). Data were expressed as the mean ± standard deviation (S.D) with S.D. represented by the vertical error bars in figures. Statistical analysis was performed using two-tailed student t-test with a p value <0.05 considered statistically significant. For orthotopic mice model, log-rank (Mantel-cox test) derived from Kaplan-Meier survival plots was used for statistical comparisons between groups. EXAMPLE 1
XIAP is overexpressed in high-risk neuroblastoma and induces apoptosis when knocked down
During development, reduction in N-Myc and XIAP expression is necessary for nerve growth factor (NGF)-withdrawal-mediated apoptosis in sympathoadrenal progenitor cells. Since dysregulation of this mechanism could prolong the survival of these progenitor cells and promote tumorigenesis (Nakagawara et al., 2018; Potts et al., 2003), the inventors examined whether neuroblastomas, which are of sympathoadrenal origin, would have a high endogenous level of XIAP especially those with MYCN amplification. To investigate if there is a correlation between XIAP and N-Myc protein levels, their expression has been screened across a panel of neuroblastoma cell lines. As the liver and bone marrow are common metastatic sites for neuroblastoma, THLE3, a liver tissue- derived cell line and HS5, a bone marrow-derived cell line, were used as representative non-cancerous cells for baseline evaluation of protein expression. As shown by Figure 1A, in general, the majority of neuroblastoma cell lines expressed higher XIAP protein levels compared to non-cancerous normal tissue-derived cell lines. Specifically, the expression of XIAP was higher in Af FC/V-amplificd neuroblastoma cells compared to non-A7FC/V-amplificd cells (positive correlation with R2 = 0.76) (Figure lA(i) and (ii)). This was supported by examining a tissue microarray of neuroblastoma patient samples where 64.3% of patients with MYCN amplification expressed moderate (2+) to high (3+) XIAP whereas the majority of iwmA7FC/V- amplified patients had null (0) to low (1+) XIAP expression (Figure IB). This suggested that XIAP is highly expressed in neuroblastoma and is positively correlated to MYCN amplification status.
Since XIAP reduction is necessary to make sympathetic neuronal progenitors vulnerable to developmental apoptosis (Potts et al., 2003), the inventors next investigated how removal of high endogenous XIAP expression would impact apoptosis in neuroblastoma cells. Using two independent lentiviral shRNAs targeting XIAP transcripts (shX/AP 78 and 79), endogenous XIAP expression was knocked down (Figure 1C). In contrast to non-targeting shRNA (sh.SC/?) controls, silencing of XIAP in high XIAP-expressing neuroblastoma cell lines BE(2)-C (Fig. 1D(i)) and KELLY (Fig. 1D(ii)) significantly induced apoptosis, indicated by increased cleavage of PARP and caspase-3 and significant increase in apoptotic cells quantified via flow cytometric analysis (Figure 1C and Figure ID). Furthermore, silencing of XIAP decreased colony formation over time (Figure IE). Consistent with previous findings, these results demonstrated that XIAP expression is necessary for survival of neuroblastoma cells.
EXAMPLE 2
Specific loss of XIAP expression, not c-IAPl, is required for mediating apoptosis in high-risk neuroblastoma cells
To investigate the efficacy of targeting XIAP as a treatment strategy for neuroblastoma, six different small molecule antagonists, each at different phases of clinical testing, and representing a variety of chemical structures and IAP targeting-specificity, were evaluated (Table 2). These six IAP antagonists include: (1) CUDC-427, a phase 1 clinically-tested monovalent pan-IAP antagonist (Tolcher et al., 2016); (2) LCL161, a phase 2 clinically-tested monovalent pan-IAP antagonist (Infante et al., 2014); (3) Debio 1143, a phase 3 clinically-tested monovalent pan-IAP antagonist (Cai et al., 2011); (4) BV6, a pre-clinical bivalent pan-IAP antagonist (Gao et al., 2007); (5) A4 and (6) B3, both pre-clinical XIAP-specific antagonists (Mamriev et al., 2020).
To determine the efficacy of the six IAP antagonists in neuroblastoma, a panel of neuroblastoma cell lines including neuroblastoma patient-derived cell lines and non- cancerous normal tissue-derived cell lines were used. The cells were treated with increasing doses of each antagonist (0-100 pM), followed by real-time measurement of cell viability every 24 hours for 72 hours. Among the six IAP antagonists, XIAP-specific antagonists A4 and B3 showed highest potency against neuroblastoma cells across the panel of cell lines with the lowest IC50 achieved (2 - 15 pM) (Table 2 and Figure 2A). Intriguingly, the remaining four pan-IAP antagonists with selective IAP affinity and activity showed poorer average efficacy, ranked by mean IC50: BV6 with IC50 of 3 - 20 pM, LCL161 and CUDC-427 with IC5020 - 60 pM, and Debio 1143 with IC50 50 - >100 pM (Table 2 and Figure 2A). Among the 4 pan-IAP antagonists with the same inhibitory mechanism, BV6 was the most potent in suppressing neuroblastoma cells. Though the overall IC50 range of A4, B3 and BV6 on neuroblastoma cells were comparable despite the different modes of action, their relative effects on non-cancerous cells have not been previously studied.
To evaluate this, non-cancerous normal tissue-derived cell lines THLE3 and HS5 described earlier, were utilized to evaluate toxicity and tolerability. XIAP-specific antagonists, A4 and B3, were observed to be the least toxic to non-cancerous cells and were the most discriminatory in killing neuroblastoma cells (Table 2 and Figure 2A). In contrast, the pan-IAP antagonists especially LCL161, CUDC-427 and Debio 1143, were more toxic towards non-cancerous cells than neuroblastoma cells and thus, deemed unsuitable for use in neuroblastoma treatment. Based on their overall potency and specificity towards neuroblastoma cells over non-cancerous normal cells, A4, B3 and BV6 were selected as the more efficacious antagonists to be studied further in subsequent experiments (Figure 2A).
Interestingly, high-risk and MYCN-amplified neuroblastoma cell lines BE(2)-C and KELLY expressing high XIAP levels (Figure 1A), were generally sensitive to XIAP- specific antagonists A4 and B3 but were highly resistant to pan-IAP antagonist BV6 (Figure 2A). This suggests that specific targeting of XIAP could be a potential treatment strategy for high-risk neuroblastoma. To further investigate this, the impact of A4, B3 and BV6 was studied on the expression of XIAP and c-IAPl and subsequent downstream effects, using BE(2)-C and KELLY as representative cell lines of high-risk neuroblastoma and HS5 as control for toxicity and tolerability.
BE(2)-C (Fig. 2b(i)) and KELLY (Fig. 2B(i)) cells treated with A4 or B3 showed loss of XIAP expression over time with intact c-IAPl expression (Figure 2B(i)-(iii)). Correspondingly, apoptosis was induced, indicated by increasing cleavage of PARP and caspase-3 as well as increased caspase-3/7 activity (Figure 2B and 2C). In contrast, BE(2)-C and KELLY cells treated with BV6 showed loss of c-IAPl expression with no change in XIAP expression (Figure 2B). BV6 induced minimal or no apoptosis in BE(2)- C and KELLY cells despite the loss of c-IAPl expression (Figure 2B and 2C). This suggested that targeting of XIAP, and not c-IAPl, is required for the killing of high-risk neuroblastoma. Consistent with the cell viability findings, evaluation of cell morphological changes confirmed that BE(2)-C and KELLY cells were sensitive to cell killing by A4 and B3 but resistant to BV6 treatment (Figure 2D).
In contrast to neuroblastoma cells, normal tissue-derived HS5 cells treated with A4 or B3 showed minimal change in XIAP expression with c-IAPl remaining intact (Figure 2B(iii)) and minimal apoptosis observed (Figure 2B(iii)). This could be attributed to the low endogenous XIAP expression of HS5 which led to lower extent of XIAP reduction and thus, higher tolerability towards A4 and B3. Similarly, BE(2)-C, KELLY and HS5 cells treated with BV6 showed loss of c-IAPl expression with no changes in XIAP expression (Figure 2B(i)-(ii)). However, unlike BE(2)-C and KELLY, BV6 induced significant apoptosis in HS5 cells (Figure 2B(iii) and 2C(iii)). Likewise, cell morphological changes showed that BV6 was toxic to HS5 cells but not A4 and B3 (Figure 2D). These results suggest an important role of c-IAPl in maintaining survival of normal cells but not neuroblastoma cells, with neuroblastoma cells exhibiting specific addiction for XIAP required for their survival. The dependence of neuroblastoma cells on XIAP, render them more sensitive to XIAP-targeting. Taken together, these findings demonstrated that the loss of XIAP expression mediated by XIAP-specific antagonists A4 and B3 - not pan-IAP antagonist BV6 - is highly selective and is necessary and sufficient to induce apoptosis in MYC-Namplified high-risk neuroblastoma cells.
To investigate whether XIAP is indeed a target of A4-and B3-mediated apoptosis, XIAP was overexpressed in KELLY cells to evaluate if this could blunt apoptosis induced by A4 or B3 treatment (Figure 2E(i)-(iiO). Decreased levels of cleaved PARP and cleaved caspase-3 in treated cells overexpressing XIAP suggest that A4 and B3 is indeed dependent on targeting XIAP to induce apoptosis (Figure 2E(i)). XIAP-overexpressing KELLY cells treated with B V6 did not undergo apoptosis, similar to control cells (Figure 2E(i)), since XIAP is less sensitive to BV6 (Figure 2B(i)-(iii)). This renders the high XIAP-expressing cells resistant to BV6, with or without XIAP overexpression. Interestingly, NLF, a low XIAP-expressing, MYC-Namplified neuroblastoma cell line, was responsive to all three antagonists which could be mitigated by XIAP overexpression (Figure 2E). This suggests that BV6 is able to reduce XIAP expression but with less potency than A4 and B3 and may explain why certain neuroblastoma cell lines remain sensitive to BV6 while lines with high XIAP and MYCN expression only respond to A4 and B3. Taken together, these results strongly suggest XIAP as a viable target for the suppression of high-risk, resistant neuroblastoma.
Table 2: IC50 (jiM) indicating the effect of individual IAP antagonists on viability of neuroblastoma and normal tissue-derived cells measured every 24 hours up to 72 hours
Figure imgf000133_0001
Figure imgf000134_0001
EXAMPLE 3
Binding and degradation of XIAP by A4 is necessary for targeting high-risk neuroblastoma cells
Having demonstrated significantly selective responses of neuroblastoma cells, to A4 and B3 versus BV6, the inventors hypothesized that these antagonists act by different mechanisms of action. To better understand the mechanisms by which these compounds target XIAP in high-risk neuroblastoma, binding interactions between the antagonists and XIAP were first determined in BE(2)-C and KELLY cells using a NanoBRET quantitative target engagement assay. When evaluating the potency of both ARTS antagonist small molecules A4 and B3, the inventors decided to focus on A4 for further investigation. As shown in Figure 3A, a dose-dependent decrease in BRET ratio was observed with increasing A4 (Fig. 3A(i)) or BV6 (Fig. 3A(ii)) concentrations. This indicates a competitive displacement of the labelled fluorescent tracer from XIAP by A4 or BV6 which suggests direct interaction between A4 and BV6 with XIAP. It was worth noting that BV6 bound more strongly to XIAP than A4 with the following apparent intracellular affinities determined - 0.21 pM (BV6) vs. 10.7 pM (A4) in BE(2)-C and 0.30 pM (BV6) vs. 9.1 pM (A4) in KELLY (Figure 3A(ii)). As the BRET signal was not completely abolished even at saturating A4 concentrations, nuclear magnetic resonance (NMR) was conducted to further examine the binding interaction between A4 and XIAP (Figure 4A-4B). Consistent with the above findings, NMR analysis on ^-^N-HSQC spectra of XIAP in the absence or presence of A4 showed a direct interaction between A4 and XIAP with chemical shift perturbations observed in the spectra (Figure 4).
Next, to determine whether direct binding of the antagonists to XIAP could lead to changes in XIAP expression as seen previously in Figure 2, a highly sensitive method to quantify the degradation of XIAP was performed. Neuroblastoma cells containing endogenous luciferase-tagged XIAP were generated using CRISPR/Cas9 gene editing to knock-in a bioluminescent tag at endogenous loci of XIAP (Figure 5A-5B). Following treatment of BE(2)-C and KELLY cells with A4 or BV6, the degradation of XIAP under endogenous condition was monitored (Figure 3B). Real-time monitoring of endogenous XIAP levels in BE(2)-C and KELLY cells revealed a rapid degradation of XIAP upon A4 treatment, taking place within 10-15 minutes post-treatment (Figure 3B(i), (iii) and Figure 6A). The extent of degradation occurs in a dose-dependent manner with quantitative DCso readout at 2 hours as follows - BE(2)-C (DCso of 22.3 pM) and KELLY (DCso of 3.9 pM) (Figure 3B and Figure 6A). In contrast, 2 hours exposure to BV6 resulted in minimal degradation of XIAP and changes in XIAP expression was only apparent at higher doses of BV6 and at a later time (Figure 3B(ii), (iv), Figure 6B and Figure 6C).
As rapid degradation of proteins is often catalyzed by the ubiquitin-proteasome system (UPS), the inventors sought to determine whether the UPS is involved in the rapid degradation of XIAP upon treatment with A4. Using a NanoBRET quantitative ubiquitination assay to examine the binding of ubiquitin to XIAP upon treatment with A4, neuroblastoma cells were observed to exhibit a clear dose-dependent increase in BRET ratio, indicating an increase binding of fluorescent-labelled ubiquitin to the endogenous luminescent-tagged XIAP (Figure 3C(i), (ii)). To determine if ubiquitination of XIAP leads to its proteasomal degradation, MG- 132 was used to inhibit the proteasome. Compared to A4 treatment alone, addition of MG- 132 was able to prevent A4-mediated degradation of XIAP (Figure 3D(i), (ii)). This suggested that removal of XIAP by A4 is indeed mediated by proteasomal degradation. Taken together, these results demonstrated different mechanisms of XI AP targeting by A4 and BV6 - the former binding and degrading XIAP via UPS and the latter binding XIAP without degradation. This suggests that binding and degradation of XIAP is required for inducing apoptosis in high-risk neuroblastoma cells.
EXAMPLE 4
Treatment with XIAP-specific antagonist A4 prolongs and improves overall survival in high-risk neuroblastoma patient-derived xenografts (PDXs)
As shown herein, the ARTS mimetic A4 compound was the most potent compound in suppressing high-risk neuroblastoma in vitro. Therefore, the effect of targeting XIAP by A4 in vivo was next examined using PDX models of MYCN -amplified high-risk neuroblastoma. Human primary tumor cells derived from AfTCA-amplified high-risk neuroblastoma patients were implanted orthotopically in the retroperitoneal peri-adrenal space of mice, which modeled the typical anatomic site and microenvironment of human disease. Four weeks after tumor implantation, PDXs were then subjected to A4 treatment for pharmacokinetic and survival analysis.
Pharmacokinetic analysis of 10 mg/kg A4 delivered via intraperitoneal injection showed good exposure of A4 in mouse plasma and tumor with a maximum modeled concentration (Cmax) of 600 and 4000 nM, respectively (Figure 7A, 7B, respectively). Tumors harvested from neuroblastoma PDXs after exposure to lOmg/kg A4 at serial time points over 48 h showed an overall reduction of XIAP tissue expression, consistent with in vitro findings (Figure 8A). Also, mice receiving lOmg/kg A4 showed no substantial weight loss or adverse effects with twice-weekly dosing for up to 3 weeks. This suggested that lOmg/kg could be a clinically suitable and sufficient dose for evaluating the potential anticancer effect of A4 in neuroblastoma PDX models.
To determine the clinical potential of A4 on high-risk neuroblastoma, PDXs generated after 4 weeks of tumor implantation were randomly distributed into treatment (10 mg/kg A4) and vehicle control (DMSO; ImL/kg) groups and injected intraperitoneally twice a week for 3 weeks. The mice were monitored over a period of 7 weeks in total from implantation to the end of treatment, and deaths within the monitoring period were recorded. Kaplan-Meier curves of MYCN-amplified neuroblastoma PDXs showed a significantly longer overall survival in the treatment group, with more than 50% of mice alive at the end of the treatment period (Figure 8B(i)). In contrast, the majority of the mice in the vehicle control group did not survive (Figure 8B(ii)). Despite the prolonged overall survival in the treatment group, there was no observable difference in regression of tumor size between treatment and vehicle control groups (Figure 8C(i),(iii)). This suggested that A4 as a single agent in vivo can extend survival and decrease the rate of disease progression at the current dose, with the potential to be a novel antineuroblastoma drug that could work well in combination with existing therapies.
EXAMPLE 5
XIAP-specific antagonist A4 works synergistically with and promotes effective dose reduction of vincristine and topotecan in vitro
For treatment of high-risk neuroblastoma, current standard-of-care utilizes intensive multimodal therapy which already involves the combination of multiple cytotoxic agents. Since treatment with A4 as a single agent did not substantially suppress tumor growth despite prolonging and improving survival in neuroblastoma PDXs, the inventors sought to evaluate the synergistic potential of A4 in combination with current standard-of-care cytotoxic agents, vincristine and topotecan, for the treatment of high-risk neuroblastoma. To analyze potential drug interactions, treatment of BE(2)-C and KELLY cells with A4 in combination with either vincristine or topotecan was examined. With the widely used Chou-Talalay method, the effects/types of drug interactions can be determined by calculating the drug combination (CI) index - Synergistic effect with CI <1; additive effect with CI =1 and antagonistic effect with CI >1. Moreover, it can also be used to calculate the dose reduction index (DRI) which reflects the fold difference of effective dose reduction of each drug when used in combination - DRI < 1 indicates an unfavorable dose reduction and DRI > 1 indicates a favorable dose reduction that leads to toxicity reduction (Chou, 2010).
Combination of A4 and vincristine revealed a synergistic interaction in BE(2)-C and KELLY, with an average combination index at 60-90% cell death (CI60-90) of 0.861 ± 0.070 and 0.384 ± 0.028, respectively (Figure 9A(i)). Similarly, a synergistic interaction between A4 and topotecan was also detected in the same cell lines, although with a lower average Cl60-90 of 0.908 ± 0.056 in BE(2)-C and 0.690 ± 0.104 in KELLY (Figure 9A(ii)). Together, these findings showed that A4 works synergistically when used in combination with either vincristine or topotecan in neuroblastoma cells. Furthermore, when A4 was used in combination with these cytotoxic agents, it promoted an effective dose reduction of vincristine, with an average dose reduction index at 60- 90% cell death (DRI6o-9o) of 1.674 ± 0.129 in BE(2)-C and 37.120 ± 31.315 in KELLY (Figure 9B(i), (ii)). This was depicted by the left-shift in dose-response curves, indicating a reduction of vincristine dose in cells treated with A4 and vincristine, compared to vincristine alone (Figure 9B). Similarly, when A4 was used in combination with topotecan, it promoted an effective dose reduction of topotecan with an average DRIeo-9o of 3.183 ± 0.378 in BE(2)-C and 6.537 ± 2.430 in KELLY (Figure 9C). Compared to cells treated with topotecan alone, a left-shift in dose -response curves of cells treated with A4 and topotecan was also observed. Taken together, XIAP-specific antagonist A4 not only prolongs and improves the overall survival of high-risk neuroblastoma PDXs as a single agent, but also shows translational potential to augment the efficacy of cytotoxic agents in killing neuroblastoma cells when used in combination.
Thus, without being bound by any theory, in high-risk neuroblastoma, as illustrated by Figure 10, there is a presence of MYCN amplification which frequently associates with high XIAP expression. With high XIAP dependency on survival, these neuroblastomas are highly sensitive to treatment with small molecule A4 but not BV6. Upon A4 treatment, it binds and triggers the ubiquitination and rapid degradation of XIAP resulting in the release of caspases for executing effective apoptosis in neuroblastoma. On the contrary, upon BV6 treatment, it preferentially binds c-IAPs over XIAP, resulting in possible sequestration of BV6 by c-IAPs and less caspase being free from XIAP (1). BV6 rapidly triggers the autoubiquitination and degradation of c-IAPs which however showed minimal effect on neuroblastoma. Furthermore, the binding and inhibition of XIAP by BV6 was also seen to be less effective in triggering apoptosis. The BV6-bound XIAP could still be functional independent on the inhibition binding site (2), thus allowing XIAP to bind to other proteins that could mediate anti-apoptotic effect (2a). As a result, BV6 mediates overall less or no apoptosis in these high-risk neuroblastoma. Taken together, XIAP-specific antagonist outperforms pan-IAP antagonist by specifically degrading and not inhibiting XIAP, thus presenting as a potential treatment strategy to overcome high-risk neuroblastoma.

Claims

CLAIMS:
1. At least one apoptosis related protein in the TGF-beta signaling pathway (ARTS) mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition comprising at least one of said ARTS mimetic compound, for use in a method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof, wherein said ARTS mimetic compound interacts and binds the Baculo viral IAP Repeat (BIR) domain 3 of X-linked inhibitor of apoptosis protein (XI AP), thereby leading to proteasomal degradation of XIAP, and wherein said neoplastic disorder is neuroblastoma.
2. The at least one ARTS mimetic compound for use according to claim 1 , wherein at least one of said ARTS mimetic compound has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof,
Figure imgf000139_0001
(Formula I); wherein:
R1 is independently selected from H, C1-C3 alkyl; R2 is independently selected from -C(=O)-X-R3, -S(=O)-X-R3';
X is a heteroatom independently selected from O; R3 and R3', independently of each other may be selected independently from H, C1-C3 alkyl;
R5 is — L1-R7-L2-RS; L1 and L2, independently of each other, are selected independently from -(CH2)n ; -NH- C(=O)-(CH2)n-, -C(=O)-NH-(CH2)n-; -S-S-(CH2)n-; -O-(CH2)n-; -NH-(CH2)n-; C(=O)- (CH2)n-; -S-(CH2)n-; -NH-S(=O)n-(CH2)n-; n is independently 1 to 3; R7 is a carbocyclic ring or a heterocyclic ring; R8 is a mono cyclic ring system having 5 to 12, optionally substituted with at least one of OH, CF3, halogen, -COOH, -NH2, CN, C1-C3alkyl; and R6 is independently selected from H, halogen, CN, NO2, C1-C3 alkoxy, straight or branched C1-C3 alkyl.
3. The at least one ARTS mimetic compound for use according to claim 2, wherein said ARTS mimetic compound of the general formula (I) is further characterized by at least one of:
(a) wherein R1 is H;
(b) wherein R2 is -C(=O)-X-R3;
(c) wherein X is O;
(d) wherein L1 and L2 independently selected independently from each other from -NH- C(=O)-(CH2)n-, -(CH2)n-; and
(e) wherein R6 is independently selected from the group consisting of H, halogen, CN, NO2.
4. The at least one ARTS mimetic compound for use according to claim 2, wherein said ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof has the general formula of any one of:
(a) formula (II)
Figure imgf000140_0001
(Formula II) wherein R1, R2, R6, R7, R8, L1 and L2 are as defined in claim 2; or
(b) formula (III)
Figure imgf000141_0001
5. The at least one ARTS mimetic compound for use according to claim 2, wherein said compound is 3-[2-(4-Benzyl-piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2- carboxylic acid methyl ester (denoted herein as “A4”), said compound has the structure of Formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000142_0001
6. The at least one ARTS mimetic compound for use according to claim 1 , wherein said ARTS mimetic compound is having the general formula (X) or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, wherein formula (X) is:
Figure imgf000142_0002
wherein
R1, R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is L1’-R3’-L1”-R3” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R2 is L2’-R4’-L2”-R4”; or R1 is L1’-R3’-L1”-R3” and R2 is L2’-R4’-L2”-R4”; wherein R3’, R3”, R4’, R4” is each independently from each other, absent or H, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, heteroaryl, heterocycloalkyl, a ring system containing five to twelve atoms, each optionally substituted; L1’, L1”, L2’ and L2” is each independently from each other, absent or -(CH2)n, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2- -S-, -S-S-, -S-(CH2)n-; n is an integer selected from any one of 0, 1, 2, 3, 4, 5, each one of L1’, L1”, L2’ and L2” independently from each other, may be optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m- halogen and m is an integer selected from 0, 1, 2, 3, 4, 5..
7. The at least one ARTS mimetic compound for use according to claim 6, wherein said compound has the structure of formula 3.2:
Figure imgf000143_0001
(S)-N 1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide (denoted herein as “B3”).
8. The at least one ARTS mimetic compound for use according to any one of claims 1 to 7, wherein said neoplastic disorder affecting the neural system and/or neural cell is high-risk neuroblastoma.
9. The at least one ARTS mimetic compound for use according to any one of claims 1 to 8, wherein said ARTS mimetic compound prolongs the overall survival (OS) of said subjects.
10. The at least one ARTS mimetic compound for use according to any one of claims 1 to 8, wherein said method further comprises the step of administering to said subject an effective amount of at least one anti-cancer agent.
11. The at least one ARTS mimetic compound for use according to claim 10, wherein said anti-cancer agent is at least one of a topoisomerase inhibitor and an alkaloid agent.
12. The at least one ARTS mimetic compound for use according to claim 11 , wherein said topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
13. The at least one ARTS mimetic compound for use according to claim 11 , wherein said alkaloid agent is anti-mitotic and/or anti-microtubule alkaloid agent, optionally, vincristine, or any derivative or formulation thereof.
14. A combined composition comprising:
(a) an effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, wherein said ARTS mimetic compound interacts and binds the BIR3 domain of XI AP, thereby leading to proteasomal degradation of XIAP; and
(b) an effective amount of at least one anti-cancer agent, said agent is at least one of a topoisomerase inhibitor and an alkaloid agent.
15. The combined composition according to claim 14, wherein at least one of said ARTS mimetic compound has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, or any vehicle, matrix, nano- or micro-particle comprising the same:
Figure imgf000145_0001
rmula I); wherein:
R1 is independently selected from H, C1-C3 alkyl;
R2 is independently selected from -C(=O)-X-R3, -S(=O)-X-R3';
X is a heteroatom independently selected from O; R3 and R3', independently of each other may be selected independently from H, C1-C3 alkyl;
R5 is — L1-R7-L2-R8; L1 and L2, independently of each other, are selected independently from -(CH2)n ; -NH- C(=O)-(CH2)n-, -C(=O)-NH-(CH2)n-; -S-S-(CH2)n-; -O-(CH2)n-; -NH-(CH2)n-; C(=O)- (CH2)n-; -S-(CH2)n-; -NH-S(=O)n-(CH2)n-; n is independently 1 to 3; R7 is a carbocyclic ring or a heterocyclic ring; R8 is a mono cyclic ring system having 5 to 12, optionally substituted with at least one of OH, CF3, halogen, -COOH, -NH2, CN, C1-C3alkyl; and R6 is independently selected from H, halogen, CN, NO2, C1-C3 alkoxy, straight or branched C1-C3 alkyl.
16. The combined composition according to claim 15, wherein said ARTS mimetic compound of the general formula (I) is further characterized by at least one of:
(a) wherein R1 is H;
(b) wherein R2 is -C(=O)-X-R3;
(c) wherein X is O;
(d) wherein L1 and L2 independently selected independently from each other from -NH- C(=O)-(CH2)n-, -(CH2)n-; and (e) wherein R6 is independently selected from the group consisting of H, halogen, CN, NO2.
17. The combined composition according to claim 15, wherein said ARTS mimetic compound, or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, has the general formula of any one of:
Figure imgf000146_0001
wherein R1, R2, R6, R7, R8, L1 and L2 are as defined in claim 15; or
Figure imgf000146_0002
(c) formula (IV)
Figure imgf000147_0001
18. The combined composition according to claim 15, wherein said compound is 3- [2-(4-Benzyl-piperazin- 1 -yl)-acetylamino] -5-chloro- 1 H-indole-2-carboxylic acid methyl ester, said compound has the structure of Formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000148_0001
19. The combined composition according to claim 14, wherein at least one of said ARTS mimetic compounds is having the general formula (X), or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, or a pharmaceutically acceptable salt or hydrate thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, wherein formula (X) is:
Figure imgf000148_0002
wherein
R1, R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is L1’-R3’-L1”-R3” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R2 is L2’-R4’-L2”-R4”; or
R1 is L1’-R3’-L1”-R3” and R2 is L2’-R4’-L2”-R4”; wherein R3', R3”, R4’ , R4” is each independently from each other, absent or H, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, heteroaryl, heterocycloalkyl, a ring system containing five to twelve atoms, each optionally substituted; L1’, L1”, L2’ and L2” is each independently from each other, absent or -(CH2)n, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2-,-S-, -S-S-, -S-(CH2)n-; n is an integer selected from any one of 0, 1, 2, 3, 4, 5, each one of L1’, L1”, L2’ and L2” independently from each other, may be optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m- halogen and m is an integer selected from 0, 1, 2, 3, 4, 5, said composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
20. The combined composition according to claim 19, wherein said compound is (S)- N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide, having the structure of formula 3.2:
Figure imgf000149_0001
(S)-N 1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide.
21. The combined composition according to any one of claims 14 to 20, wherein said topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
22. The combined composition according to any one of claims 14 to 20, wherein said alkaloid agent is anti-mitotic and/or anti-microtubule alkaloid agent, optionally, vincristine, or any derivative or formulation thereof.
23. The combined composition according to any one of claims 14 to 22, said combined composition comprises an effective amount of 3-[2-(4-Benzyl-piperazin-l-yl)- acetylamino]-5-chloro-1H-indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof.
24. The combined composition according to any one of claims 14 to 22, said combined composition comprises an effective amount of 3-[2-(4-Benzyl-piperazin-l-yl)- acetylamino]-5-chloro-1H-indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
25. The combined composition according to any one of claims 14 to 22, said combined composition comprises an effective amount of (S)-N1-(l -hydroxy-3 - phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2-carbonyl)phenyl)oxalamide, or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof.
26. The combined composition according to any one of claims 14 to 22, said combined composition comprises an effective amount of (S)-N1-(l -hydroxy-3 - phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2-carbonyl)phenyl)oxalamide, a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
27. The combined composition according to any one of claims 14 to 26, wherein said composition further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
28. A kit comprising: (a) an effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, wherein said ARTS mimetic compound interacts and binds the BIR3 domain of XIAP, thereby leading to proteasomal degradation of XIAP, optionally in a first dosage form; and
(b) an effective amount of at least one anti-cancer agent, said agent is at least one of a topoisomerase inhibitor and alkaloid agent, optionally in a second dosage form.
29. The kit composition according to claim 28, wherein at least one of said ARTS mimetic compound has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000151_0001
(Formula I); wherein:
R1 is independently selected from H, C1-C3 alkyl; R2 is independently selected from -C(=O)-X-R3, -S(=O)-X-R3' ;
X is a heteroatom independently selected from O; R3 and R33 independently of each other may be selected independently from H, C1-C3 alkyl;
Rs is — L1-R7-L2-RS; L1 and L2, independently of each other, are selected independently from -(CH2)n ; -NH- C(=O)-(CH2)n-, -C(=O)-NH-(CH2)n-; -S-S-(CH2)n-; -O-(CH2)n-; -NH-(CH2)n-; C(=O)- (CH2)n-; -S-(CH2)n-; -NH-S(=O)n-(CH2)n-; n is independently 1 to 3; R7 is a carbocyclic ring or a heterocyclic ring; R8 is a mono cyclic ring system having 5 to 12, optionally substituted with at least one of OH, CF3, halogen, -COOH, -NH2, CN, C1-C3alkyl; and R6 is independently selected from H, halogen, CN, NO2, C1-C3 alkoxy, straight or branched C1-C3 alkyl.
30. The kit according to claim 29, wherein said compound is 3-[2-(4-Benzyl- piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2-carboxylic acid methyl ester, has the structure of formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000152_0001
31. The kit according to claim 28, wherein at least one of said ARTS mimetic compounds has the general formula (X), or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, or a pharmaceutically acceptable salt or hydrate thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, wherein formula (X) is:
Figure imgf000152_0002
wherein
R1, R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is L1’-R3’-L1”-R3” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R2 is L2’-R4’-L2”-R4”; or
R1 is L1’-R3’-L1”-R3” and R2 is L2’-R4’-L2”-R4”; wherein R3’, R3”, R4’, R4” is each independently from each other, absent or H, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, heteroaryl, heterocycloalkyl, a ring system containing five to twelve atoms, each optionally substituted; L1’, L1”, L2’ and L2” is each independently from each other, absent or -(CH2)n, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2- -S-, -S-S-, -S-(CH2)n-; n is an integer selected from any one of 0, 1, 2, 3, 4, 5, each one of L1’, L1”, L2’ and L2” independently from each other, may be optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m- halogen and m is an integer selected from 0, 1, 2, 3, 4, 5, said composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
32. The kit according to claim 31, wherein said compound is (S)-N1-(1-hydroxy-3- phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2-carbonyl)phenyl)oxalamide, having the structure of formula 3.2:
Figure imgf000153_0001
(S)-N 1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide.
33. The kit according to any one of claims 28 to 32, wherein said topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
34. The kit according to any one of claims 28 to 33, wherein said alkaloid agent is anti-mitotic and/or anti-microtubule alkaloid agent, optionally, vincristine, or any derivative or formulation thereof.
35. The kit according to claim 34, comprising an effective amount of 3-[2-(4-Benzyl- piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof.
36. The kit according to claim 33, comprising an effective amount of 3-[2-(4-Benzyl- piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
37. The kit according to claim 34, comprising, comprising an effective amount of (S)- N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide, or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of vincristine, or any derivative or formulation thereof.
38. The kit according to claim 33, comprising an effective amount of (S)-N1-(1- hydroxy-3-phenylpropan-2-yl)-N2-(4-(l -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide, or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and an effective amount of Topotecan, or any derivative or formulation thereof.
39. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof, the method comprising the step of administering to said subject a therapeutically effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition and/or kit comprising at least one of said ARTS mimetic compound, wherein said ARTS mimetic compound interacts and binds the BIR3 domain of XIAP, thereby leading to proteasomal degradation of XIAP, and wherein said neoplastic disorder is neuroblastoma.
40. The method according to claim 39, wherein at least one of said ARTS mimetic compound has the general formula (I), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000155_0001
(Formula I); wherein:
R1 is independently selected from H, C1-C3 alkyl; R2 is independently selected from -C(=O)-X-R3, -S(=O)-X-R3';
X is a heteroatom independently selected from O; R3 and R33 independently of each other may be selected independently from H, C1-C3 alkyl;
Rs is — L1-R7-L2-RS; L1 and L2, independently of each other, are selected independently from -(CH2)n ; -NH- C(=O)-(CH2)n-, -C(=O)-NH-(CH2)n-; -S-S-(CH2)n-; -O-(CH2)n-; -NH-(CH2)n-; C(=O)- (CH2)n-; -S-(CH2)n-; -NH-S(=O)n-(CH2)n-; n is independently 1 to 3; R7 is a carbocyclic ring or a heterocyclic ring; R8 is a mono cyclic ring system having 5 to 12, optionally substituted with at least one of OH, CF3, halogen, -COOH, -NH2, CN, C1-C3alkyl; and R6 is independently selected from H, halogen, CN, NO2, C1-C3 alkoxy, straight or branched C1-C3 alkyl.
41. The method according to claim 40, wherein said ARTS mimetic compound of the general formula (I) is further characterized by at least one of:
(a) wherein R1 is H;
(b) wherein R2 is -C(=O)-X-R3;
(c) wherein X is O;
(d) wherein L1 and L2 independently selected independently from each other from -NH- C(=O)-(CH2)n-, -(CH2)n-; and
(e) wherein R6 is independently selected from the group consisting of H, halogen, CN, NO2.
42. The method according to claim 40, wherein said ARTS mimetic compound, or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof has the general formula of any one of:
(a) formula (II) (Formula II)
Figure imgf000156_0001
wherein R1, R2, R6, R7, R8, L1 and L2 are as defined in claim 40; or
(b) formula (III)
Figure imgf000157_0001
43. The method according to claim 40, wherein said compound is 3-[2-(4-Benzyl- piperazin-l-yl)-acetylamino]-5-chloro-1H-indole-2-carboxylic acid methyl ester, said compound has the structure of formula (g), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof:
Figure imgf000158_0001
44. The method according to claim 39, wherein at least one of said ARTS mimetic compounds has the general formula (X), or a pharmaceutically acceptable salt, solvate, hydrate, any stereoisomer thereof or physiologically functional derivative thereof, or a pharmaceutically acceptable salt or hydrate thereof, or any vehicle, matrix, nano- or micro-particle comprising the same, wherein formula (X) is:
Figure imgf000158_0002
wherein
R1, R2 are each, independently from each other, absent or alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is L1’-R3’-L1”-R3” and R2 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups; or
R1 is alkyl, alkenyl, alkynyl, alkenylene, a ring system containing five to twelve atoms, or a substituted version of any of these groups and R2 is L2’-R4’-L2”-R4”; or R1 is L1’-R3’-L1”-R3” and R2 is L2’-R4’-L2”-R4”; wherein R3’, R3”, R4’, R4” is each independently from each other, absent or H, alkyl, alkylene, alkenyl, alkenylene, alkynyl, alkynylene, aryl, arylalkyl, heteroaryl, heterocycloalkyl, a ring system containing five to twelve atoms, each optionally substituted; L1’, L1”, L2’ and L2” is each independently from each other, absent or -(CH2)n, -NH-(CH2)n- ,C(=O), C(=O)-(CH2)n-, -O-, -SO2- -S-, -S-S-, -S-(CH2)n-; n is an integer selected from any one of 0, 1, 2, 3, 4, 5, each one of L1’, L1”, L2’ and L2” independently from each other, may be optionally substituted by one or more of C1-C5alkoxy, C1-C5 carboxylic acid, -(CH2)m-OH, -(CH2)m-SH, -(CH2)m-NH2, -(CH2)m- halogen and m is an integer selected from 0, 1, 2, 3, 4, 5, said composition optionally further comprises at least one pharmaceutically acceptable carrier/s, excipient/s, auxiliaries, and/or diluent/s.
45. The method according to claim 44, wherein said compound is (S)-N1-(1-hydroxy-
3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2-carbonyl)phenyl)oxalamide, having the structure of formula 3.2:
Figure imgf000159_0001
(S)-N 1 -( 1 -hydroxy-3 -phenylpropan-2-yl)-N2-(4-( 1 -methyl- 1 H-imidazole-2- carbonyl)phenyl)oxalamide.
46. The method according to any one of claims 39 to 45, wherein said neoplastic disorder affecting the neural system and/or neural cell is a high-risk neuroblastoma.
47. The method according to any one of claims 39 to 46, wherein said ARTS mimetic compound prolongs the overall survival (OS) of said subjects.
48. The method according to any one of claims 39 to 47, wherein said method further comprises the step of administering to said subject an effective amount of at least one anti-cancer agent.
49. The method according to claim 48, wherein said anti-cancer agent is at least one of a topoisomerase inhibitor and alkaloid agent.
50. The method according to claim 49, wherein said topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
51. The method according to claim 49, wherein said alkaloid agent is anti-mitotic and/or anti-microtubule alkaloid agent, optionally, vincristine, or any derivative or formulation thereof.
52. A method for treating, preventing, inhibiting, reducing, eliminating, protecting or delaying the onset of at least one neoplastic disorder affecting the neural system and/or neural cell in a subject in need thereof, the method comprising the step of administering to said subject a therapeutically effective amount of at least one ARTS mimetic compound or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer thereof, or any vehicle, matrix, nano- or micro-particle and/or composition and/or kit comprising at least one of said ARTS mimetic compound, wherein said ARTS mimetic compound interacts and binds the BIR3 domain of XIAP, thereby leading to proteasomal degradation of XIAP, wherein said subject is a subject treated with at least one anti-cancer agent, said agent is at least one of a topoisomerase inhibitor and alkaloid agent, and wherein said neoplastic disorder is neuroblastoma.
53. The method according to claim 52, wherein said ARTS mimetic compound is 3- [2-(4-Benzyl-piperazin- 1 -yl)-acetylamino] -5-chloro- 1 H-indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and wherein said alkaloid agent is vincristine, or any derivative or formulation thereof.
54. The method according to claim 52, wherein said ARTS mimetic compound is 3- [2-(4-Benzyl-piperazin- 1 -yl)-acetylamino] -5-chloro- 1 H-indole-2-carboxylic acid methyl ester (formula (g)), or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and wherein said topoisomerase inhibitor is Topotecan, or any derivative or formulation thereof.
55. The method according to claim 52, wherein said ARTS mimetic compound is (S)- N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide, or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and wherein said alkaloid agent is vincristine.
56. The method according to claim 52, wherein said ARTS mimetic compound is (S)- N1-(1-hydroxy-3-phenylpropan-2-yl)-N2-(4-(1-methyl-1H-imidazole-2- carbonyl)phenyl)oxalamide, or a pharmaceutically acceptable salt or hydrate thereof or any stereoisomer or salt thereof, and wherein said topoisomerase inhibitor is Topotecan.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017077535A1 (en) * 2015-11-02 2017-05-11 Carmel-Haifa University Economic Corporation Ltd. Apoptosis related protein in the tgf-beta signaling pathway (arts) mimetic compounds, compositions, methods and uses thereof in induction of differentiation and/or apoptosis of premalignant and malignant cells, thereby restoring their normal-like phenotype
WO2020047668A1 (en) * 2018-09-05 2020-03-12 The University Of British Columbia Myc-max inhibitor compound therapeutics for cancer treatment, methods and uses associated therewith
WO2021064180A1 (en) * 2019-10-03 2021-04-08 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and compositions for modulating macrophages polarization

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017077535A1 (en) * 2015-11-02 2017-05-11 Carmel-Haifa University Economic Corporation Ltd. Apoptosis related protein in the tgf-beta signaling pathway (arts) mimetic compounds, compositions, methods and uses thereof in induction of differentiation and/or apoptosis of premalignant and malignant cells, thereby restoring their normal-like phenotype
WO2020047668A1 (en) * 2018-09-05 2020-03-12 The University Of British Columbia Myc-max inhibitor compound therapeutics for cancer treatment, methods and uses associated therewith
WO2021064180A1 (en) * 2019-10-03 2021-04-08 INSERM (Institut National de la Santé et de la Recherche Médicale) Methods and compositions for modulating macrophages polarization

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